CN114621996A

CN114621996A - Method for detecting activity of one or more polymerases

Info

Publication number: CN114621996A
Application number: CN202011420925.3A
Authority: CN
Inventors: 倪润芳; 黄丽婷; 邱逸东; 何昌华; 宋娜杰
Original assignee: XIAMEN ZEESAN BIOTECH CO Ltd
Current assignee: XIAMEN ZEESAN BIOTECH CO Ltd
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2022-06-14
Also published as: WO2022121754A1

Abstract

The present invention provides a method for detecting the activity of one or more polymerases using melting curve analysis. The method can roughly and quickly judge the activity of the polymerase, is simple and convenient to operate, has simple steps, and saves the detection time.

Description

Method for detecting activity of one or more polymerases

Technical Field

The present invention provides a method for detecting the activity of one or more polymerases using melting curve analysis.

Background

MMLV reverse transcriptase gene, derived from the genome of Murine Leukemia Virus (Molony Murine Leukemia Virus), MMLV is commonly used to make multiple or multiple combinatorial mutants, and reverse transcriptase with high efficiency or other biological properties can be screened by large scale testing. The activity of the reverse transcriptase can be more accurately determined by the traditional technology. For example, detection methods for reverse transcriptase activity are generally product-enhanced reverse transcriptase assay (PERT) and polymerase chain reaction based reverse transcriptase assay (PBRT). However, there is no method for roughly determining the activity of a reverse transcriptase, and if the activity of the reverse transcriptase can be distinguished quickly and easily, the efficiency can be improved.

The present application intends to use the melting curve technique for enzyme activity determination to solve the problems of the conventional methods. Melting curve refers to the extent of degradation of the double helix structure of DNA with increasing temperature. The initial melting curves used intercalating dyes to quantify products, but these dyes have the significant disadvantage of detecting both specific and non-specific nucleic acid products. By introducing a fluorescent-labeled probe, the melting curve is significantly improved. The use of these fluorescent probes allows the development of a melting curve method, allowing the detection of only specific nucleic acid products. The invention improves the analysis of the melting curve and innovatively realizes the application of the melting curve analysis in the determination of the activity of the reverse transcriptase.

Disclosure of Invention

In order to solve the problems, the present application realizes the detection of the activity of one or more polymerases by performing a melting curve analysis on a nucleic acid product of a polymerase-amplified nucleic acid molecule using a detection probe and calculating the melting peak height.

Accordingly, in a first aspect, the present application provides a method of detecting one or more polymerase activities, the method comprising:

(a) providing a sample containing nucleic acid molecules, a set of primers capable of amplifying said nucleic acid molecules, and providing one or more polymerases to be detected;

(b) providing at least one detection probe labeled with a reporter and a quencher, wherein the reporter is capable of emitting a signal and the quencher is capable of absorbing or quenching the signal emitted by the reporter; and wherein the detection probe emits a signal when hybridized to its complement that is different from the signal when not hybridized to its complement; the detection probe is capable of specifically hybridizing to a designated region of the nucleic acid molecule under conditions that allow for hybridization or annealing of the nucleic acid;

(c) amplifying the nucleic acid molecules by using the polymerase and the primer group to obtain amplification products, and respectively performing melting curve analysis on the amplification products by using the detection probes;

(d) obtaining a melting peak height (Rm) of the amplification product based on the melting curve analysis result of step (c), thereby analyzing the activity of the one or more polymerases.

In certain embodiments, wherein, in step (c), the nucleic acid molecule is mixed with the polymerase and the primer set and amplified, and then, after amplification is complete, a detection probe is added to the product of step (b) and a melting curve analysis is performed; alternatively, in the step (b), the nucleic acid molecule is mixed with the polymerase, the primer set, and the detection probe, and amplified, and then, after the amplification is completed, a melting curve analysis is performed.

In certain embodiments, in step (d), the activity of the plurality of polymerases is assayed by comparing the melting peak heights (Rm) of the amplification products of the plurality of polymerases to be tested.

In certain embodiments, in step (a), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more polymerases are provided.

In certain embodiments, in step (a), at least 2 polymerases (e.g., a first polymerase, a second polymerase) are provided. The first polymerase performs an amplification reaction on a sample containing nucleic acid molecules and obtains a first amplification product, and the detection probe is capable of hybridizing to a region of the first amplification product, passing through a first melting peak and a first Rm value. The second polymerase performs an amplification reaction on the sample containing the nucleic acid molecule and obtains a second amplification product, the detection probe being capable of hybridizing to a region of the second amplification product and generating a second melting peak and a second Rm value. When the first Rm value is greater than the second Rm value, the first polymerase is more active than the second polymerase. When the second Rm value is greater than the first Rm value, the second polymerase is more active than the first polymerase. Thus, by comparing the magnitude between different Rm values, the activity of multiple polymerases can be determined.

In certain embodiments, in step (d), the melting peak height (Rm), or raw data (e.g., T) for the melting curve, is obtained by a real-time fluorescence PCR instrument_mValue corresponding to the fluorescence signal value of the fluorescence channel) to obtain the melting peak height (Rm).

In certain embodiments, the melting peak height (Rm) is automatically output by analysis software (e.g., SLAN full-automatic medical PCR analysis system 8.2.2) associated with a fluorescence PCR instrument (e.g., a Macro-Stone fluorescence PCR instrument SLAN-96S/48P). Specifically, the slope of the tangent line of the melting curve is calculated by derivation, the maximum change position of the slope of the tangent line is the peak valley of the melting peak, the peak valley positioned on the left side of the melting peak is defined as a starting point, the peak valley positioned on the right side of the melting peak is defined as an ending point, the starting point and the ending point of the peak of the melting curve are connected into a line, the highest point of the peak is taken as a vertical extension line, and the distance from the intersection point of the starting point and the ending point to the highest point of the peak is the Rm value output by the analysis software.

In certain embodiments, the melting peak height (Rm) is the raw data (e.g., melting curve, T) output by a fluorescence PCR instrument (e.g., fluorescence PCR instrument BioRad CFX96, fluorescence PCR instrument Roche LightCycler 480) and its associated software_mValues corresponding to fluorescence signal values of the fluorescent channels).

In certain embodiments, in step (a) or (b) of the method, deoxynucleoside triphosphates (dNTPs), water, comprising an ion (e.g., Mg) are also provided²⁺) A single-stranded DNA binding protein, or any combination thereof.

In certain embodiments, the sample comprises or is DNA, RNA, or any combination thereof.

In certain embodiments, the nucleic acid molecule is selected from DNA, RNA, or any combination thereof.

In certain embodiments, the amplification product is selected from DNA, RNA, or any combination thereof. In certain embodiments, the amplification product is RNA.

In certain embodiments, the sample is derived from a eukaryote (e.g., an animal, a plant, a fungus), a prokaryote (e.g., a bacterium, an actinomycete), a virus, a bacteriophage, or any combination thereof.

In certain embodiments, each of the polymerases is independently selected from a DNA polymerase, an RNA polymerase, or any combination thereof.

In certain embodiments, the polymerases are DNA polymerases each independently obtained from a bacterium selected from the group consisting of: thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermus flavus, Thermus thermophilus, Thermus antalidanii, Thermus caldophlus, Thermus cholephilus, Thermus osiphilius, Thermus canaliculus, Thermus lutera, Thermus lactius, Thermus osidamia, Thermus ruber, Thermus rubens, Thermus scodottus, Thermus silvannus, Thermus thermophilus, Thermotoga maritima, Thermotoga neolytica, Thermosipho africans, Thermococcus littoralis, Thermococcus sporophycus, Thermococcus giganticus, Thermococcus purpurea, Thermomyces neospora purpurea, Thermomyces littoralis, Thermomyces barosissimus, Thermococcus barosissima, Thermococcus purpurea, Thermocosissima pacifica, Thermocosissima purpurea, Thermocosissima pacificum, Pyrococcus purpurea, Thermocosissimus purpurea, Thermocosissima pacificum, Pyrococcus purpurea, Pyrococcus purpurea, Thermocosidium purpurea, Thermocosissimus purpurea, Thermocosium, Pyrococcus, Thermocosium, Pyrococcus, Thermocosissimus purpurum, Thermocosium, Thermocosissimus purpurum, Thermocosium, Thermocascus, Thermocosissimus purpurum, Pyrococcus, Thermocascus, Thermocosissimus purpurum, Thermocosium, Thermocascus, Thermocosium, Pyrococcus, Thermocascus, Pyrococcus, Pyrococc.

In certain embodiments, the polymerases are DNA polymerases each independently selected from Bst DNA polymerase, T7 DNA polymerase, phi29 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, Pfu DNA polymerase, vent DNA polymerase, or any combination thereof.

In certain embodiments, the polymerase is a DNA polymerase, which includes a reverse transcriptase.

In certain embodiments, the polymerase is a reverse transcriptase, each independently selected from MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, or any combination thereof.

In certain embodiments, wherein in step (a) of the method, for each nucleic acid molecule, at least one pair of primer sets is provided, the primer sets comprising at least one forward primer and at least one reverse primer.

In certain embodiments, wherein the forward primer and the reverse primer each independently comprise or consist of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof.

In certain embodiments, wherein in step (b) of the method, at least one detection probe is provided for each amplification product (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detection probes are provided).

In certain embodiments, each detection probe has a melting point (T) that differs from the melting point of the double-stranded hybrid formed by the amplification product_m) (ii) a In certain embodiments, the melting point (T) between the detection probe and the double-stranded hybrid formed by the amplification product of the nucleic acid molecule_m) The difference is 1 deg.C (e.g., 1 deg.C, 2 deg.C, 3 deg.C) or more.

In certain embodiments, the detection probes each independently comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., Peptide Nucleic Acids (PNAs) or locked nucleic acids), or any combination thereof.

In some embodiments, the length of the detection probes is 15-1000nt, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt, 900-1000 nt.

In certain embodiments, the detection probes each independently have a 3' -OH terminus; alternatively, the 3' -end of the detection probe is blocked; for example, the 3' -end of the detection probe can be blocked by adding a chemical moiety (e.g., biotin or alkyl) to the 3' -OH of the last nucleotide of the detection probe, by removing the 3' -OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a dideoxynucleotide.

In certain embodiments, in step (d), the product of step (c) is subjected to a gradual increase or decrease in temperature and the signal emitted by the reporter group on each detection probe is monitored in real time, thereby obtaining a profile of the signal intensity of each reporter group as a function of temperature; the curve is then derived to obtain the melting curve of the product of step (d).

In certain embodiments, the reporter and quencher are separated by a distance of 10-80nt or more.

In certain embodiments, the reporter groups in the detection probes are each independently a fluorophore (e.g., ALEX-350, FAM, VIC, TET, CAL)

Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS Red, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and, a quencher is a molecule or group (e.g., DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA) capable of absorbing/quenching the fluorescence.

In certain embodiments, the detection probes each independently have the same or different reporter groups. In certain embodiments, the detection probes each independently have the same or different quencher group.

In certain embodiments, the detection probes are each independently resistant to nuclease activity (e.g., 5' nuclease activity, e.g., 5' to 3' exonuclease activity); for example, the backbone of the detection probe comprises modifications that are resistant to nuclease activity, such as phosphorothioate linkages, alkylphosphotriester linkages, arylphosphotriester linkages, alkylphosphonate linkages, arylphosphonate linkages, hydrogenphosphate linkages, alkylaminophosphate linkages, arylaminophosphate linkages, 2' -O-aminopropyl modifications, 2' -O-alkyl modifications, 2' -O-allyl modifications, 2' -O-butyl modifications, and 1- (4' -thio-PD-ribofuranosyl) modifications.

In certain embodiments, the detection probes are each independently linear or have a hairpin structure.

In a second aspect of the present application, there is provided a kit comprising: one or more nucleic acid molecules, at least one pair of primer sets capable of amplifying said nucleic acid molecules, and at least one detection probe capable of hybridizing or annealing to an amplification product of a nucleic acid molecule and labeled with a reporter and a quencher, wherein said reporter is capable of emitting a signal and said quencher is capable of absorbing or quenching the signal emitted by said reporter; and wherein the detection probe emits a signal when hybridized to its complement that is different from the signal when not hybridized to its complement.

In certain embodiments, the kit further comprises: polymerase, deoxynucleoside triphosphates (dNTPs), water, containing ions (e.g., Mg)²⁺) A single-stranded DNA binding protein, or any combination thereof.

In certain embodiments, the nucleic acid molecule is lambda DNA.

In certain embodiments, the primer set comprises at least one forward primer and at least one reverse primer.

In certain embodiments, the forward primer and the reverse primer each independently comprise or consist of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof.

In certain embodiments, the primers of the primer set have the nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2.

In certain embodiments, at least one detection probe is provided for each nucleic acid molecule (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detection probes are provided).

In certain embodiments, the detection probe is as previously defined.

In certain embodiments, the detection probe has a nucleotide sequence as set forth in SEQ ID NO 3.

In certain embodiments, the kit is used to detect one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more polymerases) polymerase activities.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, etc., used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "melting peak height (Rm)" may be used to characterize the amount of nucleic acid to be determined in a system, with the level of Rm being proportional to the amount of product over a range of concentrations. Calculating the slope of a tangent line of a melting curve by derivation, defining the maximum change position of the slope of the tangent line as the peak valley of a melting peak, defining the peak valley positioned at the left side of the melting peak as a starting point, defining the peak valley positioned at the right side of the melting peak as an ending point, connecting the starting point and the ending point of the peak of the melting curve into a line, taking the highest point of the peak as a vertical extension line, and taking the distance from the intersection point of the two to the highest point of the peak as an Rm value.

As used herein, the term "amplification product" refers to an amplified nucleic acid produced by amplification of a nucleic acid template.

As used herein, the term "polymerase", also known as polymerase, is a generic term for a class of enzymes that specifically biocatalytically synthesize deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It can be divided into the following groups (1) DNA-dependent DNA polymerases; (2) an RNA-dependent DNA polymerase; (3) a DNA-dependent RNA polymerase; (4) RNA-dependent RNA polymerases. Wherein the first two are DNA polymerases and the second two are RNA polymerases. As used herein, the term "DNA polymerase" refers to an enzyme that synthesizes a DNA strand using a nucleic acid strand as a template, and DNA polymerase synthesizes DNA using existing DNA or RNA as a template. In the method of the present invention, the DNA polymerase may be a naturally occurring DNA polymerase, or may be a variant or fragment of a natural enzyme having the above-mentioned activity. As used herein, the term "RNA polymerase" refers to an enzyme that synthesizes an RNA strand using a nucleic acid strand as a template, and RNA polymerase synthesizes RNA using existing DNA or RNA as a template. In the method of the present invention, the RNA polymerase may be a naturally occurring RNA polymerase, or may be a variant or fragment of a natural enzyme having the above-mentioned activity.

As used herein, the term "reverse transcriptase" refers to an enzyme that is capable of replicating RNA as complementary DNA or cDNA. Reverse transcription is the process of copying an RNA template into DNA. In the method of the present invention, the reverse transcriptase may be a naturally occurring RNA polymerase, or may be a variant or fragment which retains the above-mentioned activity.

As used herein, and as is generally understood by those of skill in the art, the terms "forward" and "reverse" are merely for convenience in describing and distinguishing the two primers of a primer pair; they are relative and do not have a special meaning.

As used herein, the terms "targeting sequence" and "target-specific sequence" refer to a sequence capable of selectively/specifically hybridizing or annealing to a target nucleic acid sequence under conditions that allow for hybridization, annealing, or amplification of the nucleic acid, which comprises a sequence complementary to the target nucleic acid sequence. In the present application, the terms "targeting sequence" and "target-specific sequence" have the same meaning and are used interchangeably. It is readily understood that the targeting or target-specific sequence is specific for the target nucleic acid sequence. In other words, under conditions that allow nucleic acid hybridization, annealing, or amplification, the targeting or target-specific sequence hybridizes or anneals only to a particular target nucleic acid sequence, and not to other nucleic acid sequences.

The term "complementary" as used herein means that two nucleic acid sequences are capable of forming hydrogen bonds between each other according to the base pairing principle (Watton-Crick principle) and thereby forming a duplex. In the present application, the term "complementary" includes "substantially complementary" and "fully complementary". As used herein, the term "fully complementary" means that each base in one nucleic acid sequence is capable of pairing with a base in another nucleic acid strand without mismatches or gaps. As used herein, the term "substantially complementary" means that a majority of the bases in one nucleic acid sequence are capable of pairing with bases in another nucleic acid strand, which allows for the presence of mismatches or gaps (e.g., mismatches or gaps of one or several nucleotides). Typically, two nucleic acid sequences that are "complementary" (e.g., substantially complementary or fully complementary) will selectively/specifically hybridize or anneal and form a duplex under conditions that allow the nucleic acids to hybridize, anneal, or amplify. Accordingly, the term "non-complementary" means that two nucleic acid sequences do not hybridize or anneal under conditions that allow nucleic acid hybridization, annealing or amplification, and do not form a duplex. As used herein, the term "not being fully complementary" means that the bases in one nucleic acid sequence are not capable of fully pairing with the bases in another nucleic acid strand, at least one mismatch or gap being present.

As used herein, the terms "hybridization" and "annealing" refer to the process by which complementary single-stranded nucleic acid molecules form a double-stranded nucleic acid. In the present application, "hybridization" and "annealing" have the same meaning and are used interchangeably. In general, two nucleic acid sequences that are completely or substantially complementary can hybridize or anneal. The complementarity required for two nucleic acid sequences to hybridize or anneal depends on the hybridization conditions used, particularly the temperature.

As used herein, the term "PCR reaction" has the meaning commonly understood by those skilled in the art, which refers to a reaction that uses a nucleic acid polymerase and primers to amplify a target nucleic acid (polymerase chain reaction).

As used herein, the term "detection probe" refers to an oligonucleotide that is labeled with a reporter group and a quencher group. When the probe is not hybridized to other sequences, the quencher is positioned to absorb the signal from the quenching reporter (e.g., the quencher is positioned adjacent to the reporter), thereby absorbing or quenching the signal from the reporter. In this case, the probe does not emit a signal. Further, when the probe hybridizes to its complement, the quencher is located at a position that is unable to absorb or quench the signal from the reporter (e.g., the quencher is located away from the reporter), and thus unable to absorb or quench the signal from the reporter. In this case, the probe emits a signal.

As used herein, the term "melting curve analysis" has the meaning commonly understood by those skilled in the art, and refers to a method of analyzing the presence or identity (identity) of a double-stranded nucleic acid molecule by determining the melting curve of the double-stranded nucleic acid molecule, which is commonly used to assess the dissociation characteristics of the double-stranded nucleic acid molecule during heating. Methods for performing melting curve analysis are well known to those skilled in The art (see, e.g., The Journal of Molecular Diagnostics 2009,11(2): 93-101). In the present application, the terms "melting curve analysis" and "melting analysis" have the same meaning and are used interchangeably.

In certain preferred embodiments of the present application, the melting curve analysis may be performed by using a self-quenching probe labeled with a reporter group and a quencher group. Briefly, at ambient temperature, a probe is capable of forming a duplex with its complementary sequence by base pairing. In this case, the reporter (e.g., fluorophore) and the quencher on the probe are separated from each other, and the quencher cannot absorb the signal (e.g., fluorescent signal) emitted from the reporter, and at this time, the strongest signal (e.g., fluorescent signal) can be detected. As the temperature is increased, both strands of the duplex begin to dissociate (i.e., the probe gradually dissociates from its complementary sequence), and the dissociated probe is in a single-stranded free-coiled state. In this case, the reporter (e.g., fluorophore) and the quencher on the dissociated probe are close to each other, and thus a signal (e.g., a fluorescent signal) emitted from the reporter (e.g., fluorophore) is absorbed by the quencher. Thus, as the temperature increases, the detected signal (e.g., the fluorescence signal) becomes progressively weaker. When both strands of the duplex are completely dissociated, all probes are in a single-stranded free coiled-coil state. In this case, the signal (e.g., fluorescent signal) from the reporter (e.g., fluorophore) on all of the probes is absorbed by the quencher. Thus, a signal (e.g., a fluorescent signal) emitted by a reporter (e.g., a fluorophore) is substantially undetectable. Thus, detection of a signal (e.g., a fluorescent signal) emitted by the probe-containing duplex during the temperature increase or decrease permits observation of hybridization of the probe to its complementary sequenceAnd a dissociation process, forming a curve of signal intensity as a function of temperature. Further, by performing derivative analysis on the obtained curve, a curve (i.e., melting curve of the duplex) is obtained with the rate of change of signal intensity as ordinate and the temperature as abscissa. The peak in the melting curve is the melting peak and the corresponding temperature is the melting point (T) of the duplex_m). Generally, the higher the degree of match of a probe to a complementary sequence (e.g., fewer mismatched bases, more bases paired), the T of the duplex is_mThe higher. Thus, T through the duplex_mThe presence and identity of sequences in the duplex that are complementary to the probe can be determined. As used herein, the terms "melting peak", "melting point" and "T_m"has the same meaning and is used interchangeably.

In certain preferred embodiments of the present application, the melting curve analysis may be performed by using a detection probe labeled with a reporter group and a quencher group. The detection principle is the same as that described above.

Advantageous effects of the invention

The detection method of the application is different from the traditional detection method, and realizes a method for detecting one or more polymerase activities by utilizing melting curve analysis. In particular, the methods of the present application enable simultaneous determination of the activity of multiple polymerases. And the method is simple and convenient to operate, has simple steps, saves the detection time and realizes the rough judgment of the activity of the polymerase.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

Fig. 1 shows a schematic diagram of a melting peak-to-peak value (Rm) calculation method.

FIG. 2 shows the results of the activity of MMLV-L139P using the method of the present invention.

FIG. 3 shows the results of the activity of MMLV-L435G/D524A using the method of the present invention.

FIG. 4 shows the results of the activity of MMLV-E302K/D524A using the method of the present invention.

FIG. 5 shows the results of the activity of MMLV-D524A using the method of the present invention.

FIG. 6 shows the results of the activity of MMLV-D524A/E562K using the method of the invention.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of biochemistry, molecular biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook (Sambrook), friesch (Fritsch), and manitis (manitis), "molecular cloning: a LABORATORY Manual (Molecular CLONING: A Laboratory Manual), 2 nd edition (1989); a Current Manual of MOLECULAR BIOLOGY experiments (Current PROTOCOLS IN MOLECULAR BIOLOGY BIOLOGY) (edited by F.M. Otsubel et al, (1987)); METHODS IN ENZYMOLOGY (METHODS IN Enzyology) series (academic Press Co.): PCR 2: practical methods (PCR 2: A PRACTICAL APPROACH) (M.J. Mefferson (M.J. MacPherson), B.D. Hemmers (B.D. Hames) and G.R. Taylor (edited by G.R. Taylor) (1995)).

In addition, those whose specific conditions are not specified in the examples are conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1

In this example, nucleic acid fragments with known lengths are constructed in advance, and the length of the constructed DNA fragments is detected by the method of the present invention, so as to verify the feasibility and accuracy of the method of the present invention.

1. Construction of DNA fragments to be tested

In this example, construction of a DNA fragment was carried out using lambda DNA (purchased from Life technologies (Shanghai)) as a template, and the length of the constructed DNA fragment was 2024 bp. The amplification was performed using a PCR method and corresponding primers, wherein the primers used are shown in Table 1. The enzyme used for PCR amplification is 2xTaKaRa Taq^TMHS Perfect Mix (from TaKaRa), the specific reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

TABLE 1 primers and sequences used

TABLE 2 PCR reaction System for construction of 2024 bp-long fragment

Reaction components	Volume (μ L)
		Lambda DNA template	5
2xTaKaRa Taq^TM HS Perfect Mix	25
		50μMλDNA-F	0.2
50μMλDNA-2024-R	0.2
		RNase Free Water	19.6

TABLE 3 reaction procedure for PCR

2. Determination and analysis of fragments

And respectively designing probes 1 bound to the amplification products according to the nucleotide sequences of the products amplified by the primers, wherein the probes 1 can bind to 1152nt to 1189nt of the nucleotide sequences of the amplification products, and the specific sequences are shown in Table 4.

TABLE 4 sequence of probes

The amount of the amplification product was measured by melting curve analysis using the detection probe 1. In short, after the amplification product obtained above is denatured at high temperature, the probe is hybridized with the amplification product at lower temperature, and the corresponding melting peak value (Rm value) can be obtained by calculation and analysis through gradually raising the temperature and detecting the fluorescence signal of the probe and the analysis software matched with the fluorescence PCR instrument. The schematic diagram of the Rm value is shown in fig. 1, specifically, the slope of the tangent line of the melting curve is calculated by derivation, the maximum position of the change of the slope of the tangent line is defined as the peak valley of the melting peak, the peak valley positioned at the left side of the melting peak is defined as the starting point, the peak valley positioned at the right side of the melting peak is defined as the ending point, the starting point and the ending point of the peak of the melting curve are connected to form a line, the highest point of the peak is taken as the extension line in the vertical direction, and the distance from the intersection point of the two points to the highest point of the peak is the Rm value. The Rm value is in direct proportion to the product amount in a certain concentration range, and can be used for representing the content of nucleic acid to be determined in a system. The Rm value in the embodiment is automatically output by an analysis software SLAN full-automatic medical PCR analysis system 8.2.2 matched with a macro-stone fluorescence PCR instrument (SLAN-96S/48P).

The reaction procedure for detection is shown in Table 5, and the detection system used is shown in Table 6. The preparation process of the system is operated on ice, and the prepared reaction system is placed in a fluorescent quantitative PCR instrument for reaction. Wherein, the formula of the 10x PCR buffer solution is as follows: (NH)₄)₂SO₄ 21.142g，Tris 81.164g，Tween-20 1.0mL，pH8.8。

TABLE 5 reaction procedure for DNA fragment detection

TABLE 6 reaction procedure for DNA fragment detection

The measurement results of the amplification products are shown in FIG. 1. The black solid line shows the melting peak of the detected amplification product. The Rm value output by software matched with the fluorescence PCR instrument is 92.90. The experimental results of this example demonstrate that the method of the present invention can detect the amplification product and the amount of the amplification product, and the detection results are accurate.

Example 2

In this example, amplification reactions were carried out using 5 types of reverse transcriptases, respectively, using lambda DNA (available from Life technologies (Shanghai)) as a template, and activities of the 5 types of reverse transcriptases were compared using the method of the present invention and a conventional enzyme activity measurement method, respectively.

1. Construction of RNA fragments of interest

Construction of the target RNA fragment Using HiScribe T7 Rapid high Performance RNA Synthesis kit (purchased from NEB, Beijing), the specific transcription reaction system is shown in Table 7:

TABLE 7 reaction System for construction of RNA fragments of interest

Reaction components	Volume (μ L)
		10xReaction Buffer	2
100mM ATP	2
		100mM TTP	2
100mM GTP	2
		100mM CTP	2
DNA fragment of interest	8

2. The method of the present invention measures the activity of an enzyme

The determination using the method of the present invention was carried out using MMLV standard strain and five MMLV mutants or combinations (D524A/E562K, D524A, E302K/D524A, L435G/D524A and L139P, respectively) (see Yu, Chen, Weiguo, et al. A novel and simple method for high-level promoter of conversion transcription from modification mutation (MMLV-RT) in molecular specimen [ J ]. Biotechnology Letters, 2009; MMLV mutants D524A, E302K and L435. J.A. for the construction process and specific sequence of conversion mutation K, Mizu M, Konishi A, Across. expression mutation of conversion mutation of molecular specimen [ 12 J.2010. conversion mutation of conversion mutation No. 150, J.2010. for the construction process of conversion mutation of expression strain, III strain, strain Research,2009,37(2): 473-; MMLV mutants D524A/E562K, D524A, E302K/D524A, L435G/D524A are constructed and the specific sequences are described in Konishi, Atsushi, Hisayoshi, Tetsuro, Yokokawa, Kanta, et al, amino acid residues from the RNase H catalytic site acquisition the thermal stability of the molecular tissue virus recovery fragment, biological & biological Research microorganisms, 2014,454(2):269 Comm 274). The target fragment is subjected to reverse transcription amplification, wherein the reverse transcription system is shown in Table 8, and the reverse transcription reaction conditions are shown in Table 9.

TABLE 8 reverse transcription reaction System

Components	Dosage (mu l)
		5x MMLV Buffer	4
MMLV	1
		50μMλDNA-F	1
RNA fragment of interest	1.2
		RNase Free Water	12.8

TABLE 9 reverse transcription reaction conditions

Temperature of	Time
		42℃	30min
85℃	1min

After the reaction was completed, the amounts of the products amplified by 5 types of reverse transcriptases were measured by the method of the present invention, respectively. The specific experimental process is as described in example 1, in brief, 12.5 μ L of the product is added into the PCR reaction tube to be tested, the detection probe 1 is combined with the product, Rm values of 5 enzyme-amplified products are calculated by melting curve analysis, the melting peaks of 5 products are shown in fig. 2 to 5, and the specific Rm values are shown in table 12.

3. Conventional methods for determining enzyme activity

Firstly, 5 reverse transcriptases are used to perform reverse transcription amplification on the target RNA fragment, the reaction procedure of the reverse transcription reaction is shown in Table 9, the reaction system is shown in Table 10, and the 5x MMLV Buffer contains 250mM Tris-HCl, pH8.3, 375mM KCl and 15mM MgCl₂. Storage with 1 XMMLV enzyme (20mM Tris-HCl, pH7.8, 100mM NaCl, 1mM EDTA, 1mM DTT, 5)0% glycerol (v/v)), diluting 5 MMLV mutants and MMLV standard strains to be detected to 10 ng/. mu.L, 5 ng/. mu.L and 2.5 ng/. mu.L in sequence, and placing on ice for later use. mu.L of diluted MMLV mutant, MMLV standard strain or 1 XMMLV enzyme stock solution (control) with different concentrations are added into the reaction system respectively. Gently and uniformly mixing the solution by using a 10 mu L pipette for 5 times, avoiding the generation of bubbles during operation, placing the reaction solution in a micro centrifuge, and centrifuging the reaction solution for 3 seconds for a short time. After the reaction, the product was diluted 10 times and added to each well of 25. mu.L each.

TABLE 10 reverse transcription reaction System

Components	Amount of the composition
		5x MMLV Buffer	4
Reverse transcriptase or control	2
		50μMλDNA-F	1
RNA fragment of interest	1.2
		RNase Free Water	11.8

Then, the activities of 5 types of reverse transcriptases were measured by PCR, and Quant-iT was used as a reaction solution for detecting the enzyme activities^TM PicoGreen^TMThe dsDNA Assay Kit, purchased from Invitrogen, cat # P7589, was formulated according to Kit instructions. The reaction is carried out on a macro-stone fluorescence PCR instrument (SLAN-96S/48P), fluorescence signals are collected when the reaction is finished, and analysis software (SLAN full-automatic medical PCR analysis system 8.2.2) is used for automatically outputting the end point fluorescence signals. Calculating the difference value of the fluorescence value of the MMLV mutant and the control fluorescence value, and recording the difference value as S1; calculating the difference value of the fluorescence value of the MMLV standard strain and the fluorescence value of the contrast, and recording as S2; the enzyme activity was recorded as the ratio of S1/S2.

4. The result of the detection

The results of the detection of 5 types of reverse transcriptases by the two methods are shown in Table 11. The results of the detection and differentiation of the activity of 5 reverse transcriptases by the method of the invention are as follows: the activities of D524A/E562K, D524A, E302K/D524A, L435G/D524A and L139P are sequentially improved, and the detection and differentiation result is consistent with that of the traditional method. Moreover, the method can quickly and simply achieve the detection aim.

TABLE 11 results of enzyme Activity measurement

Mutant	Enzyme activity	Rm
			MMLV-D524A/E562K	1.43	24.91
MMLV-D524A	1.75	26.89
			MMLV-E302K/D524A	1.93	47.19
MMLV-L435G/D524A	2.41	51.62
			MMLV-L139P	2.83	91.56

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail are possible in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110 Xiamen Zhishan Biotechnology Ltd

<120> A method for detecting one or more polymerase activities

<130> IDC200483

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 38

<212> DNA

<213> artificial

<220>

<223> primer

<400> 1

taatacgact cactataggg acaggtgctg aaagcgag 38

<210> 2

<211> 21

<212> DNA

<213> artificial

<220>

<223> primer

<400> 2

cagcgtctgt tcatcgtcgt g 21

<210> 3

<211> 37

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 3

tgcttttgat gatgatattg aacaggaagg ctctccg 37

Claims

1. A method of detecting one or more polymerase activities, the method comprising:

2. The method of claim 1, wherein, in step (c), the nucleic acid molecule is mixed with the polymerase and the primer set and amplified, and then, after the amplification is completed, a detection probe is added to the product of step (b) and a melting curve analysis is performed; alternatively, in the step (b), the nucleic acid molecule is mixed with the polymerase, the primer set, and the detection probe, and amplified, and then, after the amplification is completed, a melting curve analysis is performed.

3. The method of claim 1 or 2, having one or more of the following features:

(1) in the step (d), analyzing the activities of the polymerases by comparing the melting peak heights (Rm) of the amplification products of the polymerases to be detected;

(2) providing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more polymerases in step (a);

(3) in step (d), the melting peak height (Rm), or raw data (e.g., T) for the melting curve, is obtained by real-time fluorescence PCR_mValue corresponding to the fluorescence signal value of the fluorescence channel) to obtain the melting peak height (Rm).

4. The method of any one of claims 1-3, wherein in step (a) or (b) of the method, further deoxynucleoside triphosphates (dNTPs), water, comprising ions (e.g., Mg)²⁺) A single-stranded DNA binding protein, or any combination thereof.

5. The method of any one of claims 1-4, wherein the method has one or more technical features selected from the group consisting of:

(1) the sample comprises or is DNA, RNA, or any combination thereof;

(2) the nucleic acid molecule is selected from DNA, RNA, or any combination thereof;

(3) the amplification product is selected from DNA, RNA, or any combination thereof; preferably, the amplification product is RNA;

(4) the sample is derived from a eukaryote (e.g., an animal, a plant, a fungus), a prokaryote (e.g., a bacterium, an actinomycete), a virus, a bacteriophage, or any combination thereof.

6. The method of any one of claims 1-5, wherein the polymerase has one or more technical features selected from the group consisting of:

(1) each of the polymerases is independently selected from a DNA polymerase, an RNA polymerase, or any combination thereof;

(2) the polymerases are DNA polymerases each independently obtained from a bacterium selected from the group consisting of: thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermus flavus, Thermococcus literalis, Thermus antandranii, Thermus caldophlus, Thermus chloridophilus, Thermus flavus, Thermus iginiterrae, Thermus lactius, Thermus osihima, Thermus ruber, Thermus rubens, Thermus scodottus, Thermus silvannus, Thermus thermophilus, Thermotoga maritima, Thermotoga neolytica, Thermosipho africans, Thermococcus littoralis, Thermococcus barbadensis, Thermococcus purpurea, Thermotoga neolytica, Thermosiphorubium africans, Thermococcus purpurea, Thermococcus littoralis, Thermococcus barosissimus, Thermococcus purpurea, Pyrococcus purpurea, Pyrococcus purpurea, Pyrococcus purpureus, Pyrococcus;

(3) the polymerases are DNA polymerases each independently selected from Bst DNA polymerase, T7 DNA polymerase, phi29 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, Pfu DNA polymerase, vent DNA polymerase, or any combination thereof;

(4) the polymerase is a DNA polymerase, which includes a reverse transcriptase;

(5) the polymerase is a reverse transcriptase, each independently selected from MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, or any combination thereof.

7. The method of any one of claims 1-6, wherein, in step (a) of the method, for each nucleic acid molecule, at least one pair of primer sets is provided, the primer sets comprising at least one forward primer and at least one reverse primer.

8. The method of claim 7, wherein the forward primer and reverse primer each independently comprise or consist of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof.

9. The method of any one of claims 1-8, wherein, in step (b) of the method, at least one detection probe is provided for each amplification product (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detection probes are provided).

10. The method of any one of claims 1-9, wherein the detection probe has one or more technical features selected from the group consisting of:

(1) each detection probe has a different melting point (T) from the double-stranded hybrid formed by the amplification product_m) (ii) a Preferably, the melting point (T) between the detection probe and the double-stranded hybrid formed by the amplification product of the nucleic acid molecule_m) A difference of 1 deg.C (e.g., 1 deg.C, 2 deg.C, 3 deg.C) or more;

(2) the detection probes each independently comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., Peptide Nucleic Acids (PNAs) or locked nucleic acids), or any combination thereof;

(3) the length of the detection probe is 15-1000nt, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt, 900-1000 nt;

(4) the detection probes each independently have a 3' -OH terminus; alternatively, the 3' -end of the detection probe is blocked; for example, the 3' -end of the detection probe can be blocked by adding a chemical moiety (e.g., biotin or alkyl) to the 3' -OH of the last nucleotide of the detection probe, by removing the 3' -OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a dideoxynucleotide;

(5) in the step (d), gradually heating or cooling the product of the step (c) and monitoring the signal emitted by the reporter group on each detection probe in real time, thereby obtaining a curve of the signal intensity of each reporter group changing along with the change of the temperature; then, deriving the curve to obtain a melting curve of the product of step (d);

(6) the reporter and quencher are separated by a distance of 10-80nt or more;

(7) the reporter groups in the detection probes are each independently a fluorophore (e.g., ALEX-350, FAM, VIC, TET, CAL)

Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS Red, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and, the quenching group is a molecule or group capable of absorbing/quenching the fluorescence (e.g., DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA);

(8) the detection probes each independently have the same or different reporter groups; preferably, the detection probes each independently have the same or different quencher group;

(9) the detection probes are each independently resistant to nuclease activity (e.g., 5' nuclease activity, e.g., 5' to 3' exonuclease activity); for example, the backbone of the detection probe comprises modifications that are resistant to nuclease activity, such as phosphorothioate linkages, alkylphosphotriester linkages, arylphosphotriester linkages, alkylphosphonate linkages, arylphosphonate linkages, hydrogenphosphate linkages, alkylaminophosphate linkages, arylaminophosphate linkages, 2' -O-aminopropyl modifications, 2' -O-alkyl modifications, 2' -O-allyl modifications, 2' -O-butyl modifications, and 1- (4' -thio-PD-ribofuranosyl) modifications;

(10) the detection probes are each independently linear or have a hairpin structure.

11. A kit, comprising: one or more nucleic acid molecules, at least one pair of primer sets capable of amplifying said nucleic acid molecules, and at least one detection probe capable of hybridizing or annealing to an amplification product of a nucleic acid molecule and labeled with a reporter and a quencher, wherein said reporter is capable of emitting a signal and said quencher is capable of absorbing or quenching the signal emitted by said reporter; and wherein the detection probe emits a signal when hybridized to its complement that is different from the signal when not hybridized to its complement;

preferably, the kit further comprises: polymerase, deoxynucleoside triphosphates (dNTPs), water, containing ions (e.g., Mg)²⁺) A single-stranded DNA binding protein, or any combination thereof;

preferably, the kit has any one or more of the following features:

(1) the nucleic acid molecule is selected from DNA, RNA, or any combination thereof;

preferably, the sample is derived from a eukaryote (e.g., an animal, a plant, a fungus), a prokaryote (e.g., a bacterium, an actinomycete), a virus, a bacteriophage, or any combination thereof;

preferably, the nucleic acid molecule is lambda DNA;

(2) the primer set comprises at least one forward primer and at least one reverse primer;

preferably, the forward primer and the reverse primer each independently comprise or consist of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof;

preferably, the primers of the primer group have nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

(3) providing at least one detection probe for each nucleic acid molecule (e.g., providing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detection probes);

preferably, the detection probe is as defined in claim 9;

preferably, the detection probe has a nucleotide sequence shown as SEQ ID NO. 3;

preferably, the polymerase has one or more technical features selected from the group consisting of:

(4) the polymerase is a DNA polymerase, which includes a reverse transcriptase;

(5) the polymerase is a reverse transcriptase, each independently selected from the group consisting of MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, or any combination thereof;

preferably, the kit is used to detect one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more polymerases) polymerase activities.