CN114606298A - Method for detecting length of one or more nucleic acid molecule amplification products in sample - Google Patents
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Abstract
The invention relates to a melting curve analysis technology and provides a method for detecting the length of an amplification product of one or more nucleic acid molecules in a sample. The method is simple and convenient to operate, simple in steps and capable of saving detection time.
Description
Technical Field
The present invention provides a method for detecting the length of an amplification product of one or more nucleic acid molecules in a sample using melting curve analysis.
Background
The conventional method for detecting the length of nucleic acid molecules is mainly an agarose gel electrophoresis method, which is an electrophoresis method using agar or agarose as a support medium. For samples with relatively large molecular weight, such as macromolecular nucleic acids, viruses, etc., agarose gel with relatively large pore size is generally used for electrophoretic separation. Agarose gels have a network structure, and the molecules of a substance are subjected to resistance when passing through the gel, and macromolecular substances are subjected to high resistance when surging, so that in gel electrophoresis, the separation of charged particles depends not only on the nature and quantity of net charges, but also on the size of the molecules. The DNA molecule has charge effect and molecular sieve effect when running in agarose gel. DNA molecules are negatively charged in a solution above the isoelectric point and move in an electric field towards the positive electrode. Due to the repetitive nature of the sugar-phosphate backbone in structure, double-stranded DNA of the same number of nucleotides has almost the same amount of net charge, and therefore they can move in the positive direction at the same rate. The length of the nucleic acid fragment can be judged by agarose electrophoresis.
However, conventional detection methods (e.g., agarose gel electrophoresis, alkaline agarose gel) are difficult to detect in real time the product length of the nucleic acid molecule being amplified. Moreover, agarose gel electrophoresis is complicated in operation, and requires steps such as gel preparation, electrophoresis, gel irradiation, and the like. And the requirement on the electrophoresis time is strict. The alkaline agarose gel method is complex in operation and requires the steps of gel preparation, electrophoresis, neutralization, dyeing, gel irradiation and the like. And is time-consuming, requiring several hours for the electrophoresis process alone.
Therefore, it is necessary to provide a new method to solve the above problems.
Disclosure of Invention
In order to solve the above problems, the present application realizes detection of an amplification product of a nucleic acid molecule by performing melting curve analysis on the amplification product using a detection probe.
Accordingly, in a first aspect, the present application provides a method of detecting the length of an amplification product of one or more nucleic acid molecules in a sample, the method comprising:
(a) providing a polymerase and a primer set capable of amplifying the nucleic acid molecule;
(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 molecule using the polymerase and primer set to obtain an amplification product, and performing a melting curve analysis on the amplification product using the detection probe;
(d) judging the length of the amplification product according to the result of the melting curve analysis.
In certain embodiments, 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, the method further comprises, prior to step (c), providing deoxynucleoside triphosphates (dNTPs), water, comprising an ion (e.g., Mg)2+) 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 of the nucleic acid molecule is selected from DNA, RNA, or any combination thereof. In certain embodiments, the amplification product of the nucleic acid molecule is DNA.
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, the polymerase is selected from a DNA polymerase, an RNA polymerase, or any combination thereof.
In certain embodiments, the polymerase is a DNA polymerase 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 polymerase is a DNA polymerase selected from Bst DNA polymerase, T7DNA polymerase, phi29DNA polymerase, T4DNA polymerase, T5DNA 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 selected from the group consisting of MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, or any combination thereof.
In certain embodiments, 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, 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 detection probe has a nucleotide sequence that is complementary (e.g., fully complementary) to a designated region of the nucleic acid molecule. In certain embodiments, the designated region is a designated distance (e.g., 100nt, 200nt, 300nt, 400nt, 500nt, 800nt, 1000nt, 1500nt, 2000nt, 3000nt, 4000nt, 5000nt, or other designated distance) from the region to which the primer hybridizes.
In certain embodiments, in step (b) of the method, at least one detection probe is provided for each amplification product of a nucleic acid molecule (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detection probes are provided).
In certain embodiments, at least two detection probes (e.g., a first detection probe and a second detection probe) are provided for each amplification product of a nucleic acid molecule, wherein the first detection probe is capable of hybridizing to a first region of the nucleic acid molecule that is a first distance from the region to which the forward primer hybridizes, the second detection probe is capable of hybridizing to a second region of the nucleic acid molecule that is a second distance from the region to which the forward primer hybridizes, and the second distance is greater than the first distance. When the amplification of the nucleic acid molecule is completed, the amplification product is subjected to melting curve analysis, and the presence or absence of a corresponding melting peak and/or melting point (T) in the melting curve analysis is determinedm) It can be determined whether an amplification product complementary to the corresponding detection probe is present. When the amplification of the nucleic acid molecule is completed and only the first melting peak corresponding to the duplex formed by the first detection probe is detected by performing a melting curve analysis, it can be determined that the length of the amplification product is greater than the first distance and less than the second distance. When amplification of the nucleic acid molecule is completed and a melting curve analysis is performed to detect a first melting peak corresponding to a duplex formed by the first detection probe and a second melting peak corresponding to a duplex formed by the second detection probe, it can be determined that the length of the amplification product is greater than the second distance.
In certain embodiments, each detection probe is independently capable of specifically hybridizing to a different designated region of the nucleic acid molecule under conditions that allow for hybridization or annealing of the nucleic acid. In certain embodiments, each detection probe independently has a nucleotide sequence that is complementary (e.g., fully complementary) to a different designated region of the nucleic acid molecule.
In certain embodiments, in step (d) of the method, the melting curve is analyzed for the presence of a corresponding melting peak and/or melting point (T)m) To determine the length of the amplification product.
In the present application, the melting point (T) of the different duplexes formed by the binding of different detection probes to their complementary sequences can be pre-calculated, depending on the specific sequence of the detection probemValue). Thus, the presence or absence of the corresponding melting peak and/or melting point (T) in the analysis according to the melting curvem) It is possible to determine whether an amplification product complementary to the corresponding detection probe is present, and further, based on the region to which the detection probe specifically hybridizes, the length of the amplification product can be determined.
In certain embodiments, the detection probes are designed such that the duplexes formed by each detection probe have a different T from each othermThe value is obtained. Thus, by detecting the melting point (T) of different duplexes in a melting curve analysismValue) of the melting peak, the presence or absence of an amplification product complementary to the corresponding detection probe can be determined, and the length of the amplification product can be determined. In such embodiments, different detection probes may be labeled with the same or different reporter groups (e.g., fluorescent groups).
In certain embodiments, the detection probes are designed such that different detection probes label different reporter groups (e.g., fluorescent groups). The different reporter groups may be detected in different detection channels. Thus, by detecting the presence or absence of a melting peak in each detection channel in the melting curve analysis, it is possible to determine the presence or absence of an amplification product complementary to the corresponding detection probe, and further determine the length of the amplification product. In such embodiments, duplexes formed by different detection probes may have the same or different TmThe value is obtained.
In certain embodiments, the detection probes are designed such that duplexes formed by different detection probes have different melting points (T)mValues), and different detection probes are labeled with different reporter groups (e.g., fluorescent groups). In such embodiments, the detection of each of the detection channels is performed by detecting each of the detection channels in a melting curve analysisThe presence or absence of the species melting peak can be used to determine the presence or absence of an amplification product complementary to the corresponding detection probe, and thus the length of the amplification product.
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 of the nucleic acid moleculem). 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 moleculem) 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 curve of the signal intensity of each reporter group as a function of temperature; then, the curve is derived to obtain the melting curve of the product of step (d).
In certain embodiments, the detection probes are each independently labeled at their 5 'terminus or upstream with a reporter and at their 3' terminus or downstream with a quencher, or at their 3 'terminus or downstream with a reporter and at their 5' terminus or upstream with a quencher.
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 certain embodiments, the sequence of the detection probe is selected from SEQ ID NOs 6, 7, 8, 9, or any combination thereof.
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 "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 synthesizing or 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, a sequence in a detection probe that is capable of specifically hybridizing to a designated region of the nucleic acid molecule is also referred to as a "targeting sequence" or a "target-specific sequence," and the terms "targeting sequence" and "target-specific sequence" refer to a sequence that is 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, and that comprises a sequence that is 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 for hybridization, annealing, or amplification of the nucleic acids, 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 double-stranded nucleic acid molecules 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-coil state. In this case, the reporter (e.g., fluorophore) and the quencher on the probe under dissociation are brought into close proximity to each other, whereby 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) from the duplex comprising the probe during the temperature increase or decrease allows observation of the hybridization and dissociation of the probe from its complementary sequence, forming a curve of varying signal intensity with 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 corresponding to itIs the melting point (T) of the duplexm). In general, the higher the degree of match of a probe to a complementary sequence (e.g., the fewer mismatched bases, the more bases paired), the T of the duplexmThe higher. Thus, T through the duplexmThe presence and identity of the sequence in the duplex that is complementary to the probe can be determined. As used herein, the terms "melting peak", "melting point" and "Tm"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 present application is different from the conventional detection methods, and realizes the detection of the length of the amplification product of one or more nucleic acid molecules by melting curve analysis. The method of the present application enables simultaneous determination of the length of a plurality of nucleic acid molecule amplification products. And the method is simple and convenient to operate, simple in steps and capable of saving reaction time and detection time.
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 the results of melting curve analysis using a detection probe 1 to detect the length of a fragment 1; wherein the black solid line represents the detection result of fragment 1, and the gray solid line represents the detection result of the negative control.
FIG. 2 shows the results of melting curve analysis using detection probe 2 to detect the length of fragment 2; wherein the black solid line represents the detection result of fragment 2, and the gray solid line represents the detection result of the negative control.
FIG. 3 shows the results of melting curve analysis using detection probe 3 to detect the length of fragment 3; wherein the black solid line represents the detection result of fragment 3, and the gray solid line represents the detection result of the negative control.
FIG. 4 shows the results of melting curve analysis using detection probe 4 to detect the length of fragment 4; wherein the black solid line represents the detection result of fragment 4, and the gray solid line represents the detection result of the negative control.
FIG. 5 shows the results of melting curve analysis using detection probes 1-4 to detect the length of fragment 5.
FIG. 6 shows the results of melting curve analysis using detection probes 1-4 to detect the length of fragment 6.
FIG. 7 shows the results of melting curve analysis using detection probes 1-4 to measure the length of fragment 7.
FIG. 8 shows the results of melting curve analysis using detection probes 1-4 to detect the length of fragment 8.
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 Enzymology) series (academic Press): 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
This example pre-constructs 4 nucleic acid fragments of known length and tests the length of the constructed DNA fragments using the method of the present invention to verify the feasibility of the method of the present invention.
1. Construction of DNA fragments of varying lengths
This example describes the construction of fragments of different lengths using lambda DNA (available from Life technologies, Shanghai) as template. The amplification was performed using a PCR method and corresponding primers, wherein the length of amplified fragment 1 was 2024bp, the length of amplified fragment 2 was 3036bp, the length of amplified fragment 3 was 4026bp, the length of amplified fragment 4 was 5044bp, and the primers used were as shown in Table 1. The enzyme used for PCR amplification is 2xTaKaRa TaqTMHS Perfect Mix (from TaKaRa) and the specific reaction systems are shown in tables 2 to 6.
TABLE 1 primers and sequences used
TABLE 2 reaction System for PCR of fragment 1
| Reaction components | Volume (μ L) |
| Lambda DNA template | 5 |
| 2xTaKaRa TaqTM HS Perfect Mix | 25 |
| 50μMλDNA-F | 0.2 |
| 50μMλDNA-2024-R | 0.2 |
| RNase Free Water | 19.6 |
TABLE 3 reaction System for PCR of fragment 2
| Reaction components | Volume (μ L) |
| Lambda DNA template | 5 |
| 2xTaKaRa TaqTM HS Perfect Mix | 25 |
| 50μMλDNA-F | 0.2 |
| 50μMλDNA-3036-R | 0.2 |
| RNase Free Water | 19.6 |
TABLE 4 reaction System for PCR of fragment 3
| Reaction components | Volume (mu L) |
| Lambda DNA template | 5 |
| 2xTaKaRa TaqTM HS Perfect Mix | 25 |
| 50μMλDNA-F | 0.2 |
| 50μMλDNA-4026-R | 0.2 |
| RNase Free Water | 19.6 |
TABLE 5 reaction System for PCR of fragment 4
| Reaction components | Volume (μ L) |
| Lambda DNA template | 5 |
| 2xTaKaRa TaqTM HS Perfect Mix | 25 |
| 50μMλDNA-F | 0.2 |
| 50μMλDNA-5044-R | 0.2 |
| RNase Free Water | 19.6 |
TABLE 6 reaction procedure for PCR
And respectively designing probes 1-4 bound to the amplification products according to the nucleotide sequences of the products amplified by the primers, wherein the probe 1 can be bound to 1152nt to 1189nt of the nucleotide sequences of the amplification products, the probe 2 can be bound to 2108nt to 2134nt of the nucleotide sequences of the amplification products, the probe 3 can be bound to 3137nt to 3167nt of the nucleotide sequences of the amplification products, and the probe 4 can be bound to 4246nt to 4274nt of the nucleotide sequences of the amplification products.
The lengths of the fragments 1 to 4 were measured by melting curve analysis using the detection probes 1 to 4, respectively. Briefly, after denaturing the amplification product obtained above at a high temperature, the probe is hybridized thereto at a lower temperature, and the melting temperature (T) corresponding to the duplex formed by the probe and the amplification product is calculated by gradually raising the temperature and detecting the fluorescence signal thereofm) The presence or absence of a corresponding detection target is indicated by the presence or absence of a melting peak at the temperature corresponding to the probe. This implementationThe probes used in the examples are shown in Table 7, and the fluorescent quantitative PCR reaction procedures are shown in Table 8. The detection line used was 25 μ L: 2.5. mu.L of 10 XPCR buffer, 1.5. mu.L of MgCl2(25mM), 2. mu.L of 5. mu.M probe (probes 1-4), 12.5. mu.L of amplified fragment (fragments 1-4), 6.5. mu.L of RNase Free Water. 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 7 sequence of probes
TABLE 8 reaction sequence for Length detection
The results of measuring the lengths of the fragments 1 to 4 are shown in FIGS. 1 to 4, respectively. The gray solid line is the negative control, and the black solid line is the detected nucleic acid fragment. The result proves that the method can detect the length of the nucleic acid fragment and has accurate detection result.
Example 2
This example uses lambda DNA (available from Life technologies, Shanghai) as a template and the length of the synthesized product is determined after the polymerase synthesis reaction is complete using the method of the invention. Lambda DNA was amplified by PCR with primers shown in Table 9, and 4 amplifications of different product lengths were performed, wherein fragment 5 was 1463bp long, fragment 6 was 2875bp long, fragment 7 was 3362bp long, and fragment 8 was 4528bp long. 2xTaKaRa Taq was used for PCR amplificationTMHS Perfect Mix (from TaKaRa) and the specific reaction systems are shown in tables 10 to 13.
TABLE 9 primers and sequences used
TABLE 10 reaction System for fragment 5
| Reaction components | Volume (μ L) |
| Lambda DNA template | 5 |
| 2xTaKaRa TaqTM HS Perfect Mix | 25 |
| 50μMλDNA-F | 0.2 |
| Lambda DNA fragment 5-R | 0.2 |
| RNase Free Water | 19.6 |
TABLE 11 reaction System for fragment 6
| Reaction components | Volume (μ L) |
| Lambda DNA template | 5 |
| 2xTaKaRa TaqTM HS Perfect Mix | 25 |
| 50μMλDNA-F | 0.2 |
| Lambda DNA fragment 6-R | 0.2 |
| RNase Free Water | 19.6 |
TABLE 12 reaction System for fragment 7
| Reaction components | Volume (μ L) |
| Lambda DNA template | 5 |
| 2xTaKaRa TaqTM HS Perfect Mix | 25 |
| 50μMλDNA-F | 0.2 |
| Lambda DNA fragment 7-R | 0.2 |
| RNase Free Water | 19.6 |
TABLE 13 reaction System for fragment 8
| Reaction components | Volume (μ L) |
| Lambda DNA template | 5 |
| 2xTaKaRa TaqTM HS Perfect Mix | 25 |
| 50μMλDNA-F | 0.2 |
| Lambda DNA fragment 8-R | 0.2 |
| RNase Free Water | 19.6 |
The assays were performed according to the probes and methods described in example 1, and the detection systems used in this example are shown in Table 14. Wherein, the formula of the 10x PCR buffer solution is as follows: (NH)4)2SO4 21.142g，Tris 81.164g，Tween-20 1.0mL，pH 8.8。
TABLE 14 detection System
The results of measuring the lengths of the fragments 5 to 8 are shown in FIGS. 5 to 8, respectively. The results show that fragment 5 is capable of binding to probe 1, but not to the remaining probes, demonstrating that fragment 5 ranges in length from 1189nt to 2108 nt; fragment 6 was able to bind to both probe 1 and probe 2, but not to the remaining probes, demonstrating that fragment 6 ranged in length from 2134nt to 3137 nt; fragment 7 was able to bind to probes 1, 2, 3 and not to probe 4, demonstrating that fragment 7 ranged in length from 3167nt to 4246nt, fragment 8 was able to bind to probes 1-4, demonstrating that fragment 8 ranged in length from 4274nt and above. The results prove that the method can simultaneously judge the lengths of a plurality of nucleic acid molecule amplification products, and the process is simple and quick. And the result of the multiple fragment length analysis is consistent with the designed fragment length, which shows that the result is reliable and stable.
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 can be made 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
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Claims (10)
1. A method of detecting the length of an amplification product of one or more nucleic acid molecules in a sample, the method comprising:
(a) providing a polymerase and a primer set capable of amplifying the nucleic acid molecule;
(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 molecule using the polymerase and primer set to obtain an amplification product, and performing a melting curve analysis on the amplification product using the detection probe;
(d) judging the length of the amplification product according to the result of the melting curve analysis.
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 amplification is completed, a detection probe is added to the product of step (b) and 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, wherein in step (d) of the method, the melting curve is analyzed for the presence of a corresponding melting peak and/or melting point (T)m) To determine the length of the amplification product.
4. The method of any one of claims 1-3, further comprising, prior to step (c), providing 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; preferably, the amplification product of the nucleic acid molecule is DNA;
(3) the amplification product of the nucleic acid molecule is selected from DNA, RNA, or any combination thereof;
(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) the polymerase is selected from a DNA polymerase, an RNA polymerase, or any combination thereof;
(2) the polymerase is a DNA polymerase 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 polymerase is a DNA polymerase selected from Bst DNA polymerase, T7DNA polymerase, phi29DNA polymerase, T4DNA polymerase, T5DNA 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 selected from the group consisting of 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 of a nucleic acid molecule (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) the detection probe has a nucleotide sequence that is complementary (e.g., fully complementary) to a designated region of the nucleic acid molecule. In certain embodiments, the designated region is a designated distance (e.g., 100nt, 200nt, 300nt, 400nt, 500nt, 800nt, 1000nt, 1500nt, 2000nt, 3000nt, 4000nt, 5000nt, or other designated distance) from the region to which the primer hybridizes;
(2) each detection probe has a melting point (T) different from that of a double-stranded hybrid formed by the amplification product of the nucleic acid moleculem) (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 moleculem) A difference of 1 deg.C (e.g., 1 deg.C, 2 deg.C, 3 deg.C) or more;
(3) 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;
(4) 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;
(5) 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;
(6) 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);
(7) the reporter and quencher are separated by a distance of 10-80nt or more;
(8) 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);
(9) 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;
(10) 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;
(11) the detection probes are each independently linear or have a hairpin structure;
(12) the detection probes are each independently labeled with a reporter group at their 5 'end or upstream and a quencher group at their 3' end or downstream, or with a reporter group at their 3 'end or downstream and a quencher group at their 5' end or upstream.
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