JP6523874B2 - Nucleic acid amplification substrate, nucleic acid amplification method using the substrate, and nucleic acid detection kit - Google Patents

Description

  The present invention relates to a nucleic acid amplification substrate, a nucleic acid amplification method using the substrate, and a nucleic acid detection kit.

  Conventionally, a DNA microarray targeting mRNA is widely used as a means for measuring the amount of gene expression in cells. If multiple kinds of single-stranded DNA with a clear base sequence are arranged on the substrate in advance and the sample is made to react with this, the DNA strand of the sample is only in the part of the base sequence complementary to the DNA sequence of the sample. Join. The binding position can be detected by fluorescence or current, and the DNA sequence contained in the sample can be known from the initial arrangement. If the base sequence of the sample can be predicted, the sequence can be efficiently identified.

Depending on the technical content, the following two types of DNA microarrays exist.
The first is the Affymetrix GeneChip, which is made by artificially synthesizing a 20-25 mer oligonucleotide on a substrate using photolithography technology and solid phase reaction chemistry technology. This oligonucleotide is designed in advance in position, length, etc. using a computer to identify a specific base sequence of a gene. Nonspecific cross hybridization by arranging not only probe perfect match (PM) designed to be completely complementary to a specific gene, but also nonspecific base sequence called mismatch (MM) as a probe The quantitative value of can be subtracted from the signal value. When this microarray is used, mRNA is extracted from one type of sample, cDNA synthesized by reverse transcription is labeled with biotin, hybridization with DNA on the substrate is performed, and fluorescence intensity is read with a dedicated scanner manufactured by Affimetrix. . The amount of expression of mRNA can be measured by irradiating light having a wavelength specific to this fluorescent dye and measuring the ratio of light emission (see, for example, Patent Document 1).

  The second is a DNA microarray developed by Stanford University, which utilizes a high-precision dispensing machine to spot a prepared DNA on a substrate to produce a high-density DNA chip. In the spot method, the pin method by mechanical contact to the solid phase of the pin tip, the ink jet method using the principle of the ink jet printer, bubbles are generated by heating in the spotter, and the pressure is used to eject the sample There is a bubble jet (registered trademark) method, a capillary method using a capillary, and the like. In recent years, a type in which 25 mer-60 mer oligonucleotides, in which specific sequences of genes are designed on a computer as in the method of Affimetrix, is placed at high density, rather than the method of striking cDNA fragments, has become mainstream. When this microarray is used, mRNA is extracted from two different samples, and the target is prepared by labeling cDNA synthesized by reverse transcription with different fluorescent dyes. The fluorescent dye is usually Cy3 (green) or Cy5 (red), and these two samples are competitively hybridized with the probe on the glass substrate, and the fluorescence intensity is detected by the scanner (for example, patent) Reference 2).

By the way, natural nucleic acids (DNA and RNA) have a large degree of freedom in shape (degree of freedom of conformation) due to their chemical structure, mainly in an equilibrium state of a conformation called A-type and a B-type conformation. Existing. Therefore, it is thermodynamically disadvantageous in duplex formation (hybridization) between DNA-DNA, DNA-RNA and RNA-RNA, leaving room for improvement in binding affinity (hybridization ability). There is.
A bridged nucleic acid, which is an artificial synthetic nucleic acid obtained by cross-linking a natural nucleic acid molecule, restricts the “degree of freedom” of the natural nucleic acid to increase the binding affinity to the target DNA or RNA, and further nuclease (nucleolytic enzyme ) Also acquired resistance.
BNA-containing oligonucleic acids in which these artificial nucleic acids BNA are incorporated into natural oligonucleotides have selective and high binding affinity not only for single-stranded DNA or RNA but also for double-stranded DNA. In particular, 3'-amino-2 ', 4'-BNA, 5'-amino-2', 4'-BNA, 2 ', 4'-BNACOC, 2', 4'-BNANC developed by the present inventors. Oligonucleotides containing BNAs from the second generation onward are superior artificial nucleic acids that have acquired excellent binding affinity to single-stranded RNA and double-stranded DNA, and are extremely resistant to nucleases. It has high characteristics (see, for example, Patent Document 3).

U.S. Pat. No. 5,424,186 Patent No. 3272365 Patent No. 4731324

In the DNA microarrays described in Patent Documents 1 and 2, it is necessary to change the spotted oligonucleotide depending on the target nucleic acid, which is unsuitable for mass production and difficult to be customized.
Furthermore, development of a DNA chip spotted with a bridged nucleic acid-containing oligonucleotide as described in Patent Document 3 is also in progress, but this also depends on the nucleic acid targeting the oligonucleotide spotted as described above. It is necessary to change, it is not suitable for mass production, and it is difficult to meet made-to-order.
In addition, the reverse transcription reaction using the target mRNA as a template and the detection of the cDNA prepared in the reverse transcription reaction are performed in separate steps, so the operation becomes complicated and there is a risk of contamination.

  The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide a nucleic acid amplification substrate excellent in sequence specificity, a nucleic acid amplification method using the substrate, and a nucleic acid detection kit while being simple. I assume.

  MEANS TO SOLVE THE PROBLEM The present inventors discovered that a subject could be solved by using the nucleic acid amplification board | substrate which fixed the probe containing bridged nucleic acid, as a result of earnestly examining in order to solve the said subject.

That is, the present invention provides the following (1) to (14).
(1) A nucleic acid amplification substrate comprising a substrate and a probe immobilized on the substrate, wherein the probe is
A first oligonucleic acid immobilized on the substrate;
A nucleic acid A comprising a sequence capable of hybridizing to the first oligo nucleic acid in the 5 'end region, and a nucleic acid B comprising a sequence capable of becoming a forward primer for target nucleic acid amplification in the 3' end region, A second oligonucleic acid comprising a bridged nucleic acid at A,
A nucleic acid amplification substrate characterized by having:
(2) The nucleic acid amplification substrate according to (1), wherein the substrate has one or more sections, and one or more types of the probes are fixed in one section.
(3) The nucleic acid amplification substrate according to (1) or (2), wherein the proportion of the bridged nucleic acid in the second oligonucleic acid is 66% or more.
(4) The nucleic acid amplification substrate according to any one of (1) to (3), wherein the first oligonucleic acid immobilized on the substrate is DNA.
(5) The nucleic acid amplification substrate according to any one of (1) to (4), wherein the target nucleic acid is gDNA or cDNA.

(6) A nucleic acid amplification method using the nucleic acid amplification substrate according to any one of (1) to (5).
(7) A nucleic acid detection kit comprising a substrate on which a probe is immobilized and a reverse primer for target nucleic acid amplification, wherein the probe is
A first oligonucleic acid immobilized on the substrate;
A nucleic acid A comprising a sequence capable of hybridizing to the nucleic acid in the 5 'end region, and a nucleic acid B comprising a sequence capable of becoming a forward primer for target nucleic acid amplification in the 3' end region A second oligonucleic acid,
A nucleic acid detection kit characterized by having:
(8) The nucleic acid detection kit according to (7), wherein the proportion of bridged nucleic acid in the second oligonucleic acid is 66% or more.
(9) The nucleic acid detection kit according to (7) or (8), wherein the first oligonucleic acid immobilized on the substrate is DNA.
(10) The nucleic acid detection kit according to any one of (7) to (9), wherein the target nucleic acid is gDNA or cDNA.

  According to the nucleic acid amplification substrate of the present invention and the nucleic acid amplification method using the substrate, the target nucleic acid can be conveniently and highly selectively amplified. Moreover, according to the nucleic acid detection kit of the present invention, the target nucleic acid can be detected easily with high sensitivity and high accuracy.

It is a schematic diagram of 1st Embodiment of the nucleic acid amplification board | substrate which concerns on this invention. It is a schematic diagram showing an example of a section on a nucleic acid amplification substrate concerning the present invention. It is a schematic diagram of the nucleic acid amplification method using the nucleic acid amplification substrate in this embodiment. It is the graph which showed the change of the light absorbency (260 nm) by the temperature rise of double stranded BNA / DNA in Example 1. FIG. 6 is an image showing the results of non-denaturing polyacrylamide gel electrophoresis of the first oligonucleic acid, the second oligonucleic acid and the double stranded oligonucleic acid in Example 2. FIG. (A) It is the graph which showed the change of the light absorbency (260 nm) by the temperature rise of the control double stranded oligo in Example 2. FIG. (B) It is the graph which showed the change of the light absorbency (260 nm) by the temperature rise of the double stranded BNA / DNA oligo in Example 2. FIG. 5 is a graph showing the results of the Allele-specific PCR reaction in Example 2.

<Nucleic acid amplification substrate>
1. First Embodiment FIG. 1 is a schematic view of a first embodiment of the nucleic acid amplification substrate according to the present invention. The nucleic acid amplification substrate 10 of the present embodiment is a nucleic acid amplification substrate comprising the substrate 1 and the probe 2 immobilized on the substrate 1, wherein the probe 2 is a first oligonucleic acid 3 or 5 immobilized on the substrate 1. A nucleic acid A (nucleic acid 5) consisting of a sequence capable of hybridizing to the first oligo nucleic acid in the 'terminal region, and a nucleic acid B (nucleic acid 6) consisting of a sequence capable of becoming a forward primer for target nucleic acid amplification in the 3' terminal region; And a second oligonucleic acid 4 comprising a nucleic acid bridged to at least nucleic acid A (nucleic acid 5).

  The target nucleic acid in the present embodiment is not particularly limited, but is preferably a stable DNA such as gDNA or cDNA. gDNA is an abbreviation of "genomic DNA" and mainly refers to DNA in the nucleus. cDNA is an abbreviation of "complementary DNA" and means DNA synthesized from mRNA by reverse transcription using reverse transcriptase. In gDNA, because it has introns and exons, it is difficult to deduce the amino acid sequence of a protein produced from the base sequence, but since cDNA has only exons, it is not possible to deduce the amino acid sequence of the protein from the base sequence It is easy.

  The substrate is not particularly limited, but is preferably a substrate made of a heat-resistant material in order to carry out a PCR reaction, and examples thereof include a glass substrate, a silicon substrate, a plastic substrate, a metal substrate and the like.

FIG. 2 is a schematic view showing an example of a section on a nucleic acid amplification substrate according to the present invention. In the nucleic acid amplification substrate 30 of the present embodiment, the substrate 1 has one or more sections, and one or more types of one or more probes 2 are fixed to one section. By immobilizing a plurality of probes of the same type on the substrate 1, it is possible to efficiently amplify the target nucleic acid. In addition, since the probe is immobilized on the substrate, the amplified target nucleic acid can be held in the compartment.
Also, conventionally, the nucleic acid to be immobilized on the substrate and the nucleic acid that specifically recognizes the target nucleic acid are identical, and it has been necessary to fix the nucleic acid consisting of a different base sequence in each section, so it is unsuitable for mass production. , It was difficult to respond to bespoke.
However, in this embodiment, since it is the role of the second oligo nucleic acid to specifically recognize and amplify the target nucleic acid, the first oligo nucleic acid 3 is a substrate consisting of a standardized and standardized base sequence. The substrate 1 on which the first oligonucleic acid 3 is immobilized can be easily mass-produced. In addition, it becomes possible to easily cope with custom-made by changing the second oligo nucleic acid to be used.

  As shown in FIG. 1, the probe 2 consists of a first oligonucleic acid 3 and a second oligonucleic acid 4. Furthermore, the second oligonucleic acid 4 can be a nucleic acid A (nucleic acid 5) consisting of a sequence capable of hybridizing to the first oligonucleic acid in the 5 'end region and a forward primer for target nucleic acid amplification in the 3' end region. And nucleic acid B (nucleic acid 6) consisting of a sequence.

  In the present invention and in the specification of the present application, “capable of hybridizing” means that one nucleic acid such as a primer or a probe used in the present invention hybridizes to the other nucleic acid such as a target nucleic acid under stringent conditions. Mean that it does not hybridize to nucleic acid molecules other than the other nucleic acid. "Stringent conditions" include, for example, conditions described in Molecular Cloning-A LABORATORY MANUAL SECOND EDITION (Sambrook et al., Cold Spring Harbor Laboratory Press). In addition, when one nucleic acid can hybridize to the other nucleic acid, one nucleic acid preferably has a homology (identity of the base sequence) with a base sequence complementary to the base sequence constituting the other nucleic acid. %, More preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more.

In the present embodiment, the first oligonucleic acid 3 is not particularly limited, but does not have a sequence complementary to the target nucleic acid so as not to hybridize with the target nucleic acid from the viewpoint of highly selective amplification of the target nucleic acid. Is preferred. The first oligonucleic acid 3 is not particularly limited as long as it is DNA or RNA, and may be natural or synthetic, but is preferably DNA.
In the first oligonucleic acid 3, the length of the nucleic acid 3a composed of a base sequence complementary to the nucleic acid A (nucleic acid 5) in the second oligonucleic acid 4 is preferably 13 bases or more, more preferably 18 bases or more. In addition, in order for nucleic acid B (nucleic acid 6) in the second oligonucleic acid 4 to hybridize with the target nucleic acid, a molecular degree of freedom is required. The length of the immobilized nucleic acid 3b is not particularly limited, but is preferably 3 to 50 bases, and more preferably 5 to 25 bases.

In the present embodiment, the nucleic acid A (nucleic acid 5) preferably contains a bridged nucleic acid. Also, the nucleic acid B (nucleic acid 6) may contain a bridged nucleic acid. In the present invention and the present specification, "bridged nucleic acid" refers to an artificial nucleic acid in which the 2 'and 4' positions of the sugar are bridged to fix the conformation to the N type, and N type and S found in natural nucleic acid It can suppress the type of conformational conversion. Specifically, the first generation BNAs of 2 ', 4'-BNA (LNA) are preferred, and 3'-amino-2', 4'-BNA, 5'-amino-2 ', 4'-BNA, More preferred are second generation BNAs such as 2 ′, 4′-BNACOC, and even more preferred are third generation BNAs such as 2 ′, 4′-BNANC.
In addition, the bridged nucleic acid has a Tm value (the value of temperature at which 50% of double-stranded DNA separates into single-stranded DNA) as compared to a natural nucleic acid by 1 to 3 ° C. Furthermore, the bridged nucleic acid has high affinity to a nucleic acid having a hybridizable sequence, and is not easily recognized by the DNA degradation enzyme or RNA degradation enzyme.
Therefore, by including the bridged nucleic acid in the nucleic acid A (nucleic acid 5), the hybridization of the first oligonucleic acid 3 and the second oligonucleic acid 4 has heat resistance and excellent affinity. Also, in the case where the nucleic acid B (nucleic acid 6) contains a bridged nucleic acid, it can be selectively hybridized with the target nucleic acid and amplified.
Further, the length of the nucleic acid A (nucleic acid 5) is, like the nucleic acid 3a in the first oligo nucleic acid 3, preferably 13 bases or more, and more preferably 18 bases or more.

  Further, the ratio of the bridged nucleic acid in the second oligonucleic acid 4 is preferably 61% or more, and more preferably 66% or more. Furthermore, in the second oligonucleic acid 4, the ratio of the bridged nucleic acid in the nucleic acid A (nucleic acid 5) is preferably 61% or more, and more preferably 66% or more. For example, in the case of an oligo-nucleic acid containing 12 to 18 bases of bridged nucleic acid, the Tm value is 95 ° C. As described above, the hybridization of the nucleic acid A (the nucleic acid 5) containing the bridged nucleic acid and the nucleic acid 3a in the first oligonucleic acid 3 has high affinity because the Tm value is high compared to the natural nucleic acid, The entire probe 2 can be fixed on the substrate 1.

  “Can be a forward primer for target nucleic acid amplification” has a sequence complementary to the target nucleic acid, can be specifically hybridized, and can be a starting point in the extension reaction of the target nucleic acid by a nucleic acid synthetase Means to be. Therefore, the length of the nucleic acid B is preferably 5 to 40 bases, more preferably 15 to 30 bases, and particularly preferably 18 to 25 bases, from the viewpoint of a forward primer for target nucleic acid amplification. In addition, it is preferable that the Tm value does not differ significantly from the reverse primer for target nucleic acid amplification used for amplification. Furthermore, it is preferred to avoid GC-rich or AT-rich sequences, in part without any base bias, in particular in the 3'-end region. Furthermore, it is preferable to avoid a complementary sequence of 3 bases or more between the inside of the primer and the reverse primer and to avoid a sequence in which the 3 'end region is complementary to 2 bases or more.

  According to the nucleic acid amplification substrate of the present embodiment, the target nucleic acid can be easily amplified, and the detection step can be performed while maintaining the amplified target nucleic acid on the substrate.

<Nucleic acid amplification method>
Next, the nucleic acid amplification method using the nucleic acid amplification substrate of the present invention will be described in detail. First, a nucleic acid sample containing a target nucleic acid and a nucleic acid amplification reaction solution are brought into contact with a nucleic acid amplification substrate.

In the present invention, "a nucleic acid sample containing a target nucleic acid" is not particularly limited as long as it is assumed to contain a nucleic acid for which amplification is desired. When it is intended to amplify a nucleic acid having a mutation, it is not particularly limited as long as it is assumed to contain a nucleic acid having a mutation. Nucleic acid samples are typically collected from mammals, preferably humans, and may be blood, pleural fluid, bronchial lavage fluid, bone marrow fluid, fluid samples such as lymph fluid, lymph nodes, blood vessels, bone marrow, brain, spleen, Solid samples such as skin can be mentioned. Since the method of the present invention can amplify nucleic acid with very high selectivity, using a liquid sample containing only a very small amount of nucleic acid sample such as blood without directly collecting the sample from a lesion site such as cancer, for example It is possible. Therefore, a preferred nucleic acid sample is a liquid sample such as blood.
Furthermore, in the nucleic acid amplification method of the present invention, the content of the target nucleic acid in the reaction solution is preferably contained in an amount corresponding to 10 ² to 10 ⁶ molecules, for example, 0.3 ng to 3 μg for human genes It is an extent. Since the frequency of nonspecific amplification increases when the amount of target nucleic acid is too large, the content of target nucleic acid is preferably suppressed to 0.5 μg or less per 100 μL.

  In the present invention, the “reaction solution for nucleic acid amplification” is a solution containing reagents and the like necessary for carrying out the nucleic acid amplification reaction. The composition of the reaction solution for nucleic acid amplification varies depending on the method of nucleic acid amplification used, but for example, four deoxynucleoside triphosphates (dATP, dTTP, dCTP, dGTP: hereinafter collectively referred to as dNTP) as a substrate, and an enzyme The reaction solution may include a DNA polymerase, magnesium ion as a cofactor of the enzyme, and a reverse primer for extension amplification. In addition, although the details will be described later, when a method such as real-time PCR, which can simultaneously perform this step and a step of detecting an amplified nucleic acid, is used as a nucleic acid amplification method, an intercalator, a fluorescence labeling probe A suitable detection reagent such as a cycling probe is also coexistent in the reaction solution for nucleic acid amplification. Regarding the concentration of dNTP, the optimum concentration may be examined in the range of 100 to 400 μM of each of the four final concentrations. As the DNA polymerase, one having properties suitable for the nucleic acid amplification method to be used is used. For example, in the case of the PCR method, thermostable DNA polymerases such as Taq DNA polymerase, Pfu DNA polymerase, and those developed by other bioscience related companies are preferably used. For the magnesium ion concentration, an optimum concentration may be examined in the final concentration range of 1 to 6 mM. The reaction solution has an optimum pH and an optimum salt concentration at which the activity of the DNA polymerase can be obtained. Furthermore, depending on the nucleic acid amplification method to be used, it may further contain ribonuclease (RNase H), reverse transcriptase (RT) and the like.

Next, the target nucleic acid is hybridized with a nucleic acid B consisting of a sequence capable of becoming a forward primer for target nucleic acid amplification in a probe immobilized on a nucleic acid amplification substrate and a reverse primer for target nucleic acid amplification. Extension reaction proceeds. The nucleic acid B and the reverse primer are paired to amplify the target nucleic acid. Similar to nucleic acid B, the length of the reverse primer is preferably 5 to 40 bases, more preferably 15 to 30 bases, and particularly preferably 18 to 25 bases.
Further, from the viewpoint of nucleic acid amplification efficiency, the nucleic acid B comprising a sequence that can be a forward primer for target nucleic acid amplification and the reverse primer for target nucleic acid amplification are designed such that the number of bases of the amplification product is 50 to 1000. Is preferred, and more preferably 50 to 300. Moreover, it is preferable that it is 50-66 degreeC, and, as for Tm value of the nucleic acid B which consists of a sequence which can become a forward primer for target nucleic acid amplification, and the reverse primer for target nucleic acid amplification, it is preferable that it is 55-60 degreeC. In the case where the nucleic acid B recognizes a mutation of at least one base and has a nucleic acid C containing a bridged nucleic acid, the Tm value is 1 to 3 ° C. higher than that of a single bridged nucleic acid. Depending on the temperature of the normal primer will be higher.

  The nucleic acid amplification method of the present invention is not limited as long as it can amplify a target nucleic acid. For example, PCR (hot-start PCR, multiplex PCR, nested PCR, RT-PCR, real-time PCR, developed based not only on PCR based on the most basic principle, but also based on it) Various variations such as quantitative PCR such as digital PCR, NASBA (Nucleic Acid Sequence-Based Amplification) (see, for example, Patent No. 2650159), TMA (Transcription-Mediated Amplification) (eg, patent) No. 3241717), TRC (Transcription Reverse Transcription Concerted Reaction) method (e.g. 0-14400), LAMP (Loop-mediated isothermal amplification) method (see, for example, International Publication No. 2000/28082), ICAN method (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method (see, for example, Patent No. 3433929), LCR (Ligase Chain Reaction) method (for example, refer to European Patent Application No. 320328), SDA (Strand Displacement Amplification) method (for example, see Japanese Patent Publication No. 7-114718), etc. it can. Since the PCR method is the most widely used nucleic acid amplification method, various reagents and devices optimized for the method can be easily obtained, and there are various variation methods as described above, and versatility is Since it is very high, it is mentioned as a suitable method for this process. The conditions, procedures, etc. of the nucleic acid amplification reaction including PCR may be in accordance with the usual method usually carried out in this field.

FIG. 3 is a schematic view of a nucleic acid amplification method using a nucleic acid amplification substrate in the present embodiment. As shown in FIG. 3, the 3 'end region of the sense strand of the target nucleic acid is annealed (hybridized) with a nucleic acid B (nucleic acid 6) consisting of a sequence that can be a forward primer for target nucleic acid amplification (see FIG. 3 (a)). The extension reaction is carried out by the DNA polymerase to synthesize the amplification product X (amplification product 9) of the target nucleic acid (see FIG. 3 (b)). In addition, the reverse primer for target nucleic acid amplification is annealed to the 3 'end region of the antisense strand of the target nucleic acid (see FIG. 3 (a)), and the extension reaction is performed by the DNA polymerase to amplify the target nucleic acid The product 11) is synthesized (see FIG. 3 (b)).
Next, a nucleic acid B (nucleic acid 6) consisting of a sequence that can be a forward primer for target nucleic acid amplification is annealed (hybridized) to the 3 'end region of the sense strand of the target nucleic acid, and extension reaction is performed by DNA polymerase. The amplification product X (amplification product 9) is synthesized (see FIG. 3 (c)). In addition, the reverse primer for target nucleic acid amplification is annealed to the 3 'end region of the antisense strand of the target nucleic acid, the DNA polymerase performs an extension reaction, and the amplification product Y of the target nucleic acid (amplification product 11) is synthesized (Amplified Product 11) See FIG. 3 (c)).
Thereafter, by repeating the steps of synthesizing the amplification product X of the target nucleic acid and the steps of synthesizing the amplification product Y, a large number of amplification products of the target nucleic acid can be obtained.
After completion of the nucleic acid amplification reaction, the amplification product X of the target nucleic acid (amplification product 9) has the second oligonucleic acid 4 in the 5 'end region, and the nucleic acid A (nucleic acid 5) in this second oligonucleic acid 4 is Since it contains a bridged nucleic acid and is specifically hybridized with the first oligo nucleic acid 3, it is retained on the substrate (see FIG. 3 (d)). On the other hand, many amplification products Y of the target nucleic acid exist in the free state (see FIG. 3 (d)). As needed, the below-mentioned detection process is implemented.

  In this embodiment, amplification and detection of nucleic acids are carried out simultaneously or sequentially in a closed system since a probe having nucleic acid B consisting of a sequence that can be a forward primer for target nucleic acid amplification is immobilized on a substrate. It becomes possible. Therefore, a method of confirming amplification over time during amplification reaction is preferable.

  As a method of confirming amplification over time during the amplification reaction, a method of detecting an increase in fluorescence intensity reflecting the increase in amplification product over time can be mentioned. Specifically, for example, real-time PCR such as intercalator method (Higuchi et al., BioTechnology 10, 413-417 (1992)), cycling probe method (Bekkaoui et al., Biotechniques 20, 240-248 (1996)), etc. And the cycling probe method is preferred in that it is excellent in detection sensitivity and accuracy. For real-time PCR, a dedicated device integrating a thermal cycler and a spectrofluorimeter is required. Examples of such an apparatus include StepOnePlus (ABI), LightCyclerNano (Roche) and the like.

  In this embodiment, as shown in FIG. 3D, detection using the detection probe 12 which is a single-stranded nucleic acid having a base sequence completely complementary to the nucleic acid B (nucleic acid 6) in the probe 2 The method is preferred. A labeling substance is bound to the 5 'end region of the detection probe 12. Specifically, it is a method using a detection probe designed to have such a base sequence, for example, in a cycling probe method or the like. The labeled substance is described in detail in the nucleic acid detection kit described later.

  The detection probe 12 used in the present embodiment is preferably a fluorescently labeled probe which is a single-stranded nucleic acid having a base sequence completely complementary to the nucleic acid B (nucleic acid 6) in the probe 2. Generally, by increasing the length of the detection probe 12, the binding strength to the complementary sequence becomes stronger, so a certain length is required, but when the detection probe 12 is longer, the base specificity is increased. It should not be too long as it tends to be low. The length of the specific detection probe 12 is preferably 5 to 40 bases, more preferably 8 to 35 bases, and still more preferably 10 to 25 bases. Further, the detection probe 13 is configured of a nucleic acid such as DNA or RNA, or a bridged nucleic acid such as LNA or BNA, or a combination thereof. As described above, since the bridged nucleic acid is superior to DNA, RNA and the like in terms of the ability to bind to complementary sequences, the ability to recognize a base, and the ability to degrade enzymes, the detection probe 12 can contain bridged nucleic acids. preferable.

The “cycling probe method” is a highly sensitive detection method using a combination of a chimeric probe consisting of RNA and DNA and RNase H. One of the chimeric probes is labeled with a fluorescent substance (reporter) and the other with a substance that quenches fluorescence (quencher), with an RNA portion interposed. Although this chimeric probe does not fluoresce upon quenching in an intact state, it becomes strongly fluorescent when the RNA portion is cleaved by RNase H after hybridization with a complementary amplification product of the sequence. By measuring this fluorescence intensity, the amount of amplification product can be monitored. If there is a mismatch near the RNA of the cycling probe, cleavage by RNase H does not occur, so that highly specific detection that can recognize single base differences is possible.
The cycling probe can be obtained by, for example, outsourcing the design and synthesis to an appropriate vendor.

  As another detection method, an intercalator method is mentioned, and the intercalator is a reagent which specifically binds between base pairs of double stranded nucleic acid and emits fluorescence, and emits fluorescence when irradiated with excitation light. The amount of amplification product of the target nucleic acid can be known based on the detection of the fluorescence intensity derived from the intercalator. In the present embodiment, any intercalator generally used in this field can be used, and examples thereof include SYBRTM Green I (Molecular Probe), ethidium bromide, fluorene and the like.

  Therefore, according to the nucleic acid amplification method of the present embodiment, nucleic acid amplification and detection can be performed simultaneously or continuously in a closed system using the above-described mass producible nucleic acid amplification substrate. Moreover, it can carry out simply and at low cost. In addition, by conducting an examination using a nucleic acid amplification substrate tailored to an individual, it can be used for made-to-order treatment.

<Nucleic acid detection kit>
The nucleic acid detection kit of the present embodiment is a nucleic acid detection kit comprising a substrate on which a probe is immobilized and a reverse primer for target nucleic acid amplification, wherein the probe is a first oligonucleic acid immobilized on the substrate; A nucleic acid A comprising a sequence capable of hybridizing to the first oligonucleic acid in the 5 'end region, and a nucleic acid B comprising a sequence capable of becoming a forward primer for target nucleic acid amplification in the 3' end region, And a second oligonucleic acid containing a bridged nucleic acid in the nucleic acid A.

  In the present embodiment, the target nucleic acid is not particularly limited, but a stable DNA such as gDNA or cDNA is preferable.

The substrate on which the probe is immobilized is as described above for the nucleic acid amplification substrate. Moreover, also about a reverse primer, as above-mentioned, 5-40 bases are preferable, 15-30 bases are more preferable, and 18-25 bases are especially preferable.
Further, from the viewpoint of nucleic acid amplification efficiency, the nucleic acid B comprising a sequence that can be a forward primer for target nucleic acid amplification and the reverse primer for target nucleic acid amplification are designed such that the number of bases of the amplification product is 50 to 5000. Is preferred, and it is more preferred that it is designed to be 100-2000. Moreover, it is preferable that it is 50-66 degreeC, and, as for Tm value of the nucleic acid B which consists of a sequence which can become a forward primer for target nucleic acid amplification, and the reverse primer for target nucleic acid amplification, it is preferable that it is 55-60 degreeC. When the nucleic acid B recognizes a mutation of at least one base and includes a nucleic acid C containing a bridged nucleic acid, the Tm value is equal to the number of bridged nucleic acids because it is 1 to 3 ° C. higher than a single bridged nucleic acid. Depending on the temperature of the normal primer will be higher.

  Furthermore, although the nucleic acid detection kit of the present embodiment differs depending on the nucleic acid amplification method to be used, it is preferable that it contains one or more of nucleotide triphosphate as a substrate, nucleic acid synthetase, and amplification reaction buffer. The nucleotide triphosphate is a substrate (dNTP, rNTP, etc.) according to the nucleic acid synthetase. The nucleic acid synthetase is an enzyme according to the nucleic acid amplification method to be used, and is, for example, DNA polymerase, RNA polymerase, reverse transcriptase and the like. Examples of the buffer for amplification reaction include Tris buffer, phosphate buffer, Veronal buffer, borate buffer, Good buffer and the like, and pH is not particularly limited.

Labeling of the amplification product may be performed by using a previously labeled reverse primer for target nucleic acid amplification, or may be performed by labeling the amplification product situation.
Examples of the labeling substance for labeling the reverse primer for target nucleic acid amplification include fluorescent dyes, fluorescent beads, quantum dots, biotin, antibodies, antigens, energy absorbing substances, radioisotopes, chemiluminescers, enzymes and the like. Fluorescent dyes include FAM (carboxyfluorescein), JOE (6-carboxy-4 ′, 5′-dichloro 2 ′, 7′-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX (Hex) 5'-hexachloro-fluorescein-CE phosphoroamidite), Cy3, Cy5, Alexa 568, Alexa 647 and the like.
Further, as a labeling substance used for labeling an amplification product, the above-mentioned fluorescent dye, fluorescent bead, quantum dot, biotin, antibody, antigen, energy absorbing substance, radioisotope, chemiluminescer, enzyme and the like can be mentioned. Among these, as the labeling substance, a fluorescent dye is preferable, and an intercalator is more preferable. The intercalator is not particularly limited as long as it is a fluorescent or UV-detectable substance that intercalates into double-stranded DNA and fluoresces, for example, ethidium bromide, acridine orange, SYBER Green, SYBER Gold Etc. When performing nucleic acid synthesis using cDNA as a template, only by adding these intercalators 16 to the reaction solution for nucleic acid synthesis, the amplification product and the fluorescent reagent intercalate, and fluorescence detection becomes possible. By using the labeling substance described above, it is possible to quantitatively detect the target nucleic acid.

  Therefore, according to the nucleic acid detection kit of the present embodiment, nucleic acid amplification and detection can be performed simultaneously or continuously in a closed system using the above-described mass producible nucleic acid amplification substrate. Moreover, it can carry out simply and at low cost. In addition, by conducting an examination using a nucleic acid amplification substrate tailored to an individual, it can be used for made-to-order treatment.

  Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

Example 1
(Synthesis of double stranded BNA / DNA oligo)
Hereinafter, sense strands and antisense strands of (I) to (IV) listed in Table 1 were designed and synthesized. In each sense strand, A, T, G and C described in capital letters are bridged nucleic acids, A and G are BNA-NC (N-Me), C and T are BNA-NC (N-H) It is.

(Formation of double stranded BNA / DNA oligo)
Each sense strand and antisense strand were added to buffer to be 2.5 μM respectively. The composition of the buffer is 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ . After heat denaturation at 95 ° C. for 5 minutes using a thermal cycler (Applied Biosystems, Veriti), each sense strand and antisense strand were annealed while decreasing the temperature by 1 ° C. per minute to 25 ° C.

(Tm value measurement)
The temperature was raised by 1 ° C. per minute from 30 ° C. to 98 ° C., and the absorbance (260 nm) was measured every 0.2 ° C. FIG. 4 is a graph showing the change in absorbance (260 nm) with temperature rise of double-stranded BNA / DNA. The Tm values of each double-stranded BNA / DNA oligo are shown in Table 2.

It can be seen from FIG. 4 that (III) and (IV), which are 18 bases in length, have higher Tm values compared to (I) and (II), which are 12 bases in length. In addition, when those having the same base length were compared, Tm values of (II) and (IV) having a high BNA content were higher than those of (I) and (III).
Therefore, it is presumed that the longer the oligo chain and the higher the content of BNA, the higher the Tm value and the stronger the binding strength.

Example 2
(Synthesis of BNA / DNA oligo)
Hereinafter, the first oligo-nucleic acid, the second oligo-nucleic acid and the Reverse primer described in Table 3 were designed and synthesized. In the second oligonucleic acid, A, T, C described in upper case are bridged nucleic acids, A is BNA-NC (N-Me), C, T is BNA-NC (N-H) . Furthermore, in the second oligonucleic acid, 15 bases from the 1st to the 15th from the 5 'end are the nucleic acid A, which anneals to the 1st oligonucleic acid, and counts from the 16th to the 39th from the 5' end. Up to 24 bases are nucleic acid B, and it is a Forward primer complementary to the sense strand of the template (24 bases from the 145th to the 168th bases in the base sequence of Allele1 described below). In addition, Reverse primer is a reverse primer complementary to the template antisense strand (21 bases from 199th to 220th bases in the base sequence of Allele1 antisense strand described later).

(Formation of double stranded BNA / DNA oligo)
The synthesized first and second oligonucleic acids were mixed and incubated at room temperature for 30 minutes to be annealed. Subsequently, non-denaturing polyacrylamide gel electrophoresis was performed on the first oligo nucleic acid, the second oligo nucleic acid, and the double-stranded BNA / DNA oligo after annealing for confirmation of the annealing. Furthermore, the non-denaturing polyacrylamide gel after electrophoresis was photographed by irradiating it with ultraviolet light (wavelength: about 254 nm). The results are shown in FIG.
From FIG. 5, it was confirmed that the synthesized first oligonucleic acid and the second oligonucleic acid were annealed to form a double stranded oligonucleic acid.

(Tm value measurement)
The temperature was raised by 1 ° C. in 1 minute from 1 ° C. to 100 ° C., and the absorbance (260 nm) was measured every 1 ° C. FIG. 6 is a graph showing the change in absorbance (260 nm) with temperature rise of double-stranded BNA / DNA. In addition, the same experiment was performed on a control double-stranded oligo having a Tm value of 74 ° C.

From FIG. 6, in the control double-stranded oligo, a peak was observed in the change in absorbance as the temperature rose, and the Tm value was calculated to be 74.77 ° C. On the other hand, in the case of double-stranded BNA / DNA, a constant peak was not observed in the change in absorbance, and a clear Tm value was not obtained.
From the above, it was confirmed that the double strand does not dissociate within the measurement range (1 ° C. to 100 ° C.) in the double strand BNA / DNA.

(Real-time PCR)
A double-stranded DNA template (Allele 1) consisting of the nucleotide sequences of SEQ ID NO: 12 (sense strand) and SEQ ID NO: 13 (antisense strand) as a template, SEQ ID NO: 14 (sense strand) and SEQ ID NO: 15 (antisense strand) 60 ng of a mixture with a double-stranded DNA template (Allele 2) consisting of the nucleotide sequence of Allele2 is a sequence in which 15 bases from 131st to 145th of Allele 1 are deleted. As shown in Table 4 below, five types of templates in which the mixing ratio of Allele 1 and Allele 2 were changed were prepared, and PCR reactions were performed for each. The copy number in parentheses is a theoretical value when it is assumed that one copy is contained in 3 pg of gDNA.

  Subsequently, they were mixed so as to be the PCR compositions shown in Table 5 below.

  The PCR reaction cycle is as shown in Table 6. The amplification product was measured each time at 72 ° C. The results are shown in FIG. In FIG. 7, NTC is an abbreviation of No template control, and is for examining the presence or absence of a primer dimer.

  From FIG. 7, the amplification curve first rises at (V) where Allele 1 is 100%, and continues as (VI), (VII) and (VIII). It is inferred that the rise of the amplification curve is faster in proportion to the content of Allele1. Also, although amplification is also observed for (IX) in which Allele 2 is 100%, (VIII) containing Allele 1 in 0.1% has a significant difference in the rising of the amplification curve as compared with (IX). Thus, it was confirmed that the double-stranded BNA / DNA oligo can specifically amplify Allele1.

  From the above results, it is clear that according to the present invention, a target nucleic acid can be amplified conveniently and highly selectively.

  DESCRIPTION OF SYMBOLS 1 ... substrate, 2 ... probe, 3 ... 1st oligo nucleic acid, 3 a ... nucleic acid consisting of a base sequence complementary to nucleic acid A, 3 b ... nucleic acid fixed to substrate, 4 ... 2nd oligo nucleic acid, 5 ... nucleic acid A: 6 nucleic acid B 7 reverse primer for target nucleic acid amplification 8 target nucleic acid 8 a sense strand of target nucleic acid 9 amplification product of target nucleic acid X 8 b antisense strand of target nucleic acid 10 20: nucleic acid amplification substrate, 11: amplification product of target nucleic acid Y, 12: detection probe, 21: compartment

Claims (10)

A nucleic acid amplification substrate comprising a substrate and a probe immobilized on the substrate, the probe comprising
A first oligonucleic acid immobilized on the substrate;
A nucleic acid A comprising a sequence capable of hybridizing to the first oligo nucleic acid in the 5 'end region, and a nucleic acid B comprising a sequence capable of becoming a forward primer for target nucleic acid amplification in the 3' end region, A second oligonucleic acid comprising a bridged nucleic acid at A,
A nucleic acid amplification substrate characterized by having:   The nucleic acid amplification substrate according to claim 1, wherein the substrate comprises one or more sections, and one section or more of one type of the probe is fixed in one section.   The nucleic acid amplification substrate according to claim 1 or 2, wherein the proportion of bridged nucleic acid in the second oligonucleic acid is 66% or more.   The nucleic acid amplification substrate according to any one of claims 1 to 3, wherein the first oligonucleic acid is DNA.   The nucleic acid amplification substrate according to any one of claims 1 to 4, wherein the target nucleic acid is gDNA or cDNA.   A nucleic acid amplification method using the nucleic acid amplification substrate according to any one of claims 1 to 5. A nucleic acid detection kit comprising a substrate on which a probe is immobilized and a reverse primer for target nucleic acid amplification, wherein the probe is
A first oligonucleic acid immobilized on the substrate;
A nucleic acid A comprising a sequence capable of hybridizing to the first oligo nucleic acid in the 5 'end region, and a nucleic acid B comprising a sequence capable of becoming a forward primer for target nucleic acid amplification in the 3' end region, A second oligonucleic acid comprising a bridged nucleic acid at A,
A nucleic acid detection kit characterized by having:   The nucleic acid detection kit according to claim 7, wherein the proportion of bridged nucleic acid in the second oligonucleic acid is 66% or more.   The nucleic acid detection kit according to claim 7 or 8, wherein the first oligonucleic acid is DNA.   The nucleic acid detection kit according to any one of claims 7 to 9, wherein the target nucleic acid is gDNA or cDNA.