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WO2024186140A1 - Method for detecting target nucleic acid using artificial nucleic acid - Google Patents

Method for detecting target nucleic acid using artificial nucleic acid Download PDF

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Publication number
WO2024186140A1
WO2024186140A1 PCT/KR2024/002935 KR2024002935W WO2024186140A1 WO 2024186140 A1 WO2024186140 A1 WO 2024186140A1 KR 2024002935 W KR2024002935 W KR 2024002935W WO 2024186140 A1 WO2024186140 A1 WO 2024186140A1
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nucleic acid
artificial
target nucleic
mediator
sequence
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PCT/KR2024/002935
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French (fr)
Korean (ko)
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강경찬
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강경찬
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Priority claimed from KR1020230049884A external-priority patent/KR102678676B1/en
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Publication of WO2024186140A1 publication Critical patent/WO2024186140A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/6853Nucleic acid amplification reactions using modified primers or templates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a method for detecting a target nucleic acid using an artificial nucleic acid.
  • Detection of target nucleic acids is generally performed for disease diagnosis, confirmation of viral or bacterial infection, detection of biological indicator factors, etc.
  • target nucleic acids are amplified using PCR (polymerase chain reaction) to determine the presence and amount of target nucleic acids in a sample.
  • a method that detects amplified products by performing electrophoresis after PCR amplification and using intercalators (DNA binding dyes) such as ethidium bromide, SYBR Green I, and EvaGreen a method that detects amplified products simultaneously with PCR amplification.
  • the real-time PCR method is a technology that detects target nucleic acids simultaneously with amplification, allowing for more accurate quantitative analysis by accurately knowing the initial concentration of the target nucleic acids.
  • amplification and detection are performed in a sealed reaction tube, the possibility of contamination is low, resulting in high detection accuracy and reliability.
  • Real-time PCR methods use the intercalator described above to detect the amplification product of the target nucleic acid, or use a hydrolyzable probe such as a TaqMan probe (U.S. Patent Nos. 5,210,015 and 5,538,848), or use hybridization probes such as a molecular beacon (Tyagi et al, Nature Biotechnology v14 MARCH 1996), HyBeacon (French DJ et al, Mol Cell Probes, 15(6):363-374, 2001), or two hybridization probes each labeled with a donor and an acceptor (Bernad et al, 147-148 Clin Chem 2000; 46).
  • a hydrolyzable probe such as a TaqMan probe (U.S. Patent Nos. 5,210,015 and 5,538,848)
  • hybridization probes such as a molecular beacon (Tyagi et al, Nature Biotechnology v14 MARCH 1996), HyBeacon (French DJ et
  • the molecular beacons or Taqman probes when detecting target nucleic acids using hydrolyzable probes such as Taqman probes in real-time PCR or hybridizable probes such as molecular beacons, the molecular beacons or Taqman probes must be designed and manufactured to have complementary sequences according to the target nucleic acid sequence.
  • the target nucleic acids of the sample to be detected exist in nature, their sequences are already determined, and when designing and manufacturing Taqman probes or molecular beacons, it is necessary to search for suitable sites to which these Taqman probes or molecular beacons will bind.
  • the amplified products of the multiple target nucleic acids are detected by performing melting curve analysis so that they have different Tm (Melting temperatures).
  • Tm Melting temperatures
  • the different Tm can be controlled only by the length as long as the target nucleic acid sequence is already determined.
  • W16041591 (Title: Nucleic acid target identification by structure based probe cleavage) disclose methods for detecting target nucleic acids by detecting flap fragments or their processed products by exonucleases, etc., which are generated only when a target nucleic acid is present, using a flap oligonucleotide, through mass spectroscopy, etc.
  • the flap portion of the flap oligonucleotide is composed of an artificial sequence that is non-complementary to the target nucleic acid, but the portion that acts as the probe must be manufactured to have a complementary sequence specific to the target nucleic acid.
  • FIG. 1(A) discloses a method for detecting a target nucleic acid by using a flap oligonucleotide to construct a sequence such that a U-shaped region complementary to each other exists at the 3' end of a flap fragment (referred to as Snapback oligonucleotide in the specification of this patent) that is generated only when a target nucleic acid is present, and further constructing a sequence such that a hydrolyzable probe binds to a single-stranded region of the flap fragment, so that the hydrolyzable probe is decomposed as the sequence is extended from the 3' end of the U-shaped region to generate a signal.
  • the flap oligonucleotide must be manufactured so that its probe region has a complementary sequence specific to the target nucleic acid.
  • WO13115442A1 (Name: Detection of target nucleic acid squences by PTO cleavage and extension-dependentant signaling oligonucleotide hybridization assay), International Patent Publication No. WO13133561A1 (Name: Detection of nucleotide variation on target nucleic acid sequence by by PTO cleavage and extension assay), International Patent Publication No.
  • WO13157821A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent signaling oligonucleotide cleavage), Internationally published patent WO13187628A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent transcription), Internationally published patent WO14104818A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent non-hybridization assay), International Publication Patent WO15008985A1 (Name: Detection of target nucleic acid squences by PTO cleavage and extension-dependant immobilized oligonucleotide hybridization), International Publication Patent WO15057008A2 (Detection of target nucleic acid squences on solid phase by PTO cleavage and extension using HCTO assay) , international open patent WO
  • Wadle S et al. Real-time PCR probe optimization using design of experiments approach, Biomolecular Detection and Quantification 7 (2016), 1-8] Lehnert M et al., Fluorescence signal-to-noise optimization for real-time PCR using universal reporter oligonucleotides, June 2018Analytical Methods 10(28), DOI:10.1039/C8AY00812D], Wadle S et al., Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters, Biotechniques (2016), Vol. 61, no.
  • the mediator oligonucleotide (which is the same as the flap oligonucleotide) is composed of a probe portion that binds complementarily to the target nucleic acid and an mediator portion (B) that is comprised of an artificial random sequence that is cleaved by a thermostable DNA polymerase having nuclease activity and acts as a primer for an artificial template.
  • the mediator oligonucleotide must be designed and manufactured to have a probe portion that is complementary to the target nucleic acid to be detected.
  • the present invention discloses a method for detecting a target nucleic acid using a mediator oligonucleotide, which is an artificial nucleic acid that is not limited by the sequence of the target nucleic acid to be detected (generic), without the need to design and manufacture the mediator oligonucleotide (i.e., a flap oligonucleotide) according to each target nucleic acid.
  • a mediator oligonucleotide which is an artificial nucleic acid that is not limited by the sequence of the target nucleic acid to be detected (generic), without the need to design and manufacture the mediator oligonucleotide (i.e., a flap oligonucleotide) according to each target nucleic acid.
  • the purpose of the present invention is to provide a method for detecting a target nucleic acid by detecting mediator fragments, which are artificial nucleic acids, through mass spectrometry or the like.
  • Another object of the present invention is to provide a method for detecting a target nucleic acid by detecting a signal generated in the process of converting a target nucleic acid into an artificial nucleic acid using a mediator fragment.
  • Another object of the present invention is to provide a method for detecting a target nucleic acid by detecting an artificial nucleic acid converted by the above method.
  • Another object of the present invention is to provide a method for detecting a target nucleic acid by detecting an additional artificial nucleic acid produced by relying on an artificial nucleic acid converted by the above method, or by detecting a signal generated when such an additional artificial nucleic acid is produced.
  • the present invention relates to a method for detecting a target nucleic acid using a mediator fragment (or flap fragment) in one aspect.
  • flap oligonucleotide and mediator oligonucleotide are used interchangeably with the same meaning, and the terms flap fragment and mediator fragment are also used interchangeably with the same meaning.
  • the method for detecting a target nucleic acid using a mediator fragment of the present invention is characterized by using, as illustrated in FIG. 2, a forward primer having an artificial random sequence tag (hereinafter, "5' tag sequence” or “5' tag portion”) at the 5' end, a conventional reverse primer, and a single-stranded mediator oligonucleotide including a probe portion (A, probe region) that binds complementarily to the 5' tag sequence and a mediator portion (B, mediator region or flap region) consisting of an artificial random sequence.
  • the mediator portion (B, flap region) may be located at the 5' side (upstream) or the 3' side (downstream) of the probe portion (A, probe region), or may be located at both sides.
  • the mediator portion (B, flap region) is preferably located at the 5' side (upstream) of the probe portion (A, probe region).
  • the 5' tag sequence is shown in FIG. 2 as being present in the forward primer, the 5' tag sequence may be present in either or both of the forward primer and the reverse primer.
  • the artificial random sequence refers to an artificially determined random sequence.
  • This artificial random sequence may be a sequence different from the target nucleic acid to be detected or a sequence different from any and all nucleic acids including the target nucleic acid present in the sample.
  • the sequence different from any and all nucleic acids including the target nucleic acid present in the sample may be not only an artificially constructed sequence but also a sequence existing in nature.
  • sequences of photosynthesis-related gene sequences of plants are sequences existing in nature.
  • the mediator oligonucleotide complementarily binds to the 5' tag sequence of the primer through its probe portion.
  • a minor groove binder MGB
  • MGB minor groove binder
  • the MGB moiety increases the Tm, thereby increasing the binding affinity of the probe to the target nucleic acid (in the present invention, the binding affinity between the 5' tag sequence and the probe portion of the mediator oligonucleotide), and ultimately increasing the binding specificity.
  • MGB is usually a small molecule, and examples of such compounds include polyamide, netropsin, distamycin, and thiazotropsin A.
  • a strand having a 5' tag sequence is synthesized by a forward primer having a 5' tag sequence.
  • a primer i.e., a reverse primer
  • the mediator oligonucleotide binds to the 5' tag sequence of the strand through the probe portion (A), which is a portion complementary to the 5' tag sequence.
  • the mediator oligonucleotide bound to the 5' tag sequence of the strand is decomposed, thereby releasing a fragment (mediator fragment) of the mediator portion (B) consisting of an artificial random sequence.
  • a DNA polymerase such as Taq polymerase having 5'-->3' exonuclease activity cleaves the bond between the first and second nucleotides of the probe region (cuts between AT in FIG. 3), so the intermediate fragment includes one nucleotide (A in FIG.
  • intermediate fragment means that, in addition to the sequence of the intermediate region (B), one nucleotide at the 5' end of the probe region is included at its 3' end.
  • DNA polymerases such as Taq polymerase having the above 5'-->3' exonuclease activity also have bifurcated duplex structure-specific flap endonuclease activity. Unless otherwise specifically stated, the nuclease activity is used herein as a term meaning both the 5'-->3' exonuclease activity and the flap endonuclease activity.
  • the mediator oligonucleotide is not decomposed, the mediator portion is not cleaved, and thus the mediator fragment is not released.
  • the mediator portion (B) in the mediator oligonucleotide may be located at the 3' side (downstream) of the probe portion (A) or may be located on both sides (upstream and downstream) of the probe portion (A). Even if the mediator portion (B) is located at the 3' side or both sides of the probe portion (A), if the target nucleic acid is present in the sample, the mediator fragment is extracted, and if the target nucleic acid is not present, the mediator fragment is not extracted. Therefore, the mediator portion (B) in the mediator oligonucleotide may be located at the 3' side or both sides of the probe portion (A). If located at both sides, the probe portions (B) may have the same sequence, different sequences, or complementary sequences.
  • the target nucleic acid can be detected by determining the presence or absence of the flap fragments by detecting the base sequence (nucleotide order) and/or the length of the released mediator fragment.
  • the target nucleic acid can be detected by determining the presence or absence of the flap fragments by detecting the base sequence (nucleotide order) and/or the length of the released mediator fragment.
  • multiple detection is possible if different mediator oligonucleotides are used according to the target nucleic acid. If different mediator oligonucleotides are used according to the target nucleic acid, the 5' tag site to which the probe site of the mediator oligonucleotide specifically binds will also vary depending on the target nucleic acid.
  • Detection of released mediator fragments can be accomplished by performing mass spectrometry, electrophoresis, liquid chromatography, sequence analysis, microarray, etc.
  • Mass spectrometry can use methods such as MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry), Tandem Mass Spectrometry (Tandem MS), ESI-TOF (Electrospray Ionization-Time of Flight), Ion Trap Mass Spectrometry, LC-MS (Liquid Chromatography Mass Spectrometry), GC-MS (Gas Chromatography Mass Spectrometry), and IM-MS (Ion Mobility Mass Spectrometry).
  • a method for detecting a target nucleic acid by detecting a processed product of the intermediate fragment by mass spectrometry is disclosed. Therefore, the methods disclosed in the above international published patent can be applied to detecting a target nucleic acid by detecting the intermediate fragment of the present invention.
  • Electrophoresis allows the mediator oligonucleotides, which are not cleaved at the mediator site, to be detected separately from the mediator fragments that are produced only when the target nucleic acid is present, since they are of a different size.
  • This electrophoresis can also utilize the single-strand conformation polymorphism (SSCP) method (Natural Protocols, 1(6):3121-3128, 2006).
  • SSCP single-strand conformation polymorphism
  • Next generation sequencing (NGS) technology can be used to analyze the sequence of the mediator fragments. If next generation sequencing technology is used, the mediator fragments are counted, so not only qualitative detection of the target nucleic acid but also quantitative detection can be possible.
  • US Patent Publication No. 2008-0241838 (title: Methods and systems for detecting nucleic acids) can be used.
  • the method disclosed in US Patent Publication No. 2008-0241838 is a method for detecting a flap fragment by labeling a signal substance at the 5' end (the 5' end of the flap portion) of a flap oligonucleotide and fixing a capture probe complementary to the flap portion to a support.
  • the length of the capture probe is adjusted short so that when a target nucleic acid is present in a sample, the flap fragment binds to the capture probe, and when the target nucleic acid is not present, the uncleaved flap oligonucleotide does not bind, thereby detecting the flap fragment (see FIG. 4 of US Patent Publication No. 2008-0241838).
  • the mediator fragment may be capable of detecting an artificial nucleic acid generated by extension from the mediator fragment, as described below, or detecting a signal that changes during the process of generating the artificial nucleic acid, or generating an additional artificial nucleic acid (a second artificial nucleic acid) dependent on the artificial nucleic acid (the first artificial nucleic acid) and detecting the additional artificial nucleic acid generated dependent on the artificial nucleic acid, or detecting a signal that changes during the generation of the additional artificial nucleic acid.
  • the artificial nucleic acid generated by extension from the mediator fragment is also generated only when a target nucleic acid is present in the sample and the mediator fragment is generated, thereby enabling detection of the mediator fragment. In this way, when the target nucleic acid is converted into an artificial nucleic acid or an additional artificial nucleic acid and detected using the mediator fragment, PCR-based real-time detection is possible.
  • the method for detecting the target nucleic acid of the present invention by converting it into an artificial nucleic acid or an additional artificial nucleic acid is characterized by using, as illustrated in FIG. 5, a forward primer having a 5' tag portion, a conventional reverse primer, a single-stranded mediator oligonucleotide including a probe portion (A, probe region) that binds complementarily to the 5' tag sequence, a mediator portion (B, mediator region) consisting of an artificial random sequence, and an artificial template.
  • the artificial template may not exist separately as in the method disclosed in the international patent publication WO06122208 (title: Snapback oligonucleotide probe) disclosed in Fig. 1(A), but the cleaved mediator itself may act as the artificial template.
  • the 3' end of the cleaved mediator is extended, thereby decomposing the hydrolyzable probe and changing the signal, and the target nucleic acid can be detected by detecting the changed signal.
  • the cleaved mediator itself can act as an artificial template, as illustrated in FIG. 5, it is preferable to use a single-stranded nucleic acid of a separate molecule as an artificial template.
  • the artificial template has a priming region (B') having a sequence complementary to a mediator fragment that is cleaved from the mediator oligonucleotide by a DNA polymerase having nuclease activity during the amplification process of the target nucleic acid and that acts as a primer for the production of an artificial nucleic acid, and also has a template region (C') that acts as a template for an artificial nucleic acid (C, artificial nucleic acid), which is a newly synthesized and extended sequence.
  • B' priming region having a sequence complementary to a mediator fragment that is cleaved from the mediator oligonucleotide by a DNA polymerase having nuclease activity during the amplification process of the target nucleic acid and that acts as a primer for the production
  • the mediator oligonucleotide bound to the 5' tag sequence of the strand is decomposed, releasing mediator fragments consisting of an artificial random sequence.
  • the mediator oligonucleotide is not decomposed, so the mediator fragment is not released, and then an artificial nucleic acid having a sequence (C) complementary to the template portion (C') of the artificial template is not newly generated.
  • the mediator oligonucleotide is not decomposed, so even if it binds to the artificial template, the template portion (C') of the artificial template and the probe portion (A) that complementarily binds to the 5' tag sequence of the mediator oligonucleotide are not complementary to each other, so they remain unbound, and thus an artificial nucleic acid having a sequence (C) complementary to the template region (C') of the artificial template is not newly synthesized.
  • ddNTP di-deoxynucleotide triphosphate
  • inverted dNTP inverted dNTP
  • HOG hexathylene glycol
  • a mediator fragment consisting of an artificial random sequence is released from a mediator oligonucleotide in proportion to the concentration of the target nucleic acid in the sample, and therefore, when this mediator fragment acts as a primer for the artificial template, an artificial nucleic acid which is a newly generated sequence (C) complementary to the template portion (C') of the artificial template is also generated in proportion to the concentration of the target nucleic acid in the sample.
  • the mediator oligonucleotide does not have a sequence complementary to the target nucleic acid, but has a sequence complementary to a 5' tag sequence consisting of an artificial random sequence of the forward primer. Therefore, by configuring this 5' tag sequence as an artificial sequence, the mediator oligonucleotide sequence can be artificially controlled and configured, and the mediator oligonucleotide sequence controlled in this way can be universally used regardless of the target nucleic acid sequence to be detected, so that the mediator oligonucleotide sequence (in particular, the sequence of the probe portion (A) complementarily binding to the target nucleic acid among these sequences) does not need to be individually designed and manufactured according to the target nucleic acid sequence to be detected.
  • the target nucleic acid when a target nucleic acid is present in a sample, a mediator oligonucleotide is decomposed, and a mediator fragment of an artificial random sequence released therefrom acts as a primer on an artificial template and is extended, thereby converting the target nucleic acid into an artificial nucleic acid.
  • the target nucleic acid can be detected by (i) detecting a signal generated during the conversion (i.e., a signal generated when the artificial nucleic acid is generated), (ii) detecting the converted artificial nucleic acid, (iii) detecting an additional artificial nucleic acid generated depending on the converted artificial nucleic acid, or detecting a signal generated when such an additional artificial nucleic acid is generated.
  • the template portion of the artificial template has a hairpin structure (stem-loop structure)
  • the mediator fragment released from the mediator oligonucleotide binds to the artificial template and acts as a primer to extend the sequence therefrom
  • the hairpin structure unfolds
  • the reporter molecule is separated from the quencher molecule, and a signal is generated. Since the amount of this signal generation is proportional to the target nucleic acid in the sample, quantitative information on the target nucleic acid can be obtained by measuring this signal.
  • the 5' tag sequence of the forward primer is configured differently depending on the target nucleic acids
  • the mediator sequence of the mediator oligonucleotide that will act as a primer in the artificial template is configured differently depending on the target nucleic acids
  • the priming site to which the mediator binds in the artificial template is configured differently
  • the reporter molecule is configured to generate different fluorescence signals at the template site (C') of the artificial template having a hairpin structure (by labeling the template site (C') of the artificial template with reporter molecules having different emission wavelengths), thereby enabling detection of multiple target nucleic acids.
  • the template site of the artificial template may not vary its sequence depending on the target nucleic acids but may be configured with the same sequence. Even if they are composed of the same sequence, different signals are generated depending on the target nucleic acid.
  • the length and/or GC content of the sequence of the template portion (C') of the artificial template taking a hairpin structure is configured differently depending on the target nucleic acids, and the hybrids of the template portion (C') of the artificial template and the artificial nucleic acid (a sequence newly extended complementary to the template portion) are configured to have different Tms, thereby performing melting curve analysis, so that detection of multiple target nucleic acids becomes possible even when a single fluorescence signal is used.
  • the sequence of the template portion (C') of the artificial template can be artificially controlled, it can be very easy to design each hybridization derived from each target nucleic acid in the sample to have a different Tm. This melting curve analysis is performed by lowering the temperature after the completion of the PCR reaction so that all target nucleic acids become two-stranded hybrids, and then gradually raising the temperature.
  • the above melting curve analysis detects multiple target nucleic acids by using a single fluorescence signal after the end of a PCR reaction, but as illustrated in FIG. 8 and disclosed in International Application No. PCT/KR2014/012074 (WO 2015 147412, titled: Detection of Target Nucleic Acid Sequences Using Different Detection Temperatures) and International Application No. PCT/KR2019/004780 (WO 2019 203623, titled: Method and Apparatus for Detecting a Plurality of Target Nucleic acid Sequences in Sample), it is also possible to detect multiple target nucleic acids by using a single fluorescence signal in real time during a PCR reaction.
  • the length and/or GC content of the template portion (C') of the artificial template that takes a hairpin structure are configured differently depending on the target nucleic acids, so that the hybridization states of the artificial template and the artificial nucleic acid for the two target nucleic acids are both maintained at a relatively low temperature, and one of the hybrids dissociates at a relatively high temperature (at this time, if dissociated, it takes on a hairpin structure again and the signal disappears), so that they have different Tms.
  • the signal detected at the relatively low temperature becomes a signal for the abundance of both target nucleic acids
  • the signal detected at the relatively high temperature becomes a signal for the abundance of one of the two target nucleic acids. Therefore, by subtracting the signal detected at the relatively high temperature from the signal detected at the relatively low temperature, a detection signal for the abundance of the remaining target nucleic acid can be obtained. By repeating this signal detection and subtraction for each cycle, two target nucleic acids can be detected simultaneously in real time.
  • detection of three or more target nucleic acids can also be possible.
  • the mediator fragment released from the mediator oligonucleotide binds to the artificial template, acts as a primer, and the sequence is extended, and a DNA polymerase such as Taq polymerase exhibits 5'-->3' exonuclease activity to decompose the hydrolyzable probe bound to the template portion (C').
  • a DNA polymerase such as Taq polymerase exhibits 5'-->3' exonuclease activity to decompose the hydrolyzable probe bound to the template portion (C').
  • the reporter molecule that was being quenched by the quencher molecule is separated from the quencher molecule, generating a signal. Since the amount of this signal generation is also proportional to the target nucleic acid in the sample, quantitative information on the target nucleic acid can be obtained by measuring this signal.
  • the artificial template and the hydrolyzable probe may be configured as a single strand in which the 5' end of the artificial template and the 3' end of the hydrolyzable probe are connected to each other. Even if configured as a single strand in this way, when the mediator fragment binds to the artificial template and acts as a primer to extend the sequence, the reporter molecule, which was being quenched by the quencher molecule, is separated from the quencher molecule by the 5'-->3' exonuclease activity of a DNA polymerase such as Taq polymerase, thereby generating a signal.
  • a DNA polymerase such as Taq polymerase
  • the reporter molecule in the hydrolyzable probe is configured to generate different fluorescence signals (by labeling the hydrolyzable probe with reporter molecules having different emission wavelengths), detection of multiple target nucleic acids becomes possible.
  • a hydrolyzable probe such as a TaqMan probe
  • an artificial nucleic acid which is a new extended sequence
  • a primer (C-p) complementary to the new extended sequence is used to extend the sequence
  • a signal is generated when the hydrolyzable probe is decomposed by a DNA polymerase, such as Taq polymerase, and by measuring the generated signal, quantitative information on the target nucleic acid can be obtained.
  • the primer (C-p) complementary to the new extended sequence, which is an artificial nucleic acid binds to the new extended sequence, which is an artificial nucleic acid, only when the artificial nucleic acid is generated, and the extension reaction occurs, and since the artificial nucleic acid is generated only when the target nucleic acid exists in the sample, the signal generated when the hydrolyzable probe bound to the artificial nucleic acid is decomposed reflects quantitative information on the target nucleic acid in the sample.
  • the new extended sequence which is an artificial nucleic acid
  • a reactant including the new extended sequence with a complementary sequence fixed to a support (substrate, bead, etc.) and then treating the reactant with a probe labeled with a detection signal, and then washing and removing unbound nucleic acids, etc. with a washing solution and measuring the detection signal
  • the new extended sequence which is an artificial nucleic acid
  • This is a method that applies a sandwich detection method of a microarray known in the art.
  • any method known in the art for nucleic acid detection may be applied.
  • an intercalator such as ethidium bromide may be used to detect the double strand of the artificial template and the artificial nucleic acid during the PCR reaction and/or after the completion of the PCR reaction.
  • the length and GC of the double strand of the newly generated artificial template and the artificial nucleic acid may be adjusted by using an artificial template according to each target nucleic acid.
  • a 5' tag portion (D, 5' tag portion of the mediator oligonucleotide) of an artificial sequence is additionally introduced at the 5' end of the mediator oligonucleotide
  • a portion (D', complementary portion of the 5' tag portion) complementary to the 5' tag portion (D) can also be generated only if a target nucleic acid exists.
  • an artificial nucleic acid (C) which is a new extended sequence, is generated only when a target nucleic acid exists in the sample, and only when this artificial nucleic acid (C) is generated can a complementary primer (C-p) bind to it, and when this primer (C-p) binds, sequence extension occurs therefrom, so that a complementary portion (D') of the 5' tag portion (D) of the mediator oligonucleotide can be generated.
  • the complementary portion (D') of the 5' tag portion (D) of the mediator oligonucleotide is an additional artificial nucleic acid (D', a second artificial nucleic acid) generated depending on the artificial nucleic acid (C, the first artificial nucleic acid), which is a new extended sequence, and when this additional artificial nucleic acid is detected or a signal that changes as this additional artificial nucleic acid is generated is detected by applying the method described with reference to FIGS. 8 to 12, qualitative and quantitative information about the target nucleic acid in the sample can be obtained. For example, this is a case where the method illustrated in FIG.
  • an additional artificial nucleic acid (a second artificial nucleic acid) can also be generated by extending from the 3' end of the artificial nucleic acid (the first artificial nucleic acid) using an additional artificial template (indicated as Second CTO in the drawing), as illustrated in FIG. 2 of international patent publication WO19066461A2 (title: Detection of target nucleic acid sequences by PTO cleavage and extension-dependent extension assay).
  • Wadle S et al. Real-time PCR probe optimization using design of experiments approach, Biomolecular Detection and Quantification 7 (2016), 1-8] Lehnert M et al., Fluorescence signal-to-noise optimization for real-time PCR using universal reporter oligonucleotides, June 2018Analytical Methods 10(28), DOI:10.1039/C8AY00812D], Wadle S et al., Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters, Biotechniques (2016), Vol. 61, no.
  • the mediator oligonucleotide in the present invention binds to the 5' tag site of an artificial random sequence
  • the only difference in the international published patents and papers is that the mediator oligonucleotide in the present invention binds to a sequence (internal sequence) between the sites where the forward primer and the reverse primer bind in the target nucleic acid. Therefore, in the above international published patents and papers, as in the present invention, when a target nucleic acid is present in a sample, a mediator oligonucleotide is cleaved and the cleaved mediator fragment acts as a primer in an artificial template.
  • the target nucleic acid in the sample can be detected by (i) detecting a signal generated by extension from the mediator fragment (i.e., a signal generated by generating an artificial nucleic acid), (ii) detecting the artificial nucleic acid, or (iii) detecting an additional artificial nucleic acid generated depending on the artificial nucleic acid, or detecting a signal generated by generating such an additional artificial nucleic acid.
  • a signal generated by extension from the mediator fragment i.e., a signal generated by generating an artificial nucleic acid
  • the reverse primer may have a tag sequence composed of an artificial random sequence at the 5' end.
  • the reverse primer having the 5' tag sequence is extended in the first cycle of PCR, and then the forward primer binds to the extended strand in the next cycle of PCR, and further, when a mediator oligonucleotide binds to the 5' tag sequence of the extended strand and an extension reaction occurs by a DNA polymerase such as Taq polymerase, the mediator fragment is cleaved from the mediator oligonucleotide bound to the 5' tag sequence by the nuclease activity of the polymerase such as Taq polymerase, so that the generation of artificial nucleic acids increases by two times compared to when the 5' tag sequence is present only in the forward primer, and thus the sensitivity of detection can be doubled.
  • the 5' tag sequence of the reverse primer is composed of the same sequence as the 5' tag sequence in the forward primer
  • the mediator oligonucleotide sequence that binds thereto is composed of the same sequence as the mediator oligonucleotide sequence that binds to the 5' tag sequence of the forward primer.
  • two or more 5' tag sequences may be configured in the forward primer and/or the reverse primer.
  • the binding site of the mediator oligonucleotide increases depending on the number of 5' tag sequences present, so the production of artificial nucleic acids increases proportionally (i.e., the copy number of the artificial nucleic acids increases), thereby further increasing the sensitivity of detection.
  • the target nucleic acid can be converted into an artificial nucleic acid by using an auxiliary forward primer having the same sequence as the 5' tag sequence and an additional tag sequence (5' additional tag sequence) at the 5' end of the 5' tag sequence, and the target nucleic acid can be detected.
  • the 5' additional tag sequence can be composed of the same sequence as the 5' tag sequence or a different sequence.
  • the auxiliary primer is used for the reverse primer when the 5' tag sequence is present in the reverse primer.
  • the 5' additional tag sequence is configured with the same sequence as the 5' tag sequence, the binding sites of the mediator oligonucleotide increase, and the production of artificial nucleic acids ultimately increases, making it easier to detect the target nucleic acid.
  • the 5' additional tag sequence is composed of a sequence different from the 5' tag sequence, and the probe portion has a different sequence but the intermediate portion has the same sequence, the specificity increases and the production of artificial nucleic acids increases, so that the detection sensitivity of the target nucleic acid can be increased.
  • an auxiliary forward primer of a different configuration can be used.
  • This auxiliary forward primer of a different configuration is a short primer having the same sequence as the 5' tag sequence, and will act as a forward primer competitively with the forward primer having the 5' tag sequence for the complementary strand to the strand to which the 5' tag sequence is attached, the copy number of which gradually increases as the number of amplification cycles increases, but will act as a primer more efficiently due to its shorter sequence length.
  • a spacer consisting of 1 to 10 nucleotides may be present between the 5' tag sequence and the region complementary to the target nucleic acid.
  • two or more types of a forward primer having a 5' tag portion and/or a reverse primer having a 5' tag portion may be used with different specific binding sites of each of these primers for one target nucleic acid.
  • These two or more types of primers only differ in their specific binding sites with respect to the target nucleic acid, and the 5' tag portions are all composed of the same sequence, so that when a mediator oligonucleotide having the same sequence is used, the binding sites of the mediator oligonucleotide increase, and thus the production of artificial nucleic acids increases, which can ultimately have the effect of amplifying the detection signal.
  • binding sites of two or more mediator oligonucleotides are created without determining the binding sites of the mediator oligonucleotides in the sequence (internal sequence) between the forward primer and the reverse primer, so that it can be useful for the detection of target nucleic acids with high mutations, such as RNA viruses, and can also be useful for the detection of target nucleic acids with a low copy number in a sample (i.e., target nucleic acids with a low concentration in a sample).
  • the sensitivity of detection can be increased for the detection of a single infectious microorganism (bacteria or virus), such as in the detection of coronavirus (SARS-CoV-2, severe acute respiratory syndrome type 2), which ultimately enables detection even when the number of target microorganisms in the sample is small.
  • bacteria or virus such as in the detection of coronavirus (SARS-CoV-2, severe acute respiratory syndrome type 2)
  • the present invention can be understood in one aspect as a method for detecting a target nucleic acid using a flap fragment (or a mediator fragment).
  • a single-stranded mediator oligonucleotide comprising a probe portion (A) that binds complementarily to the 5' tag sequence and a mediator portion (B) consisting of an artificial random sequence specific for a target nucleic acid;
  • step (b) a step of placing the mixture of step (a) into a reaction vessel and performing a PCR reaction so that the target nucleic acid is amplified and at the same time, a mediator fragment is cleaved from the mediator oligonucleotide by a DNA polymerase having the nuclease activity, and
  • the mediator portion consisting of an artificial random sequence specific to the target nucleic acid means that at least one of the base sequence (nucleotide order) and size is specific to the target nucleic acid.
  • the 5' tag portion of the primer will also be complementarily configured according to the mediator oligonucleotide sequence (particularly, the sequence of the probe portion).
  • the detection of the mediator fragment can be performed by mass spectrometry, electrophoresis, liquid chromatography, sequence analysis, microarray, artificial nucleic acid generated by extension from the mediator fragment, etc., as described above.
  • the artificial nucleic acid generated by extension from the mediator fragment can be generated by (i) extending from the 3' end of the mediator fragment by the mediator fragment itself acting as an artificial template as described above with reference to Fig. 1(A), or (ii) extending from the 3' end of the mediator fragment by the mediator fragment complementarily binding to an artificial template of a separate molecule to act as a primer as described with reference to Fig. 6.
  • the artificial template of the separate molecule is additionally included in the mixture for the PCR reaction, and the artificial template has a priming site complementary to the mediator segment of the mediator oligonucleotide and also has a template site (C') that acts as a template for the artificial nucleic acid generated by extension from the mediator segment.
  • C' template site
  • the present invention relates to a method for converting a target nucleic acid into an artificial nucleic acid.
  • the method for converting the artificial nucleic acid of the present invention comprises steps (a) and (b) below.
  • a single-stranded mediator oligonucleotide comprising a probe portion (A) complementarily binding to the 5' tag sequence and a mediator portion (B) consisting of an artificial random sequence;
  • an artificial template having a priming site (B') complementary to the mediator site (B) of the above mediator oligonucleotide and a template site (C') that acts as a template for an artificial nucleic acid, which is a newly extended sequence (C);
  • step (b) a step of placing the mixture of step (a) into a reaction vessel, performing a PCR reaction, so that the target nucleic acid is amplified and at the same time, a mediator fragment is generated from the mediator oligonucleotide by the DNA polymerase having the nuclease activity, and the mediator fragment binds to the priming site of the artificial template and is extended using the template site of the artificial template as a template, thereby generating an artificial nucleic acid having a sequence complementary to the template site of the artificial template.
  • the present invention can be understood as a method for detecting a target nucleic acid of a sample to be detected by converting it into an artificial nucleic acid.
  • the method for detecting a target nucleic acid of the present invention comprises steps (a) to (c) below.
  • a single-stranded mediator oligonucleotide comprising a probe portion (A) complementarily binding to the 5' tag sequence and a mediator portion (B) consisting of an artificial random sequence;
  • step (b) a step of placing the mixture of step (a) into a reaction vessel, performing a PCR reaction, so that the target nucleic acid is amplified and at the same time, a mediator fragment is generated from the mediator oligonucleotide by the DNA polymerase having the nuclease activity, and the mediator fragment binds to the priming site of the artificial template and is extended using the template site of the artificial template as a template, thereby generating an artificial nucleic acid having a sequence complementary to the template site of the artificial template;
  • a step of detecting a target nucleic acid in a sample by (i) detecting a signal that changes as the artificial nucleic acid is generated or (ii) detecting the generated artificial nucleic acid.
  • the 5' tag sequence may be present in one or more of the forward and reverse primers, or in both of these primers.
  • all of the 5' tag sequences may be composed of the same sequence so as to enable the use of one type of mediator oligonucleotide.
  • a step is included of generating an artificial nucleic acid (second artificial nucleic acid) dependent on the artificial nucleic acid (first artificial nucleic acid) or an additional artificial nucleic acid from the artificial nucleic acid, and detection of the target nucleic acid may be accomplished by (i) detecting the additional artificial nucleic acid generated dependent on the artificial nucleic acid, or (ii) detecting a signal that changes as the additional artificial nucleic acid is generated.
  • the nuclease activity includes 5'-->3' exonuclease activity and flap endonuclease activity.
  • detecting a signal that changes while an artificial nucleic acid (first artificial nucleic acid) is generated specifically includes the following cases (i) to (iii).
  • the artificial template and the hydrolyzable probe are composed of a single strand in which the 5' end of the artificial template and the 3' end of the hydrolyzable probe are connected to each other, and the mediator fragment binds to the priming site of the artificial template and is extended by a DNA polymerase having a 5'-->3' exonuclease activity, thereby detecting a signal generated by the hydrolyzable probe being decomposed.
  • detecting an artificial nucleic acid specifically includes cases (i) to (iii) below.
  • the detection of a signal that changes as the additional artificial nucleic acid (second artificial nucleic acid) is generated is specifically as follows:
  • a hydrolyzable probe that specifically binds to the 5' tag portion of the intermediate oligonucleotide and a primer that specifically binds to the artificial nucleic acid are included in the PCR reaction mixture, so that the primer bound to the artificial nucleic acid is extended by a DNA polymerase having a 5'-->3' exonuclease activity, thereby detecting a signal generated by decomposition of the hydrolyzable probe bound to the 5' tag portion of the intermediate oligonucleotide.
  • detecting the additional artificial nucleic acid is as follows: using the intermediate oligonucleotide having an artificial sequence tag portion (5' tag portion) introduced at the 5' end thereof, a molecular beacon that specifically binds to a sequence portion complementary to the 5' tag portion (complementary portion of the 5' tag portion) and a primer that specifically binds to the artificial nucleic acid (first artificial nucleic acid) are included in the PCR reaction mixture, so that the primer bound to the artificial nucleic acid (first artificial nucleic acid) is extended depending on the artificial nucleic acid (first artificial nucleic acid) using the artificial nucleic acid (first artificial nucleic acid) as a template, thereby generating an additional artificial nucleic acid (second artificial nucleic acid) including a complementary portion of the 5' tag portion, and detecting a signal generated by the specific binding of the molecular beacon to the complementary portion of the 5' tag portion.
  • the signal change may be caused by a change in the distance between the reporter molecule and the quencher molecule or by a change in the distance between the donor and the acceptor.
  • the PCR reaction mixture may contain an auxiliary primer having the same sequence as the 5' tag sequence, as illustrated in FIG. 16.
  • the PCR reaction mixture may contain two or more primers having the 5' tag portion with different specific binding positions for each target nucleic acid.
  • the 5' tag portions all have the same sequence, the same mediator oligonucleotide is bound to all of them, and as a result, the detection of a target nucleic acid with a low copy number or a severe mutation in the sample can be facilitated.
  • the target nucleic acids are two or more, and for each target nucleic acid, a forward primer and a reverse primer are included in the mixture, wherein at least one primer among the forward primers and reverse primers for each target nucleic acid has a 5' tag portion, and all of the 5' tag portions may be composed of the same sequence.
  • all of the 5' tag portions have the same sequence, all of the same mediator oligonucleotides bind, and as a result, even if the number of target infectious agent microorganisms (viruses or bacteria) in the sample is small, detection can be easy because the detection signals of two or more target nucleic acids are generated as the same signal.
  • the portion of the forward and/or reverse primers that bind to the target nucleic acid, the 5' tag portion of the forward and/or reverse primers, the mediator portion that acts as a primer for an artificial template in the mediator oligonucleotide, and the probe portion complementary to the 5' tag portion in the mediator oligonucleotide may each have any length from 15 to 50 nucleotides as long as sequence specificity is ensured (i.e., as long as it can specifically bind to the complementary sequence). It may preferably have a length of 15 to 25 nucleotides.
  • the primer, the mediator oligonucleotide, the 5' tag moiety, etc. may be RNA as well as DNA as long as it does not affect their function in the present invention (when the oligonucleotide of the present invention is RNA, "T" in the base sequence is read as "U"), and may also be nucleic acid analogues such as PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid), HNA (Hexitol Nucleic Acids), ANA (Altritol Nucleic Acids), MNA (Mannitol Nucleic Acids) which are stable to nuclease or heat.
  • PNA Peptide Nucleic Acid
  • LNA Locked Nucleic Acid
  • HNA Heexitol Nucleic Acids
  • ANA Altritol Nucleic Acids
  • MNA Merannitol Nucleic Acids
  • the primer, the mediator oligonucleotide, the 5' tag moiety, etc. may also include modified nucleotides as well as natural nucleotides as long as it does not affect their function in the present invention.
  • modified nucleotides may be those modified in their sugars, phosphates and/or bases. Nucleotides modified in such sugars, phosphates and/or bases are specifically known in the art, including methods for their preparation.
  • nucleotides modified in sugars include those in which the hydroxyl group (OH group) of the sugar is modified with a halogen group, an aliphatic group, an ether group, an amine group, etc., those in which the sugar ribose or deoxyribose itself can be replaced by sugar analogues such as ⁇ -anomeric sugars, arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, etc.
  • modifications in phosphate include modifications of phosphate to P(O)S(thioate), P(S)S(dithioate), P(O)NR2(amidate), P(O)R, P(O)OR', CO or CH2(formacetal).
  • R or R' is H or substituted or unsubstituted alkyl, etc., and when modified in phosphate, the linking group becomes -O-, -N-, -S- or -C-, and adjacent nucleotides are bonded to each other through this linking group.
  • the complementary sequence of the above primer, mediator oligonucleotide, etc. to its 5' tag portion, target nucleic acid, artificial template, etc. need not be 100% complementary as long as it confers specific binding ability, and may be at least about 80%, preferably at least about 85%, more preferably at least about 90%, 95% or 99%.
  • the sample may be any mixture or solution that contains or is suspected of containing the target nucleic acid to be detected and thus has a need for detection.
  • the sample may be a biological sample obtained from a human or animal, as well as a processed sample obtained by processing such a biological sample to increase the concentration of the target nucleic acid, and further may be a sample that requires inspection, such as a sample suspected of containing an environmental pollutant or toxic factor, such as water, food, or industrial wastewater that contains or is suspected of containing the target nucleic acid, or an organic matter sample obtained from a food processing device.
  • a sample may include an appropriate diluent, a buffer solution, and when it is desired to detect the presence of bacteria or viruses, it may be a bacterial culture or virus culture containing a medium or a medium component.
  • the sample may preferably be a biological sample obtained from a human or animal or a processed sample thereof.
  • the biological sample may be obtained from a human or animal that contains or is suspected of containing the target nucleic acid to be detected, such as whole blood, serum, plasma, umbilical cord blood, urine, feces, saliva, nasal mucus, semen, amniotic fluid, lavage fluid (bronchial alveolar, stomach, peritoneal, ear, etc.), lymph, sputum, tissue, cell, etc., and thus has a need for detection.
  • the processed sample may be, for example, plasma, serum, a biological sample whose concentration of nucleic acid (DNA and/or RNA) is increased using a nucleic acid extraction kit, a tissue extract, a cell obtained from a tissue, a cell lysate, a cell culture, a bacterial culture, a virus culture, etc.
  • a nucleic acid extraction kit for example, plasma, serum, a biological sample whose concentration of nucleic acid (DNA and/or RNA) is increased using a nucleic acid extraction kit, a tissue extract, a cell obtained from a tissue, a cell lysate, a cell culture, a bacterial culture, a virus culture, etc.
  • the target nucleic acid refers to any nucleic acid whose detection can bring about some usefulness, such as diagnosis or prognosis of a disease, confirmation of a biological indicator, etc., by detecting or measuring the presence and/or amount of the target nucleic acid.
  • This target nucleic acid may be understood as a double-stranded DNA containing sequences complementary to forward and reverse primers.
  • This double-stranded DNA may be genomic DNA in a sample, particularly a biological sample, a nucleic acid synthesized from a primer for detecting SNP (Single nucleotide polymerization), a nucleic acid of a sample treated with bisulfite for detecting the presence or absence of DNA methylation, or cDNA generated by reverse transcription from RNA such as mRNA, rRNA, or micro RNA.
  • a sample particularly a biological sample
  • a nucleic acid of a sample treated with bisulfite for detecting the presence or absence of DNA methylation or cDNA generated by reverse transcription from RNA such as mRNA, rRNA, or micro RNA.
  • the primer is an oligonucleotide capable of inducing the initiation of synthesis of a target nucleic acid, and is composed of a forward primer and a reverse primer for each target nucleic acid.
  • the primer is designed to specifically bind to the target nucleic acid (the actual target nucleic acid in the sample or the artificial nucleic acid generated depending on the target nucleic acid) and to have a melting temperature sufficient to maintain binding to the target nucleic acid during the extension reaction of the PCR reaction.
  • the target nucleic acid the actual target nucleic acid in the sample or the artificial nucleic acid generated depending on the target nucleic acid
  • a melting temperature sufficient to maintain binding to the target nucleic acid during the extension reaction of the PCR reaction.
  • the mediator oligonucleotide is designed to have a sequence complementary to the 5' tag sequence of the forward and/or reverse primers so as to specifically bind and have a melting temperature sufficient to maintain binding to the target nucleic acid during the extension reaction of the PCR reaction.
  • the design of such mediator oligonucleotides can be carried out according to the methods known in the art with respect to probe design, such as [Unit Evol. Genet. 2005; 5:1-9]; [Afr. Jo. Biotechnol. 2003; 2:91-95], [Methods Mol. Biol. 1993; 15:31-40], [PCR Methods Appl. 1993; 3:S30-S37], [Nucleic Acids Res.
  • one cycle of the PCR reaction may consist of two steps, a denaturation step and annealing and extension steps, or three steps, a denaturation step, annealing and extension steps.
  • the denaturation step may be performed at a specific temperature in the range of 93 to 98°C
  • the annealing and extension steps may be performed at a specific temperature in the range of 54 to 75°C.
  • the denaturation step may be performed at a specific temperature in the range of 93 to 98°C
  • the annealing step may be performed at a specific temperature in the range of 54 to 60°C
  • the extension step may be performed at a specific temperature in the range of 70 to 75°C.
  • the reporter molecule is a substance that generates a signal, wherein the signal is generally a fluorescent signal.
  • any fluorescent substance for generating a fluorescent signal as a reporter molecule can be appropriately selected and used from among those known in the art by taking into consideration the detection wavelength of each detection channel of the PCR device, etc.
  • Known fluorescent substances include, for example, Cy2TM (506), YO-PROTM-1 (509), YOYOTM-1 (509), Calcein (517), FITC (518), FluorXTM (519), AlexaTM (520), Rhodamine 110 (520), Oregon GreenTM 500 (522), Oregon GreenTM 488 (524), RiboGreenTM (525), Rhodamine GreenTM (527), Rhodamine 123 (529), Magnesium GreenTM (531), Calcium GreenTM (533), TO-PROTM-1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY TMR (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3TM (570), AlexaTM 546 (570
  • the above reporter molecule can be used as a pair of donor and receptor by selecting two appropriate ones with different emission wavelengths.
  • the fluorescence emitted from the donor is converted into fluorescence of a lower wavelength by FRET (Fluorescence Resonance Energy Transfer) at the acceptor adjacent to the donor, and then emitted and extinguished.
  • FRET Fluorescence Resonance Energy Transfer
  • the emission wavelength of the donor is usually measured to measure the signal change according to the presence or absence of the target nucleic acid.
  • a quencher molecule is a substance that absorbs, generates heat, and extinguishes fluorescence emitted by a reporter molecule when it is adjacent to the reporter molecule.
  • Many such quencher molecules are known in the art. Among these known quencher molecules, one having an appropriate absorption wavelength can be selected and used in consideration of the emission wavelength of the reporter molecule used.
  • Known quencher molecules include Dabcyl (453), QSY 35 (475), BHQ-0 (495), Eclipse (530), BHQ-1 (534), QSY 7 (560), QSY 9 (562), BHQ-2 (579), ElleQuencher (630), Iowa Black (651), QSY 21 (661), BHQ-3 (672), etc.
  • the numbers in parentheses are the maximum absorption wavelengths in nanometers.
  • Cy5/BHQ-2, Cy5/BHQ-1, TexRed/BHQ-2, TexRed/QSY 7, TexRed/BHQ-1, TexRed/Dabcyl, Cy3/BHQ2, Cy3/QSY 7, Cy3/BHQ-1, Cy3/Dabcyl, TET/BHQ-2, TET/QSY 7, TET/TAMRA, TET/BHQ-1, FAM/BHQ-2, FAM/QSY 7, FAM/TAMRA, FAM/BHQ-1, FAM/Dabcyl are known to have high quenching efficiency ( Nucliec Acids Res. 30, e122, 2002), so it may be desirable to select and use one of these pairs.
  • Such references include: Methods Mol Biol. 335, 17-29, 2006; Biotechniques 31, 1106-1121, 2001; J. Am Chem. Soc. 124, 6950-6956, 2002; Chem. Eur. J. 9, 3466-3471, 2003; Nucleic Acids Res. 30, e122, 2002; J. Phys. Chem. 100, 5541-5553, 1996;
  • the mixture for the PCR reaction of the step (a) includes, in addition to the primer set, mediator oligonucleotide, and artificial template as described above, four NTPs, DNA polymerase, and other factors such as Tris-HCl as a pH stabilizer, MgCl2 or MgSO4 or (NH4)2SO4 as an auxiliary factor for promoting enzyme activity, BSA (Bovine Serum Albumin), gelatin, glycerol, or PEG 6000 as a factor for stabilizing DNA polymerase, KCl as a factor for promoting binding of primers to target nucleic acids, Tween 20 or Triton X-100 as a nonionic surfactant for suppressing nonspecific amplification, spermidine for reducing nonspecific binding of DNA polymerase to nucleic acids, urea or DMSO (dimethylsulfoxide) as a factor for promoting annealing of primers to target nucleic acids, or It may also contain additional ingredients such as D
  • the DNA polymerase uses a thermostable polymerase having 5'-->3' exonuclease activity and flap endonuclease activity, and this thermostable polymerase is selected from the group consisting of Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococc
  • DNA polymerase may be used in a modified form as long as it has 5'-->3' exonuclease activity and flap nuclease activity.
  • Modified polymerases include those in which some sequences are artificially modified or naturally modified (mutated).
  • the DNA polymerase is preferably Taq polymerase.
  • the PCR reaction amplifies the target nucleic acid by repeatedly performing the denaturation step, the annealing step, and the extension step for two or more cycles, usually 15 to 50 cycles.
  • An initial denaturation step may be included before performing these cycles.
  • This initial denaturation step is performed to activate DNA polymerase and denature template DNA.
  • This initial denaturation step may be performed within a range of 30 seconds to 20 minutes, taking into account the GC content of the template DNA, etc.
  • an elongation step may be included. This elongation step is for the DNA polymerase to finish its final elongation reaction, and is usually performed at the same temperature as the elongation step of the above cycle for about 1 to 20 minutes.
  • a mediator oligonucleotide used for converting a target nucleic acid in a sample into an artificial nucleic acid can be universally configured without being limited by the sequence of the target nucleic acid to be detected, and while universally configured, the sequence can be artificially controlled, and an artificial nucleic acid generated therefrom can also be artificially controlled, so that it is possible to artificially configure the sequence so that the detection effect is optimally and maximally increased (sensitivity and specificity are increased).
  • Figure 1 is a conceptual diagram of a method for detecting a target nucleic acid by converting the target nucleic acid into an artificial nucleic acid, as disclosed in international published patents, such as international published patents WO06122208 and WO12096430A1.
  • Figure 2 illustrates the structures of a forward primer, a reverse primer, and a mediator oligonucleotide having a 5' tag portion, which are components of the present invention.
  • Figure 3 shows that a mediator fragment is generated when a target nucleic acid is present in a sample in a method according to the present invention.
  • Figure 4 shows that in the method according to the present invention, artificial nucleic acids are not generated when no target nucleic acid exists in the sample.
  • Figure 5 illustrates the components of the present invention (structures of a forward primer having a 5' tag portion, a reverse primer, a mediator oligonucleotide, and an artificial template) when detecting a target nucleic acid by converting the target nucleic acid into an artificial nucleic acid.
  • Figure 6 is a conceptual diagram of a method for detecting a target nucleic acid by converting it into an artificial nucleic acid.
  • Figure 7 shows that in the method according to the present invention, artificial nucleic acids are not generated when no target nucleic acid exists in the sample.
  • Figures 8 and 9 illustrate conceptual diagrams of methods for detecting target nucleic acids in a sample by detecting signals generated when artificial nucleic acids are produced.
  • Figures 10 to 12 illustrate conceptual diagrams of methods for detecting target nucleic acids in a sample by detecting generated artificial nucleic acids.
  • Figure 13 is a conceptual diagram illustrating a method for detecting a target nucleic acid in a sample by detecting additional artificial nucleic acids produced dependent on artificial nucleic acids or by detecting signals that change during the process of producing such additional artificial nucleic acids.
  • Figure 14 is a conceptual diagram showing that, in addition to the forward primer, the reverse primer can also have a tag site at the 5' end.
  • Figure 15 is a conceptual diagram showing that more than two 5' tag sites may be present in the forward primer and/or the reverse primer.
  • Figure 16 is a conceptual diagram of a method for detecting a target nucleic acid by converting the target nucleic acid into an artificial nucleic acid using an auxiliary forward primer having the same sequence as the 5' tag sequence and an additional tag sequence at the 5' side of the 5' tag sequence or an auxiliary forward primer having the same sequence as the 5' tag sequence.
  • Figure 17 is a conceptual diagram of using two or more forward primers having a 5' tag portion and/or reverse primers having a 5' tag portion for one target nucleic acid, with different binding positions with respect to the target nucleic acid.
  • Figure 18 is a conceptual diagram of using a forward primer having a 5' tag portion and/or a reverse primer having a 5' tag portion for two target nucleic acids, respectively.
  • FIG. 19 is a result showing that a target nucleic acid can be detected by detecting a signal generated in the process of converting a target nucleic acid into an artificial nucleic acid according to the conceptual diagram of the present invention of FIGS. 9 and 17.
  • FIG. 20 is a result showing that a target nucleic acid can be detected by detecting an artificial nucleic acid generated from a target nucleic acid according to the conceptual diagram of the present invention of FIG. 11.
  • FIG. 21 is a result showing that a target nucleic acid can be detected by detecting a signal generated in the process of generating an additional artificial nucleic acid that is produced depending on the artificial nucleic acid according to the conceptual diagram of the present invention of FIG. 13.
  • FIG. 22 is a result showing that when two forward primers specific for each target nucleic acid, each having an identical 5' tag portion, are used together to detect two target nucleic acids, the detection signal is amplified and detection can be facilitated, according to the conceptual diagram of the present invention of FIGS. 9 and 18.
  • the target nucleic acid was synthesized from the gene of Neisseria gonorrhoeae (NG), the causative agent of sexually transmitted diseases.
  • the sequence of the target nucleic acid is as follows.
  • the 5' tag portion of the forward primer used an artificially constructed sequence, 5' -GCTACCCAGCCGGCTACAAG-3'.
  • sequence of the complementary portion of the forward primer to the target nucleic acid was 5'-TCCGACGTTTCGGTTGTG-3'.
  • the forward primer sequence with a 5' tag portion is 5' - GCTACCCAGCCGGCTACAAG TCCGACGTTTCGGTTGTGTT-3', where the portion in bold is the 5' tag portion.
  • the reverse primer used was 5'-GCTCCTTATTCGGTTTGACCGG-3'.
  • the mediator oligonucleotide is composed of a region complementary to the 5' tag region as 5' - CTTGTAGCCGGCTGGGTAGC -3', and a mediator sequence that is released when a target nucleic acid is present in the sample and acts as a primer for the artificial template as 5' - ACGACGGCTTGGCATTGC -3'. Therefore, the entire sequence of the mediator oligonucleotide is as follows.
  • the underlined portion is a mediator sequence that is cleaved and released by the nuclease activity of DNA polymerase when a target nucleic acid is present.
  • the portion in bold is a sequence complementary to the 5' tag portion of the forward primer.
  • a total reaction volume of 20 ⁇ l containing 1x PCR buffer, 150 pg/ ⁇ l of synthetic template for the NG gene, 3 ⁇ l of each of forward and reverse primers with 5' tag sites, 5 ⁇ l of 5'-labeled mediator oligonucleotide, 3 mM MgCl2, 400 ⁇ l of dNTPs, and 1.5 unit of Taq Polymerase was used as follows: the reaction mixture was denatured at 95 ° C. for 10 minutes, followed by 30 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 60 ° C. for 60 seconds, and extension, and a final extension for 3 minutes.
  • the PCR reaction product was purified through a desalting process, and mass analysis was performed using MALTI-TOF MS.
  • the PCR reaction product was purified through a desalting process, and mass analysis was performed using MALTI-TOF MS.
  • the target nucleic acid and forward and reverse primers were the same as those used in Example 1.
  • the mediator oligonucleotide was used as follows, the 3' end of which was blocked with HEG (hexathylene glycol) to prevent extension reaction.
  • the underlined portion is a mediator sequence that acts as a primer for each artificial template and complementarily binds to the 3' end of the artificial template.
  • the bold portion is a sequence complementary to the 5' tag portion of the forward primer.
  • the 3' end of this mediator oligonucleotide is blocked with HEG (hexathylene glycol) so that an extension reaction does not occur.
  • the template portion (C') is the portion in bold, and the underlined portion is the portion where the mediator sequence, which is released from the mediator oligonucleotide and acts as a primer for the artificial template, binds.
  • the 3' end is blocked with HEG (hexathylene glycol).
  • a TaqMan probe having a sequence complementary to the underlined region of the template region (C') and a reporter molecule and a quencher molecule of the following configuration was used.
  • Taxman probe sequence and composition are as follows.
  • the second forward primer sequence with a 5' tag region is as follows.
  • the 5' tag portion (bold text) of the second forward primer has the same sequence as the 5' tag portion of the forward primer (first forward primer).
  • the fluorescence signal was measured for each cycle and is shown in Figure 19.
  • Fig. 19 (A) shows the result when a forward primer without a 5' tag portion was used
  • Fig. 19 (B) shows the result when no mediator oligonucleotide was present in the PCR reaction
  • Fig. 19 (C) shows the result when no artificial template was present in the PCR reaction
  • Fig. 19 (D) shows the result when a forward primer with a tag portion was used and both a mediator oligonucleotide and an artificial template were used
  • Fig. 19 (E) shows the result when both a mediator oligonucleotide and an artificial template were used according to the schematic diagram of Fig. 17 and two types of forward primers with tag portions were used with different binding positions to the target nucleic acid.
  • a target nucleic acid can be detected by measuring a signal generated when it is converted into an artificial nucleic acid, and that, when two forward primers having tag portions are used at different binding positions with respect to the target nucleic acid, a signal can appear more quickly (a signal can be amplified).
  • a forward primer (first forward primer) having a 5' tag portion of the same configuration as in Example 2 above, a mediator oligonucleotide, and an artificial template were used, and a TaqMan probe having the following sequence and configuration was used.
  • a primer (C-p) specific for an artificial nucleic acid having the following sequence was used.
  • Taxman Probe 5' -[BHQ-1] CTCCTCCTCCTCCTCCTCCA [FAM] -3'
  • TaxMan probe is the sequence of the underlined portion in the artificial template of Example 2.
  • the PCR reaction was performed in the same manner as in Example 2, and the results are shown in Fig. 20.
  • Fig. 20 (A) shows the result when a forward primer without a 5' tag portion was used
  • Fig. 20 (B) shows the result when a mediator oligonucleotide was not present in the PCR reaction
  • Fig. 20 (C) shows the result when an artificial template was not present in the PCR reaction
  • Fig. 20 (D) shows the result when a primer (C-p) specific for an artificial nucleic acid was not present in the PCR reaction
  • Fig. 20 (E) shows the result when a forward primer having a tag portion, a mediator oligonucleotide, an artificial template, a TaqMan probe, and a primer (C-p) specific for an artificial nucleic acid were all present in the PCR reaction.
  • Figure 20 shows that, according to the present invention, a target nucleic acid can be detected by detecting an artificial nucleic acid.
  • a target nucleic acid could be detected by detecting a signal generated in the process of converting an artificial nucleic acid (first artificial nucleic acid) into an additional artificial nucleic acid (second artificial nucleic acid) using a TaqMan probe specific to the 5' tag portion (D) of a mediator oligonucleotide.
  • a forward primer having a 5' tag portion of the same configuration as in Example 2 above, a mediator oligonucleotide (which has a 5' tag portion (D)), an artificial template were used, and the 5' tag portion (D) of the mediator oligonucleotide and a TaqMan probe specific thereto were used having the sequence and configuration below, and in addition, a primer (C-p) specific for an artificial nucleic acid having the sequence below was used.
  • Taxman Probe 5'-[BHQ-1] ATGGCGTATGGCTAGCTG [FAM]-3'
  • the PCR reaction was performed in the same manner as in Example 2, and the results are shown in Fig. 21.
  • Figure 21 (A) shows the result when a mediator oligonucleotide without a 5' tag portion (D) of the mediator oligonucleotide was used
  • Figure 18 (B) shows the result when a mediator oligonucleotide with a 5' tag portion (D) was used.
  • Figure 21 shows that, according to the present invention, a target nucleic acid can be detected by detecting a signal generated in the process of generating additional artificial nucleic acids from artificial nucleic acids.
  • PCR was performed according to the schematic diagram of Fig. 9, as in Example 2, using two forward primers specific for each target nucleic acid, each having an identical 5' tag portion, with a partial sequence of the LacZ gene (target sequence 1) and a partial sequence of the GAPDH gene (target sequence 2) as target nucleic acids, separately or together.
  • sequences of target sequences 1 and 2 are as follows.
  • the 5'- GCTACCCAGCCGGCTACAAG sequence above is the 5' tag sequence.
  • the 5'- GCTACCCAGCCGGCTACAAG sequence above is the 5' tag sequence.
  • the PCR reaction was performed in the same manner as in Example 2 using 150 pg/ul of target sequences 1 and 2, respectively, and the fluorescence signal was measured at each cycle and is shown in Figure 22.
  • Figure 22 (A) is a PCR result of target sequence 1, which is a partial sequence of the LacZ gene
  • Figure 22 (B) is a PCR result of target sequence 2, which is a partial sequence of the GAPDH gene
  • Figure 22 (C) is a PCR result of using two forward primers specific for each target nucleic acid, each having the same 5' tag portion, for target sequence 1, which is a partial sequence of the LacZ gene, and target sequence 2, which is a partial sequence of the GAPDH gene.
  • the present invention can be applied to PCR-based molecular diagnosis, especially to in vitro diagnostic medical devices for nucleic acid detection.

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Abstract

Disclosed is a method for detecting a target nucleic acid using an artificial nucleic acid. Particularly, disclosed is a method for detecting a target nucleic acid, in which, by using a primer with a 5' tag and using a flap oligonucleotide (i.e., mediator oligonucleotide) with a sequence complementary to the 5' tag, flap fragments (i.e., mediator fragments), which are cleaved only when the target nucleic acid is present in a sample, are detected.

Description

인공핵산을 이용한 표적핵산의 검출 방법Method for detecting target nucleic acid using artificial nucleic acid
본 발명은 인공핵산(artificial nucleic acid)을 이용한 표적핵산의 검출 방법에 관한 것이다.The present invention relates to a method for detecting a target nucleic acid using an artificial nucleic acid.
표적핵산의 검출은 일반적으로 질병 진단, 바이러스나 세균의 감염 여부의 확인, 생물학적 지표 인자 검출 등을 위하여 수행된다. 표적핵산을 검출하기 위해서는 표적핵산을 PCR(polymerase chain reaction)로 증폭하여 시료 중의 표적핵산에 대한 존재 여부와 존재량을 결정하게 된다.Detection of target nucleic acids is generally performed for disease diagnosis, confirmation of viral or bacterial infection, detection of biological indicator factors, etc. To detect target nucleic acids, target nucleic acids are amplified using PCR (polymerase chain reaction) to determine the presence and amount of target nucleic acids in a sample.
표적핵산의 검출에는 PCR 증폭 후 전기영동하고 브롬화 에티듐(ethidium bromide), 사이버그린Ⅰ(SYBR Green I), 에바그린(EvaGreen) 등 인터칼레이터(intercalator, DNA binding dyes)를 사용하여 증폭 산물을 검출하는 방법과, PCR 증폭과 동시에 증폭 산물을 검출하는 실시간 PCR(real-time PCR 또는 quantitative PCR) 방법이 있다. For detection of target nucleic acids, there are two methods: a method that detects amplified products by performing electrophoresis after PCR amplification and using intercalators (DNA binding dyes) such as ethidium bromide, SYBR Green I, and EvaGreen, and a real-time PCR (or quantitative PCR) method that detects amplified products simultaneously with PCR amplification.
실시간 PCR 방법은 표적핵산을 증폭과 동시에 검출하는 기술로, 표적핵산의 초기 농도를 정확하게 알 수 있어 더욱 정확한 정량적 분석을 가능하게 하고, 또한 반응 튜브가 밀폐된 상태에서 증폭과 검출이 이루어지므로 오염의 가능성이 낮아 검출의 정확성과 신뢰성이 높다.The real-time PCR method is a technology that detects target nucleic acids simultaneously with amplification, allowing for more accurate quantitative analysis by accurately knowing the initial concentration of the target nucleic acids. In addition, since amplification and detection are performed in a sealed reaction tube, the possibility of contamination is low, resulting in high detection accuracy and reliability.
실시간 PCR 방법은 표적핵산의 증폭 산물 검출을 위하여 전술한 바의 인터칼레이터를 사용하거나, 택맨 프로브(TaqMan probe)(미국 특허 제5,210,015호 및 제5,538,848호) 등의 가수분해성 프로브를 사용하거나, 분자비콘(Molecular beacon)(Tyagi et al, Nature Biotechnology v14 MARCH 1996), 하이비콘(HyBeacon) (French DJ et al, Mol Cell Probes, 15(6):363-374, 2001), 공여체 및 수용체로 각각 표지된 2개의 혼성화 프로브(Bernad et al, 147-148 Clin Chem 2000; 46) 등의 혼성화성 프로브(hybridization probes) 등을 사용한다.Real-time PCR methods use the intercalator described above to detect the amplification product of the target nucleic acid, or use a hydrolyzable probe such as a TaqMan probe (U.S. Patent Nos. 5,210,015 and 5,538,848), or use hybridization probes such as a molecular beacon (Tyagi et al, Nature Biotechnology v14 MARCH 1996), HyBeacon (French DJ et al, Mol Cell Probes, 15(6):363-374, 2001), or two hybridization probes each labeled with a donor and an acceptor (Bernad et al, 147-148 Clin Chem 2000; 46).
그런데 실시간 PCR 등에서 택맨 프로브(Taqman probe) 등 가수분해성 프로브를 이용하거나 분자비콘(molecular beacon) 등 혼성화성 프로브를 이용하여 표적핵산을 검출할 경우, 표적핵산 서열에 따라 이에 상보적 서열을 갖도록 분자비콘이나 택맨 프로브를 설계, 제작하여야 한다. 그런데 검출하고자 하는 시료의 표적핵산은 자연계에 존재하는 것이므로 그 서열이 이미 정해져 있어 택맨 프로브나 분자비콘의 설계, 제작 시에 이들 택맨 프로브나 분자비콘이 결합할 적합한 부위를 탐색하는 것이 필요하다. However, when detecting target nucleic acids using hydrolyzable probes such as Taqman probes in real-time PCR or hybridizable probes such as molecular beacons, the molecular beacons or Taqman probes must be designed and manufactured to have complementary sequences according to the target nucleic acid sequence. However, since the target nucleic acids of the sample to be detected exist in nature, their sequences are already determined, and when designing and manufacturing Taqman probes or molecular beacons, it is necessary to search for suitable sites to which these Taqman probes or molecular beacons will bind.
또한 PCR 종료 후 인터칼레이터를 이용하여 2개 이상의 다중의 표적핵산을 검출할 경우 그 다중 표적핵산의 증폭물이 서로 다른 Tm(Melting temperature)을 가지도록 하여 융해 곡선 분석(melting curve analysis)을 수행하여 검출하게 되는데, 이 경우 서로 다른 Tm은, 표적핵산 서열이 이미 정해져 있는 이상 오로지 길이에 의해서만 조절할 수 있다. In addition, when detecting two or more multiple target nucleic acids using an intercalator after the completion of PCR, the amplified products of the multiple target nucleic acids are detected by performing melting curve analysis so that they have different Tm (Melting temperatures). In this case, the different Tm can be controlled only by the length as long as the target nucleic acid sequence is already determined.
또한 분자비콘을 이용하여 2개 이상의 다중의 표적핵산을, 융해 곡선 분석을 통해 단일 형광 신호로 검출할 경우에도 표적핵산과 분자비콘 혼성화물이 서로 다른 Tm을 가지도록 그 분자비콘을 설계, 제작하는 것이 필요한데, 이러한 설계, 제작에 있어서도 표적핵산 서열이 이미 정해져 있기 때문에 제한이 따른다. In addition, when detecting two or more multiple target nucleic acids with a single fluorescence signal through melting curve analysis using a molecular beacon, it is necessary to design and manufacture the molecular beacon so that the target nucleic acids and molecular beacon hybrids have different Tms. However, there are limitations in this design and manufacture because the target nucleic acid sequence is already determined.
따라서 표적핵산을 인위적 임의 서열과 길이를 가진 인공핵산으로 전환하여 검출할 경우, 그 인공핵산의 서열과 길이를 인위적으로 조절하여 표적핵산에 대한 식별자(identifier)로 사용하는 것이 가능하고 또 표적핵산 서열에 따라 택맨 프로브나 분자비콘 등을 그때 그때 설계, 제작할 필요가 없게 되는 등, 표적핵산을 인공핵산으로 전환시켜 검출하는 것은 여러 장점을 가질 수 있다.Therefore, when a target nucleic acid is converted into an artificial nucleic acid with an artificial random sequence and length and then detected, it is possible to artificially control the sequence and length of the artificial nucleic acid and use it as an identifier for the target nucleic acid, and there is no need to design and manufacture a TaxMan probe or molecular beacon on demand according to the target nucleic acid sequence, etc., so there can be several advantages to converting the target nucleic acid into an artificial nucleic acid and then detecting it.
국제 공개특허 WO08136868(명칭: Detecting and quantification of biomolecules using mass spectrometry), 국제 공개특허 WO09073251(명칭: Detecting and quantification of biomolecules using mass spectrometry), 국제 공개특허 WO14140147(명칭: Nucleic acid target identification by structure based probe cleavage), 국제 공개특허 WO15091765(명칭: Mutiplexed Nucleic acid target identification by structure based probe cleavage), 국제 공개특허 W16041591(명칭: Nucleic acid target identification by structure based probe cleavage)는 플랩(flap) 올리고뉴클레오티드를 이용하여 표적핵산이 존재할 경우에만 생성되는 플랩 절편이나 그것의 엑소뉴클레아제 등에 의한 가공물을 질량 분석(mass spectroscopy) 등을 통해 검출하여 표적핵산을 검출하는 방법을 개시하고 있다. 그러나 이 검출 방법에서 플랩(flap) 올리고뉴클레오티드의 플랩 부위는 표적핵산에 비상보적인 인공서열로 구성되지만, 프로브로 작용하는 부위는 표적핵산에 특이적인 상보적인 서열을 가지도록 제작되어야 한다.International Patent Publication No. WO08136868 (Title: Detecting and quantification of biomolecules using mass spectrometry), International Patent Publication No. WO09073251 (Title: Detecting and quantification of biomolecules using mass spectrometry), International Patent Publication No. WO14140147 (Title: Nucleic acid target identification by structure based probe cleavage), International Patent Publication No. WO15091765 (Title: Mutiplexed Nucleic acid target identification by structure based probe cleavage), International Patent Publication No. W16041591 (Title: Nucleic acid target identification by structure based probe cleavage) disclose methods for detecting target nucleic acids by detecting flap fragments or their processed products by exonucleases, etc., which are generated only when a target nucleic acid is present, using a flap oligonucleotide, through mass spectroscopy, etc. However, in this detection method, the flap portion of the flap oligonucleotide is composed of an artificial sequence that is non-complementary to the target nucleic acid, but the portion that acts as the probe must be manufactured to have a complementary sequence specific to the target nucleic acid.
국제 공개특허 WO06122208(명칭: Snapback oligonucleotide probe)는 도 1(A)에 도시된 바와 같이, 플랩(flap) 올리고뉴클레오티드를 사용하여 표적핵산이 존재할 경우에만 생성되는 플랩 절편(이 특허의 명세서에서는 Snapback oligonucleotide로 언급되어 있음)에 대해서, 그 3' 부위에 서로 상보적인 U자형 부위가 존재하도록 서열을 구성하고 또 가수분해성 프로브가 그 플랩 절편의 단일 가닥 부위에 결합하도록 서열을 구성하여, 상기 U자형 부위의 3' 말단에서 서열이 연장되면서 가수분해성 프로브가 분해되어 신호가 발생하는 원리를 이용한 표적핵산의 검출 방법을 개시하고 있다. 이 방법에 있어서도 플랩(flap) 올리고뉴클레오티드는 그 프로브 부위가 표적핵산에 특이적인 상보적인 서열을 가지도록 제작되어야 한다. International Patent Publication No. WO06122208 (Title: Snapback oligonucleotide probe), as illustrated in FIG. 1(A), discloses a method for detecting a target nucleic acid by using a flap oligonucleotide to construct a sequence such that a U-shaped region complementary to each other exists at the 3' end of a flap fragment (referred to as Snapback oligonucleotide in the specification of this patent) that is generated only when a target nucleic acid is present, and further constructing a sequence such that a hydrolyzable probe binds to a single-stranded region of the flap fragment, so that the hydrolyzable probe is decomposed as the sequence is extended from the 3' end of the U-shaped region to generate a signal. In this method as well, the flap oligonucleotide must be manufactured so that its probe region has a complementary sequence specific to the target nucleic acid.
국제 공개특허 WO12096430A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension assay), 국제 공개특허 WO12096523A2(명칭: Detection of target nucleic acid squences by PTO cleavage and extension assay), 국제 공개특허 WO12134195A2(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant cleavage), 국제 공개특허 WO13115442A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant signaling oligonucleotide hybridization assay), 국제 공개특허 WO13133561A1(명칭: Detection of nucleotide variation on target nucleic acid sequence by by PTO cleavage and extension assay), 국제 공개특허 WO13157821A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant signaling oligonucleotide cleavage), 국제 공개특허 WO13187628A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant transcription), 국제 공개특허 WO14104818A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant non-hybridization assay), 국제 공개특허 WO15008985A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant immobilized oligonucleotide hybridization), 국제 공개특허 WO15057008A2(Detection of target nucleic acid squences on solid phase by PTO cleavage and extension using HCTO assay), 국제 공개특허 WO19066461A2(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant extension assay), 국제 공개특허 WO2018196842(명칭: Method for detecting target nucleic acid sequences), 문헌[Bernd Faltin et al., Mediator Probe PCR: A Novel Approach for Detection of Real-Time PCR Based on Label-Free Primary Probes and Standardized Secondary Universal Fluorogenic Reporters, Clinical Chemistry 58:11 1546-1556 (2012)], 문헌[Wadle S et al., Mediator Probe PCR: Detection of Real-Time PCR by Label-Free Probes and a Universal Fluorogenic Reporter, Methods Mol Biol. (2014), 1160:55-73], 문헌[Wadle S et al., Real-time PCR probe optimization using design of experiments approach, Biomolecular Detection and Quantification 7 (2016), 1-8], 문헌[Lehnert M et al., Fluorescence signal-to-noise optimisation for real-time PCR using universal reporter oligonucleotides, June 2018Analytical Methods 10(28), DOI:10.1039/C8AY00812D], 문헌[Wadle S et al., Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters, Biotechniques (2018), Vol. 61, No. 3], 문헌[Elena Kipf et al., Advanced Minimal Residual Disease Monitoring for Acute Lymphoblastic Leukemia with Multiplex Mediator Probe PCR, J Mol Diagn. 2022 Jan;24(1):57-68], 문헌[Qiuying Huang et al., Highly multiplex PCR assays by coupling the 5'-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters, Proc Natl Acad Sci USA. 2022 Mar 1;119(9):e2110672119] 등은 모두, 도 1(B)에 도시된 바와 같이, 매개체 올리고뉴클레오티드(mediator oligonucleotide)와 인공 주형(artificial template)을 이용하여 표적핵산(target nucleic acid)을 인공핵산(artificial nucleic acid)으로 전환시키고, 그 전환 과정에서 발생하는 신호를 검출하거나, 그 전환된 인공핵산을 검출하거나, 그 인공핵산(제1의 인공핵산)에 의존하여 생성되는 추가의 인공핵산(제2의 인공핵산)을 검출하거나, 그 추가의 인공핵산이 생성되면서 발생하는 신호를 검출함에 의하여 표적핵산을 검출하는 방법을 개시하고 있다. 여기서 매개체 올리고뉴클레오티드(이는 상기 플랩 올리고뉴클레오티드와 같음)는 표적핵산에 상보적으로 결합하는 프로브 부위와 뉴클레아제 활성을 가지는 열안정성 DNA 중합효소에 의하여 절단되어 인공주형에 대해 프라이머로 작용하는 인위적 임의 서열로 이루어진 매개체 부위(B)로 구성된다.International Patent Publication No. WO12096430A1 (Name: Detection of target nucleic acid squences by PTO cleavage and extension assay), International Patent Publication No. WO12096523A2 (Name: Detection of target nucleic acid squences by PTO cleavage and extension assay), International Patent Publication No. WO12134195A2 (Name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent cleavage), International Patent Publication No. WO13115442A1 (Name: Detection of target nucleic acid squences by PTO cleavage and extension-dependentant signaling oligonucleotide hybridization assay), International Patent Publication No. WO13133561A1 (Name: Detection of nucleotide variation on target nucleic acid sequence by by PTO cleavage and extension assay), International Patent Publication No. WO13157821A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent signaling oligonucleotide cleavage), Internationally published patent WO13187628A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent transcription), Internationally published patent WO14104818A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent non-hybridization assay), International Publication Patent WO15008985A1 (Name: Detection of target nucleic acid squences by PTO cleavage and extension-dependant immobilized oligonucleotide hybridization), International Publication Patent WO15057008A2 (Detection of target nucleic acid squences on solid phase by PTO cleavage and extension using HCTO assay) , international open patent WO19066461A2 (Name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent extension assay), International Patent WO2018196842 (Name: Method for detecting target nucleic acid sequences), Literature [Bernd Faltin et al., Mediator Probe PCR: A Novel Approach for Detection of Real-Time PCR Based on Label-Free Primary Probes and Standardized Secondary Universal Fluorogenic Reporters , Clinical Chemistry 58:11 1546-1556 (2012)], Wadle S et al., Mediator Probe PCR: Detection of Real-Time PCR by Label-Free Probes and a Universal Fluorogenic Reporter, Methods Mol Biol. (2014), 1160:55-73], Wadle S et al., Real-time PCR probe optimization using design of experiments approach, Biomolecular Detection and Quantification 7 (2016), 1-8], Lehnert M et al., Fluorescence signal-to-noise optimization for real-time PCR using universal reporter oligonucleotides, June 2018Analytical Methods 10(28), DOI:10.1039/C8AY00812D], Wadle S et al., Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters, Biotechniques (2018), Vol. 61, no. 3], Elena Kipf et al., Advanced Minimal Residual Disease Monitoring for Acute Lymphoblastic Leukemia with Multiplex Mediator Probe PCR, J Mol Diagn. 2022 Jan;24(1):57-68], Qiuying Huang et al., Highly multiplex PCR assays by coupling the 5'-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters, Proc Natl Acad Sci USA. 2022 Mar 1;119(9):e2110672119], etc., all disclose methods for detecting a target nucleic acid by converting a target nucleic acid into an artificial nucleic acid using a mediator oligonucleotide and an artificial template, as illustrated in FIG. 1(B), and detecting a signal generated during the conversion process, detecting the converted artificial nucleic acid, detecting an additional artificial nucleic acid (second artificial nucleic acid) generated dependent on the artificial nucleic acid (first artificial nucleic acid), or detecting a signal generated when the additional artificial nucleic acid is generated. Here, the mediator oligonucleotide (which is the same as the flap oligonucleotide) is composed of a probe portion that binds complementarily to the target nucleic acid and an mediator portion (B) that is comprised of an artificial random sequence that is cleaved by a thermostable DNA polymerase having nuclease activity and acts as a primer for an artificial template.
그러나 이들 방법에서도 모두 매개체 올리고뉴클레오티드는 검출하고자 하는 표적핵산에 상보적인 부분인 프로브 부위를 가지도록 설계, 제작되어야 한다.However, in all of these methods, the mediator oligonucleotide must be designed and manufactured to have a probe portion that is complementary to the target nucleic acid to be detected.
본 발명은 각각의 표적핵산에 따라 상기 매개체 올리고뉴클레오티드(즉 플랩 올리고뉴클레오티드임)를 설계, 제작할 필요 없이, 검출하고자 하는 표적핵산의 서열에 제한을 받지 않는(generic), 인공핵산인 매개체 올리고뉴클레오티드를 이용하여 표적핵산을 검출하는 방법을 개시한다. The present invention discloses a method for detecting a target nucleic acid using a mediator oligonucleotide, which is an artificial nucleic acid that is not limited by the sequence of the target nucleic acid to be detected (generic), without the need to design and manufacture the mediator oligonucleotide (i.e., a flap oligonucleotide) according to each target nucleic acid.
본 발명의 목적은 인공핵산인 매개체 절편(mediator fragments)을 질량 분석 등을 통해 검출함에 의한 표적핵산의 검출 방법을 제공하는 데 있다.The purpose of the present invention is to provide a method for detecting a target nucleic acid by detecting mediator fragments, which are artificial nucleic acids, through mass spectrometry or the like.
본 발명의 다른 목적은 매개체 절편을 이용하여 표적핵산을 인공핵산으로 전환시키는 과정에서 발생하는 신호를 검출하여 표적핵산을 검출하는 방법을 제공하는 데 있다.Another object of the present invention is to provide a method for detecting a target nucleic acid by detecting a signal generated in the process of converting a target nucleic acid into an artificial nucleic acid using a mediator fragment.
본 발명의 또 다른 목적은 상기 방법으로 전환된 인공핵산을 검출하여 표적핵산을 검출하는 방법을 제공하는 데 있다.Another object of the present invention is to provide a method for detecting a target nucleic acid by detecting an artificial nucleic acid converted by the above method.
본 발명의 또 다른 목적은 상기 방법으로 전환된 인공핵산에 의존하여 생성되는 추가의 인공핵산을 검출하거나, 이러한 추가의 인공핵산이 생성되면서 발생하는 신호를 검출하여 표적핵산을 검출하는 방법을 제공하는 데 있다.Another object of the present invention is to provide a method for detecting a target nucleic acid by detecting an additional artificial nucleic acid produced by relying on an artificial nucleic acid converted by the above method, or by detecting a signal generated when such an additional artificial nucleic acid is produced.
본 발명의 다른 목적이나 구체적인 목적은 이하에서 제시될 것이다.Other purposes or specific purposes of the present invention will be presented below.
본 발명은 일 측면에 있어서 매개체 절편(또는 플랩 절편)을 이용한 표적핵산의 검출 방법에 관한 것이다.The present invention relates to a method for detecting a target nucleic acid using a mediator fragment (or flap fragment) in one aspect.
본 명세서에서 플랩 올리고뉴클레오티드와 매개체 올리고뉴클레오티드는 서로 같은 의미로 혼용되어 사용되고, 플랩 절편과 매개체 절편도 같은 의미로 서로 혼용되어 사용된다.In this specification, the terms flap oligonucleotide and mediator oligonucleotide are used interchangeably with the same meaning, and the terms flap fragment and mediator fragment are also used interchangeably with the same meaning.
본 발명의 매개체 절편을 이용한 표적핵산의 검출 방법은 도 2에 도시된 바와같이, 5' 말단 쪽에 인위적 임의 서열의 태그(이하 "5' 태그 서열" 또는 "5' 태그 부위")를 가진 정방향 프라이머와, 통상의 역방향 프라이머, 그리고 상기 5' 태그 서열에 상보적으로 결합하는 프로브 부위(A, probe region)와 인위적 임의 서열로 이루어진 매개체 부위(B, mediator region or flap region)을 포함하는 단일 가닥의 매개체 올리고뉴클레오티드(mediator oligonucleotide)를 사용함을 특징으로 한다. 매개체 부위(B, flap region)는 프로브 부위(A, probe region)의 5' 쪽(upstream)이나 3' 쪽(dowmstream)에 위치할 수 있고, 양쪽 모두에 위치할 수도 있다. 다만 후술하는 바의 별도 분자의 인공주형을 사용하여 매개체 올리고뉴클레오티드를 검출할 경우는 매개체 부위(B, flap region)는 프로브 부위(A, probe region)의 5' 쪽(upstream)에 위치하는 것이 바람직하다. The method for detecting a target nucleic acid using a mediator fragment of the present invention is characterized by using, as illustrated in FIG. 2, a forward primer having an artificial random sequence tag (hereinafter, "5' tag sequence" or "5' tag portion") at the 5' end, a conventional reverse primer, and a single-stranded mediator oligonucleotide including a probe portion (A, probe region) that binds complementarily to the 5' tag sequence and a mediator portion (B, mediator region or flap region) consisting of an artificial random sequence. The mediator portion (B, flap region) may be located at the 5' side (upstream) or the 3' side (downstream) of the probe portion (A, probe region), or may be located at both sides. However, when detecting the mediator oligonucleotide using an artificial template of a separate molecule as described below, the mediator portion (B, flap region) is preferably located at the 5' side (upstream) of the probe portion (A, probe region).
도 2에서 5' 태그 서열이 정방향 프라이머에 존재하는 것으로 도시되어 있지만 5' 태그 서열은 정방향 프라이머 및 역방향 프라이머 중 어느 하나 이상의 프라이머에 존재할 수 있다.Although the 5' tag sequence is shown in FIG. 2 as being present in the forward primer, the 5' tag sequence may be present in either or both of the forward primer and the reverse primer.
본 발명에서, 상기 프라이머의 5' 태그 서열이나 매개체 올리고뉴클레오티드(특히 매개체 부위)에서, 인위적 임의 서열이란 인위적으로 정할 수 있는 임의 서열을 의미한다. 이러한 인위적 임의 서열은 검출하고자하는 표적핵산과는 다른 서열이거나 시료 중에 존재하는 표적핵산을 포함한 임의의 모든 핵산과 다른 서열일 수 있다. 시료 중에 존재하는 표적핵산을 포함한 임의의 모든 핵산과 다른 서열이란 인위적으로 구성한 서열뿐만 아니라 자연계에 존재하는 서열일 수도 있다. 예컨대 인간 유래 시료에 존재하는 표적핵산을 포함한 임의의 모든 핵산과 다른 서열로서 식물의 광합성 관련 유전자 서열의 일부 서열은 자연계에 존재하는 서열이다. In the present invention, in the 5' tag sequence of the primer or the mediator oligonucleotide (especially the mediator portion), the artificial random sequence refers to an artificially determined random sequence. This artificial random sequence may be a sequence different from the target nucleic acid to be detected or a sequence different from any and all nucleic acids including the target nucleic acid present in the sample. The sequence different from any and all nucleic acids including the target nucleic acid present in the sample may be not only an artificially constructed sequence but also a sequence existing in nature. For example, as a sequence different from any and all nucleic acids including the target nucleic acid present in a human-derived sample, some sequences of photosynthesis-related gene sequences of plants are sequences existing in nature.
본 발명에서, 매개체 올리고뉴클레오티드는 그 프로브 부위를 통해 상기 프라이머의 5' 태그 서열에 상보적으로 결합하는데, 이 경우 이들 결합의 특이성을 높이기 위하여 매개체 올리고뉴클레오티드의 3' 말단(즉 프로브 부위의 3' 말단)에 MGB(minor groove binder)를 도입할 수도 있다(즉 접합시킬 수 있다). 당업계에서는 MGB 부분(MGB moiety)이 Tm을 높여 프로브의 표적핵산과의 결합력(본 발명에서는 5' 태그 서열과 매개체 올리고뉴클레오티드의 프로브 부위의 결합력)을 높이고 결국 결합 특이성을 높여준다는 것이 당업계에 잘 알려져 있다. MGB는 통상 저분자 화합물(small molecule)인데, 그러한 화합물로서 폴리아미드(polyamide), 네트롭신(netropsin), 디스타마이신(distamycin), 티아조트롭신 A(thiazotropsin A) 등이 예시될 수 있다.In the present invention, the mediator oligonucleotide complementarily binds to the 5' tag sequence of the primer through its probe portion. In this case, in order to increase the specificity of this binding, a minor groove binder (MGB) may be introduced (i.e., conjugated) to the 3' end of the mediator oligonucleotide (i.e., the 3' end of the probe portion). It is well known in the art that the MGB moiety increases the Tm, thereby increasing the binding affinity of the probe to the target nucleic acid (in the present invention, the binding affinity between the 5' tag sequence and the probe portion of the mediator oligonucleotide), and ultimately increasing the binding specificity. MGB is usually a small molecule, and examples of such compounds include polyamide, netropsin, distamycin, and thiazotropsin A.
MGB의 사용과 관련하여 구체적인 것은 문헌[Igor V. Kutyavin, et al., 3'-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures, Nucleic Acids Res. 2000 Jan 15; 28(2): 655-661], 문헌[Hasan Y Alniss, Thermodynamics of DNA Minor Groove Binders, J Med Chem. 2019 Jan 24;62(2):385-402] 등을 참조할 수 있다.For specific details regarding the use of MGB, see [Igor V. Kutyavin, et al., 3'-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures, Nucleic Acids Res. 2000 Jan 15; 28(2): 655-661], [Hasan Y Alniss, Thermodynamics of DNA Minor Groove Binders, J Med Chem. 2019 Jan 24;62(2):385-402].
본 발명에서 전술한 바와 같은 프라이머 세트와 매개체 올리고뉴클레오티드를 사용할 경우, 도 3의 (A)에 도시된 바와 같이, PCR의 제1 cycle에서는 5' 태그 서열을 가진 정방향 프라이머에 의해 5' 태그 서열을 가진 가닥이 합성되게 된다. 다음 제2 cycle에서는 그 cycle의 어닐링(annealing) 단계에서 그 5' 태그 서열을 가진 가닥의 3' 말단에 프라이머(즉 역방향 프라이머)가 결합함과 함께 그 가닥의 5' 태그 서열에 매개체 올리고뉴클레오티드가, 상기 5' 태그 서열에 상보적인 부위인 프로브 부위(A)를 통해 결합하게 되며, 이후 Taq 폴리머라아제 등 DNA 중합효소에 의해 프라이머(역방향 프라이머)가 연장(extension)되게 되면, 그 가닥의 5' 태그 서열에 결합하고 있던 매개체 올리고뉴클레오티드가 분해되면서 인위적 임의 서열로 이루어진 매개체 부위(B)의 절편(매개체 절편)이 방출되게 된다. 여기서 도 3의 (B)에 도시된 바와 같이 5'-->3' 엑소뉴클레아제 활성(5′-->3′ exonuclease activity)을 가지는 Taq 폴리머라아제 등 DNA 중합효소는 프로브 부위의 첫 번째와 두 번째 뉴클레오티드 사이의 결합을 자르기 때문에(도 3에서 AT 사이를 자름), 매개체 절편은 매개체 부위(B)의 서열 이외에 프로브 부위의 5' 말단의 1개의 뉴클레오티드(도 3에서 A임)를 그 3' 말단에 포함하게 된다. 따라서 본 명세서에서 "매개체 절편"은 매개체 부위(B)의 서열 이외에 그 3' 말단에, 프로브 부위의 5' 말단의 1개의 뉴클레오티드가 포함된 것을 의미한다. In the case of using the primer set and mediator oligonucleotide as described above in the present invention, as illustrated in (A) of FIG. 3, in the first cycle of PCR, a strand having a 5' tag sequence is synthesized by a forward primer having a 5' tag sequence. In the second cycle, in the annealing step of the cycle, a primer (i.e., a reverse primer) binds to the 3' end of the strand having the 5' tag sequence, and the mediator oligonucleotide binds to the 5' tag sequence of the strand through the probe portion (A), which is a portion complementary to the 5' tag sequence. Thereafter, when the primer (reverse primer) is extended by a DNA polymerase such as Taq polymerase, the mediator oligonucleotide bound to the 5' tag sequence of the strand is decomposed, thereby releasing a fragment (mediator fragment) of the mediator portion (B) consisting of an artificial random sequence. Here, as shown in (B) of FIG. 3, a DNA polymerase such as Taq polymerase having 5'-->3' exonuclease activity cleaves the bond between the first and second nucleotides of the probe region (cuts between AT in FIG. 3), so the intermediate fragment includes one nucleotide (A in FIG. 3) at the 5' end of the probe region in addition to the sequence of the intermediate region (B) at its 3' end. Therefore, in the present specification, "intermediate fragment" means that, in addition to the sequence of the intermediate region (B), one nucleotide at the 5' end of the probe region is included at its 3' end.
상기 5'-->3' 엑소뉴클레아제 활성(5′-->3′ exonuclease activity)을 가지는 Taq 폴리머라아제 등 DNA 중합효소는 분할된 이중가닥 구조 특이적인, 플랩(flap) 엔도뉴클레아제 활성(bifurcated duplex structure-specific flap endonuclease activity)를 함께 갖는다. 본 명세서에서 특별한 명시적 언급이 없는 한 뉴클레아제 활성은 5'-->3' 엑소뉴클레아제 활성과 플랩 엔도뉴클레아제 활성 모두를 의미하는 용어로 사용된다. DNA polymerases such as Taq polymerase having the above 5'-->3' exonuclease activity also have bifurcated duplex structure-specific flap endonuclease activity. Unless otherwise specifically stated, the nuclease activity is used herein as a term meaning both the 5'-->3' exonuclease activity and the flap endonuclease activity.
만일 시료 중에 표적핵산이 존재하지 않을 경우, 도 4에서 보는 바와 같이, 매개체 올리고뉴클레오티드가 분해되지 않아 매개체 부위가 절단되지 않으므로 매개체 절편이 방출되지 않게 된다. If the target nucleic acid is not present in the sample, as shown in Fig. 4, the mediator oligonucleotide is not decomposed, the mediator portion is not cleaved, and thus the mediator fragment is not released.
도 2에 도시된 바와 같이, 매개체 올리고뉴클레오티드에서 매개체 부위(B)가 프로브 부위(A)의 3' 쪽(downstream)에 위치하거나 프로브 부위(A) 양쪽(upstream과 downstream) 모두에 위치할 수도 있다. 매개체 부위(B)가 프로브 부위(A)의 3' 쪽이나 양쪽 모두에 위치하더라도 시료 중 표적핵산이 존재할 경우에 매개체 절편이 발출되고 표적핵산이 존재하지 않을 경우에는 매개체 절편이 발출되지 않으므로, 매개체 올리고뉴클레오티드에서 매개체 부위(B)가 프로브 부위(A)의 3' 쪽이나 양쪽 모두에 위치할 수도 있다. 양쪽 모두에 위치할 경우 프로브 부위(B)는 동일 서열이거나 서로 다른 서열이거나 서로 상보적인 서열일 수 있다. As illustrated in FIG. 2, the mediator portion (B) in the mediator oligonucleotide may be located at the 3' side (downstream) of the probe portion (A) or may be located on both sides (upstream and downstream) of the probe portion (A). Even if the mediator portion (B) is located at the 3' side or both sides of the probe portion (A), if the target nucleic acid is present in the sample, the mediator fragment is extracted, and if the target nucleic acid is not present, the mediator fragment is not extracted. Therefore, the mediator portion (B) in the mediator oligonucleotide may be located at the 3' side or both sides of the probe portion (A). If located at both sides, the probe portions (B) may have the same sequence, different sequences, or complementary sequences.
이와 같이 매개체 올리고뉴클레오티드의 매개체 절편은 시료 중 표적핵산이 존재할 경우에만 방출되므로, 이 방출된 매개체 절편의 염기서열(뉴클레오티드 순서) 및/또는 그 길이를 검출하여 그 존부(presence or absence of the flap fragments)를 결정함에 의하여 표적핵산을 검출할 수 있게 된다. 이때 매개체 올리고뉴클레오티드를 표적핵산에 맞춰 각기 다른 것을 사용할 경우 다중 검출이 가능하게 된다. 매개체 올리고뉴클레오티드를 표적핵산에 맞춰 각기 다른 것을 사용할 경우 이 매개체 올리고뉴클레오티드의 프로브 부위가 특이적으로 결합하는 5' 태그 부위도 표적핵산에 따라 달라지게 될 것이다.In this way, since the mediator fragment of the mediator oligonucleotide is released only when the target nucleic acid is present in the sample, the target nucleic acid can be detected by determining the presence or absence of the flap fragments by detecting the base sequence (nucleotide order) and/or the length of the released mediator fragment. At this time, multiple detection is possible if different mediator oligonucleotides are used according to the target nucleic acid. If different mediator oligonucleotides are used according to the target nucleic acid, the 5' tag site to which the probe site of the mediator oligonucleotide specifically binds will also vary depending on the target nucleic acid.
방출된 매개체 절편의 검출은, 질량 분석(Mass Spectrometry)이나 전기영동(Electrophoresis)이나 액체 크로마토그래피(Liquid chromatography)를 수행하거나, 서열 분석이나 마이크로어레이 등을 수행하여 이루어질 수 있다.Detection of released mediator fragments can be accomplished by performing mass spectrometry, electrophoresis, liquid chromatography, sequence analysis, microarray, etc.
질량 분석(Mass Spectrometry)에는 MALDI-TOF MS(Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry), 탠덤 질량 분석(Tandem MS), ESI-TOF(Electrospray Ionization-Time of Flight), 이온 포집 질량 분석(Ion Trap Mass Spectrometry), LC-MS(Liquid Chromatography Mass Spectrometry), GC-MS(Gas Chromatography Mass Spectrometry) IM-MS(Ion Mobility Mass Spectrometry) 등의 방법이 사용될 수 있다.Mass spectrometry can use methods such as MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry), Tandem Mass Spectrometry (Tandem MS), ESI-TOF (Electrospray Ionization-Time of Flight), Ion Trap Mass Spectrometry, LC-MS (Liquid Chromatography Mass Spectrometry), GC-MS (Gas Chromatography Mass Spectrometry), and IM-MS (Ion Mobility Mass Spectrometry).
한편 국제 공개특허 WO08136868(명칭: Detecting and quantification of biomolecules using mass spectrometry), 국제 공개특허 WO09073251(명칭: Detecting and quantification of biomolecules using mass spectrometry), 국제 공개특허 WO14140147(명칭: Nucleic acid target identification by structure based probe cleavage), 국제 공개특허 WO15091765(명칭: Mutiplexed Nucleic acid target identification by structure based probe cleavage), 국제 공개특허 W16041591(명칭: Nucleic acid target identification by structure based probe cleavage)는 모두 본 발명과 같이 표적핵산이 존재할 경우에만 생성되는 매개체 올리고뉴클레오티드의 매개체 절편을 질량 분석(mass spectroscopy)을 통해 검출하거나, 3'-->5' 엑소뉴클레아제나 5'-->3' 엑소뉴클레아제 등의 처리에 의한 그 매개체 절편의 가공물을 질량 분석을 통해 검출하여 표적핵산을 검출하는 방법을 개시하고 있다. 따라서 상기 국제 공개특허에 개시된 방법들은 본 발명의 매개체 절편 검출을 통한 표적핵산이 검출에 적용될 수 있다. Meanwhile, international patent publication WO08136868 (title: Detecting and quantification of biomolecules using mass spectrometry), international patent publication WO09073251 (title: Detecting and quantification of biomolecules using mass spectrometry), international patent publication WO14140147 (title: Nucleic acid target identification by structure based probe cleavage), international patent publication WO15091765 (title: Mutiplexed Nucleic acid target identification by structure based probe cleavage), international patent publication W16041591 (title: Nucleic acid target identification by structure based probe cleavage) all detect mediator fragments of mediator oligonucleotides that are generated only when a target nucleic acid is present, as in the present invention, through mass spectroscopy, or by treatment with 3'-->5' exonuclease or 5'-->3' exonuclease. A method for detecting a target nucleic acid by detecting a processed product of the intermediate fragment by mass spectrometry is disclosed. Therefore, the methods disclosed in the above international published patent can be applied to detecting a target nucleic acid by detecting the intermediate fragment of the present invention.
전기영동은 매개체 부위가 절단되지 아니한 매개체 올리고뉴클레오티드가 표적핵산이 존재할 경우에만 생성되는 매개체 절편과는 크기가 다르므로 서로 구분되어 검출될 수 있도록 한다. 이러한 전기영동은 SSCP(Single-strand conformation polymorphism) 방법(Natural Protocols, 1(6):3121-3128, 2006)을 이용할 수도 있다.Electrophoresis allows the mediator oligonucleotides, which are not cleaved at the mediator site, to be detected separately from the mediator fragments that are produced only when the target nucleic acid is present, since they are of a different size. This electrophoresis can also utilize the single-strand conformation polymorphism (SSCP) method (Natural Protocols, 1(6):3121-3128, 2006).
매개체 절편의 서열 분석에는 차세대서열분석 기술(Next generation sequencing, NGS) 등을 이용할 수 있으며, 차세대서열분석 기술을 이용할 경우 매개체 절편이 계수되므로 이로부터 표적핵산에 대한 정성적 검출뿐만 아니라 정량적 검출까지 가능할 수 있다. Next generation sequencing (NGS) technology can be used to analyze the sequence of the mediator fragments. If next generation sequencing technology is used, the mediator fragments are counted, so not only qualitative detection of the target nucleic acid but also quantitative detection can be possible.
매개체 절편의 검출에 마이크로어레이를 이용할 경우, 미국 공개특허 제2008-0241838호에 개시된 방법(명칭: Methods and systems for detecting nucleic acids) 등을 이용할 수 있다. 미국 공개특허 제2008-0241838호에 개시된 방법은 플랩 올리고뉴클레오티드의 5' 말단(플랩 부위의 5' 말단)에 신호 물질을 표지화시키고 이 플랩 부위에 상보적인 캡쳐 프로브를 지지체에 고정시켜 플랩 절편을 검출하는 방법인데, 캡쳐 프로브의 길이를 짧게 조절하여 시료 중 표적핵산이 존재할 경우 플랩 절편은 캡쳐 프로브와 결합하도록 하고 표적핵산이 존재하지 않을 경우는 절단되지 아니한 플랩 올리고뉴클레오티드는 결합하지 않도록 하여 플랩 절편을 검출하는 방법이다(미국 공개특허 제2008-0241838호의 도 4 참조). When using a microarray to detect a mediator fragment, the method disclosed in US Patent Publication No. 2008-0241838 (title: Methods and systems for detecting nucleic acids) can be used. The method disclosed in US Patent Publication No. 2008-0241838 is a method for detecting a flap fragment by labeling a signal substance at the 5' end (the 5' end of the flap portion) of a flap oligonucleotide and fixing a capture probe complementary to the flap portion to a support. In this method, the length of the capture probe is adjusted short so that when a target nucleic acid is present in a sample, the flap fragment binds to the capture probe, and when the target nucleic acid is not present, the uncleaved flap oligonucleotide does not bind, thereby detecting the flap fragment (see FIG. 4 of US Patent Publication No. 2008-0241838).
또한 매개체 절편은 특히 후술하는 바와 같이 그 매개체 절편으로부터 연장되어 생성된 인공핵산을 검출하거나 그 인공핵산이 생성되는 과정에서 변화하는 신호를 검출하거나 그 인공핵산(제1의 인공핵산)에 의존하여 추가의 인공핵산(제2의 인공핵산)이 생성되도록 하여 상기 인공핵산에 의존하여 생성되는 추가의 인공핵산을 검출하거나 그 추가의 인공핵산이 생성되면서 변화하는 신호를 검출함에 의해 가능할 수 있다. 매개체 절편으로부터 연장되어 생성된 인공핵산도, 시료 중에 표적핵산이 존재하여 매개체 절편이 생성되어야만 그 매개체 절편으로부터 연장되어 생성되므로 매개체 절편의 검출을 가능하게 한다. 이렇게 매개체 절편을 사용하여 표적핵산을 인공핵산이나 추가의 인공핵산으로 전환시켜 검출할 경우 PCR 기반의 실시간 검출이 가능하다. In addition, the mediator fragment may be capable of detecting an artificial nucleic acid generated by extension from the mediator fragment, as described below, or detecting a signal that changes during the process of generating the artificial nucleic acid, or generating an additional artificial nucleic acid (a second artificial nucleic acid) dependent on the artificial nucleic acid (the first artificial nucleic acid) and detecting the additional artificial nucleic acid generated dependent on the artificial nucleic acid, or detecting a signal that changes during the generation of the additional artificial nucleic acid. The artificial nucleic acid generated by extension from the mediator fragment is also generated only when a target nucleic acid is present in the sample and the mediator fragment is generated, thereby enabling detection of the mediator fragment. In this way, when the target nucleic acid is converted into an artificial nucleic acid or an additional artificial nucleic acid and detected using the mediator fragment, PCR-based real-time detection is possible.
이하 매개체 절편을 이용하여 표적핵산을 인공핵산이나 추가의 인공핵산으로 전환시켜 검출하는 본 발명의 방법에 대해서 상세히 설명한다. Hereinafter, the method of the present invention for detecting a target nucleic acid by converting it into an artificial nucleic acid or an additional artificial nucleic acid using a mediator fragment is described in detail.
본 발명의 표적핵산을 인공핵산이나 추가의 인공핵산으로 전환시켜 검출하는 방법은, 도 5에 도시된 바와 같이, 5' 태그 부위를 가진 정방향 프라이머와, 통상의 역방향 프라이머, 그리고 상기 5' 태그 서열에 상보적으로 결합하는 프로브 부위(A, probe region)와 인위적 임의 서열로 이루어진 매개체 부위(B, mediator region)을 포함하는 단일 가닥의 매개체 올리고뉴클레오티드(mediator oligonucleotide) 그리고 인공 주형(artificial template)을 사용함을 특징으로 한다. The method for detecting the target nucleic acid of the present invention by converting it into an artificial nucleic acid or an additional artificial nucleic acid is characterized by using, as illustrated in FIG. 5, a forward primer having a 5' tag portion, a conventional reverse primer, a single-stranded mediator oligonucleotide including a probe portion (A, probe region) that binds complementarily to the 5' tag sequence, a mediator portion (B, mediator region) consisting of an artificial random sequence, and an artificial template.
여기서 인공 주형은, 도 1(A)에 개시된 상기 국제 공개특허 WO06122208(명칭: Snapback oligonucleotide probe)에서 개시된 방법에서처럼 별도로 존재하지 않고 절단된 매개체 자체가 인공주형으로 작용할 수 있다. 이 경우 절단된 매개체의 3' 말단이 연장됨으로써 가수분해성 프로브가 분해되어 신호가 변화하게 되고 그 변화하는 신호를 검출하여 표적핵산을 검출할 수 있다. Here, the artificial template may not exist separately as in the method disclosed in the international patent publication WO06122208 (title: Snapback oligonucleotide probe) disclosed in Fig. 1(A), but the cleaved mediator itself may act as the artificial template. In this case, the 3' end of the cleaved mediator is extended, thereby decomposing the hydrolyzable probe and changing the signal, and the target nucleic acid can be detected by detecting the changed signal.
이처럼 절단된 매개체 자체가 인공 주형으로 작용할 수 있으나, 도 5에 도시된 바와 같이, 별도 분자의 단일 가닥 핵산을 인공주형으로 사용하는 것이 바람직하다. 이 경우 인공 주형은, 표적핵산의 증폭 과정 중에 상기 매개체 올리고뉴클레오티드로부터 뉴클레아제 활성을 가지는 DNA 중합효소에 의하여 절단되어 생성되는 매개체(mediator, B) 절편이 결합하여 인공핵산 생성에 프라이머로 작용하도록 그 매개체 절편에 상보적인 서열을 가진 프라이밍 부위(B', priming region)를 가지고, 또한 신규로 합성되어 연장되는 서열인 인공핵산(C, artificial nucleic acid)에 대해 주형으로 작용하는 주형 부위(C')를 가진다. Although the cleaved mediator itself can act as an artificial template, as illustrated in FIG. 5, it is preferable to use a single-stranded nucleic acid of a separate molecule as an artificial template. In this case, the artificial template has a priming region (B') having a sequence complementary to a mediator fragment that is cleaved from the mediator oligonucleotide by a DNA polymerase having nuclease activity during the amplification process of the target nucleic acid and that acts as a primer for the production of an artificial nucleic acid, and also has a template region (C') that acts as a template for an artificial nucleic acid (C, artificial nucleic acid), which is a newly synthesized and extended sequence.
본 발명에서 전술한 바와 같은 프라이머 세트와 매개체 올리고뉴클레오티드 그리고 별도 분자의 인공 주형을 사용할 경우, 도 6에 도시된 바와 같이, PCR의 제1 cycle에서는 5' 태그 서열을 가진 정방향 프라이머에 의해 5' 태그 서열을 가진 가닥이 합성되게 된다. 다음 제2 cycle에서는 그 cycle의 어닐링(annealing) 단계에서 그 5' 태그 서열을 가진 가닥의 3' 말단에 프라이머(즉 역방향 프라이머)가 결합함과 함께 그 가닥의 5' 태그 서열에 매개체 올리고뉴클레오티드가, 상기 5' 태그 서열에 상보적인 부위인 프로브 부위(A)를 통해 결합하게 되며, 이후 Taq 폴리머라아제 등 DNA 중합효소에 의해 프라이머(역방향 프라이머)가 연장(extension)되게 되면, 그 가닥의 5' 태그 서열에 결합하고 있던 매개체 올리고뉴클레오티드가 분해되면서 인위적 임의 서열로 이루어진 매개체 절편(mediatro fragments)이 방출되게 된다. In the present invention, when using the primer set, the mediator oligonucleotide, and the artificial template of a separate molecule as described above, as illustrated in FIG. 6, in the first cycle of PCR, a strand having a 5' tag sequence is synthesized by the forward primer having the 5' tag sequence. In the second cycle, in the annealing step of the cycle, a primer (i.e., the reverse primer) binds to the 3' end of the strand having the 5' tag sequence, and the mediator oligonucleotide binds to the 5' tag sequence of the strand through the probe portion (A), which is a portion complementary to the 5' tag sequence. Thereafter, when the primer (reverse primer) is extended by a DNA polymerase such as Taq polymerase, the mediator oligonucleotide bound to the 5' tag sequence of the strand is decomposed, releasing mediator fragments consisting of an artificial random sequence.
만일 시료 중에 표적핵산이 존재하지 않을 경우는, 도 7에서 보는 바와 같이, 매개체 올리고뉴클레오티드가 분해되지 않아 매개체 절편이 방출되지 않게 되고, 그러면 인공 주형의 주형 부위(C')에 상보적인 서열(C)인 인공핵산이 신규로 생성되지 않게 된다. 즉 시료 중에 표적핵산이 존재하지 않을 경우에는 매개체 올리고뉴클레오티드는 분해되지 않은 상태이기 때문에 인공 주형과 결합하여도, 인공 주형의 주형 부위(C')와 매개체 올리고뉴클레오티드의 5'태그 서열에 상보적으로 결합하는 프로브 부위(A)가 서로 상보성을 띠지 않아 결합되지 않은 상태로 있으므로 인공 주형의 주형 영역(C')에 상보적인 서열(C)인 인공핵산이 신규로 합성되지 않는다. 그럼에도 인공 주형의 3'말단이 연장되거나 매개체 올리고뉴클레오티드의 3'말단이 연장되는 것을 방지하기 위하여 이들 3'말단이 아민기(amine group), 인산기(phosphate group), 알킬기(alkyl group), 알케인-다이올(alkane-diol), 포스포로사이오에이트(phophorothioate), 바이오틴(biotin), 비염기성 링커(non-nucleotide linker), C3-18 스페이서(C3-18 spacer), 디-데옥시뉴클레오티드 트리포스페이트(di-deoxynucleotide triphosphate, ddNTP), 역방향 데옥시뉴클레오티드 트리포스페이트(inverted deoxynucleotide triphosphate, inverted dNTP), HEG(hexathylene glycol) 등에 의해 블록킹(blocking)되는 것이 바람직할 수 있다. If the target nucleic acid does not exist in the sample, as shown in Fig. 7, the mediator oligonucleotide is not decomposed, so the mediator fragment is not released, and then an artificial nucleic acid having a sequence (C) complementary to the template portion (C') of the artificial template is not newly generated. In other words, if the target nucleic acid does not exist in the sample, the mediator oligonucleotide is not decomposed, so even if it binds to the artificial template, the template portion (C') of the artificial template and the probe portion (A) that complementarily binds to the 5' tag sequence of the mediator oligonucleotide are not complementary to each other, so they remain unbound, and thus an artificial nucleic acid having a sequence (C) complementary to the template region (C') of the artificial template is not newly synthesized. However, in order to prevent the 3' terminus of the artificial template from being extended or the 3' terminus of the mediator oligonucleotide from being extended, it may be desirable for these 3' termini to be blocked by an amine group, a phosphate group, an alkyl group, an alkane-diol, a phosphorothioate, biotin, a non-nucleotide linker, a C3-18 spacer, a di-deoxynucleotide triphosphate (ddNTP), an inverted deoxynucleotide triphosphate (inverted dNTP), hexathylene glycol (HEG), or the like.
시료 중 표적핵산이 존재할 경우에는 매개체 올리고뉴클레오티드에서 인위적 임의 서열로 이루어진 매개체 절편이 표적핵산의 시료 중의 농도에 비례하여 방출되고, 따라서 이 매개체 절편이 인공 주형에 대해 프라이머로 작용함으로써 인공 주형의 주형 부위(C')에 대해 상보적으로 신규 생성되는 서열(C)인 인공핵산도 표적핵산의 시료 중의 농도에 비례하여 생성되게 되므로, 그 인공핵산이 생성되는 과정에서 발생하는 신호를 검출하거나, 이러한 인공핵산(C) 등을 검출하면 시료 중 표적핵산에 대한 정성적 정보(표적핵산의 존재 여부) 뿐만 아니라 정량적 정보(표적핵산의 존재량)까지 얻을 수 있게 된다. When a target nucleic acid exists in a sample, a mediator fragment consisting of an artificial random sequence is released from a mediator oligonucleotide in proportion to the concentration of the target nucleic acid in the sample, and therefore, when this mediator fragment acts as a primer for the artificial template, an artificial nucleic acid which is a newly generated sequence (C) complementary to the template portion (C') of the artificial template is also generated in proportion to the concentration of the target nucleic acid in the sample. Therefore, by detecting a signal generated in the process of generating the artificial nucleic acid or detecting such artificial nucleic acid (C), it is possible to obtain not only qualitative information (whether the target nucleic acid exists) but also quantitative information (the amount of the target nucleic acid present) about the target nucleic acid in the sample.
본 발명의 방법에서, 매개체 올리고뉴클레오티드는 표적핵산에 상보적인 서열을 가진 것이 아니라 정방향 프라이머의 인위적 임의 서열로 이루어진 5' 태그 서열에 상보적인 서열을 가진다. 따라서 이 5' 태그 서열을 인위적 서열로 구성함으로써 그 매개체 올리고뉴클레오티드 서열을 인위적으로 조절하여 구성할 수 있으며, 이렇게 조절된 매개체 올리고뉴클레오티드 서열은, 검출하고자 하는 표적핵산 서열에 상관없이 범용적으로 사용될 수 있으므로 검출하고자 하는 표적핵산에 따라 그 매개체 올리고뉴클레오티드 서열(특히 이 서열 중 표적핵산과 상보적으로 결합하는 프로브 부위(A)의 서열)을 표적핵산 서열에 따라 일일이 설계, 제작할 필요가 없게 된다. In the method of the present invention, the mediator oligonucleotide does not have a sequence complementary to the target nucleic acid, but has a sequence complementary to a 5' tag sequence consisting of an artificial random sequence of the forward primer. Therefore, by configuring this 5' tag sequence as an artificial sequence, the mediator oligonucleotide sequence can be artificially controlled and configured, and the mediator oligonucleotide sequence controlled in this way can be universally used regardless of the target nucleic acid sequence to be detected, so that the mediator oligonucleotide sequence (in particular, the sequence of the probe portion (A) complementarily binding to the target nucleic acid among these sequences) does not need to be individually designed and manufactured according to the target nucleic acid sequence to be detected.
본 발명의 방법에서, 시료 중에 표적핵산이 존재할 경우 매개체 올리고뉴클레오티드가 분해되면서 이로부터 방출되는 인위적 임의 서열의 매개체 절편이 인공 주형에 프라이머로 작용하여 연장되면서 표적핵산이 인공핵산으로 전환되게 되는데, 이때 (i) 전환되면서 발생하는 신호(즉 인공핵산이 생성되면서 발생하는 신호)를 검출하거나 (ii) 그 전환된 인공핵산을 검출하거나, (iii) 그 전환된 인공핵산에 의존하여 생성되는 추가의 인공핵산을 검출하거나, 또는 이러한 추가의 인공핵산이 생성되면서 발생하는 신호를 검출함으로써 표적핵산을 검출할 수 있게 된다.In the method of the present invention, when a target nucleic acid is present in a sample, a mediator oligonucleotide is decomposed, and a mediator fragment of an artificial random sequence released therefrom acts as a primer on an artificial template and is extended, thereby converting the target nucleic acid into an artificial nucleic acid. At this time, the target nucleic acid can be detected by (i) detecting a signal generated during the conversion (i.e., a signal generated when the artificial nucleic acid is generated), (ii) detecting the converted artificial nucleic acid, (iii) detecting an additional artificial nucleic acid generated depending on the converted artificial nucleic acid, or detecting a signal generated when such an additional artificial nucleic acid is generated.
1. 인공핵산이 생성되면서 변화하는 신호 검출1. Detection of changing signals as artificial nucleic acids are produced
도 8에 도시된 바와 같이, 인공 주형의 주형 부위가 헤어핀 구조(스템 루프 구조, stem-loop structure)를 취하고 있어, 매개체 올리고뉴클레오티드에서 방출된 매개체 절편이 인공 주형에 결합, 프라이머로서 작용하여 이로부터 서열이 연장되게 되면, 상기 헤어핀 구조가 펴지면서 리포터 분자가 퀜처 분자로부터 분리되어 신호가 발생하게 된다. 이 신호 발생량은 시료 중의 표적핵산에 비례하므로 이 신호를 측정하면 표적핵산에 대한 정량적 정보를 얻을 수 있다. As illustrated in Fig. 8, since the template portion of the artificial template has a hairpin structure (stem-loop structure), when the mediator fragment released from the mediator oligonucleotide binds to the artificial template and acts as a primer to extend the sequence therefrom, the hairpin structure unfolds, the reporter molecule is separated from the quencher molecule, and a signal is generated. Since the amount of this signal generation is proportional to the target nucleic acid in the sample, quantitative information on the target nucleic acid can be obtained by measuring this signal.
이 경우 시료 중 2개 이상 표적핵산을 다중으로 검출할 경우, 정방향 프라이머의 5' 태그 서열을 표적핵산에 따라 달리 구성하고 또 인공 주형에서 프라이머로 작용할 매개체 올리고뉴클레오티드의 매개체 서열을 표적핵산에 따라 달리 구성하며, 또 이에 맞춰 인공 주형에서 상기 매개체가 결합하는 프라이밍 부위를 달리 구성함과 함께 헤어핀 구조를 취하는 인공 주형의 주형 부위(C')에서 그 리포터 분자가 서로 다른 형광 신호를 발생시키도록 구성하면(서로 다른 방출 파장을 가진 리포터 분자로 인공 주형의 주형 부위(C')를 표지화시키면), 다중의 표적핵산 검출이 가능해진다. 이렇게 정방향 프라이머의 5' 태그 서열과, 그에 따른 매개체 서열 그리고 인공 주형의 프라이밍 부위 서열을 달리하고 리포터 분자를 달리하여, 본 발명의 방법을 다중의 표적핵산에 검출에 이용하더라도, 인공 주형의 주형 부위는 표적핵산에 따라 그 서열을 달리하지 않고 동일한 서열로 구성하더라도 무방하다. 동일한 서열로 구성하더라도 표적핵산에 따라 서로 다른 신호가 발생하게 된다.In this case, when detecting two or more target nucleic acids in a sample, the 5' tag sequence of the forward primer is configured differently depending on the target nucleic acids, and the mediator sequence of the mediator oligonucleotide that will act as a primer in the artificial template is configured differently depending on the target nucleic acids, and accordingly, the priming site to which the mediator binds in the artificial template is configured differently, and the reporter molecule is configured to generate different fluorescence signals at the template site (C') of the artificial template having a hairpin structure (by labeling the template site (C') of the artificial template with reporter molecules having different emission wavelengths), thereby enabling detection of multiple target nucleic acids. Even if the method of the present invention is used to detect multiple target nucleic acids by varying the 5' tag sequence of the forward primer, the mediator sequence corresponding thereto, and the priming site sequence of the artificial template, and varying the reporter molecules, the template site of the artificial template may not vary its sequence depending on the target nucleic acids but may be configured with the same sequence. Even if they are composed of the same sequence, different signals are generated depending on the target nucleic acid.
또한 다중 표적핵산의 검출에서, 헤어핀 구조를 취하는 인공 주형의 주형 부위(C')의 서열의 길이 및/또는 GC 함량을 표적핵산에 따라 달리 구성하고, 이 인공 주형의 주형 부위(C')의 인공핵산(주형 부위에 상보적으로 신규 연장된 서열임)과의 혼성화물이 서로 다른 Tm을 가지도록 구성하여, 융해 곡선 분석(melting curve analysis)을 수행하면 단일 형광 신호를 사용하더라도 다중 표적핵산의 검출이 가능해진다. 특히 인공 주형의 주형 부위(C')는 인위적으로 그 서열을 조절할 수 있으므로 시료 중의 각 표적핵산으로부터 유래하는 각 혼성화물이 서로 다른 Tm을 가지도록 설계하는 것이 매우 용이할 수 있다. 이러한 융해 곡선 분석은 PCR 반응 종료 후 모든 표적핵산이 2가닥의 혼성화물이 되도록 온도를 낮춘 후 온도를 점차 올려가며 수행하게 된다.In addition, in the detection of multiple target nucleic acids, the length and/or GC content of the sequence of the template portion (C') of the artificial template taking a hairpin structure is configured differently depending on the target nucleic acids, and the hybrids of the template portion (C') of the artificial template and the artificial nucleic acid (a sequence newly extended complementary to the template portion) are configured to have different Tms, thereby performing melting curve analysis, so that detection of multiple target nucleic acids becomes possible even when a single fluorescence signal is used. In particular, since the sequence of the template portion (C') of the artificial template can be artificially controlled, it can be very easy to design each hybridization derived from each target nucleic acid in the sample to have a different Tm. This melting curve analysis is performed by lowering the temperature after the completion of the PCR reaction so that all target nucleic acids become two-stranded hybrids, and then gradually raising the temperature.
상기 융해 곡선 분석은 PCR 반응 종료 후 단일 형광 신호를 이용하여 융해 곡선 분석을 통해 다중 표적 핵산을 검출하는 것이지만, 도 8에 도시되고, 국제출원 제PCT/KR2014/012074호(WO 2015 147412, 명칭: Detection of Target Nucleic Acid Sequences Using Different Detection Temperatures) 및 국제출원 제PCT/KR2019/004780호(WO 2019 203623, 명칭: Method and Apparatus for Detecting a Plurality of Target Nucleic acid Sequences in Sample)에 개시된 바와 같이, PCR 반응 중 실시간으로 단일 형광 신호를 이용하여 다중 표적 핵산을 검출하는 것도 가능하다. The above melting curve analysis detects multiple target nucleic acids by using a single fluorescence signal after the end of a PCR reaction, but as illustrated in FIG. 8 and disclosed in International Application No. PCT/KR2014/012074 (WO 2015 147412, titled: Detection of Target Nucleic Acid Sequences Using Different Detection Temperatures) and International Application No. PCT/KR2019/004780 (WO 2019 203623, titled: Method and Apparatus for Detecting a Plurality of Target Nucleic acid Sequences in Sample), it is also possible to detect multiple target nucleic acids by using a single fluorescence signal in real time during a PCR reaction.
예컨대 2개의 표적 핵산을 검출할 경우 융해 곡선 분석에서와 같이 헤어핀 구조를 취하는 인공주형의 주형 부위(C')의 서열의 길이 및/또는 GC 함량을 표적핵산에 따라 달리 구성하여, 2개의 표적 핵산에 대한 인공주형과 인공핵산의 혼성화물이 상대적으로 낮은 온도에서는 그 혼성화 상태가 2개 모두 유지되도록 하고, 상대적으로 높은 온도에서는 그 중 어느 하나의 혼성화물이 해리되도록(이때 해리되면 다시 헤어핀 구조를 취하게 되어 신호가 소멸함), 서로 다른 Tm을 가지도록 구성하면, 상대적으로 낮은 온도에서 검출된 신호는 2가지 표적핵산 모두의 존재량에 대한 신호가 되고, 상대적으로 높은 온도에서 검출된 신호는 2가지 표적핵산 중 어느 하나의 표적핵산의 존재량에 대한 신호가 되므로, 상대적 낮은 온도에서 검출된 신호에서 상대적 높은 온도에서 검출된 신호를 차감하면 나머지 하나의 표적핵산 존재량에 대한 검출 신호를 얻을 수 있게 된다. 이러한 신호 검출과 차감을 매 사이클마다 반복하게 되면 실시간으로 2개의 표적 핵산을 동시에 검출할 수 있다. 여기서 상대적으로 낮은 온도에서 어닐링 단계와 연장 단계를 모두 수행하고 상대적 높은 온도에서는 신호 검출만을 수행하는 것이 바람직할 수 있다. 여기서 인공주형과 인공핵산의 혼성화물이 서로 다른 Tm을 가지도록 구성하고 상대적 높은 온도를 2개 이상 설정하면 3개 이상의 표적핵산의 검출도 가능하게 된다. For example, in the case of detecting two target nucleic acids, the length and/or GC content of the template portion (C') of the artificial template that takes a hairpin structure, as in melting curve analysis, are configured differently depending on the target nucleic acids, so that the hybridization states of the artificial template and the artificial nucleic acid for the two target nucleic acids are both maintained at a relatively low temperature, and one of the hybrids dissociates at a relatively high temperature (at this time, if dissociated, it takes on a hairpin structure again and the signal disappears), so that they have different Tms. Therefore, the signal detected at the relatively low temperature becomes a signal for the abundance of both target nucleic acids, and the signal detected at the relatively high temperature becomes a signal for the abundance of one of the two target nucleic acids. Therefore, by subtracting the signal detected at the relatively high temperature from the signal detected at the relatively low temperature, a detection signal for the abundance of the remaining target nucleic acid can be obtained. By repeating this signal detection and subtraction for each cycle, two target nucleic acids can be detected simultaneously in real time. Here, it may be desirable to perform both the annealing step and the extension step at a relatively low temperature and perform only the signal detection at a relatively high temperature. Here, by configuring the hybrid of the artificial template and the artificial nucleic acid to have different Tms and setting two or more relatively high temperatures, detection of three or more target nucleic acids can also be possible.
한편 도 9에 도시된 바와 같이, 인공 주형의 주형 부위(C')에 택맨(TaqMan) 프로브 등 가수분해성 프로브가 결합하고 있을 경우에는, 매개체 올리고뉴클레오티드에서 방출된 매개체 절편이 인공 주형에 결합, 프라이머로서 작용하여 서열이 연장되게 되면 Taq 폴리머라아제 등의 DNA 중합효소가 5'-->3' 엑소뉴클레아제 활성을 나타내어 상기 주형 부위(C')에 결합하고 있던 가수분해성 프로브을 분해시키게 된다. 그러면 퀜처 분자에 의해 소광(Quenching)되고 있던 리포터 분자가 퀜처 분자로부터 떨어지게 되어 신호가 발생한다. 이 신호 발생량도 시료 중의 표적핵산에 비례하므로 이 신호를 측정하면 표적핵산에 대한 정량적 정보를 얻을 수 있다.Meanwhile, as illustrated in FIG. 9, when a hydrolyzable probe such as a TaqMan probe is bound to the template portion (C') of the artificial template, the mediator fragment released from the mediator oligonucleotide binds to the artificial template, acts as a primer, and the sequence is extended, and a DNA polymerase such as Taq polymerase exhibits 5'-->3' exonuclease activity to decompose the hydrolyzable probe bound to the template portion (C'). Then, the reporter molecule that was being quenched by the quencher molecule is separated from the quencher molecule, generating a signal. Since the amount of this signal generation is also proportional to the target nucleic acid in the sample, quantitative information on the target nucleic acid can be obtained by measuring this signal.
또 한편 도 9 하단에 도시된 바와 같이, 인공 주형과 가수분해성 프로브는 그 인공 주형의 5' 말단과 가수분해성 프로브의 3' 말단이 서로 연결된 단일 가닥으로 구성할 수도 있다. 이렇게 단일 가닥으로 구성하더라도 매개체 절편이 인공 주형에 결합, 프라이머로서 작용하여 서열이 연장되게 되면 Taq 폴리머라아제 등 DNA 중합효소가 5'-->3' 엑소뉴클레아제 활성에 의하여 퀜처 분자에 의해 소광(Quenching)되고 있던 리포터 분자가 퀜처 분자로부터 떨어지게 되어 신호가 발생한다.On the other hand, as illustrated in the lower part of Fig. 9, the artificial template and the hydrolyzable probe may be configured as a single strand in which the 5' end of the artificial template and the 3' end of the hydrolyzable probe are connected to each other. Even if configured as a single strand in this way, when the mediator fragment binds to the artificial template and acts as a primer to extend the sequence, the reporter molecule, which was being quenched by the quencher molecule, is separated from the quencher molecule by the 5'-->3' exonuclease activity of a DNA polymerase such as Taq polymerase, thereby generating a signal.
한편 가수분해성 프로브를 사용할 경우에도, 정방향 프라이머의 5' 태그 서열을 표적핵산에 따라 달리 구성하고 또 인공 주형에서 프라이머로 작용할 매개체 서열을 표적핵산에 따라 달리 구성하며, 또 이에 맞춰 인공 주형에서 상기 매개체절편이 결합하는 프라이밍 부위를 달리 구성함과 함께 가수분해성 프로브에서 그 리포터 분자가 서로 다른 형광 신호를 발생시키도록 구성하면(서로 다른 방출 파장을 가진 리포터 분자로 가수분해성 프로브를 표지화시키면), 다중의 표적핵산 검출이 가능해진다.Meanwhile, even when using a hydrolyzable probe, if the 5' tag sequence of the forward primer is configured differently depending on the target nucleic acid, and the mediator sequence that will act as a primer in the artificial template is configured differently depending on the target nucleic acid, and the priming site to which the mediator fragment binds in the artificial template is configured differently accordingly, and the reporter molecule in the hydrolyzable probe is configured to generate different fluorescence signals (by labeling the hydrolyzable probe with reporter molecules having different emission wavelengths), detection of multiple target nucleic acids becomes possible.
2. 신규 생성된 인공핵산의 검출2. Detection of newly generated artificial nucleic acids
도 10에 도시된 바와 같이, 신규 생성된 인공핵산에, 이 인공핵산 서열에 특이적인 분자비콘을 결합시켜 발생하는 신호를 측정하여 표적핵산에 대한 정량 정보를 얻을 수 있다. As illustrated in Figure 10, quantitative information on the target nucleic acid can be obtained by measuring the signal generated by binding a molecular beacon specific to the artificial nucleic acid sequence to the newly generated artificial nucleic acid.
이 경우에도 전술한 바의 인공 주형의 주형 부위(C')가 헤어핀 구조를 취하는 경우에 있어서와 같은 방식으로, 서로 다른 형광 신호를 이용하거나 단일 형광 신호로 증폭 후 또는 실시간으로 서로 다른 Tm을 이용하여 융해 곡선 분석 등을 수행하면 다중 표적핵산 분석이 가능하다. In this case, as well, in the same manner as in the case where the template portion (C') of the artificial template described above takes on a hairpin structure, multiple target nucleic acid analysis is possible by performing melting curve analysis, etc. using different fluorescence signals, or after amplification with a single fluorescence signal, or using different Tm in real time.
또 도 11에 도시된 바와 같이, 신규 연장된 서열인 인공핵산에, 이 서열의 일부에 특이적인 택맨 프로브 등 가수분해성 프로브를 결합시키고 이 신규 연장된 서열에 상보적인 프라이머(C-p)를 사용하여 연장시키면, Taq 폴리머라아제 등 DNA 중합효소에 의해 가수분해성 프로브가 분해되면서 신호가 발생하게 되고, 그 발생하는 신호를 측정하면 표적핵산에 대한 정량 정보를 얻을 수 있게 된다. 여기서 이 신규 연장된 서열인 인공핵산에 상보적인 프라이머(C-p)는 신규 연장된 서열인 인공핵산이 생성되어야만 여기에 결합하여 연장 반응이 일어나게 되고, 인공핵산은 시료 중에 표적핵산이 존재하여야만 생성되므로, 이 인공핵산에 결합한 가수분해성 프로브가 분해되면서 발생하는 신호는 시료 중에 표적핵산에 대한 정량 정보를 반영한다.Also, as illustrated in FIG. 11, when a hydrolyzable probe, such as a TaqMan probe, specific for a part of the sequence is bound to an artificial nucleic acid, which is a new extended sequence, and a primer (C-p) complementary to the new extended sequence is used to extend the sequence, a signal is generated when the hydrolyzable probe is decomposed by a DNA polymerase, such as Taq polymerase, and by measuring the generated signal, quantitative information on the target nucleic acid can be obtained. Here, the primer (C-p) complementary to the new extended sequence, which is an artificial nucleic acid, binds to the new extended sequence, which is an artificial nucleic acid, only when the artificial nucleic acid is generated, and the extension reaction occurs, and since the artificial nucleic acid is generated only when the target nucleic acid exists in the sample, the signal generated when the hydrolyzable probe bound to the artificial nucleic acid is decomposed reflects quantitative information on the target nucleic acid in the sample.
또 도 12에 도시된 바와 같이, 신규 연장된 서열인 인공핵산에 상보적인 서열이 고정되어 있는 지지체(기판, 비드 등)에 상기 신규 연장된 서열이 포함된 반응물을 처리하고 이에 검출 신호로 표지화된 프로브를 처리한 후에 세척용액으로 결합하지 아니한 핵산 등을 세척하고 제거하여 검출 신호를 측정하면 신규 연장된 서열인 인공핵산을 검출할 수도 있다. 이는 당업계 공지된 마이크러어레이의 샌드위치 검출법을 적용한 방법이다.Also, as illustrated in FIG. 12, by treating a reactant including the new extended sequence, which is an artificial nucleic acid, with a complementary sequence fixed to a support (substrate, bead, etc.) and then treating the reactant with a probe labeled with a detection signal, and then washing and removing unbound nucleic acids, etc. with a washing solution and measuring the detection signal, the new extended sequence, which is an artificial nucleic acid, can be detected. This is a method that applies a sandwich detection method of a microarray known in the art.
도 8 내지 도 12에 도시된 방법 이외에도 핵산 검출에 사용되는 당업계에 공지된 임의의 방법을 모두 적용할 수 있다. 예컨대 브롬화 에티듐(ethidium bromide) 등의 인터칼레이터를 사용하여 인공주형과 인공핵산의 이중가닥을 PCR 반응 중에 및/또는 PCR 반응 종료 후에 검출할 수도 있다. 이때 2가지 이상의 표적핵산을 검출하기 위한 다중 검출의 경우 각 표적핵산에 따른 인공주형을 사용하여, 신규 생성되는 인공주형과 인공핵산의 이중가닥의 길이와 GC를 조정함으로써 검출할 수 있다. In addition to the methods illustrated in FIGS. 8 to 12, any method known in the art for nucleic acid detection may be applied. For example, an intercalator such as ethidium bromide may be used to detect the double strand of the artificial template and the artificial nucleic acid during the PCR reaction and/or after the completion of the PCR reaction. In this case, in the case of multiple detection for detecting two or more target nucleic acids, the length and GC of the double strand of the newly generated artificial template and the artificial nucleic acid may be adjusted by using an artificial template according to each target nucleic acid.
3. 인공핵산에 의존하여 생성되는 추가의 인공핵산 검출 또는 추가의 인공핵산이 생성되면서 변화하는 신호 검출3. Detection of additional artificial nucleic acids produced by relying on artificial nucleic acids or detection of signals that change as additional artificial nucleic acids are produced.
도 13에 도시된 바와 같이, 매개체 올리고뉴클레오티드에, 그 5' 말단 쪽에 인위적 서열의 5' 태그 부위(D, 매개체 올리오뉴클레오티드의 5' 태그 부위)가 추가로 도입되게 되면, 그 5' 태그 부위(D)에 상보적인 부위(D', 5' 태그 부위의 상보적 부위)도 표적핵산이 존재해야만 생성될 수 있다. 구체적으로 시료 중에 표적핵산이 존재해야만 신규 연장된 서열인 인공핵산(C)이 생성되고, 이 인공핵산(C)이 생성되어야 이에 상보적인 프라이머(C-p)가 결합하게 되며, 이 프라이머(C-p)가 결합하면 이로부터 서열 연장이 이루어져 매개체 올리고뉴클레오티드의 5' 태그 부위(D)의 상보적 부위(D')가 생성될 수 있게 된다. 따라서 이러한 매개체 올리고뉴클레오티드의 5' 태그 부위(D)의 상보적 부위(D')는 신규 연장된 서열인 인공핵산(C, 제1의 인공핵산)에 의존하여 생성된 추가의 인공핵산(D', 제2의 인공핵산)이며, 이 추가의 인공핵산을 검출하거나 이 추가의 인공핵산이 생성되면서 변화하는 신호를 상기 도 8 내지 도 12를 참조하여 설명한 바의 방법을 적용하여 검출할 경우, 시료 중의 표적핵산에 대한 정성적, 정량적 정보를 얻을 수 있다. 예컨대 도 9에 도시된 방법을 응용하여 매개체 올리고뉴클레오티드의 5' 태그 부위(D)에 상보적인 가수분해성 프로브를 사용하여 검출하거나, 매개체 올리고뉴클레오티드의 5' 태그 부위(D)의 상보적 부위(D')를 이에 특이적으로 결합하는 분자비콘을 이용하여 검출하는 경우이다. As illustrated in FIG. 13, when a 5' tag portion (D, 5' tag portion of the mediator oligonucleotide) of an artificial sequence is additionally introduced at the 5' end of the mediator oligonucleotide, a portion (D', complementary portion of the 5' tag portion) complementary to the 5' tag portion (D) can also be generated only if a target nucleic acid exists. Specifically, an artificial nucleic acid (C), which is a new extended sequence, is generated only when a target nucleic acid exists in the sample, and only when this artificial nucleic acid (C) is generated can a complementary primer (C-p) bind to it, and when this primer (C-p) binds, sequence extension occurs therefrom, so that a complementary portion (D') of the 5' tag portion (D) of the mediator oligonucleotide can be generated. Therefore, the complementary portion (D') of the 5' tag portion (D) of the mediator oligonucleotide is an additional artificial nucleic acid (D', a second artificial nucleic acid) generated depending on the artificial nucleic acid (C, the first artificial nucleic acid), which is a new extended sequence, and when this additional artificial nucleic acid is detected or a signal that changes as this additional artificial nucleic acid is generated is detected by applying the method described with reference to FIGS. 8 to 12, qualitative and quantitative information about the target nucleic acid in the sample can be obtained. For example, this is a case where the method illustrated in FIG. 9 is applied to detect using a hydrolyzable probe complementary to the 5' tag portion (D) of the mediator oligonucleotide, or detecting using a molecular beacon that specifically binds to the complementary portion (D') of the 5' tag portion (D) of the mediator oligonucleotide.
또한 도 13에서 확인되는 바와 같이, 매개체 올리고큐늘레오티드의 5' 태그 부위(D)의 상보적인 부위(D')에 특이적인 프라이머(D'-p)를 도입하면 시료 중 표적핵산이 존재해야만 생성되는 인공핵산(C)과 추가의 인공핵산(D')이 증폭되게 되는 효과를 얻을 수 있다. 증폭될 경우 시료 중의 표적핵산에 대한 정성적, 정량적 정보를 제공하는 인공핵산(C)과 추가의 인공핵산(D')의 검출이 보다 용이할 수 있다.In addition, as confirmed in Fig. 13, when a specific primer (D'-p) is introduced to the complementary region (D') of the 5' tag region (D) of the mediator oligoquinuloline, an effect of amplifying an artificial nucleic acid (C) and an additional artificial nucleic acid (D') that are generated only when a target nucleic acid is present in the sample can be obtained. When amplified, the artificial nucleic acid (C) and the additional artificial nucleic acid (D'), which provide qualitative and quantitative information on the target nucleic acid in the sample, can be more easily detected.
또한 추가의 인공핵산(제2의 인공핵산)은 국제 공개특허 WO19066461A2(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant extension assay)의 도 2에 도시된 바와 같이, 추가의 인공주형(해당 도면에서는 Second CTO로 표기됨)을 사용하여 인공핵산(제1의 인공핵산)의 3' 말단에서 연장되어 생성될 수도 있다.Additionally, an additional artificial nucleic acid (a second artificial nucleic acid) can also be generated by extending from the 3' end of the artificial nucleic acid (the first artificial nucleic acid) using an additional artificial template (indicated as Second CTO in the drawing), as illustrated in FIG. 2 of international patent publication WO19066461A2 (title: Detection of target nucleic acid sequences by PTO cleavage and extension-dependent extension assay).
4. 기타의 방법4. Other methods
한편 배경기술에 언급한 국제 공개특허 WO12096430A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension assay), 국제 공개특허 WO12096523A2(명칭: Detection of target nucleic acid squences by PTO cleavage and extension assay), 국제 공개특허 WO12134195A2(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant cleavage), 국제 공개특허 WO13115442A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant signaling oligonucleotide hybridization assay), 국제 공개특허 WO13133561A1(명칭: Detection of nucleotide variation on target nucleic acid sequence by by PTO cleavage and extension assay), 국제 공개특허 WO13157821A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant signaling oligonucleotide cleavage), 국제 공개특허 WO13187628A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant transcription), 국제 공개특허 WO14104818A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant non-hybridization assay), 국제 공개특허 WO15008985A1(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant immobilized oligonucleotide hybridization), 국제 공개특허 WO15057008A2(Detection of target nucleic acid squences on solid phase by PTO cleavage and extension using HCTO assay), 국제 공개특허 WO19066461A2(명칭: Detection of target nucleic acid squences by PTO cleavage and extension-dependant extension assay), 국제 공개특허 WO2018196842(명칭: Method for detecting target nucleic acid sequences), 문헌[Bernd Faltin et al., Mediator Probe PCR: A Novel Approach for Detection of Real-Time PCR Based on Label-Free Primary Probes and Standardized Secondary Universal Fluorogenic Reporters, Clinical Chemistry 58:11 1546-1556 (2012)], 문헌[Wadle S et al., Mediator Probe PCR: Detection of Real-Time PCR by Label-Free Probes and a Universal Fluorogenic Reporter, Methods Mol Biol. (2014), 1160:55-73], 문헌[Wadle S et al., Real-time PCR probe optimization using design of experiments approach, Biomolecular Detection and Quantification 7 (2016), 1-8], 문헌[Lehnert M et al., Fluorescence signal-to-noise optimisation for real-time PCR using universal reporter oligonucleotides, June 2018Analytical Methods 10(28), DOI:10.1039/C8AY00812D], 문헌[Wadle S et al., Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters, Biotechniques (2018), Vol. 61, No. 3], 문헌[Elena Kipf et al., Advanced Minimal Residual Disease Monitoring for Acute Lymphoblastic Leukemia with Multiplex Mediator Probe PCR, J Mol Diagn. 2022 Jan;24(1):57-68], 문헌[Qiuying Huang et al., Highly multiplex PCR assays by coupling the 5'-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters, Proc Natl Acad Sci USA. 2022 Mar 1;119(9):e2110672119] 등은 모두 도 1(B)에 도시된 바와 같이, 본 발명의 매개체 올리고뉴클레오티드와 인공 주형을 이용하며, 이 점에서 본 발명과 공통된다. 다만 본 발명에서 매개체 올리고뉴클레이티드는 인위적 임의 서열의 5' 태그 부위에 결합하지만, 상기 국제 공개특허들 및 논문들에서는 본 발명에서 매개체 올리고뉴클레티드가 표적핵산에서 정방향 프라이머와 역방향 프라이머가 결합하는 부위 사이의 서열(internal sequence)에 결합하는 점만이 다르다. 따라서 상기 국제 공개특허들 및 논문들에서도 본 발명과 마찬가지로 시료 중에 표적핵산이 존재할 때 매개체 올리고뉴클레오티드가 절단되고 절단된 매개체 절편이 인공주형에서 프라이머로 작용하게 되는데, 이때 (i) 매개체 절편으로부터 연장되면서 발생하는 신호(즉 인공핵산이 생성되면서 발생하는 신호)를 검출하거나 (ii) 그 인공핵산을 검출하거나 또는 (iii) 그 인공핵산에 의존하여 생성되는 추가의 인공핵산을 검출하거나, 또는 이러한 추가의 인공핵산이 생성되면서 발생하는 신호를 검출함으로써 시료 중 표적핵산을 검출할 수 있게 된다. 상기 국제 공개특허들 및 논문들에 개시된 발명은 본 발명과는 매개체 올리고뉴클레티드가 결합하는 부위만 다를 뿐, 나머지 작용 방식은 서로 동일하므로 상기 국제 공개특허들 및 논문들에 개시된 검출 방법들은 모두 그대로 본 발명에 적용될 수 있다. Meanwhile, international patent publication WO12096430A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension assay), international patent publication WO12096523A2 (name: Detection of target nucleic acid squences by PTO cleavage and extension assay), international patent publication WO12134195A2 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent cleavage), international patent publication WO13115442A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent signaling oligonucleotide hybridization assay), international patent publication WO13133561A1 (name: Detection of nucleotide variation on target nucleic acid sequence by by PTO cleavage and extension assay), international patent publication WO12096430A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension assay), international patent publication WO12096523A2 (name: Detection of target nucleic acid squences by PTO cleavage and extension assay), international patent publication WO12134195A2 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependentant cleavage), international patent publication WO13115442A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependentant signaling oligonucleotide hybridization assay), international patent publication WO13133561A1 (name: Detection of nucleotide variation on target nucleic acid sequence by by PTO cleavage and extension assay), international patent publication WO12096430A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension assay), international patent publication WO12096523A2 (name: Detection of target nucleic acid squences by PTO cleavage and extension assay), international patent publication WO12096 WO13157821A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent signaling oligonucleotide cleavage), Internationally published patent WO13187628A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent transcription), Internationally published patent WO14104818A1 (name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent non-hybridization assay), International Publication Patent WO15008985A1 (Name: Detection of target nucleic acid squences by PTO cleavage and extension-dependant immobilized oligonucleotide hybridization), International Publication Patent WO15057008A2 (Detection of target nucleic acid squences on solid phase by PTO cleavage and extension using HCTO assay) , international open patent WO19066461A2 (Name: Detection of target nucleic acid squences by PTO cleavage and extension-dependent extension assay), International Patent WO2018196842 (Name: Method for detecting target nucleic acid sequences), Literature [Bernd Faltin et al., Mediator Probe PCR: A Novel Approach for Detection of Real-Time PCR Based on Label-Free Primary Probes and Standardized Secondary Universal Fluorogenic Reporters , Clinical Chemistry 58:11 1546-1556 (2012)], Wadle S et al., Mediator Probe PCR: Detection of Real-Time PCR by Label-Free Probes and a Universal Fluorogenic Reporter, Methods Mol Biol. (2014), 1160:55-73], Wadle S et al., Real-time PCR probe optimization using design of experiments approach, Biomolecular Detection and Quantification 7 (2016), 1-8], Lehnert M et al., Fluorescence signal-to-noise optimization for real-time PCR using universal reporter oligonucleotides, June 2018Analytical Methods 10(28), DOI:10.1039/C8AY00812D], Wadle S et al., Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters, Biotechniques (2018), Vol. 61, no. 3], Elena Kipf et al., Advanced Minimal Residual Disease Monitoring for Acute Lymphoblastic Leukemia with Multiplex Mediator Probe PCR, J Mol Diagn. 2022 Jan;24(1):57-68], the literature [Qiuying Huang et al., Highly multiplex PCR assays by coupling the 5'-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters, Proc Natl Acad Sci USA. 2022 Mar 1;119(9):e2110672119], etc. all use the mediator oligonucleotide of the present invention and an artificial template, as shown in Fig. 1(B), and in this respect, they have something in common with the present invention. However, while the mediator oligonucleotide in the present invention binds to the 5' tag site of an artificial random sequence, the only difference in the international published patents and papers is that the mediator oligonucleotide in the present invention binds to a sequence (internal sequence) between the sites where the forward primer and the reverse primer bind in the target nucleic acid. Therefore, in the above international published patents and papers, as in the present invention, when a target nucleic acid is present in a sample, a mediator oligonucleotide is cleaved and the cleaved mediator fragment acts as a primer in an artificial template. At this time, the target nucleic acid in the sample can be detected by (i) detecting a signal generated by extension from the mediator fragment (i.e., a signal generated by generating an artificial nucleic acid), (ii) detecting the artificial nucleic acid, or (iii) detecting an additional artificial nucleic acid generated depending on the artificial nucleic acid, or detecting a signal generated by generating such an additional artificial nucleic acid. The inventions disclosed in the above international published patents and papers differ only in the site to which the mediator oligonucleotide binds from the present invention, and the remaining modes of action are the same, so that all detection methods disclosed in the above international published patents and papers can be applied as they are to the present invention.
한편 도 14에서 보여지는 바와 같이, 역방향 프라이머도 정방향 프라이머와 마찬가지로 5' 말단에 인위적 임의 서열로 구성된 태그 서열을 가질 수 있다. 이럴 경우 시료 중에 표적핵산이 존재하게 되면, PCR의 제1 cycle에서 5' 태그 서열을 가진 역방향 프라이머가 연장되게 되고, 그러면 PCR의 다음 cycle에서 이 연장된 가닥에 정방향 프라이머가 결합하고, 더불어 그 연장된 가닥의 5' 태그 서열에 매개체 올리고뉴클레오티드가 결합하여 Taq 폴리머라아제 등 DNA 중합효소에 의해 연장반응이 일어나면, Taq 폴리머라아제 등 중합효소의 뉴클레아아제 활성에 의해 5' 태그 서열에 결합하고 있던 매개체 올리고뉴클레오티드에서 매개체 절편이 절단되어 발생하므로, 인공핵산의 생성이 정방향 프라이머에만 5' 태그 서열이 존재할 경우에 비해 2배로 증가하게 되므로 검출의 민감성이 2배로 높아질 수 있다. Meanwhile, as shown in Fig. 14, the reverse primer, like the forward primer, may have a tag sequence composed of an artificial random sequence at the 5' end. In this case, if a target nucleic acid is present in the sample, the reverse primer having the 5' tag sequence is extended in the first cycle of PCR, and then the forward primer binds to the extended strand in the next cycle of PCR, and further, when a mediator oligonucleotide binds to the 5' tag sequence of the extended strand and an extension reaction occurs by a DNA polymerase such as Taq polymerase, the mediator fragment is cleaved from the mediator oligonucleotide bound to the 5' tag sequence by the nuclease activity of the polymerase such as Taq polymerase, so that the generation of artificial nucleic acids increases by two times compared to when the 5' tag sequence is present only in the forward primer, and thus the sensitivity of detection can be doubled.
이 경우, 역방향 프라이머의 5' 태그 서열은 정방향 프라이머에 있는 5' 태그 서열과 같은 서열로 구성하고 또한 이에 결합하는 매개체 올리고뉴클레오티드 서열도 정방향 프라이머의 5' 태그 서열에 결합하는 매개체 올리고뉴클레오티드 서열과 같은 서열로 구성하게 된다. In this case, the 5' tag sequence of the reverse primer is composed of the same sequence as the 5' tag sequence in the forward primer, and also the mediator oligonucleotide sequence that binds thereto is composed of the same sequence as the mediator oligonucleotide sequence that binds to the 5' tag sequence of the forward primer.
또 한편 도 15에서 보여지는 바와 같이, 정방향 프라이머 및/또는 역방향 프라이머에 5' 태그 서열을 2개 이상 구성할 수도 있다. 이럴 경우 5' 태그 서열이 존재하는 개수에 의존하여 매개체 올리고뉴클레오티드의 결합 부위가 증가하므로 이에 비례하여 인공핵산의 생성이 증가하게 되므로(즉 인공핵산의 copy 수가 많아지게 되므로) 검출의 민감성이 더욱 높일 수 있는 효과가 있다.On the other hand, as shown in Fig. 15, two or more 5' tag sequences may be configured in the forward primer and/or the reverse primer. In this case, the binding site of the mediator oligonucleotide increases depending on the number of 5' tag sequences present, so the production of artificial nucleic acids increases proportionally (i.e., the copy number of the artificial nucleic acids increases), thereby further increasing the sensitivity of detection.
또 한편 도 16에서 보여지는 바와 같이, 5' 태그 서열과 같은 서열을 가지고 그 5' 태그 서열의 5'쪽에 추가의 태그 서열(5' 추가 태그 서열)을 가진 보조의 정방향 프라이머를 이용하여 표적핵산을 인공핵산으로 전환시켜 표적핵산을 검출할 수도 있다. 이 때 5' 추가 태그 서열은 5' 태그 서열과 동일 서열로 구성하거나 다른 서열로 구성할 수도 있다. 여기서 보조의 프라이머는 5' 태그 서열이 역방향 프라이머에 존재할 경우 역방향 프라이머에 대해 사용한다.On the other hand, as shown in Fig. 16, the target nucleic acid can be converted into an artificial nucleic acid by using an auxiliary forward primer having the same sequence as the 5' tag sequence and an additional tag sequence (5' additional tag sequence) at the 5' end of the 5' tag sequence, and the target nucleic acid can be detected. At this time, the 5' additional tag sequence can be composed of the same sequence as the 5' tag sequence or a different sequence. Here, the auxiliary primer is used for the reverse primer when the 5' tag sequence is present in the reverse primer.
5' 추가의 태그 서열을 5' 태그 서열과 동일 서열로 구성할 경우 매개체 올리고뉴클레오티드의 결합 부위가 많아지고 결국 생성되는 인공핵산의 생성이 증가하여 표적핵산의 검출이 용이해질 수 있다.If the 5' additional tag sequence is configured with the same sequence as the 5' tag sequence, the binding sites of the mediator oligonucleotide increase, and the production of artificial nucleic acids ultimately increases, making it easier to detect the target nucleic acid.
5' 추가 태그 서열을 5' 태그 서열과 다른 서열로 구성할 경우, 프로브 부위의 서열은 서로 다른 서열을 가지지만 매개체 부위의 서열은 같은 서열을 가진 매개체 올리고뉴클레오티드를 사용하게 되면, 특이성이 증가하고 더불어 생성되는 인공핵산의 생성이 증가하여 표적핵산의 검출 민감도가 상승할 수 있다. When the 5' additional tag sequence is composed of a sequence different from the 5' tag sequence, and the probe portion has a different sequence but the intermediate portion has the same sequence, the specificity increases and the production of artificial nucleic acids increases, so that the detection sensitivity of the target nucleic acid can be increased.
또한 도 16에서 보여지는 바와 같이, 다른 구성의 보조의 정방향 프라이머를 사용할 수도 있다. 이 다른 구성의 보조의 정방향 프라이머는 5' 태그 서열과 같은 서열 가지는 짧은 길이로 된 프라이머로서, 증폭 cycle 회수가 증가함에 따라 점점 copy 수가 증가하는, 5' 태그 서열이 붙어있는 가닥에의 상보적인 가닥에 대해, 5'태그 서열을 가진 정방향 프라이머와 경쟁적으로 정방향 프라이머로서 작용하지만, 서열 길이가 짧아 보다 효율적으로 프라이머로서 작용하게 될 것이다. Also, as shown in Fig. 16, an auxiliary forward primer of a different configuration can be used. This auxiliary forward primer of a different configuration is a short primer having the same sequence as the 5' tag sequence, and will act as a forward primer competitively with the forward primer having the 5' tag sequence for the complementary strand to the strand to which the 5' tag sequence is attached, the copy number of which gradually increases as the number of amplification cycles increases, but will act as a primer more efficiently due to its shorter sequence length.
5' 태그 서열이 정방향 프라이머 및/또는 역방향 프라이머에 존재할 경우, 그 5' 태그 서열과 표적핵산에 상보적인 영역 사이에, 1 내지 10 뉴클레오티드로 구성되는 스페이서(spacer)가 존재할 수 있다. When a 5' tag sequence is present in the forward primer and/or the reverse primer, a spacer consisting of 1 to 10 nucleotides may be present between the 5' tag sequence and the region complementary to the target nucleic acid.
한편 도 17에 도시된 바와 같이, 5' 태그 부위를 가진 정방향 프라이머 및/또는 5' 태그 부위를 가진 역방향 프라이머를, 하나의 표적핵산에 대해서 이들 프라이머의 각 특이적 결합 위치를 달리하여 2종 이상 사용할 수도 있다. 이들 2종 이상의 프라이머는 표적핵산과의 특이적 결합 위치만이 다르고, 5' 태그 부위는 모두 같은 서열로 구성하여, 같은 서열을 가진 매개체 올리고뉴클레오티드를 사용하게 되면, 매개체 올리고뉴클레오티드의 결합 부위가 많아지고, 이에 따라 생성되는 인공핵산의 생성이 증가하게 되어 되어, 결국 검출 신호가 증폭되는 효과를 가져올 수 있다. Meanwhile, as illustrated in FIG. 17, two or more types of a forward primer having a 5' tag portion and/or a reverse primer having a 5' tag portion may be used with different specific binding sites of each of these primers for one target nucleic acid. These two or more types of primers only differ in their specific binding sites with respect to the target nucleic acid, and the 5' tag portions are all composed of the same sequence, so that when a mediator oligonucleotide having the same sequence is used, the binding sites of the mediator oligonucleotide increase, and thus the production of artificial nucleic acids increases, which can ultimately have the effect of amplifying the detection signal.
이렇게 5' 태그 부위를 가진 정방향 프라이머 및/또는 5' 태그 부위를 가진 역방향 프라이머를 2종 이상 사용할 경우, 도 1에 도시된 기존의 방법과 달리 정방향 프라이머와 역방향 프라이머 사이의 서열(internal sequence)에서 매개체 올리고뉴클레오티드의 결합 부위를 정하지 않고도, 2개 이상의 매개체 올리고뉴클레오티드의 결합 부위가 생겨나므로 RNA 바이러스 등의 표적핵산처럼 변이가 심한 표적핵산의 검출에 유용할 수 있고, 또 시료 중 카피 수(copy number)가 적은 표적핵산(즉 시료 중 농도가 낮은 표적핵산)의 검출에도 유용할 수 있다. In this way, when two or more types of forward primers having a 5' tag portion and/or reverse primers having a 5' tag portion are used, unlike the conventional method illustrated in FIG. 1, binding sites of two or more mediator oligonucleotides are created without determining the binding sites of the mediator oligonucleotides in the sequence (internal sequence) between the forward primer and the reverse primer, so that it can be useful for the detection of target nucleic acids with high mutations, such as RNA viruses, and can also be useful for the detection of target nucleic acids with a low copy number in a sample (i.e., target nucleic acids with a low concentration in a sample).
아래의 실시예는 Neisseria gonorrhoeae (NG)의 유전자를 표적핵산으로 하여 동일한 5' 태그 부위를 가진 2종의 정방향 프라이머를, 표적핵산과의 특이적 결합 위치를 달리하여 사용할 경우 5' 태그 부위를 가진 1종의 정방향 프라이머를 사용할 경우에 비해 검출 신호가 더 빨리 나타남을 보여준다. The examples below show that when two forward primers having the same 5' tag portion are used with different specific binding positions to the target nucleic acid, using a gene of Neisseria gonorrhoeae (NG) as a target nucleic acid, a detection signal appears faster than when one forward primer having a 5' tag portion is used.
한편 도 18에 도시된 바와 같이, 5' 태그 부위를 가진 정방향 프라이머 및/또는 5' 태그 부위를 가진 역방향 프라이머를, 표적핵산을 달리하여 사용할 수도 있다. 이때 표적핵산은 2종 이상일 수 있다. 물론 이 경우에 있어서도 2종 이상의 프라이머는 그것이 결합하는 표적핵산에 따라 다르지만, 5' 태그 부위는 모두 같은 서열로 구성하여 같은 서열의 매개체 올리고뉴클레오티드를 사용하게 되면, 매개체 올리고뉴클레오티드의 결합 부위가 많아지고, 이에 따라 생성되는 인공핵산의 생성이 증가하게 되어, 결국 검출 신호가 증폭되는 효과를 가져올 수 있다. Meanwhile, as illustrated in FIG. 18, a forward primer having a 5' tag portion and/or a reverse primer having a 5' tag portion may be used with different target nucleic acids. At this time, there may be two or more target nucleic acids. Of course, even in this case, the two or more primers are different depending on the target nucleic acids to which they bind, but if the 5' tag portions are all composed of the same sequence and a mediator oligonucleotide of the same sequence is used, the binding sites of the mediator oligonucleotide increase, and accordingly, the production of artificial nucleic acids increases, which can ultimately have the effect of amplifying the detection signal.
이렇게 구성할 경우, 코로나 바이러스(SARS-CoV-2, 제2형 중증급성호흡기증후군) 검출에서처럼 1종 감염 미생물(세균 또는 바이러스)의 검출에 있어서, 검출의 민감성을 높여 결국 시료 중 검출 대상 미생물의 숫자가 적은 경우에도 검출이 가능해질 수 있다.When configured in this way, the sensitivity of detection can be increased for the detection of a single infectious microorganism (bacteria or virus), such as in the detection of coronavirus (SARS-CoV-2, severe acute respiratory syndrome type 2), which ultimately enables detection even when the number of target microorganisms in the sample is small.
아래의 실시예는 LacZ의 유전자의 일부 서열(표적서열 1)과 GAPDH 유전자의 일부 서열(표적서열 2)을 표적핵산으로 하여 동일한 5' 태그 부위를 가진, 각각의 표적핵산에 특이적인 2종의 정방향 프라이머를 사용할 경우, 5' 태그 부위를 가진 1종의 정방향 프라이머를 사용하여 각각의 표적핵산을 검출할 경우에 비해 검출 신호가 더 빨리 나타남을 보여준다. The examples below show that when two forward primers, each specific for a target nucleic acid having the same 5' tag portion, are used, targeting a part of a LacZ gene (target sequence 1) and a part of a GAPDH gene (target sequence 2), a detection signal appears faster than when each target nucleic acid is detected using one forward primer having a 5' tag portion.
전술한 바를 종합할 때, 본 발명은 일 측면에 있어서, 플랩 절편(또는 매개체 절편)을 이용한 표적핵산의 검출 방법으로 파악할 수 있다.In summary of the above, the present invention can be understood in one aspect as a method for detecting a target nucleic acid using a flap fragment (or a mediator fragment).
본 발명의 매개체 절편을 이용한 표적핵산의 검출 방법은 아래의 (a) 단계 내지 (c) 단계를 포함한다.The method for detecting a target nucleic acid using a mediator fragment of the present invention comprises steps (a) to (c) below.
(a) 검출 대상 시료와 4가지 NTP(nucleoside triphosphate) 및 뉴클레아제 활성을 가지는 DNA 중합효소를 포함하고, 아래의 (i) 내지 (iii)를 포함하는 PCR 반응을 위한 혼합물을 준비하는 단계(a) a step of preparing a mixture for PCR reaction comprising a sample to be detected, four NTPs (nucleoside triphosphates) and a DNA polymerase having nuclease activity, and including (i) to (iii) below
(i) 표적핵산을 증폭시키기 위한 한 세트의 정방향 및 역방향 프라이머, 여기서 정방향 프라이머 및 역방향 프라이머 중 하나 이상은 그 5' 말단 부위에 인위적 임의 서열로 구성된 5' 태그 서열을 하나 이상 가짐; 및(i) a set of forward and reverse primers for amplifying a target nucleic acid, wherein at least one of the forward primer and the reverse primer has at least one 5' tag sequence consisting of an artificial random sequence at its 5' terminal portion; and
(ii) 상기 5' 태그 서열에 상보적으로 결합하는 프로브 부위(A)와 표적핵산에 특이적인 인위적 임의 서열로 이루어진 매개체 부위(B)을 포함하는 단일 가닥의 매개체 올리고뉴클레오티드(ii) a single-stranded mediator oligonucleotide comprising a probe portion (A) that binds complementarily to the 5' tag sequence and a mediator portion (B) consisting of an artificial random sequence specific for a target nucleic acid;
(b) 상기 (a) 단계의 혼합물을 반응 용기에 넣고, PCR 반응을 수행하여, 표적핵산이 증폭됨과 동시에, 상기 뉴클레아제 활성을 갖는 DNA 중합효소에 의해 매개체 올리고뉴클레오티드에서 매개체 절편이 절단되어 생성되도록 하는 단계, 및(b) a step of placing the mixture of step (a) into a reaction vessel and performing a PCR reaction so that the target nucleic acid is amplified and at the same time, a mediator fragment is cleaved from the mediator oligonucleotide by a DNA polymerase having the nuclease activity, and
(c) 상기 매개체 절편을 검출하여 표적핵산을 검출하는 단계.(c) A step of detecting the target nucleic acid by detecting the above medium fragment.
본 발명의 방법에서, 상기 표적핵산에 특이적인 인위적 임의 서열로 이루어진 매개체 부위는 염기서열(뉴클레오티드 순서) 및 크기 중 어느 하나 이상이 표적핵산에 특이적인 것을 의미한다.In the method of the present invention, the mediator portion consisting of an artificial random sequence specific to the target nucleic acid means that at least one of the base sequence (nucleotide order) and size is specific to the target nucleic acid.
또 본 발명의 방법에서, 상기 매개체 올리고뉴클레오티드를 2가지 이상의 표적핵산에 따라 각기 다른 것을 사용함으로써 2가지 표적핵산을 하나의 반응으로 검출할 수도 있다. 이 경우 프라이머의 5' 태그 부위도 상기 매개체 올리고뉴클레오티드 서열(특히 프로브 부위의 서열)에 따라 상보적으로 구성될 것이다.In addition, in the method of the present invention, by using different mediator oligonucleotides according to two or more target nucleic acids, two target nucleic acids can be detected in one reaction. In this case, the 5' tag portion of the primer will also be complementarily configured according to the mediator oligonucleotide sequence (particularly, the sequence of the probe portion).
또 본 발명의 방법에서, 상기 매개체 절편의 검출은 전술한 바와 같이, 질량 분석(mass spectrometry), 전기영동(electrophoresis), 액체 크로마토그래피(liquid chromatography), 서열분석, 마이크로어레이, 매개체 절편으로부터 연장되어 생성되는 인공핵산 등에 의하여 수행될 수 있다. 여기서 상기 매개체 절편으로부터 연장되어 생성되는 인공핵산은 (i) 앞서 도 1(A)를 참조하여 설명한 바와 같이, 그 매개체 절편 자체가 인공주형으로 작용하여 그 매개체 절편의 3' 말단에서 연장되어 생성되거나, (ii) 도 6을 참조하여 설명한 바와 같이, 별도 분자의 인공주형에 상기 매개체 절편이 상보적으로 결합하여 프라이머로 작용함으로써 그 매개체 절편의 3' 말단에서 연장되어 생성될 수 있다. In addition, in the method of the present invention, the detection of the mediator fragment can be performed by mass spectrometry, electrophoresis, liquid chromatography, sequence analysis, microarray, artificial nucleic acid generated by extension from the mediator fragment, etc., as described above. Here, the artificial nucleic acid generated by extension from the mediator fragment can be generated by (i) extending from the 3' end of the mediator fragment by the mediator fragment itself acting as an artificial template as described above with reference to Fig. 1(A), or (ii) extending from the 3' end of the mediator fragment by the mediator fragment complementarily binding to an artificial template of a separate molecule to act as a primer as described with reference to Fig. 6.
별도 분자의 인공주형으로부터 인공핵산이 매개체 절편으로부터 연장되어 생성될 경우 상기 PCR 반응을 위한 혼합물에 상기 별도 분자의 인공주형이 추가로 포함되고, 그 인공주형은 상기 매개체 올리고뉴클레오티드의 매개체 부위에 대해 상보적인 프라이밍 부위를 가지고 또한 상기 매개체 절편으로부터 연장되어 생성되는 인공핵산에 대해 주형으로 작용하는 주형 부위(C' )를 가진다. When an artificial nucleic acid is generated by extension from a mediator segment from an artificial template of a separate molecule, the artificial template of the separate molecule is additionally included in the mixture for the PCR reaction, and the artificial template has a priming site complementary to the mediator segment of the mediator oligonucleotide and also has a template site (C') that acts as a template for the artificial nucleic acid generated by extension from the mediator segment.
다른 측면에 있어서, 본 발명은 표적핵산을 인공핵산으로 전환하는 방법에 관한 것이다.In another aspect, the present invention relates to a method for converting a target nucleic acid into an artificial nucleic acid.
본 발명의 인공핵산으로의 전환 방법은 아래의 (a) 단계 및 (b) 단계를 포함한다.The method for converting the artificial nucleic acid of the present invention comprises steps (a) and (b) below.
(a) 검출 대상 시료와 4가지 NTP(nucleoside triphosphate, 뉴클레오시드 3인산) 및 뉴클레아제 활성을 가지는 DNA 중합효소를 포함하고, 아래의 (i) 내지 (iii)를 포함하는 PCR 반응을 위한 혼합물을 준비하는 단계(a) a step of preparing a mixture for a PCR reaction comprising a sample to be detected, four NTPs (nucleoside triphosphates) and a DNA polymerase having nuclease activity, and including (i) to (iii) below
(i) 표적핵산을 증폭시키기 위한 한 세트의 정방향 및 역방향 프라이머, 여기서 정방향 프라이머 및 역방향 프라이머 중 하나 이상은 그 5' 말단 부위에 인위적 임의 서열로 구성된 태그 서열을 하나 이상 가짐; (i) a set of forward and reverse primers for amplifying a target nucleic acid, wherein at least one of the forward primer and the reverse primer has at least one tag sequence consisting of an artificial random sequence at its 5' end;
(ii) 상기 5' 태그 서열에 상보적으로 결합하는 프로브 부위(A)와 인위적 임의 서열로 이루어진 매개체 부위(B)을 포함하는 단일 가닥의 매개체 올리고뉴클레오티드; 및(ii) a single-stranded mediator oligonucleotide comprising a probe portion (A) complementarily binding to the 5' tag sequence and a mediator portion (B) consisting of an artificial random sequence; and
(iii) 상기 매개체 올리고뉴클레오티드의 매개체 부위(B)에 대해 상보적인 프라이밍 부위(B' )를 가지고 또한 신규로 연장되는 서열(C)인 인공핵산에 대해 주형으로 작용하는 주형 부위(C' )를 가진 인공 주형, (iii) an artificial template having a priming site (B') complementary to the mediator site (B) of the above mediator oligonucleotide and a template site (C') that acts as a template for an artificial nucleic acid, which is a newly extended sequence (C);
(b) 상기 (a) 단계의 혼합물을 반응 용기에 넣고, PCR 반응을 수행하여, 표적핵산이 증폭됨과 동시에, 상기 뉴클레아제 활성을 갖는 DNA 중합효소에 의해 매개체 올리고뉴클레오티드에서 매개체 절편이 생성되도록 하고, 그 매개체 절편이 인공주형의 프라이밍 부위에 결합하여 인공주형의 주형 부위를 주형으로 하여 연장됨으로써 상기 인공주형의 주형 부위에 상보적인 서열을 가지는 인공핵산이 생성되도록 하는 단계.(b) a step of placing the mixture of step (a) into a reaction vessel, performing a PCR reaction, so that the target nucleic acid is amplified and at the same time, a mediator fragment is generated from the mediator oligonucleotide by the DNA polymerase having the nuclease activity, and the mediator fragment binds to the priming site of the artificial template and is extended using the template site of the artificial template as a template, thereby generating an artificial nucleic acid having a sequence complementary to the template site of the artificial template.
본 발명은 다른 측면에 있어서, 검출 대상 시료의 표적핵산을 인공핵산으로 전환시켜 검출하는 방법으로 파악할 수 있다.In another aspect, the present invention can be understood as a method for detecting a target nucleic acid of a sample to be detected by converting it into an artificial nucleic acid.
본 발명의 표적핵산의 검출 방법은 아래의 (a) 단계 내지 (c) 단계를 포함한다.The method for detecting a target nucleic acid of the present invention comprises steps (a) to (c) below.
(a) 검출 대상 시료와 4가지 NTP(nucleoside triphosphate, 뉴클레오시드 3인산) 및 뉴클레아제 활성을 가지는 DNA 중합효소를 포함하고, 아래의 (i) 내지 (iii)를 포함하는 PCR 반응을 위한 혼합물을 준비하는 단계(a) a step of preparing a mixture for a PCR reaction comprising a sample to be detected, four NTPs (nucleoside triphosphates) and a DNA polymerase having nuclease activity, and including (i) to (iii) below
(i) 표적핵산을 증폭시키기 위한 한 세트의 정방향 및 역방향 프라이머, 여기서 정방향 프라이머 및 역방향 프라이머 중 하나 이상은 그 5' 말단 부위에 인위적 임의 서열로 구성된 태그 서열을 하나 이상 가짐; (i) a set of forward and reverse primers for amplifying a target nucleic acid, wherein at least one of the forward primer and the reverse primer has at least one tag sequence consisting of an artificial random sequence at its 5' end;
(ii) 상기 5' 태그 서열에 상보적으로 결합하는 프로브 부위(A)와 인위적 임의 서열로 이루어진 매개체 부위(B)을 포함하는 단일 가닥의 매개체 올리고뉴클레오티드; 및(ii) a single-stranded mediator oligonucleotide comprising a probe portion (A) complementarily binding to the 5' tag sequence and a mediator portion (B) consisting of an artificial random sequence; and
(iii) 상기 매개체 올리고뉴클레오티드의 매개체 부위(B)에 대해 상보적인 프라이밍 부위(B' )를 가지고 또한 신규로 연장되는 서열(C)인 인공핵산에 대해 주형으로 작용하는 주형 부위(C' )를 가진 인공 주형, (iii) an artificial template having a priming site (B') complementary to the mediator site (B) of the above mediator oligonucleotide and a template site (C') that acts as a template for an artificial nucleic acid, which is a newly extended sequence (C);
(b) 상기 (a) 단계의 혼합물을 반응 용기에 넣고, PCR 반응을 수행하여, 표적핵산이 증폭됨과 동시에, 상기 뉴클레아제 활성을 갖는 DNA 중합효소에 의해 매개체 올리고뉴클레오티드에서 매개체 절편이 생성되도록 하고, 그 매개체 절편이 인공주형의 프라이밍 부위에 결합하여 인공주형의 주형 부위를 주형으로 하여 연장됨으로써 상기 인공주형의 주형 부위에 상보적인 서열을 가지는 인공핵산이 생성되도록 하는 단계, 및(b) a step of placing the mixture of step (a) into a reaction vessel, performing a PCR reaction, so that the target nucleic acid is amplified and at the same time, a mediator fragment is generated from the mediator oligonucleotide by the DNA polymerase having the nuclease activity, and the mediator fragment binds to the priming site of the artificial template and is extended using the template site of the artificial template as a template, thereby generating an artificial nucleic acid having a sequence complementary to the template site of the artificial template; and
(c) (i) 상기 인공핵산이 생성되면서 변화하는 신호를 검출하거나 (ii) 상기 생성된 인공핵산을 검출함에 의해 시료 중의 표적핵산을 검출하는 단계.(c) a step of detecting a target nucleic acid in a sample by (i) detecting a signal that changes as the artificial nucleic acid is generated or (ii) detecting the generated artificial nucleic acid.
본 발명의 방법에서, 상기 5' 태그 서열은 정방향 및 역방향 프라이머 중 하나에 또는 이들 프라이머 모두에, 1개 이상 존재할 수 있다. 이 경우 한 종류의 매개체 올리고뉴클레오티드를 사용할 수 있도록 하기 위해 5' 태그 서열을 모두 동일 서열로 구성할 수 있다.In the method of the present invention, the 5' tag sequence may be present in one or more of the forward and reverse primers, or in both of these primers. In this case, all of the 5' tag sequences may be composed of the same sequence so as to enable the use of one type of mediator oligonucleotide.
본 발명의 방법에서, 상기 (b) 단계 후에 상기 인공핵산에서, 그 인공핵산(제1의 인공핵산)에 의존하거나 추가의 인공핵산(제2의 인공핵산)이 생성되도록 하는 단계가 포함되고, 표적핵산의 검출은 (i) 상기 인공핵산에 의존하여 생성되는 추가의 인공핵산을 검출하거나, 또는 (ii) 상기 추가의 인공핵산이 생성되면서 변화하는 신호를 검출함에 의해 이루어질 수도 있다. In the method of the present invention, after the step (b), a step is included of generating an artificial nucleic acid (second artificial nucleic acid) dependent on the artificial nucleic acid (first artificial nucleic acid) or an additional artificial nucleic acid from the artificial nucleic acid, and detection of the target nucleic acid may be accomplished by (i) detecting the additional artificial nucleic acid generated dependent on the artificial nucleic acid, or (ii) detecting a signal that changes as the additional artificial nucleic acid is generated.
본 발명의 방법에서, 상기 뉴클레아제 활성은 5'-->3' 엑소뉴클레아제 활성 및 플랩(flap) 엔도뉴클레아제 활성을 포함한다.In the method of the present invention, the nuclease activity includes 5'-->3' exonuclease activity and flap endonuclease activity.
본 발명의 방법에서, 인공핵산(제1의 인공핵산)이 생성되면서 변화하는 신호를 검출하는 것은 구체적으로 아래 (i) 내지 (iii)의 경우를 포함한다.In the method of the present invention, detecting a signal that changes while an artificial nucleic acid (first artificial nucleic acid) is generated specifically includes the following cases (i) to (iii).
(i) 상기 인공주형이 헤어핀 구조의 분자비콘이고, 상기 매개체 절편이 상기 헤어핀 구조의 분자비콘에 특이적으로 결합하여 연장됨으로써 상기 헤어핀 구조가 펴지면서 리포터 분자가 퀜처 분자로부터 분리되어 발생하는 신호를 검출하는 것,(i) detecting a signal generated by the separation of a reporter molecule from a quencher molecule as the artificial template is a molecular beacon of a hairpin structure, and the mediator fragment specifically binds to and extends the molecular beacon of the hairpin structure, thereby unfolding the hairpin structure;
(ii) 상기 인공주형의 주형 부위에 가수분해성 프로브가 결합하고, 상기 매개체 절편이 인공 주형의 프라이밍 부위에 결합하여, 상기 5'-->3' 엑소뉴클레아제 활성을 가지는 DNA 중합효소에 의해 연장됨으로써 상기 가수분해성 프로브가 분해되어 발생하는 신호를 검출하는 것,(ii) detecting a signal generated by the hydrolyzable probe being decomposed by binding to the template portion of the artificial template, the mediator fragment being bound to the priming portion of the artificial template, and being extended by a DNA polymerase having the 5'-->3' exonuclease activity;
(iii) 상기 인공 주형과 가수분해성 프로브는 그 인공 주형의 5' 말단과 가수분해성 프로브의 3' 말단이 서로 연결된 단일 가닥으로 구성되어, 상기 매개체 절편이 인공 주형의 프라이밍 부위에 결합하여 5'-->3' 엑소뉴클레아제 활성을 가지는 DNA 중합효소에 의해 연장됨으로써 상기 가수분해성 프로브가 분해되어 발생하는 신호를 검출하는 것,(iii) The artificial template and the hydrolyzable probe are composed of a single strand in which the 5' end of the artificial template and the 3' end of the hydrolyzable probe are connected to each other, and the mediator fragment binds to the priming site of the artificial template and is extended by a DNA polymerase having a 5'-->3' exonuclease activity, thereby detecting a signal generated by the hydrolyzable probe being decomposed.
또 본 발명의 방법에서, 인공핵산(제1의 인공핵산)을 검출하는 것은 구체적으로 아래 (i) 내지 (iii)의 경우를 포함한다.In addition, in the method of the present invention, detecting an artificial nucleic acid (first artificial nucleic acid) specifically includes cases (i) to (iii) below.
(i) 상기 인공핵산에, 상기 인공핵산 서열에 특이적인 분자비콘을 결합시켜 발생하는 신호를 검출하는 것,(i) detecting a signal generated by binding a molecular beacon specific to the artificial nucleic acid sequence to the artificial nucleic acid;
(ii) 상기 인공핵산에, 상기 인공핵산 서열에 특이적인 가수분해성 프로브를 결합시키고, 상기 인공핵산에 상보적인 프라이머를 결합시켜 5'-->3' 엑소뉴클레아제 활성을 가지는 DNA 중합효소에 의해 연장시킴으로써 상기 가수분해성 프로브가 분해되어 발생하는 신호를 검출하는 것,(ii) detecting a signal generated by decomposition of the hydrolyzable probe by binding a hydrolyzable probe specific to the artificial nucleic acid sequence to the artificial nucleic acid, binding a complementary primer to the artificial nucleic acid, and extending the probe by a DNA polymerase having 5'-->3' exonuclease activity;
(iii) 상기 인공핵산에 상보적인 서열을 가지는 캡쳐 프로브(제1 프로브)가 고정되어 있는 지지체에 상기 인공핵산을 결합시키고, 상기 인공핵산 또는 상기 매개체 절편에 검출 신호로 표지화된 검출 프로브(제2 프로브)를 상보적으로 결합시키고 미반응물을 세척하여 제거한 후 그 프로브의 검출 신호를 검출하는 것, 여기서 상기 매개체 절편은 그것이 프라이머로서 작용함으로써 그로부터 인공핵산이 연장되어 있으므로 검출 신호를 가진 프로브를 그 매개체 절편에 상보적으로 결합시키더라도 인공핵산이 검출됨(도 12 참조). (iii) binding the artificial nucleic acid to a support on which a capture probe (first probe) having a complementary sequence to the artificial nucleic acid is fixed, complementarily binding a detection probe (second probe) labeled with a detection signal to the artificial nucleic acid or the mediator fragment, washing away unreacted matter, and then detecting the detection signal of the probe, wherein the mediator fragment acts as a primer from which the artificial nucleic acid is extended, so that even if a probe having a detection signal is complementarily bound to the mediator fragment, the artificial nucleic acid is detected (see FIG. 12).
또 본 발명의 방법에서, 상기 추가의 인공핵산(제2의 인공핵산)이 생성되면서 변화하는 신호를 검출하는 것은 구체적으로 다음과 같다: 상기 매개체 올리고뉴클레오티드로 그 5' 말단 쪽에 인위적 서열의 태그 부위(매개체 올리고뉴클레오티드의 5' 태그 부위)가 도입된 것을 사용하여, 상기 매개체 올리고뉴클레오티드의 5' 태그 부위에 특이적으로 결합하는 가수분해성 프로브와 상기 인공핵산에 특이적으로 결합하는 프라이머를 상기 PCR 반응 혼합물에 포함시킴으로써, 상기 인공핵산에 결합한 프라이머가 5'-->3' 엑소뉴클레아제 활성을 가지는 DNA 중합효소에 의해 연장됨으로써 상기 매개체 올리고뉴클레오티드의 5' 태그 부위에 결합하고 있던 가수분해성 프로브가 분해되어 발생하는 신호를 검출하는 것.In addition, in the method of the present invention, the detection of a signal that changes as the additional artificial nucleic acid (second artificial nucleic acid) is generated is specifically as follows: By using the intermediate oligonucleotide to which an artificial sequence tag portion (5' tag portion of the intermediate oligonucleotide) is introduced at the 5' end, a hydrolyzable probe that specifically binds to the 5' tag portion of the intermediate oligonucleotide and a primer that specifically binds to the artificial nucleic acid are included in the PCR reaction mixture, so that the primer bound to the artificial nucleic acid is extended by a DNA polymerase having a 5'-->3' exonuclease activity, thereby detecting a signal generated by decomposition of the hydrolyzable probe bound to the 5' tag portion of the intermediate oligonucleotide.
또 본 발명의 방법에서, 상기 추가의 인공핵산(제2의 인공핵산)을 검출하는 것은 다음과 같다: 상기 매개체 올리고뉴클레오티드로 그 5' 말단 쪽에 인위적 서열의 태그 부위(5' 태그 부위)가 도입된 것을 사용하여, 상기 5' 태그 부위에 대해 상보적인 서열 부위(5' 태그 부위의 상보적 부위)에 특이적으로 결합하는 분자비콘과 상기 인공핵산(제1의 인공핵산)에 특이적으로 결합하는 프라이머를 상기 PCR 반응 혼합물에 포함시킴으로써, 상기 인공핵산(제1의 인공핵산)에 결합한 프라이머가 인공핵산(제1의 인공핵산)을 주형으로 그 인공핵산(제1의 인공핵산)에 의존하여 연장됨으로써 상기 5' 태그 부위의 상보적 부위를 포함하는 추가의 인공핵산(제2의 인공핵산)이 생성되고, 상기 5' 태그 부위의 상보적 부위에 분자비콘이 특이적으로 결합함으로써 발생하는 신호를 검출하는 것.In addition, in the method of the present invention, detecting the additional artificial nucleic acid (second artificial nucleic acid) is as follows: using the intermediate oligonucleotide having an artificial sequence tag portion (5' tag portion) introduced at the 5' end thereof, a molecular beacon that specifically binds to a sequence portion complementary to the 5' tag portion (complementary portion of the 5' tag portion) and a primer that specifically binds to the artificial nucleic acid (first artificial nucleic acid) are included in the PCR reaction mixture, so that the primer bound to the artificial nucleic acid (first artificial nucleic acid) is extended depending on the artificial nucleic acid (first artificial nucleic acid) using the artificial nucleic acid (first artificial nucleic acid) as a template, thereby generating an additional artificial nucleic acid (second artificial nucleic acid) including a complementary portion of the 5' tag portion, and detecting a signal generated by the specific binding of the molecular beacon to the complementary portion of the 5' tag portion.
본 발명의 방법에서, 상기 신호 변화는 리포터 분자와 퀜처 분자의 거리가 변화함에 의해서 생기거나 또는 공여체와 수용체의 거리가 변화함에 의해서 생길 수 있다. In the method of the present invention, the signal change may be caused by a change in the distance between the reporter molecule and the quencher molecule or by a change in the distance between the donor and the acceptor.
또 본 발명의 방법에서, 상기 PCR 반응 혼합물에는, 도 16에 도시된 바와 같이, 상기 5' 태그 서열과 같은 서열을 가지는 보조의 프라이머가 포함될 수 있다.In addition, in the method of the present invention, the PCR reaction mixture may contain an auxiliary primer having the same sequence as the 5' tag sequence, as illustrated in FIG. 16.
또 본 발명의 방법에서, 상기 PCR 반응 혼합물에는, 상기 5' 태그 부위를 가진 프라이머가 하나의 표적핵산에 대해서 각 특이적 결합 위치를 달리하여 2가지 이상 포함될 수 있다. 이 경우 상기 5' 태그 부위는 모두 같은 서열을 가지므로, 모두 같은 매개체 올리고뉴클레오티드가 결합하게 되고, 결과적으로 시료 중 카피 수(copy number)가 적거나 변이가 심한 표적핵산의 검출이 용이할 수 있다. In addition, in the method of the present invention, the PCR reaction mixture may contain two or more primers having the 5' tag portion with different specific binding positions for each target nucleic acid. In this case, since the 5' tag portions all have the same sequence, the same mediator oligonucleotide is bound to all of them, and as a result, the detection of a target nucleic acid with a low copy number or a severe mutation in the sample can be facilitated.
또 본 발명의 방법에서, 상기 표적핵산은 2가지 이상이고, 각 표적핵산에 대해서 각각의 정방향 프라이머와 역방향 프라이머가 상기 혼합물에 포함되되, 상기 각 표적핵산에 대한 각각의 정방향 프라이머 및 역방향 프라이머 중 하나 이상의 프라이머는 5' 태그 부위를 가지고, 상기 5' 태그 부위는 모두 동일 서열로 이루어질 수 있다. 이 경우도 상기 5' 태그 부위는 모두 동일 서열을 가지므로, 모두 같은 매개체 올리고뉴클레오티드가 결합하게 되고, 결과적으로 시료 중 검출 대상 감염증 원인 미생물(바이러스 또는 세균)이 적더라도 2개 이상 표적핵산의 검출 신호가 같은 신호로 발생하므로 검출이 용이할 수 있다. In addition, in the method of the present invention, the target nucleic acids are two or more, and for each target nucleic acid, a forward primer and a reverse primer are included in the mixture, wherein at least one primer among the forward primers and reverse primers for each target nucleic acid has a 5' tag portion, and all of the 5' tag portions may be composed of the same sequence. In this case, since all of the 5' tag portions have the same sequence, all of the same mediator oligonucleotides bind, and as a result, even if the number of target infectious agent microorganisms (viruses or bacteria) in the sample is small, detection can be easy because the detection signals of two or more target nucleic acids are generated as the same signal.
또 본 발명의 방법에서, 정방향 및/또는 역방향 프라이머에서 이들이 표적핵산과 결합하는 부위, 정방향 및/또는 역방향 프라이머의 5' 태그 부위, 또 매개체 올리고뉴클레오티드에서 인공 주형에 대해 프라이머로 작용할 매개체 부위, 또 매개체 올리고뉴클레오티드에서 상기 5' 태그 부위에 상보적인 프로브 부위 등은 각각 서열 특이성이 담보되는 한(즉 그 상보적인 서열에 특이적으로 결합할 수 있는 한) 그 길이를 15~50 뉴클레오티드의 길이 중 임의의 길이로 할 수 있다. 바람직하게는 15 내지 25 뉴클레오티드로 할 수 있다. In addition, in the method of the present invention, the portion of the forward and/or reverse primers that bind to the target nucleic acid, the 5' tag portion of the forward and/or reverse primers, the mediator portion that acts as a primer for an artificial template in the mediator oligonucleotide, and the probe portion complementary to the 5' tag portion in the mediator oligonucleotide may each have any length from 15 to 50 nucleotides as long as sequence specificity is ensured (i.e., as long as it can specifically bind to the complementary sequence). It may preferably have a length of 15 to 25 nucleotides.
또 본 발명의 방법에서, 상기 프라이머, 매개체 올리고뉴클레오티드, 5' 태그 부위 등은 그것들의 본 발명에서의 작용에 영향을 미치지 않는 한 DNA 뿐만 아니라 RNA일 수 있으며(본 발명의 올리고뉴클레오티드 등이 RNA일 경우 그 염기서열에서 "T"는 "U"로 읽힌다), 또한 뉴클레아제나 열에 안정한 PNA(Peptide Nucleic Acid), LNA(Locked Nucleic Acid), HNA(Hexitol Nucleic Acids), ANA(Altritol Nucleic Acids), MNA(Mannitol Nucleic Acids) 등의 핵산 유사체일 수도 있다. 프라이머나 매개체 올리고뉴클레오티드, 5' 태그 부위 등은 또한 이들의 본 발명에서의 작용에 영향을 미치지 않는 한 자연적 뉴클레오티드(natural nucleotide) 뿐만 아니라 변형된 뉴클레오티드(modified nucleotide)를 포함할 수 있다. 이러한 변형된 뉴클레오티드는 그 당, 그 포스페이트 및/또는 그 염기에서 변형된 것일 수 있다. 이러한 당, 포스페이트 및/또는 염기에서 변형된 뉴클레오티드는 당업계에 그 제조방법을 포함하여 구체적으로 공지되어 있다. 예컨대 당에서 변형된 뉴클레오티드는, 그 당의 하이드록실 기(OH group)가 할로겐 기, 지방족 기, 에테르 기, 아민 기 등으로 수식된 것, 당인 리보스 또는 디옥시 리보오스 자체가 이를 대신할 수 있는 당 유사체 α-아노머 당(α-anomeric sugars), 아라비노스(arabinose), 자일로스(xyloses) 또는 릭소오스(lyxoses)와 같은 에피머 당(epimeric sugars), 피라노오스 당(pyranose sugars), 퓨라노오스 당(furanose sugars) 등으로 치환된 것을 들수 있다. 또한 예컨대 포스페이트에서의 변형은 포스페이트가 P(O)S(thioate), P(S)S(dithioate), P(O)NR2(amidate), P(O)R, P(O)OR', CO 또는 CH2(formacetal)로의 변형된 것 등을 들수 있다. 여기서 상기 R 또는 R'는 H 또는 치환되거나 치환되지 않은 알킬 등이며, 포스페이트에서 변형될 경우 그 연결기는 -O-, -N-, -S- 또는 -C-가 되어, 이러한 연결기를 통해 인접 뉴클레오티드가 서로 결합하게 된다.In addition, in the method of the present invention, the primer, the mediator oligonucleotide, the 5' tag moiety, etc. may be RNA as well as DNA as long as it does not affect their function in the present invention (when the oligonucleotide of the present invention is RNA, "T" in the base sequence is read as "U"), and may also be nucleic acid analogues such as PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid), HNA (Hexitol Nucleic Acids), ANA (Altritol Nucleic Acids), MNA (Mannitol Nucleic Acids) which are stable to nuclease or heat. The primer, the mediator oligonucleotide, the 5' tag moiety, etc. may also include modified nucleotides as well as natural nucleotides as long as it does not affect their function in the present invention. Such modified nucleotides may be those modified in their sugars, phosphates and/or bases. Nucleotides modified in such sugars, phosphates and/or bases are specifically known in the art, including methods for their preparation. For example, nucleotides modified in sugars include those in which the hydroxyl group (OH group) of the sugar is modified with a halogen group, an aliphatic group, an ether group, an amine group, etc., those in which the sugar ribose or deoxyribose itself can be replaced by sugar analogues such as α-anomeric sugars, arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, etc. Also, for example, modifications in phosphate include modifications of phosphate to P(O)S(thioate), P(S)S(dithioate), P(O)NR2(amidate), P(O)R, P(O)OR', CO or CH2(formacetal). Here, R or R' is H or substituted or unsubstituted alkyl, etc., and when modified in phosphate, the linking group becomes -O-, -N-, -S- or -C-, and adjacent nucleotides are bonded to each other through this linking group.
상기 프라이머, 매개체 올리고뉴클레오티드 등은 그것의 5' 태그 부위, 표적핵산, 인공주형 등에의 상보적인 서열은 그것이 특이적 결합 능력을 부여하는 한 100% 상보적인 필요는 없으며, 적어도 약 80%, 바람직하게는 적어도 약 85%, 보다 바람직하게는 적어도 약 90%, 95% 또는 99%이어도 무방하다.The complementary sequence of the above primer, mediator oligonucleotide, etc. to its 5' tag portion, target nucleic acid, artificial template, etc. need not be 100% complementary as long as it confers specific binding ability, and may be at least about 80%, preferably at least about 85%, more preferably at least about 90%, 95% or 99%.
본 발명의 방법에서, 시료(검출 대상 시료 또는 분석 대상 시료)는 검출하고자 하는 표적핵산을 포함하거나 포함하고 있을 것으로 의심되어 검출의 필요성을 가지는 임의의 혼합물 또는 용액일 수 있다. 시료는 인체 또는 동물로부터 얻어진 생체시료뿐만 아니라 그러한 생체시료를 가공하여 표적핵산의 농도를 높인 가공시료일 수 있고, 나아가 표적핵산이 포함되어 있거나 있을 것으로 여겨지는 물, 식품, 산업폐수 등 환경오염인자나 독성인자를 포함할 것으로 의심되는 시료나 식품 가공 기기로부터 얻어진 유기물 시료 등 검사가 필요한 시료일 수도 있다. 이러한 시료는 적정 희석제, 완충용액을 포함할 수 있으며, 세균이나 바이러스 존재 여부를 검출하고자 할 때에는 배지나 배지 성분이 포함되어 있는 세균 배양물, 바이러스 배양물일 수도 있다.In the method of the present invention, the sample (sample to be detected or sample to be analyzed) may be any mixture or solution that contains or is suspected of containing the target nucleic acid to be detected and thus has a need for detection. The sample may be a biological sample obtained from a human or animal, as well as a processed sample obtained by processing such a biological sample to increase the concentration of the target nucleic acid, and further may be a sample that requires inspection, such as a sample suspected of containing an environmental pollutant or toxic factor, such as water, food, or industrial wastewater that contains or is suspected of containing the target nucleic acid, or an organic matter sample obtained from a food processing device. Such a sample may include an appropriate diluent, a buffer solution, and when it is desired to detect the presence of bacteria or viruses, it may be a bacterial culture or virus culture containing a medium or a medium component.
또 본 발명에서, 시료는 바람직하게는 인체 또는 동물로부터 얻어진 생체시료 또는 그 가공시료일 수 있다. 생체시료는 전혈, 혈청, 혈장, 제대혈, 소변, 분변, 타액, 코점액, 정액, 양수, 세척액(기관지 폐포, 위, 복강, 귀 등), 림프액, 객담, 조직, 세포 등 검출하고자 하는 표적핵산을 포함하거나 포함하고 있을 것으로 의심되어 검출의 필요성을 가지는 인체 또는 동물로부터 얻어진 것일 수 있다. 가공시료는 예컨대 혈장, 혈청, 생체시료를 핵산 추출 키트를 이용하여 핵산(DNA 및/또는 RNA) 농도를 높인 시료, 조직 추출물, 조직에서 얻어진 세포, 세포 용해물, 세포 배양물, 세균 배양물, 바이러스 배양물 등일 수 있다.In addition, in the present invention, the sample may preferably be a biological sample obtained from a human or animal or a processed sample thereof. The biological sample may be obtained from a human or animal that contains or is suspected of containing the target nucleic acid to be detected, such as whole blood, serum, plasma, umbilical cord blood, urine, feces, saliva, nasal mucus, semen, amniotic fluid, lavage fluid (bronchial alveolar, stomach, peritoneal, ear, etc.), lymph, sputum, tissue, cell, etc., and thus has a need for detection. The processed sample may be, for example, plasma, serum, a biological sample whose concentration of nucleic acid (DNA and/or RNA) is increased using a nucleic acid extraction kit, a tissue extract, a cell obtained from a tissue, a cell lysate, a cell culture, a bacterial culture, a virus culture, etc.
또 본 발명에서, 표적핵산은 검출 대상 핵산으로 그 존재 및/또는 존재량이 검출되거나 측정됨으로써 질병의 진단이나 예후, 생물학적 지표의 확인 등, 그 검출이 어떤 유용함을 가져올 수 있는 임의의 핵산을 의미한다. 이러한 표적핵산은 정방향 및 역방항 프라이머에 상보적인 서열을 포함하는 이중가닥 DNA으로 이해될 수도 있다. 이 이중가닥 DNA는 시료, 특히 생체 시료 중의 게놈 DNA이거나, SNP(Single nucleotide polymerphism) 검출을 위한 프라이머로부터 합성된 핵산이거나, DNA 메틸화 유무를 검출하기 위하여 바이설파이트(bisulfate)를 처리한 시료의 핵산이거나, mRNA, rRNA, 마이크로 RNA 등 RNA에서 역전사에 의해 생성된 cDNA일 수도 있다. In addition, in the present invention, the target nucleic acid refers to any nucleic acid whose detection can bring about some usefulness, such as diagnosis or prognosis of a disease, confirmation of a biological indicator, etc., by detecting or measuring the presence and/or amount of the target nucleic acid. This target nucleic acid may be understood as a double-stranded DNA containing sequences complementary to forward and reverse primers. This double-stranded DNA may be genomic DNA in a sample, particularly a biological sample, a nucleic acid synthesized from a primer for detecting SNP (Single nucleotide polymerization), a nucleic acid of a sample treated with bisulfite for detecting the presence or absence of DNA methylation, or cDNA generated by reverse transcription from RNA such as mRNA, rRNA, or micro RNA.
본 발명에 있어서, 프라이머는 표적핵산의 합성 개시를 유도할 수 있는 올리고뉴클레오티드로서, 각 표적핵산에 대해 정방향 프라이머와 역방향 프라이머로 구성된다. 프라이머는 표적핵산(시료 중의 실제 표적핵산 또는 그에 의존하여 생성되는 인공핵산)에 특이적으로 결합하고 PCR 반응의 연장 반응에서 표적핵산과 결합을 유지할 정도의 융해온도를 가지도록 설계된다. 이러한 프라이머 설계에는 문헌[PCR Methods Appl . 1993 Dec;3(3):S30-7], 문헌[Biomolecular Detection and Quantification 14 (2017) 19-28] 등을 참조하거나 시판되는 소프트웨어(예컨대 Visual OMP, Invitrogen OligoPerfect Designer) 등을 활용할 수 있다.In the present invention, the primer is an oligonucleotide capable of inducing the initiation of synthesis of a target nucleic acid, and is composed of a forward primer and a reverse primer for each target nucleic acid. The primer is designed to specifically bind to the target nucleic acid (the actual target nucleic acid in the sample or the artificial nucleic acid generated depending on the target nucleic acid) and to have a melting temperature sufficient to maintain binding to the target nucleic acid during the extension reaction of the PCR reaction. For the design of such primers, reference may be made to the literature [PCR Methods Appl. 1993 Dec;3(3):S30-7], the literature [Biomolecular Detection and Quantification 14 (2017) 19-28], etc., or commercially available software (e.g., Visual OMP, Invitrogen OligoPerfect Designer), etc. may be utilized.
또 본 발명에서, 매개체 올리고뉴클레오티드도 정방향 및/또는 역방향 프라이머의 5' 태그 서열에 상보적인 서열을 가져 특이적으로 결합하고 PCR 반응의 연장 반응에서 표적핵산과 결합을 유지할 정도의 융해온도를 가지도록 설계된다. 이러한 매개체 올리고뉴클레오티드의 설계에는 프로브 설계와 관련하여 당업계에 공지된 문헌[Unit Evol. Genet. 2005; 5:1-9]; 문헌[Afr. Jo. Biotechnol. 2003; 2:91-95], 문헌[Methods Mol. Biol. 1993; 15:31-40], 문헌[PCR Methods Appl. 1993; 3:S30-S37], 문헌[Nucleic Acids Res. 1994; 22:2760-2768] 등 다양한 문헌을 참조할 수 있고, Visual OMP™ 소프트웨어 (DNA Software, Inc, Ann Arbor, Mich), Oli2go™ 소프트웨어(Nucleic Acids Res. 2018 Jul 2; 46(Web Server issue): W252-W256) 등 시판되는 프로그램의 활용이 바람직할 수 있다. In addition, in the present invention, the mediator oligonucleotide is designed to have a sequence complementary to the 5' tag sequence of the forward and/or reverse primers so as to specifically bind and have a melting temperature sufficient to maintain binding to the target nucleic acid during the extension reaction of the PCR reaction. The design of such mediator oligonucleotides can be carried out according to the methods known in the art with respect to probe design, such as [Unit Evol. Genet. 2005; 5:1-9]; [Afr. Jo. Biotechnol. 2003; 2:91-95], [Methods Mol. Biol. 1993; 15:31-40], [PCR Methods Appl. 1993; 3:S30-S37], [Nucleic Acids Res. 1994; 22:2760-2768], and it may be desirable to utilize commercially available programs such as Visual OMP™ software (DNA Software, Inc, Ann Arbor, Mich) and Oli2go™ software (Nucleic Acids Res. 2018 Jul 2; 46 (Web Server issue): W252-W256).
본 발명의 방법에서, PCR 반응의 1 cycle은 변성 단계 및 어닐링과 연장 단계인 2 단계로 구성되거나, 변성 단계, 어닐링 단계 및 연장 단계인 3 단계로 구성될 수 있다. 2 단계로 구성될 경우, 변성 단계는 그 온도가 93~98℃ 범위의 특정 온도에서 이루어지고, 어닐링과 연장 단계는 그 온도가 54~75℃ 범위의 특정 온도에서 이루어질 수 있다. 3 단계로 구성될 경우에는 변성 단계는 그 온도가 93~98℃ 범위의 특정 온도에서 이루어지고, 어닐링 단계는 그 온도가 54~60℃ 범위의 특정 온도에서 이루어지며, 연장 단계는 그 온도가 70~75℃ 범위의 특정 온도에서 이루어질 수 있다. In the method of the present invention, one cycle of the PCR reaction may consist of two steps, a denaturation step and annealing and extension steps, or three steps, a denaturation step, annealing and extension steps. When consisting of two steps, the denaturation step may be performed at a specific temperature in the range of 93 to 98°C, and the annealing and extension steps may be performed at a specific temperature in the range of 54 to 75°C. When consisting of three steps, the denaturation step may be performed at a specific temperature in the range of 93 to 98°C, the annealing step may be performed at a specific temperature in the range of 54 to 60°C, and the extension step may be performed at a specific temperature in the range of 70 to 75°C.
본 발명의 방법에서, 리포터 분자는 신호를 발생시키는 물질인데, 이때 신호는 일반적으로 형광 신호이다. In the method of the present invention, the reporter molecule is a substance that generates a signal, wherein the signal is generally a fluorescent signal.
리포터 분자와 퀜처 분자를 프로브 등에 표지화시키는 방법이나 그러한 프로브를 제조하는 방법은 고체상 1단계 화학적 표지화 방법(Direct chemical labeling via solid-phase synthesis), 고체상 2단계 화학적 표지화 방법(Two-step chemical labeling via solid-phase synthesis) 등을 포함하여 다양한 방법이 당업계에 공지되어 있고, 또한 문헌[Chem. Soc. Rev., 2020, 49, 8749-8773], 문헌[ACS Cent. Sci., 2017, 3, 701-707], 문헌[Curr. Opin. Biotechnol., 2015, 31, 42-49], 문헌[Annual Review of Physiology, ed. D. Julius, 2017, vol. 79, pp. 93-117] 등 다양한 문헌이 공지되어 있다. 표지화 방법과 관련하여 구체적인 것은 이들 문헌을 참조할 수 있다.There are various methods known in the art for labeling reporter molecules and quencher molecules on probes, etc., or for preparing such probes, including a one-step solid-phase chemical labeling method (direct chemical labeling via solid-phase synthesis), a two-step solid-phase chemical labeling method (two-step chemical labeling via solid-phase synthesis), and also various references are known, such as [Chem. Soc. Rev., 2020, 49, 8749-8773], [ACS Cent. Sci., 2017, 3, 701-707], [Curr. Opin. Biotechnol., 2015, 31, 42-49], and [Annual Review of Physiology, ed. D. Julius, 2017, vol. 79, pp. 93-117]. For specific details regarding labeling methods, refer to these references.
본 발명에서, 리포터 분자로서 형광 신호를 발생시키기 위한 형광 물질은 당업계에 공지된 것들 중에서 임의의 것을 PCR 기기의 각 검출 채널의 검출 파장 등을 고려하여 적절히 선택하여 사용할 수 있다. 공지된 형광 물질로서는 예컨대 Cy2™(506), YO-PRO™-1(509), YOYO™-1(509), Calcein(517), FITC(518), FluorX™(519), Alexa™(520), Rhodamine 110(520), Oregon Green™ 500(522), Oregon Green™ 488(524), RiboGreen™(525), Rhodamine Green™(527), Rhodamine 123(529), Magnesium Green™(531), Calcium Green™(533), TO-PRO™-1(533), TOTO1(533), JOE(548), BODIPY530/550(550), Dil(565), BODIPY TMR(568), BODIPY558/568(568), BODIPY564/570(570), Cy3™(570), Alexa™ 546(570), TRITC(572), Magnesium O범위™(575), Phycoerythrin R&B(575), Rhodamine Phalloidin(575), Calcium O범위™(576), Pyronin Y(580), Rhodamine B(580), TAMRA(582), Rhodamine Red™(590), Cy35™(596), ROX(608), Calcium Crimson™(615), Alexa™ 594(615), TexRed(615), Nile Red(628), YO-PRO™-3(631), YOYO™-3(631), R-phycocyanin(642), C-Phycocyanin(648), TO-PRO™-3(660), TOTO3(660), DiD DilC(5)(665), Cy5™(670), Thiadicarbocyanine(671), Cy55(694), HEX(556), TET(536), Biosearch Blue(447), CAL Fluor Gold 540(544), CAL Fluor Orange 560(559), CAL Fluor Red 590(591), CAL Fluor Red 610(610), CAL Fluor Red 635(637), FAM(520), Fluorescein(520), Fluorescein-C3(520), Pulsar 650(566), Quasar 570(667), Quasar 670(705) 및 Quasar 705(610) 등을 들 수 있다. 괄호 안의 숫자는 나노미터 단위의 최대 방출 파장이다.In the present invention, any fluorescent substance for generating a fluorescent signal as a reporter molecule can be appropriately selected and used from among those known in the art by taking into consideration the detection wavelength of each detection channel of the PCR device, etc. Known fluorescent substances include, for example, Cy2™ (506), YO-PRO™-1 (509), YOYO™-1 (509), Calcein (517), FITC (518), FluorX™ (519), Alexa™ (520), Rhodamine 110 (520), Oregon Green™ 500 (522), Oregon Green™ 488 (524), RiboGreen™ (525), Rhodamine Green™ (527), Rhodamine 123 (529), Magnesium Green™ (531), Calcium Green™ (533), TO-PRO™-1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY TMR (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3™ (570), Alexa™ 546 (570), TRITC (572), Magnesium O range™ (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium O range™ (576), Pyronin Y (580), Rhodamine B (580), RA (582), Rhodamine Red™ (590), Cy35™ (596), ROX (608), Calcium Crimson™ (615), Alexa™ 594 (615), TexRed (615), Nile Red (628), YO-PRO™-3 (631), YOYO™-3 (631), R-phycocyanin (642), anin(648), TO-PRO™-3(660), TOTO3(660), DiD DilC(5)(665), Cy5™(670), Thiadicarbocyanine(671), Cy55(694), HEX(556), TET(536), Biosearch Blue(447), CAL Fluor Gold 540(544), CAL Fluor Orange 560(559), CAL Fluor Red 590(591), CAL Fluor Red 610(610), CAL Fluor Red 635(637), FAM(520), Fluorescein(520), Fluorescein-C3(520), Pulsar 650(566), Quasar 570(667), Quasar 670(705), and Quasar 705(610). The numbers in parentheses are the maximum emission wavelengths in nanometers.
상기 리포터 분자는 방출 파장이 서로 다른 적절한 2가지를 선택하여 공여체(donor)와 수용체(receptor)의 쌍으로 사용할 수도 있다. 이 경우 공여체에서 발산된 형광은 FRET(Fluorescence Resonance Energy Transfer)에 의하여 그 공여체와 인접하고 있는 수용체에서 더 낮은 파장의 형광으로 전환되어 방출되고 소멸되게 된다. 이 경우 통상 공여체의 방출 파장을 측정하여 표적핵산의 존부에 따른 신호 변화를 측정하게 된다. The above reporter molecule can be used as a pair of donor and receptor by selecting two appropriate ones with different emission wavelengths. In this case, the fluorescence emitted from the donor is converted into fluorescence of a lower wavelength by FRET (Fluorescence Resonance Energy Transfer) at the acceptor adjacent to the donor, and then emitted and extinguished. In this case, the emission wavelength of the donor is usually measured to measure the signal change according to the presence or absence of the target nucleic acid.
퀜처 분자는 그것이 리포터 분자와 인접하여 있을 때 리포터 분자에 의하여 방출된 형광을 흡수하고 발열시켜 소멸시키는 물질로서 이러한 퀜처 분자도 당업계에 많은 것들이 공지되어 있는데, 그러한 공지된 것들 중에서 사용되는 리포터 분자의 방출 파장을 고려하여 적절한 흡수 파장을 가진 것을 선택하여 사용할 수 있다. 공지된 퀜처 분자로서는 Dabcyl(453), QSY 35(475), BHQ-0(495), Eclipse(530), BHQ-1(534), QSY 7(560), QSY 9(562), BHQ-2(579), ElleQuencher(630), Iowa Black(651), QSY 21(661), BHQ-3(672) 등을 들 수 있다. 괄호 안의 숫자는 나노미터 단위의 최대 흡수 파장이다.A quencher molecule is a substance that absorbs, generates heat, and extinguishes fluorescence emitted by a reporter molecule when it is adjacent to the reporter molecule. Many such quencher molecules are known in the art. Among these known quencher molecules, one having an appropriate absorption wavelength can be selected and used in consideration of the emission wavelength of the reporter molecule used. Known quencher molecules include Dabcyl (453), QSY 35 (475), BHQ-0 (495), Eclipse (530), BHQ-1 (534), QSY 7 (560), QSY 9 (562), BHQ-2 (579), ElleQuencher (630), Iowa Black (651), QSY 21 (661), BHQ-3 (672), etc. The numbers in parentheses are the maximum absorption wavelengths in nanometers.
리포터 분자와 퀜처 분자의 적합한 쌍(pair)과 관련하여 불필요한 잡음 신호없이 정확한 검출을 위해서 소광 효율(Quenching efficiency)을 고려할 필요가 있는데, Cy5/BHQ-2, Cy5/BHQ-1, TexRed/BHQ-2, TexRed/QSY 7, TexRed/BHQ-1, TexRed/Dabcyl, Cy3/BHQ2, Cy3/QSY 7, Cy3/BHQ-1, Cy3/Dabcyl, TET/BHQ-2, TET/QSY 7, TET/TAMRA, TET/BHQ-1, FAM/BHQ-2, FAM/QSY 7, FAM/TAMRA, FAM/BHQ-1, FAM/Dabcyl 등이 소광 효율이 높다고 알려져 있어(Nucliec Acids Res. 30, e122, 2002), 이들 쌍 중에서 선택하여 사용하는 것이 바람직할 수 있다.In order to accurately detect without unnecessary noise signals, it is necessary to consider quenching efficiency in relation to a suitable pair of reporter molecules and quencher molecules. Cy5/BHQ-2, Cy5/BHQ-1, TexRed/BHQ-2, TexRed/QSY 7, TexRed/BHQ-1, TexRed/Dabcyl, Cy3/BHQ2, Cy3/QSY 7, Cy3/BHQ-1, Cy3/Dabcyl, TET/BHQ-2, TET/QSY 7, TET/TAMRA, TET/BHQ-1, FAM/BHQ-2, FAM/QSY 7, FAM/TAMRA, FAM/BHQ-1, FAM/Dabcyl are known to have high quenching efficiency ( Nucliec Acids Res. 30, e122, 2002), so it may be desirable to select and use one of these pairs.
리포터 분자와 퀜처 분자, 공여체와 수용체에 대해 그리고 이들 적합한 쌍(pair)의 선택에 대해 당업계에 많은 문헌이 공지되어 있으며, 그러한 공지된 문헌을 참조할 수 있다. 그러한 문헌으로서는 문헌[Methods Mol Biol. 335, 17-29, 2006], 문헌[Biotechniques 31, 1106-1121, 2001], 문헌[J. Am Chem. Soc. 124, 6950-6956, 2002], 문헌[Chem. Eur. J. 9, 3466-3471, 2003], 문헌[Nucliec Acids Res. 30, e122, 2002], 문헌[J. Phys. Chem. 100, 5541-5553, 1996] 등을 들 수 있다.There are many references known in the art regarding reporter molecules and quencher molecules, donors and acceptors, and the selection of suitable pairs thereof, and reference may be made to such references. Such references include: Methods Mol Biol. 335, 17-29, 2006; Biotechniques 31, 1106-1121, 2001; J. Am Chem. Soc. 124, 6950-6956, 2002; Chem. Eur. J. 9, 3466-3471, 2003; Nucleic Acids Res. 30, e122, 2002; J. Phys. Chem. 100, 5541-5553, 1996;
본 발명에서 있어서, 상기 (a) 단계의 PCR 반응을 위한 혼합물에는 전술한 바의 프라이머 세트와 매개체 올리고뉴클레오티드, 인공주형이 포함되는 이외에, 4가지 NTP, DNA 중합효소를 포함하며, 또한 기타 인자로서 pH 안정화제인 Tris-HCl, 효소 활성을 촉진하기 위한 보조 인자인 MgCl2나 MgSO4 또는 (NH4)2SO4, DNA 중합효소 안정화를 위한 인자인 BSA(Bovine Serum Albumin)나 젤라틴이나 글리세롤 또는 PEG 6000, 프라이머의 표적핵산에의 결합을 촉진하는 인자인 KCl, 비특이적 증폭을 억제하기 위한 비이온성 계면활성제인 Tween 20이나 Triton X-100, DNA 중합효소의 핵산에의 비특이적 결합을 감소시키기 위한 스퍼미딘(spermidine), 프라이머의 표적핵산과의 어닐닝을 촉진하기 위한 인자인 우레아(urea)나 DMSO(dimethylsulfoxide) 또는 DMF(dimethylformamid), Betain), DNA 폴리머라아제의 응집을 방지하기 위한 인자인 SDS(Sodium Dodecyl Sulfate)나 Triton X-100 등을 추가로 포함할 수도 있다.In the present invention, the mixture for the PCR reaction of the step (a) includes, in addition to the primer set, mediator oligonucleotide, and artificial template as described above, four NTPs, DNA polymerase, and other factors such as Tris-HCl as a pH stabilizer, MgCl2 or MgSO4 or (NH4)2SO4 as an auxiliary factor for promoting enzyme activity, BSA (Bovine Serum Albumin), gelatin, glycerol, or PEG 6000 as a factor for stabilizing DNA polymerase, KCl as a factor for promoting binding of primers to target nucleic acids, Tween 20 or Triton X-100 as a nonionic surfactant for suppressing nonspecific amplification, spermidine for reducing nonspecific binding of DNA polymerase to nucleic acids, urea or DMSO (dimethylsulfoxide) as a factor for promoting annealing of primers to target nucleic acids, or It may also contain additional ingredients such as DMF (dimethylformamid), Betain, SDS (Sodium Dodecyl Sulfate), or Triton X-100, which are factors to prevent aggregation of DNA polymerase.
본 발명에서 DNA 폴리머라아제는 5'-->3'엑소뉴클레아제 활성과 플랩(flap) 엔도뉴클레아제 활성을 가진 열안정성 폴리머라아제를 사용하는데, 이러한 열안정성 폴리머라아제는 Thermus aquaticus(Taq), Thermus thermophilus(Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcus litoralis, Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifex pyrophilusAquifex aeolieus 등 다양한 박테리아 종으로부터 직접 분리하여 사용하거나 유전자 재조합 기술에 의해 제조하여 사용하거나 분리 또는 제조되어 시판되는 것을 구입하여 사용할 수 있다. In the present invention, the DNA polymerase uses a thermostable polymerase having 5'-->3' exonuclease activity and flap endonuclease activity, and this thermostable polymerase is selected from the group consisting of Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcus litoralis, It can be used by directly isolating from various bacterial species such as Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifex pyrophilus , and Aquifex aeolieus, or by manufacturing it using genetic recombination technology, or by purchasing it commercially after being isolated or manufactured.
DNA 폴리머라아제는 5'-->3'엑소뉴클레아제 활성과 플랩(flap) 뉴클레아제 활성을 가지는 한 변형된 것을 사용하여도 무방하다. 변형된 폴리머라아제는 일부 서열이 인위적으로 변형되거나 자연적으로 변형된(돌연변이된) 것을 포함한다. 이러한 변형된 폴리머라아제로서는 G46E E678G CS5 DNA 폴리머라아제, G46E L329A E678G CS5 DNA 폴리머라아제, G46E L329A D640G S671F CS5 DNA 폴리머라아제, G46E L329A D640G S671F E678G CS5 DNA 폴리머라아제, G46E E678G CS6 DNA 폴리머라아제, Z05 DNA 폴리머라아제, ΔZ05 폴리머라아제, ΔZ05-Gold 폴리머라아제, ΔZ05R 폴리머라아제, E615G Taq DNA 폴리머라아제, E678G TMA-25 폴리머라아제, E678G TMA-30 폴리머라아제 등이 예시될 수 있다.DNA polymerase may be used in a modified form as long as it has 5'-->3' exonuclease activity and flap nuclease activity. Modified polymerases include those in which some sequences are artificially modified or naturally modified (mutated). Examples of such modified polymerases include G46E E678G CS5 DNA polymerase, G46E L329A E678G CS5 DNA polymerase, G46E L329A D640G S671F CS5 DNA polymerase, G46E L329A D640G S671F E678G CS5 DNA polymerase, G46E E678G CS6 DNA polymerase, Z05 DNA polymerase, ΔZ05 polymerase, ΔZ05-Gold polymerase, ΔZ05R polymerase, E615G Taq DNA polymerase, E678G TMA-25 polymerase, and E678G TMA-30 polymerase.
DNA 폴리머라아제는 바람직하게는 Taq 폴리머라아제이다.The DNA polymerase is preferably Taq polymerase.
본 발명에서 PCR 반응은 변성 단계, 어닐링 단계 및 연장 단계를 2 사이클 이상, 통상 15 내지 50 사이클을 반복적으로 수행하여 표적 핵산을 증폭시키는데, 이러한 사이클 수행 전에 초기 변성 단계(Initial Denaturation)가 포함될 수 있다. 이 초기 변성 단계는 DNA 폴리머라아제를 활성화시키고 주형 DNA를 변성시키기 위하여 수행된다. 이러한 초기 변성 단계는 주형 DNA의 GC 함량 등을 고려하여 30초 내지 20분 범위 내에서 수행될 수 있다. In the present invention, the PCR reaction amplifies the target nucleic acid by repeatedly performing the denaturation step, the annealing step, and the extension step for two or more cycles, usually 15 to 50 cycles. An initial denaturation step may be included before performing these cycles. This initial denaturation step is performed to activate DNA polymerase and denature template DNA. This initial denaturation step may be performed within a range of 30 seconds to 20 minutes, taking into account the GC content of the template DNA, etc.
또한 상기 cycle의 반복 반응 후에는 연장 단계(Final elongation step)가 포함될 수 있다. 이 연장 단계는 DNA 폴리머라아제가 그 마지막 연장 반응을 마무리하기 위한 것으로 통상 상기 cycle의 연장 단계와 같은 온도에서 1~20분 정도 수행된다.In addition, after the repeated reaction of the above cycle, an elongation step may be included. This elongation step is for the DNA polymerase to finish its final elongation reaction, and is usually performed at the same temperature as the elongation step of the above cycle for about 1 to 20 minutes.
PCR 반응과 관련하여 더 구체적인 것은 미국 특허 번호 제4,683,195호, 제4,683,202호, 문헌[PCR Protocols: A Guide to Methods and Applications, Innis et al, eds, 1990], 문헌[BioTechniques 69: 317?325 (October 2020)], 문헌[Front Microbiol. 2017 Feb 2;8:108], 문헌[J Invest Dermatol. 2013 Mar; 133(3): e6] 등을 참조할 수 있다.For more specific information regarding PCR reactions, see U.S. Patent Nos. 4,683,195, 4,683,202, PCR Protocols: A Guide to Methods and Applications, Innis et al, eds, 1990, BioTechniques 69: 317–325 (October 2020), Front Microbiol. 2017 Feb 2;8:108, J Invest Dermatol. 2013 Mar; 133(3): e6, etc.
전술한 바와 같이, 본 발명에 따르면, 표적핵산을 인공핵산으로 전환시키는 방법 및 이를 이용한 표적핵산의 검출 방법을 제공할 수 있다. 본 발명의 방법에 따르면 시료 중의 표적핵산을 인공핵산으로 전환시키는 데 사용되는 매개체 올리고뉴클레오티드를 검출하고자 하는 표적핵산의 서열에 제한을 받지 않게 범용적으로 구성할 수 있으며, 범용적으로 구성하면서도 그 서열을 인위적으로 조절하는 것이 가능하고 또 그로부터 생성되는 인공핵산도 인위적으로 조절하는 것이 가능하므로, 검출 효과가 최적으로, 최대로 높아지도록(민감성과 특이성이 높아지도록), 서열을 인위적으로 구성하는 것이 가능할 수 있다. As described above, according to the present invention, a method for converting a target nucleic acid into an artificial nucleic acid and a method for detecting the target nucleic acid using the same can be provided. According to the method of the present invention, a mediator oligonucleotide used for converting a target nucleic acid in a sample into an artificial nucleic acid can be universally configured without being limited by the sequence of the target nucleic acid to be detected, and while universally configured, the sequence can be artificially controlled, and an artificial nucleic acid generated therefrom can also be artificially controlled, so that it is possible to artificially configure the sequence so that the detection effect is optimally and maximally increased (sensitivity and specificity are increased).
도 1은 국제 공개특허, 국제 공개특허 WO06122208, WO12096430A1 등이 개시하는 표적핵산을 인공핵산으로 전환시켜 표적핵산을 검출하는 방법의 개념도이다.Figure 1 is a conceptual diagram of a method for detecting a target nucleic acid by converting the target nucleic acid into an artificial nucleic acid, as disclosed in international published patents, such as international published patents WO06122208 and WO12096430A1.
도 2는 본 발명의 구성요소인 5' 태그 부위를 가진 정방향 프라이머, 역방향 프라이머 및 매개체 올리고뉴클레오티드의 구조를 나타낸 것이다.Figure 2 illustrates the structures of a forward primer, a reverse primer, and a mediator oligonucleotide having a 5' tag portion, which are components of the present invention.
도 3은 본 발명에 따른 방법에서 시료 중 표적핵산이 존재할 경우 매개체 절편이 생성됨을 나타낸 것이다.Figure 3 shows that a mediator fragment is generated when a target nucleic acid is present in a sample in a method according to the present invention.
도 4는 본 발명에 따른 방법에서 시료 중에 표적핵산이 존재하지 않을 경우 인공핵산이 생성되지 않음을 나타낸 것이다.Figure 4 shows that in the method according to the present invention, artificial nucleic acids are not generated when no target nucleic acid exists in the sample.
도 5는 표적핵산을 인공핵산으로 전환시켜 표적핵산을 검출할 경우 본 발명의 구성요소(5' 태그 부위를 가진 정방향 프라이머, 역방향 프라이머, 매개체 올리고뉴클레오티드 및 인공 주형의 구조)를 나타낸 것이다. Figure 5 illustrates the components of the present invention (structures of a forward primer having a 5' tag portion, a reverse primer, a mediator oligonucleotide, and an artificial template) when detecting a target nucleic acid by converting the target nucleic acid into an artificial nucleic acid.
도 6은 표적핵산을 인공핵산으로 전환시켜 검출하는 방법의 개념도이다.Figure 6 is a conceptual diagram of a method for detecting a target nucleic acid by converting it into an artificial nucleic acid.
도 7은 본 발명에 따른 방법에서 시료 중에 표적핵산이 존재하지 않을 경우 인공핵산이 생성되지 않음을 나타낸 것이다.Figure 7 shows that in the method according to the present invention, artificial nucleic acids are not generated when no target nucleic acid exists in the sample.
도 8 및 도 9는 인공핵산이 생성되면서 발생하는 신호를 검출하여 시료 중의 표적핵산을 검출하는 방법들의 개념도를 나타낸 것이다.Figures 8 and 9 illustrate conceptual diagrams of methods for detecting target nucleic acids in a sample by detecting signals generated when artificial nucleic acids are produced.
도 10 내지 도 12는 생성된 인공핵산을 검출하여 시료 중의 표적핵산을 검출하는 방법들의 개념도를 나타낸 것이다.Figures 10 to 12 illustrate conceptual diagrams of methods for detecting target nucleic acids in a sample by detecting generated artificial nucleic acids.
도 13은 인공핵산에 의존하여 생성되는 추가의 인공핵산을 검출하거나 또는 이러한 추가의 인공핵산이 생성되는 과정에서 변화하는 신호를 검출하여 시료 중의 표적핵산을 검출하는 방법의 개념도를 나타낸 것이다.Figure 13 is a conceptual diagram illustrating a method for detecting a target nucleic acid in a sample by detecting additional artificial nucleic acids produced dependent on artificial nucleic acids or by detecting signals that change during the process of producing such additional artificial nucleic acids.
도 14는 정방향 프라이머 이외에 역방향 프라이머도 5' 말단 부위에 태그 부위를 가질 수 있음을 보여주는 개념도이다. Figure 14 is a conceptual diagram showing that, in addition to the forward primer, the reverse primer can also have a tag site at the 5' end.
도 15는 정방향 프라이머 및/또는 역방향 프라이머에 5' 태그 부위가 2개 이상 존재할 수 있음을 보여주는 개념도이다.Figure 15 is a conceptual diagram showing that more than two 5' tag sites may be present in the forward primer and/or the reverse primer.
도 16은 5' 태그 서열과 동일 서열을 가지고 그 5' 태그 서열의 5' 쪽에 추가의 태그 서열을 가진 보조의 정방향 프라이머 또는 5' 태그 서열과 동일 서열을 가지는 보조의 정방향 프라이머를 이용하여 표적핵산을 인공핵산으로 전환시켜 표적핵산을 검출하는 방법의 개념도이다.Figure 16 is a conceptual diagram of a method for detecting a target nucleic acid by converting the target nucleic acid into an artificial nucleic acid using an auxiliary forward primer having the same sequence as the 5' tag sequence and an additional tag sequence at the 5' side of the 5' tag sequence or an auxiliary forward primer having the same sequence as the 5' tag sequence.
도 17은 하나의 표적핵산에 대해서 5' 태그 부위를 가진 정방향 프라이머 및/또는 5' 태그 부위를 가진 역방향 프라이머를, 그것의 표적핵산과의 결합 위치를 달리하여 2가지 이상 사용하는 것의 개념도이다.Figure 17 is a conceptual diagram of using two or more forward primers having a 5' tag portion and/or reverse primers having a 5' tag portion for one target nucleic acid, with different binding positions with respect to the target nucleic acid.
도 18은 2가지의 표적핵산에 대해서 5' 태그 부위를 가진 정방향 프라이머 및/또는 5' 태그 부위를 가진 역방향 프라이머를, 그 2가지의 표적핵산 각각에 사용하는 것의 개념도이다. Figure 18 is a conceptual diagram of using a forward primer having a 5' tag portion and/or a reverse primer having a 5' tag portion for two target nucleic acids, respectively.
도 19는 도 9 및 도 17의 본 발명의 개념도에 따라, 표적핵산을 인공핵산으로 전환시키는 과정에서 발생한 신호를 검출하여 표적핵산을 검출할 수 있음을 보여주는 결과이다.FIG. 19 is a result showing that a target nucleic acid can be detected by detecting a signal generated in the process of converting a target nucleic acid into an artificial nucleic acid according to the conceptual diagram of the present invention of FIGS. 9 and 17.
도 20은 도 11의 본 발명의 개념도에 따라, 표적핵산으로부터 생성된 인공핵산을 검출하여 표적핵산을 검출할 수 있음을 보여주는 결과이다.FIG. 20 is a result showing that a target nucleic acid can be detected by detecting an artificial nucleic acid generated from a target nucleic acid according to the conceptual diagram of the present invention of FIG. 11.
도 21은 도 13의 본 발명의 개념도에 따라 인공핵산에 의존하여 생성되는 추가의 인공핵산이 생성되는 과정에서 발생한 신호를 검출하여 표적핵산을 검출할 수 있음을 보여주는 결과이다. FIG. 21 is a result showing that a target nucleic acid can be detected by detecting a signal generated in the process of generating an additional artificial nucleic acid that is produced depending on the artificial nucleic acid according to the conceptual diagram of the present invention of FIG. 13.
도 22는 도 9 및 도 18의 본 발명의 개념도에 따라, 2가지 표적핵산를 검출할 때 동일한 5' 태그 부위를 가진, 각각의 표적핵산에 특이적인 2가지의 정방향 프라이머를 함께 사용하여 검출할 경우 검출 신호가 증폭되어 검출이 용이할 수 있음을 보여주는 결과이다.FIG. 22 is a result showing that when two forward primers specific for each target nucleic acid, each having an identical 5' tag portion, are used together to detect two target nucleic acids, the detection signal is amplified and detection can be facilitated, according to the conceptual diagram of the present invention of FIGS. 9 and 18.
이하 본 발명을 실시예를 참조하여 설명한다. 그러나 본 발명의 범위가 이러한 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described with reference to examples. However, the scope of the present invention is not limited to these examples.
<실시예> 인공핵산을 이용한 표적핵산의 검출<Example> Detection of target nucleic acid using artificial nucleic acid
<실시예 1> 매개체 절편 검출을 통한 표적핵산의 검출<Example 1> Detection of target nucleic acid through detection of mediator fragments
1. 표적핵산, 프라이머 및 매개체 올리고뉴클레오티드의 준비 1. Preparation of target nucleic acid, primer and mediator oligonucleotides
본 실험에는 표적핵산은 성매개 질환의 원인체인 Neisseria gonorrhoeae (NG)의 유전자를 합성하여 이용하였다. 그 표적핵산의 서열은 아래와 같다.In this experiment, the target nucleic acid was synthesized from the gene of Neisseria gonorrhoeae (NG), the causative agent of sexually transmitted diseases. The sequence of the target nucleic acid is as follows.
5' -TCCGACGTTTCGGTTGTGTTGAAACACCGCCCGGAACCCGATATAATCCGCCCTTCAACATCAGTGAAAATCTTTTTTTTAACCGGTCAAACCGAATAAGGAGC-3' 5' - TCCGACGTTTCGGTTTGTG TTGAAACACCGCCCGGAACCCGATATAATCCGCCCTTCAACATCAGTGAAAATCTTTTTTTTAA CCGGTCAAACCGAATAAGGAGC -3'
상기에서 밑줄 친 부분은 아래의 정방향 프라이머와 역방향 프라이머가 상보적으로 결합하는 부위이다. The underlined portion above is the site where the forward primer and reverse primer below complementarily bind.
정방향 프라이머의 5' 태그 부위는 인위적으로 구성한 5' -GCTACCCAGCCGGCTACAAG-3' 서열을 사용하였다.The 5' tag portion of the forward primer used an artificially constructed sequence, 5' -GCTACCCAGCCGGCTACAAG-3'.
또 정방향 프라이머의 표적핵산과의 상보적인 부위의 서열은 5' -TCCGACGTTTCGGTTGTG-3' 을 사용하였다.In addition, the sequence of the complementary portion of the forward primer to the target nucleic acid was 5'-TCCGACGTTTCGGTTGTG-3'.
따라서 5' 태그 부위를 가진 정방향 프라이머 서열은 5' -GCTACCCAGCCGGCTACAAGTCCGACGTTTCGGTTGTGTT-3' 이다. 여기서 굵은 글씨로 표시된 부분이 5' 태그 부위이다. Therefore, the forward primer sequence with a 5' tag portion is 5' - GCTACCCAGCCGGCTACAAG TCCGACGTTTCGGTTGTGTT-3', where the portion in bold is the 5' tag portion.
역방향 프라이머는 5'-GCTCCTTATTCGGTTTGACCGG-3'를 사용하였다.The reverse primer used was 5'-GCTCCTTATTCGGTTTGACCGG-3'.
매개체 올리고뉴클레오티드는 상기 5' 태그 부위와 상보적인 부위를 5' -CTTGTAGCCGGCTGGGTAGC-3' 로 구성하고, 그리고 시료 중 표적핵산이 있을 경우 방출되어 인공 주형에 대해 프라이머로 작용하는 매개체 서열을 5' -ACGACGGCTTGGCATTGC-3' 로 구성하였다. 따라서 매개체 올리고뉴클레오티드의 전체 서열은 아래와 같다.The mediator oligonucleotide is composed of a region complementary to the 5' tag region as 5' - CTTGTAGCCGGCTGGGTAGC -3', and a mediator sequence that is released when a target nucleic acid is present in the sample and acts as a primer for the artificial template as 5' - ACGACGGCTTGGCATTGC -3'. Therefore, the entire sequence of the mediator oligonucleotide is as follows.
5'-ACGACGGCTTGGCATTGC CTTGTAGCCGGCTGGGTAGC-3' 5'- ACGACGGCTTGGCATTGC CTTGTAGCCGGCTGGGTAGC -3'
여기서 상기 밑줄친 부분은 매개체 서열로서 표적핵산이 존재할 경우 DNA 중합효소의 뉴클레아제의 활성에 의해 절단되어 방출된다. 상기 굵은 글씨로 된 부분은 정방향 프라이머의 5' 태그 부위에 상보적인 서열이다. Here, the underlined portion is a mediator sequence that is cleaved and released by the nuclease activity of DNA polymerase when a target nucleic acid is present. The portion in bold is a sequence complementary to the 5' tag portion of the forward primer.
2. PCR 반응2. PCR reaction
1x PCR 버퍼, NG 유전자에 대한 합성 주형 150 pg/ul, 5' 태그 부위를 가진 정방향 프라이머와 역방향 프라이머 각각 3 μM, 5' 표지된 매개체 올리고뉴클레오티드 5 μM, 3 mM MgCl2, dNTPs 400 μM 및 Taq 폴리머라아제(Taq Polymerase) 1.5 unit을 함유한, 총 반응액 20 ㎕를 다음의 조건으로 다음과 같이 PCR 반응을 수행하였다: 상기 반응 혼합물을 95℃에서 10 분간 변성시키고, 95℃에서 30초 변성, 60℃에서 60초 어닐링과 연장 cycle을 30 cycle 반복, 마지막 연장 3분.A total reaction volume of 20 ㎕ containing 1x PCR buffer, 150 pg/㎕ of synthetic template for the NG gene, 3 ㎕ of each of forward and reverse primers with 5' tag sites, 5 ㎕ of 5'-labeled mediator oligonucleotide, 3 mM MgCl2, 400 ㎕ of dNTPs, and 1.5 unit of Taq Polymerase was used as follows: the reaction mixture was denatured at 95 ° C. for 10 minutes, followed by 30 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 60 ° C. for 60 seconds, and extension, and a final extension for 3 minutes.
3. 질량분석에 의한 매개체 절편의 검출3. Detection of mediator fragments by mass spectrometry
상기 PCR 반응물에서 증폭 산물과 매개체 절편의 질량분석을 위해, PCR 반응물은 탈염과정을 통해 정제하고, MALTI-TOF MS를 통해 질량분석을 수행하였다. 상기 PCR 반응물에서 증폭 산물과 매개체 절편의 질량분석을 위해, PCR 반응물은 탈염과정을 통해 정제하고, MALTI-TOF MS를 통해 질량분석을 수행하였다.For mass analysis of the amplified product and mediator fragments in the above PCR reaction product, the PCR reaction product was purified through a desalting process, and mass analysis was performed using MALTI-TOF MS. For mass analysis of the amplified product and mediator fragments in the above PCR reaction product, the PCR reaction product was purified through a desalting process, and mass analysis was performed using MALTI-TOF MS.
그 결과, 잘려져 나온 매개체 절편에 해당하는 5515.28Da, 잘리지 않은 매개체(태그 + 플랩)에 해당하는 11,741.9Da, 그리고, 사용되지 않은 정방향 프라이머와 역방향 프라이머에 해당하는 12,254Da과 6708Da에서 피크 등이 주요 피크로 검출됨을 확인하였다.As a result, it was confirmed that the main peaks detected were peaks at 5515.28 Da corresponding to the cleaved mediator fragment, 11,741.9 Da corresponding to the uncleaved mediator (tag + flap), and 12,254 Da and 6708 Da corresponding to the unused forward and reverse primers.
<실시예 2> 표적핵산의 인공핵산으로의 전환을 통한 표적핵산의 검출<Example 2> Detection of target nucleic acid through conversion of target nucleic acid into artificial nucleic acid
1. 표적핵산, 프라이머, 매개체 올리고뉴클레오티드 및 인공 주형의 준비 1. Preparation of target nucleic acid, primer, mediator oligonucleotide and artificial template
본 실험에서 표적핵산과 정방향, 역방향 프라이머는 상기 실시예 1과 동일한 것을 사용하였다. 매개체 올리고뉴클레오티드는 연장반응이 일어나지 않도록 그 3' 말단은 HEG(hexathylene glycol)로 블로킹시킨 아래의 것을 사용하였다. In this experiment, the target nucleic acid and forward and reverse primers were the same as those used in Example 1. The mediator oligonucleotide was used as follows, the 3' end of which was blocked with HEG (hexathylene glycol) to prevent extension reaction.
매개체 올리고뉴클레오티드의 전체 서열:Full sequence of the mediator oligonucleotide:
5' -ACGACGGCTTGGCATTGC CTTGTAGCCGGCTGGGTAGC-[HEG]-3' 5' - ACGACGGCTTGGCATTGC CTTGTAGCCGGCTGGGTAGC -[HEG]-3'
여기서 상기 밑줄친 부분은 각각 인공 주형에 대해 프라이머로 작용하는 매개체 서열로서 인공 주형의 3' 말단 쪽에 상보적으로 결합한다. 상기 굵은 글씨로 된 부분은 정방향 프라이머의 5' 태그 부위에 상보적인 서열이다. 이 매개체 올리고뉴클레오티드는 연장반응이 일어나지 않도록 그 3' 말단이 HEG(hexathylene glycol)로 블로킹되어 있다. Here, the underlined portion is a mediator sequence that acts as a primer for each artificial template and complementarily binds to the 3' end of the artificial template. The bold portion is a sequence complementary to the 5' tag portion of the forward primer. The 3' end of this mediator oligonucleotide is blocked with HEG (hexathylene glycol) so that an extension reaction does not occur.
그리고 인공 주형은 아래와 같이 구성된 서열을 사용하였다.And the artificial mold used a sequence composed as follows.
5'-CCTCCTCCTCCTCCTCCTC CTCCTCCTCCTCCTCCTCCA GTAAAGGCAATGCCAAGCCGTCGT-[HEG]-3' 5'- CCTCCTCCTCCTCCTCCTC CTCCTCCTCCTCCTCCTCCA GTAAA G GCAATGCCAAGCCGTCGT -[HEG]-3'
상기 인공 주형에서 주형 부위(C' )는 굵은 글씨로 되어 있는 부분이고, 상기 밑줄친 부분은 매개체 올리고뉴클레오티드에서 방출되어 인공 주형에 대해 프라이머로서 작용하는 매개체 서열이 결합하는 부위이다. 여기서도 그 3' 말단이 HEG(hexathylene glycol)로 블로킹되어 있다. In the above artificial template, the template portion (C') is the portion in bold, and the underlined portion is the portion where the mediator sequence, which is released from the mediator oligonucleotide and acts as a primer for the artificial template, binds. Here too, the 3' end is blocked with HEG (hexathylene glycol).
그리고 도 9에서 도시된 바와 같이, 매개체 절편이 인공 주형에 프라이머로서 결합하여 인공핵산이 새로이 합성되면서 신호가 발생하도록 하기 위해 상기 주형 부위(C' ) 밑줄친 부위에 상보적인 서열을 가지고 리포터 분자와 퀜처 분자를 가진 아래 구성의 택맨 프로브를 사용하였다.And as illustrated in Fig. 9, in order to generate a signal when the artificial nucleic acid is newly synthesized by binding the mediator fragment as a primer to the artificial template, a TaqMan probe having a sequence complementary to the underlined region of the template region (C') and a reporter molecule and a quencher molecule of the following configuration was used.
택맨 프로브 서열과 구성은 아래와 같다.The Taxman probe sequence and composition are as follows.
5' - [BHQ-1] TGGAGGAGGAGGAGGAGGAG [FAM] -3' 5' - [BHQ-1] TGGAGGAGGAGGAGGAGGAG [FAM] -3'
또한 도 17의 개념도에 따라 동일한 서열의 태그 부위를 가진 정방향 프라이머 2가지를 그 표적핵산과의 결합 위치를 달리하여 사용하였을 때의 검출 결과를 확인하기 위하여, 아래 표적핵산 서열에서 굵은 글씨로 밑줄친 부위에 대한 제2의 정방향 프라이머를 상기 정방향 프라이머(제1의 정방향 프라이머)와 함께 사용하였다.In addition, in order to confirm the detection results when two forward primers having the same sequence of tag portions were used with different binding positions to the target nucleic acid according to the conceptual diagram of Fig. 17, a second forward primer for the portion underlined in bold in the target nucleic acid sequence below was used together with the forward primer (first forward primer).
5' -TCCGACGTTTCGGTTGTGTTGAAACACCGCCCGGAA CCCGATATAATCCGCCCT TCAACATCAGTGAAAATCTTTTTTTTAACCGGTCAAACCGAATAAGGAGC-3' 5' - TCCGACGTTTCGGTTTGTG TTGAAACACCGCCCGGAA CCCGATATAATCCGCCCT TCAACATCAGTGAAAATCTTTTTTTTAA CCGGTCAAACCGAATAAGGAGC -3'
5' 태그 부위를 가진 제2의 정방향 프라이머 서열은 아래와 같다.The second forward primer sequence with a 5' tag region is as follows.
5' -GCTACCCAGCCGGCTACAAGCCCGATATAATCCGCCCT-3'5' - GCTACCCAGCCGGCTACAAG CCCGATATAATCCGCCCT-3'
상기 제2의 정방향 프라이머의 5' 태그 부위(굵은 글씨)는 상기 정방향 프라이머(제1의 정방향 프라이머)의 5' 태그 부위와 그 서열이 같다.The 5' tag portion (bold text) of the second forward primer has the same sequence as the 5' tag portion of the forward primer (first forward primer).
2. PCR 반응과 인공핵산을 통한 표적핵산의 검출2. Detection of target nucleic acid through PCR reaction and artificial nucleic acid
1x PCR 버퍼, NG 유전자에 대한 합성 주형 150 pg/ul, 5' 태그 부위를 가진 정방향 프라이머와 역방향 프라이머 각각 3 μM(제1과 제2의 정향방 프라이머를 함께 사용할 경우는 각각 3 μM), 매개체 올리고뉴클레오티드 5 μM, 인공 주형 150 pg/ul, 3 mM MgCl2, dNTPs 400 μM, 택맨 프로브 5 μM, 및 Taq 폴리머라아제(Taq Polymerase) 1.5 unit을 함유한, 총 반응액 20 ㎕를 다음의 조건으로 다음과 같이 PCR 반응을 수행하였다: 상기 반응 혼합물을 95℃에서 10 분간 변성시키고, 95℃에서 30초 변성, 60℃에서 60초 어닐링과 연장 cycle을 40 cycle 반복, 마지막 연장 3분. A total reaction volume of 20 ㎕ containing 1x PCR buffer, 150 pg/ul of synthetic template for NG gene, 3 ㎕ of forward primer and reverse primer with 5' tag region (3 ㎕ each when the first and second forward primers are used together), 5 ㎕ of mediator oligonucleotide, 150 pg/ul of artificial template, 3 mM MgCl2, 400 ㎕ of dNTPs, 5 ㎕ of Taq probe, and 1.5 unit of Taq polymerase was used as follows: the reaction mixture was denatured at 95 ° C. for 10 minutes, and 40 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 60 ° C. for 60 seconds, and extension, and a final extension for 3 minutes.
각 cycle마다 형광 신호를 측정하고 도 19에 나타내었다.The fluorescence signal was measured for each cycle and is shown in Figure 19.
도 19 (A)는 5' 태그 부위가 없는 정방향 프라이머를 사용하였을 때의 결과이고, 도 19 (B)는 매개체 올리고뉴클레오티드가 PCR 반응물 중에 없을 때의 결과이며, 도 19 (C)는 인공 주형이 PCR 반응물 중에 없을 때의 결과이다. 그리고 도 19 (D)는 태그 부위가 있는 정방향 프라이머를 사용하고, 또 매개체 올리고뉴클레오티드와 인공 주형 모두를 사용하였을 때의 결과이며, 도 19(E)는 도 17의 개념도에 따라 매개체 올리고뉴클레오티드와 인공 주형 모두를 사용함과 더불어 태그 부위가 있는 정방향 프라이머 2가지를 그 표적핵산과의 결합 위치를 달리하여 사용하였을 때의 결과이다.Fig. 19 (A) shows the result when a forward primer without a 5' tag portion was used, Fig. 19 (B) shows the result when no mediator oligonucleotide was present in the PCR reaction, and Fig. 19 (C) shows the result when no artificial template was present in the PCR reaction. Fig. 19 (D) shows the result when a forward primer with a tag portion was used and both a mediator oligonucleotide and an artificial template were used, and Fig. 19 (E) shows the result when both a mediator oligonucleotide and an artificial template were used according to the schematic diagram of Fig. 17 and two types of forward primers with tag portions were used with different binding positions to the target nucleic acid.
도 19를 참조하여 보면, 본 발명에 따라 표적핵산이, 인공핵산으로 전환되면서 발생한 신호를 측정함으로써 검출될 수 있고, 또한 태그 부위가 있는 정방향 프라이머 2가지를 그 표적핵산과의 결합 위치를 달리하여 사용할 경우 신호가 더 빨리 나타날 수 있음(신호가 증폭될 수 있음)을 보여준다.Referring to FIG. 19, it is shown that, according to the present invention, a target nucleic acid can be detected by measuring a signal generated when it is converted into an artificial nucleic acid, and that, when two forward primers having tag portions are used at different binding positions with respect to the target nucleic acid, a signal can appear more quickly (a signal can be amplified).
<실시예 3> 인공핵산의 검출을 통한 표적핵산의 검출<Example 3> Detection of target nucleic acid through detection of artificial nucleic acid
본 실시예에서는 도 11의 개념도에 따라 택맨 프로브를 이용하여 인공핵산을 검출하여 표적핵산을 검출할 수 있는지를 확인하였다.In this example, it was confirmed whether a target nucleic acid could be detected by detecting an artificial nucleic acid using a Taxman probe according to the conceptual diagram of Fig. 11.
상기 실시예 2와 동일한 구성의 5' 태그 부위를 가진 정방향 프라이머(제1의 정방향 프라이머), 매개체 올리고뉴클레오티드, 인공 주형을 사용하고, 택맨 프로브는 아래의 서열과 구성을 가진 것을 사용하였으며, 이와 더불어 아래의 서열을 가진 인공핵산에 특이적인 프라이머(C-p)을 사용하였다.A forward primer (first forward primer) having a 5' tag portion of the same configuration as in Example 2 above, a mediator oligonucleotide, and an artificial template were used, and a TaqMan probe having the following sequence and configuration was used. In addition, a primer (C-p) specific for an artificial nucleic acid having the following sequence was used.
택맨 프로브: 5' -[BHQ-1] CTCCTCCTCCTCCTCCTCCA [FAM]-3' Taxman Probe: 5' -[BHQ-1] CTCCTCCTCCTCCTCCTCCA [FAM] -3'
프라이머(C-p): 5' -CCTCCTCCTCCTCCTCC-3' Primer (C-p): 5' -CCTCCTCCTCCTCCTCC-3'
상기 택맨 프로브는 상기 실시예 2의 인공주형에서 밑줄친 부위의 서열이다.The above TaxMan probe is the sequence of the underlined portion in the artificial template of Example 2.
PCR 반응을 상기 실시예 2와 동일하게 수행하였으며, 그 결과를 도 20에 나타내었다.The PCR reaction was performed in the same manner as in Example 2, and the results are shown in Fig. 20.
도 20 (A)는 5' 태그 부위가 없는 정방향 프라이머를 사용하였을 때의 결과이고, 도 20 (B)는 매개체 올리고뉴클레오티드가 PCR 반응물 중에 없을 때의 결과이며, 도 20 (C)는 인공 주형이 PCR 반응물 중에 없을 때의 결과이다. 그리고 도 20 (D)는 인공핵산에 특이적인 프라이머(C-p)가 PCR 반응물 중에 없을 때의 결과이고, 도 20 (E)는 태그 부위가 있는 정방향 프라이머와, 매개체 올리고뉴클레오티드, 인공 주형, 택맨 프로브 그리고 인공핵산에 특이적인 프라이머(C-p)가 모두 PCR 반응물 중에 있을 때의 결과이다. Fig. 20 (A) shows the result when a forward primer without a 5' tag portion was used, Fig. 20 (B) shows the result when a mediator oligonucleotide was not present in the PCR reaction, and Fig. 20 (C) shows the result when an artificial template was not present in the PCR reaction. In addition, Fig. 20 (D) shows the result when a primer (C-p) specific for an artificial nucleic acid was not present in the PCR reaction, and Fig. 20 (E) shows the result when a forward primer having a tag portion, a mediator oligonucleotide, an artificial template, a TaqMan probe, and a primer (C-p) specific for an artificial nucleic acid were all present in the PCR reaction.
도 20은 본 발명에 따라 표적핵산을, 인공핵산을 검출함으로써 검출할 수 있음을 보여준다. Figure 20 shows that, according to the present invention, a target nucleic acid can be detected by detecting an artificial nucleic acid.
<실시예 4> 인공핵산의 추가의 인공핵산으로의 전환을 통한 표적핵산의 검출<Example 4> Detection of target nucleic acid through conversion of artificial nucleic acid into additional artificial nucleic acid
본 실시예에서는 도 13의 개념도에 따라, 매개체 올리고뉴클레오티드의 5' 태그 부위(D)에 특이적인 택맨 프로브를 이용하여, 인공핵산(제1의 인공핵산)을 추가의 인공핵산(제2의 인공핵산)으로 전환하는 과정에서 발생한 신호를 검출하여 표적핵산을 검출할 수 있는지를 확인하였다.In this example, according to the conceptual diagram of Fig. 13, it was confirmed whether a target nucleic acid could be detected by detecting a signal generated in the process of converting an artificial nucleic acid (first artificial nucleic acid) into an additional artificial nucleic acid (second artificial nucleic acid) using a TaqMan probe specific to the 5' tag portion (D) of a mediator oligonucleotide.
상기 실시예 2와 동일한 구성의 5' 태그 부위를 가진 정방향 프라이머, 매개체 올리고뉴클레오티드(이는 5' 태그 부위(D)를 가짐), 인공 주형을 사용하고, 매개체 올리고뉴클레오티드의 5' 태그 부위(D)와 이에 특이적인 택맨 프로브는 아래의 서열과 구성을 가진 것을 사용하였으며, 이와 더불어 아래의 서열을 가진 인공핵산에 특이적인 프라이머(C-p)을 사용하였다.A forward primer having a 5' tag portion of the same configuration as in Example 2 above, a mediator oligonucleotide (which has a 5' tag portion (D)), an artificial template were used, and the 5' tag portion (D) of the mediator oligonucleotide and a TaqMan probe specific thereto were used having the sequence and configuration below, and in addition, a primer (C-p) specific for an artificial nucleic acid having the sequence below was used.
매개체 올리고뉴클레오티드의 5' 태그 부위(D): 5'-CAGCTAGCCATACGCCAT-3'5' tag portion of the mediator oligonucleotide (D): 5'-CAGCTAGCCATACGCCAT-3'
택맨 프로브: 5'-[BHQ-1] ATGGCGTATGGCTAGCTG [FAM]-3' Taxman Probe: 5'-[BHQ-1] ATGGCGTATGGCTAGCTG [FAM]-3'
프라이머(C-p): 5'-CCTCCTCCTCCTCCTCC-3' Primer (C-p): 5'-CCTCCTCCTCCTCCTCC-3'
PCR 반응을 상기 실시예 2와 동일하게 수행하였으며, 그 결과를 도 21에 나타내었다.The PCR reaction was performed in the same manner as in Example 2, and the results are shown in Fig. 21.
도 21 (A)는 매개체 올리고뉴클레오티드의 5' 태그 부위(D)가 없는 매개체 올리고뉴클레오티드를 사용하였을 때의 결과이고, 도 18 (B)는 5' 태그 부위(D)가 있는 매개체 올리고뉴클레오티드를 사용하였을 때의 결과이다. Figure 21 (A) shows the result when a mediator oligonucleotide without a 5' tag portion (D) of the mediator oligonucleotide was used, and Figure 18 (B) shows the result when a mediator oligonucleotide with a 5' tag portion (D) was used.
도 21은 본 발명에 따라 표적핵산을, 인공핵산으로부터 추가의 인공핵산이 생성되는 과정에서 발생하는 신호를 검출함으로써 검출할 수 있음을 보여준다. Figure 21 shows that, according to the present invention, a target nucleic acid can be detected by detecting a signal generated in the process of generating additional artificial nucleic acids from artificial nucleic acids.
<실시예 5> 인공핵산으로의 전환을 통한 2가지 표적핵산의 검출<Example 5> Detection of two target nucleic acids through conversion to artificial nucleic acids
도 18의 개념도에 따라, LacZ의 유전자의 일부 서열(표적서열 1)과 GAPDH 유전자의 일부 서열(표적서열 2)을 표적핵산으로 하여 동일한 5' 태그 부위를 가진, 각각의 표적핵산에 특이적인 2가지의 정방향 프라이머를, 각각 또는 함께 사용하여, 상기 실시예 2와 같이 도 9의 개념도에 따라 PCR를 수행하였다.According to the schematic diagram of Fig. 18, PCR was performed according to the schematic diagram of Fig. 9, as in Example 2, using two forward primers specific for each target nucleic acid, each having an identical 5' tag portion, with a partial sequence of the LacZ gene (target sequence 1) and a partial sequence of the GAPDH gene (target sequence 2) as target nucleic acids, separately or together.
표적서열 1과 2, 태그 부위를 가진 정방향 프라이머, 역방향 프라이머 서열은 아래와 같다. The sequences of target sequences 1 and 2, the forward primer with the tag region, and the reverse primer are as follows.
(1) 표적서열 1:(1) Target sequence 1:
TAATCGCTTTTACGGGGCGT GCTGCCAGTGCAATTTTATTGTCACTGCCGTTAATGATGCTGTCAGGATTTACCGGCCAGTCCAGCATTTCCC AGCCCAGGCCCTGATACATA TAATCGCTTTTACGGGGCGT GCTGCCAGTGCAATTTTATTGTCACTGCCGTTAATGATGCTGTCAGGATTTACCGGCCAGTCCAGCATTTCCC AGCCCAGGCCCTGATACATA
(2) 태그서열을 가진 표적서열 1에 대한 정방향 프라이머:(2) Forward primer for target sequence 1 with tag sequence:
5'-GCTACCCAGCCGGCTACAAGTAATCGCTTTTACGGGGCGT -3'5'- GCTACCCAGCCGGCTACAAG TAATCGCTTTTACGGGGCGT -3'
상기에서 5'-GCTACCCAGCCGGCTACAAG 서열이 5' 태그 서열임.The 5'- GCTACCCAGCCGGCTACAAG sequence above is the 5' tag sequence.
(3) 표적서열 1에 대한 역방향 프라이머:(3) Reverse primer for target sequence 1:
5'-TATGTATCAGGGCCTGGGCT-3' 5'-TATGTATCAGGGCCTGGGCT-3'
(4) 표적서열 2:(4) Target sequence 2:
GTTTAGCTCCTATTGCCAAC GTATTGGAAAAAAACTTTGGTATTGAAAAAGGATTTATGACAACAGTCCACTCATATACAGCAGACCAAAG ATTACAAGATGCTCCACACA GTTTAGCTCCTATTGCCAAC GTATTGGAAAAAAACTTTGGTATTGAAAAGGATTTATGACAACAGTCCACTCATATACAGCAGACCAAAG ATTACAAGATGCTCCACACA
(5) 태그서열을 가진 표적서열 2에 대한 정방향 프라이머:(5) Forward primer for target sequence 2 with tag sequence:
5'-GCTACCCAGCCGGCTACAAGGTTTAGCTCCTATTGCCAAC -3'5'- GCTACCCAGCCGGCTACAAG GTTTAGCTCCTATTGCCAAC -3'
상기에서 5'-GCTACCCAGCCGGCTACAAG 서열이 5' 태그 서열임.The 5'- GCTACCCAGCCGGCTACAAG sequence above is the 5' tag sequence.
(6) 표적서열 2에 대한 역방향 프라이머:(6) Reverse primer for target sequence 2:
5'- TGTGTGGAGCATCTTGTAAT -3'5'- TGTGTGGAGCATCTTGTAAT -3'
다음 매개체 올리고뉴클레오티드 서열과 구성, 인공 주형의 서열과 구성, 택맨 프로브의 서열과 구성은 상기 실시예 2와 동일하게 구성하였다.The following intermediate oligonucleotide sequence and composition, the sequence and composition of the artificial template, and the sequence and composition of the TaxMan probe were configured identically to those in Example 2.
PCR 반응은 표적서열 1과 2 각각 150 pg/ul 사용하여 상기 실시예 2와 동일하게 수행하였으며, 각 cycle마다 형광 신호를 측정하고 도 22에 나타내었다.The PCR reaction was performed in the same manner as in Example 2 using 150 pg/ul of target sequences 1 and 2, respectively, and the fluorescence signal was measured at each cycle and is shown in Figure 22.
도 22 (A)는 LacZ의 유전자의 일부 서열인 표적서열 1의 PCR 결과이고 도 22 (B)는 GAPDH 유전자의 일부 서열인 표적서열 2의 PCR 결과이며, 도 22 (C)는 LacZ의 유전자의 일부 서열인 표적서열 1과 GAPDH 유전자의 일부 서열인 표적서열 2에 대해 동일한 5' 태그 부위를 가진, 각각의 표적핵산에 특이적인 2가지의 정방향 프라이머를 함께 사용하였을 때의 PCR 결과이다. Figure 22 (A) is a PCR result of target sequence 1, which is a partial sequence of the LacZ gene, Figure 22 (B) is a PCR result of target sequence 2, which is a partial sequence of the GAPDH gene, and Figure 22 (C) is a PCR result of using two forward primers specific for each target nucleic acid, each having the same 5' tag portion, for target sequence 1, which is a partial sequence of the LacZ gene, and target sequence 2, which is a partial sequence of the GAPDH gene.
도 22를 참조하여 보면, 동일한 5' 태그 부위를 가진, 각각의 표적핵산에 특이적인 2가지의 정방향 프라이머를 함께 사용하였을 때가 각각의 정방향 프라이머를 사용하였을 때에 비하여 더 빨리 신호가 생성됨을 알 수 있다.Referring to Figure 22, it can be seen that when two forward primers specific for each target nucleic acid with the same 5' tag portion are used together, a signal is generated faster than when each forward primer is used.
본 발명은 PCR 기반의 분자진단, 특히 핵산 검출을 위한 체외진단용 의료기기 등에 적용 가능하다. The present invention can be applied to PCR-based molecular diagnosis, especially to in vitro diagnostic medical devices for nucleic acid detection.

Claims (20)

  1. (a) 검출 대상 시료와 4가지 NTP(nucleoside triphosphate) 및 뉴클레아제 활성을 가지는 DNA 중합효소를 포함하고, 아래의 (i) 내지 (iii)를 포함하는 PCR 반응을 위한 혼합물을 준비하는 단계(a) a step of preparing a mixture for PCR reaction comprising a sample to be detected, four NTPs (nucleoside triphosphates) and a DNA polymerase having nuclease activity, and including (i) to (iii) below
    (i) 표적핵산을 증폭시키기 위한 한 세트의 정방향 및 역방향 프라이머, 여기서 정방향 프라이머 및 역방향 프라이머 중 하나 이상은 그 5' 말단 부위에 인위적 임의 서열로 구성된 5' 태그 서열을 하나 이상 가짐; 및(i) a set of forward and reverse primers for amplifying a target nucleic acid, wherein at least one of the forward primer and the reverse primer has at least one 5' tag sequence consisting of an artificial random sequence at its 5' terminal portion; and
    (ii) 상기 5' 태그 서열에 상보적으로 결합하는 프로브 부위(A)와 표적핵산에 특이적인 인위적 임의 서열로 이루어진 매개체 부위(B)을 포함하는 단일 가닥의 매개체 올리고뉴클레오티드 (ii) a single-stranded mediator oligonucleotide comprising a probe portion (A) that binds complementarily to the 5' tag sequence and a mediator portion (B) consisting of an artificial random sequence specific for a target nucleic acid;
    (b) 상기 (a) 단계의 혼합물을 반응 용기에 넣고, PCR 반응을 수행하여, 표적핵산이 증폭됨과 동시에, 상기 뉴클레아제 활성을 갖는 DNA 중합효소에 의해 매개체 올리고뉴클레오티드에서 매개체 절편이 절단되어 생성되도록 하는 단계, 및(b) a step of placing the mixture of step (a) into a reaction vessel and performing a PCR reaction so that the target nucleic acid is amplified and at the same time, a mediator fragment is cleaved from the mediator oligonucleotide by a DNA polymerase having the nuclease activity, and
    (c) 상기 매개체 절편을 검출하여 표적핵산을 검출하는 단계를 포함하는(c) a step of detecting the target nucleic acid by detecting the above medium fragment;
    표적핵산의 검출 방법.Method for detecting target nucleic acid.
  2. 제1항에 있어서,In the first paragraph,
    상기 표적핵산에 특이적인 인위적 임의 서열로 이루어진 매개체 부위는 염기서열(뉴클레오티드 순서) 및 크기 중 어느 하나 이상이 표적핵산에 특이적인 것이고,The mediator portion consisting of an artificial random sequence specific to the above target nucleic acid is specific to the target nucleic acid in at least one of the base sequence (nucleotide order) and size,
    상기 매개체 올리고뉴클레오티드를 2가지 이상의 표적핵산에 따라 각기 다른 것을 사용함으로써 2가지 표적핵산을 하나의 반응에서 검출할 수 있는 것을 특징으로 하는 방법.A method characterized in that two target nucleic acids can be detected in a single reaction by using different mediator oligonucleotides according to two or more target nucleic acids.
  3. 제1항에 있어서,In the first paragraph,
    상기 매개체 절편의 검출은 질량 분석(mass spectrometry), 전기영동(electrophoresis), 액체 크로마토그래피(liquid chromatography), 서열분석, 마이크로어레이, 그 매개체 절편으로부터 연장되어 생성되는 인공핵산을 검출하거나, 또는 그 인공핵산이 생성되면서 변화하는 신호를 검출함에 의하여 수행되는 것을 특징으로 하는 방법.A method characterized in that the detection of the above mediator fragment is performed by mass spectrometry, electrophoresis, liquid chromatography, sequence analysis, microarray, detecting an artificial nucleic acid generated by extending from the mediator fragment, or detecting a signal that changes as the artificial nucleic acid is generated.
  4. 제3항에 있어서,In the third paragraph,
    상기 질량 분석(mass spectrometry)은 MALDI-TOF MS(Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry), 탠덤 질량 분석(Tandem MS), ESI-TOF(Electrospray Ionization-Time of Flight) 또는 이온 포집 질량 분석(Ion Trap Mass Spectrometry), LC-MS(Liquid Chromatography Mass Spectrometry), GC-MS(Gas Chromatography Mass Spectrometry) 또는 IM-MS(Ion Mobility Mass Spectrometry)에 의하여 수행되는 것을 특징으로 하는 방법.A method characterized in that the above mass spectrometry is performed by MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry), tandem mass spectrometry (Tandem MS), ESI-TOF (Electrospray Ionization-Time of Flight) or ion trap mass spectrometry (Ion Trap Mass Spectrometry), LC-MS (Liquid Chromatography Mass Spectrometry), GC-MS (Gas Chromatography Mass Spectrometry) or IM-MS (Ion Mobility Mass Spectrometry).
  5. 제3항에 있어서,In the third paragraph,
    상기 매개체 절편으로부터 연장되어 생성되는 인공핵산은 그 매개체 절편 자체가 인공주형으로 작용하여 그 매개체 절편의 3' 말단에서 연장되어 생성되는 것을 특징으로 하는 방법. A method wherein an artificial nucleic acid produced by extension from the above-mentioned intermediate fragment is produced by extension from the 3' end of the intermediate fragment using the intermediate fragment itself as an artificial template.
  6. 제3항에 있어서,In the third paragraph,
    상기 매개체 절편의 검출은 그 매개체 절편으로부터 연장되어 생성되는 인공핵산을 검출하거나, 또는 그 인공핵산이 생성되면서 변화하는 신호를 검출함에 의하여 수행되고,The detection of the above mediator fragment is performed by detecting an artificial nucleic acid that is extended from the mediator fragment and generated, or by detecting a signal that changes as the artificial nucleic acid is generated.
    상기 PCR 반응을 위한 혼합물에 별도 분자의 인공주형이 추가로 포함되고,An artificial template of a separate molecule is additionally included in the mixture for the above PCR reaction,
    상기 인공주형은 상기 매개체 올리고뉴클레오티드의 매개체 부위에 대해 상보적인 프라이밍 부위를 가지고 또한 상기 매개체 절편으로부터 연장되어 생성되는 인공핵산에 대해 주형으로 작용하는 주형 부위(C' )를 가지며,The above artificial template has a priming site complementary to the mediator site of the mediator oligonucleotide and also has a template site (C') that acts as a template for an artificial nucleic acid that is extended from the mediator fragment.
    상기 매개체 절편으로부터 연장되어 생성되는 인공핵산은 상기 별도 분자의 인공주형에 상기 매개체 절편이 상보적으로 결합하여 프라이머로 작용함으로써 그 매개체 절편의 3' 말단에서 연장되어 생성되는 것을 특징으로 하는 방법. A method characterized in that an artificial nucleic acid produced by extending from the above-mentioned intermediate fragment is produced by extending from the 3' end of the intermediate fragment by the intermediate fragment complementarily binding to an artificial template of the above-mentioned separate molecule to act as a primer.
  7. 제6항에 있어서,In Article 6,
    상기 인공핵산이 생성되면서 변화하는 신호를 검출하는 것은, Detecting the signal that changes as the artificial nucleic acid is generated is
    상기 인공주형이 헤어핀 구조의 분자비콘이고, 상기 매개체 절편은 상기 헤어핀 구조의 분자비콘에 특이적으로 결합하여 연장됨으로써 상기 헤어핀 구조가 펴지면서 리포터 분자가 퀜처 분자로부터 분리되어 발생하는 신호를 검출하여 이루어지는 것을 특징으로 하는 방법.A method characterized in that the artificial template is a molecular beacon having a hairpin structure, and the mediator fragment specifically binds to and extends the molecular beacon having the hairpin structure, thereby detecting a signal generated when the reporter molecule is separated from the quencher molecule as the hairpin structure unfolds.
  8. 제6항에 있어서,In Article 6,
    상기 인공핵산이 생성되면서 변화하는 신호를 검출하는 것은, Detecting the signal that changes as the artificial nucleic acid is generated is
    상기 인공주형의 주형 부위에 가수분해성 프로브가 결합하고,A hydrolyzable probe is bound to the mold portion of the above artificial mold,
    상기 매개체 절편이 인공 주형의 프라이밍 부위에 결합하여 5'-->3' 엑소뉴클레아제 활성을 가지는 DNA 중합효소에 의해 연장됨으로써 상기 가수분해성 프로브가 분해되어 발생하는 신호를 검출하여 이루어지는 것을 특징으로 하는 방법.A method characterized in that the above-mentioned intermediate fragment binds to the priming site of an artificial template and is extended by a DNA polymerase having 5'-->3' exonuclease activity, thereby detecting a signal generated by decomposition of the hydrolyzable probe.
  9. 제8항에 있어서,In Article 8,
    상기 인공핵산이 생성되면서 변화하는 신호를 검출하는 것은, Detecting the signal that changes as the artificial nucleic acid is generated is
    상기 인공 주형과 가수분해성 프로브는 그 인공 주형의 5' 말단과 가수분해성 프로브의 3' 말단이 서로 연결된 단일 가닥으로 구성되어,The above artificial template and hydrolyzable probe are composed of a single strand in which the 5' end of the artificial template and the 3' end of the hydrolyzable probe are connected to each other.
    상기 매개체 절편이 인공 주형의 프라이밍 부위에 결합하여 5'-->3' 엑소뉴클레아제 활성을 가지는 DNA 중합효소에 의해 연장됨으로써 상기 가수분해성 프로브가 분해되어 발생하는 신호를 검출하여 이루어지는 것을 특징으로 하는 방법.A method characterized in that the above-mentioned intermediate fragment binds to the priming site of an artificial template and is extended by a DNA polymerase having 5'-->3' exonuclease activity, thereby detecting a signal generated by decomposition of the hydrolyzable probe.
  10. 제6항에 있어서,In Article 6,
    상기 인공핵산을 검출하는 것은 Detecting the above artificial nucleic acid
    상기 인공핵산에, 상기 인공핵산 서열에 특이적인 분자비콘을 결합시켜 발생하는 신호를 측정하여 이루어지는 것을 특징으로 하는 방법.A method characterized by measuring a signal generated by binding a molecular beacon specific to the artificial nucleic acid sequence to the artificial nucleic acid.
  11. 제6항에 있어서,In Article 6,
    상기 인공핵산을 검출하는 것은 Detecting the above artificial nucleic acid
    상기 인공핵산에, 상기 인공핵산 서열에 특이적인 가수분해성 프로브를 결합시키고, 상기 인공핵산에 상보적인 프라이머를 결합시켜 5'-->3' 엑소뉴클레아제 활성을 가지는 DNA 중합효소에 의해 연장시킴으로써 상기 가수분해성 프로브가 분해되어 발생하는 신호를 검출하여 이루어지는 것을 특징으로 하는 방법.A method characterized in that the method comprises detecting a signal generated by decomposition of the hydrolyzable probe by binding a hydrolyzable probe specific for the artificial nucleic acid sequence to the artificial nucleic acid, binding a complementary primer to the artificial nucleic acid, and extending the probe by a DNA polymerase having a 5'-->3' exonuclease activity.
  12. 제6항에 있어서,In Article 6,
    상기 인공핵산을 검출하는 것은 Detecting the above artificial nucleic acid
    상기 인공핵산에 대해 상보적인 서열을 가지는 캡쳐 프로브(제1 프로브)가 고정되어 있는 지지체에 상기 인공핵산을 결합시키고, 상기 인공핵산 또는 상기 매개체 절편에 검출 신호로 표지화된 검출 프로브(제2 프로브)를 상보적으로 결합시키고 미반응물을 세척하여 제거한 후 그 프로브의 검출 신호를 검출하여 이루어지는 것을 특징으로 하는 방법.A method characterized in that the method comprises binding the artificial nucleic acid to a support on which a capture probe (first probe) having a complementary sequence to the artificial nucleic acid is fixed, complementarily binding a detection probe (second probe) labeled with a detection signal to the artificial nucleic acid or the mediator fragment, washing away unreacted matter, and then detecting the detection signal of the probe.
  13. 제6항에 있어서,In Article 6,
    상기 추가의 인공핵산이 생성되면서 발생하는 신호를 검출하는 것은Detecting the signal generated when the above additional artificial nucleic acid is produced
    상기 매개체 올리고뉴클레오티드로 그 5' 말단 쪽에 인위적 서열의 태그 부위(매개체 올리고뉴클레오티드의 5' 태그 부위)가 도입된 것을 사용하여, 상기 매개체 올리고뉴클레오티드의 5' 태그 부위에 특이적으로 결합하는 가수분해성 프로브와 상기 인공핵산에 특이적으로 결합하는 프라이머를 상기 PCR 반응 혼합물에 포함시킴으로써, 상기 인공핵산에 결합한 프라이머가 5'-->3' 엑소뉴클레아제 활성을 가지는 DNA 중합효소에 의해 연장됨으로써 상기 매개체 올리고뉴클레오티드의 5' 태그 부위에 결합하고 있던 가수분해성 프로브가 분해되어 발생하는 신호를 검출하여 이루어지는 것을 특징으로 하는 방법.A method characterized in that the method comprises detecting a signal generated by using the above-mentioned intermediate oligonucleotide to which an artificial sequence tag portion (5' tag portion of the intermediate oligonucleotide) is introduced at the 5' end, by including a hydrolyzable probe that specifically binds to the 5' tag portion of the intermediate oligonucleotide and a primer that specifically binds to the artificial nucleic acid in the PCR reaction mixture, so that the primer bound to the artificial nucleic acid is extended by a DNA polymerase having a 5'-->3' exonuclease activity, thereby decomposing the hydrolyzable probe bound to the 5' tag portion of the intermediate oligonucleotide.
  14. 제6항에 있어서,In Article 6,
    상기 5' 태그 서열은 정방향 및 역방향 프라이머 중 하나에 또는 이들 프라이머 모두에 1개 이상 존재하는 것을 특징으로 하는 방법A method characterized in that the above 5' tag sequence is present in at least one of the forward and reverse primers or in both of these primers.
  15. 제6항에 있어서,In Article 6,
    상기 시료는 검출하고자 하는 표적핵산을 포함하거나 포함하고 있을 것으로 의심되어 검출의 필요성을 가지는 혼합물 또는 용액인 것을 특징으로 하는 방법.A method characterized in that the sample is a mixture or solution that contains or is suspected of containing a target nucleic acid to be detected and thus has a need for detection.
  16. 제6항에 있어서,In Article 6,
    상기 인공핵산이 생성되면서 변화하는 신호는 리포터 분자와 퀜처 분자의 거리가 변화함에 의해서 생기거나 또는 공여체와 수용체의 거리가 변화함에 의해서 생기는 것을 특징으로 하는 방법.A method characterized in that a signal that changes as the artificial nucleic acid is generated is generated by a change in the distance between a reporter molecule and a quencher molecule or by a change in the distance between a donor and an acceptor.
  17. 제6항에 있어서,In Article 6,
    상기 혼합물에는, 상기 5' 태그 서열과 같은 서열을 가지는 보조의 프라이머가 포함되는 것을 특징으로 하는 방법.A method characterized in that the mixture comprises an auxiliary primer having the same sequence as the 5' tag sequence.
  18. 제6항에 있어서,In Article 6,
    상기 5' 태그 부위를 가진 프라이머는 하나의 표적핵산에 대해서 각 특이적 결합 위치를 달리하여 2가지 이상 상기 PCR 반응 혼합물에 포함되는 것을 특징으로 하는 방법.A method characterized in that two or more primers having the above 5' tag portion are included in the PCR reaction mixture with different specific binding positions for each target nucleic acid.
  19. 제6항에 있어서,In Article 6,
    상기 표적핵산은 2가지 이상이고,The above target nucleic acids are two or more,
    상기 각 표적핵산에 대해서 각각의 정방향 프라이머와 역방향 프라이머가 상기 혼합물에 포함되되,For each of the above target nucleic acids, the forward primer and reverse primer are included in the mixture,
    상기 각 표적핵산에 대한 각각의 정방향 프라이머 및 역방향 프라이머 중 하나 이상의 프라이머는 5' 태그 부위를 가지고,At least one of the forward primer and reverse primer for each of the above target nucleic acids has a 5' tag portion,
    상기 5' 태그 부위는 모두 동일 서열로 이루어지는 것을 특징으로 하는 방법.A method characterized in that all of the above 5' tag regions are composed of the same sequence.
  20. 제6항에 있어서,In Article 6,
    상기 인공핵산에 의존하여 추가의 인공핵산(제2의 인공핵산)이 생성되도록 하는 단계가 포함되고,A step is included for producing an additional artificial nucleic acid (a second artificial nucleic acid) by relying on the artificial nucleic acid mentioned above,
    상기 인공핵산에 의존하여 생성되는 추가의 인공핵산을 검출하거나 상기 추가의 인공핵산이 생성되면서 변화하는 신호를 검출함에 의해, 표적핵산을 검출하는 단계를 포함하는 것을 특징으로 하는 방법.A method characterized by comprising a step of detecting a target nucleic acid by detecting an additional artificial nucleic acid produced depending on the artificial nucleic acid or by detecting a signal that changes as the additional artificial nucleic acid is produced.
PCT/KR2024/002935 2023-03-07 2024-03-07 Method for detecting target nucleic acid using artificial nucleic acid WO2024186140A1 (en)

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