WO2022120914A1 - Method for measuring length of amplification product of one or more nucleic acid molecules in sample - Google Patents

Abstract

A method for measuring the length of an amplification product of one or more nucleic acid molecules in a sample. The method uses a melting curve analysis technology. The method is convenient to operate and simple in steps, and also reduces the measurement time.

Description

A method for detecting the length of one or more nucleic acid molecule amplification products in a sample technical field

The present invention provides a method for detecting the length of amplification products of one or more nucleic acid molecules in a sample using melting curve analysis.

Background technique

The traditional method for detecting the length of nucleic acid molecules mainly includes agarose gel electrophoresis, which is an electrophoresis method using agar or agarose as a support medium. For samples with larger molecular weights, such as macromolecular nucleic acids, viruses, etc., agarose gels with larger pore sizes can generally be used for electrophoresis separation. Agarose gel has a network structure, substance molecules will be resisted when passing through, and macromolecular substances will receive great resistance when surging. Therefore, in gel electrophoresis, the separation of charged particles depends not only on the nature and number of net charges, but also on the Also depends on molecular size. DNA molecules have charge effect and molecular sieve effect when they migrate in agarose gel. DNA molecules are negatively charged in solution above the isoelectric point and move towards the positive pole in an electric field. Due to the repetitive nature of the sugar-phosphate backbone, double-stranded DNA with the same number of nucleotides has almost the same amount of net charge, so they can move toward the positive pole at the same rate. Nucleic acid fragment lengths can be determined using agarose electrophoresis.

However, traditional detection methods (eg, agarose gel electrophoresis, alkaline agarose gel method) are difficult to detect in real time the product length of nucleic acid molecules being amplified. In addition, the operation of agarose gel electrophoresis is complicated, and steps such as gel preparation, electrophoresis, and gelation are required. And the requirements for electrophoresis time are more strict. The operation of the alkaline agarose gel method is complicated, and steps such as gel preparation, electrophoresis, neutralization, staining, and gelation are required. And it is time-consuming, and the electrophoresis process only takes several hours.

Therefore, it is necessary to provide a new method to solve the above problems.

SUMMARY OF THE INVENTION

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the operation steps such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA used in this paper are conventional steps widely used in the corresponding fields. . Meanwhile, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

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

As used herein, the term "polymerase", also known as polymerase, is a general term for a class of enzymes specialized in the biocatalytic synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It can be divided into the following groups: (1) DNA-dependent DNA polymerases; (2) RNA-dependent DNA polymerases; (3) DNA-dependent RNA polymerases; (4) RNA-dependent RNA polymerases. Among them, the first two are DNA polymerases, and the latter two are RNA polymerases. As used herein, the term "DNA polymerase" refers to an enzyme that uses nucleic acid strands as templates to synthesize DNA strands. DNA polymerases use existing DNA or RNA as templates to synthesize DNA. In the method of the present invention, the DNA polymerase may be a naturally occurring DNA polymerase, or a variant or fragment of a natural enzyme having the above-mentioned activities. As used herein, the term "RNA polymerase" refers to an enzyme that uses nucleic acid strands as templates to synthesize RNA strands. RNA polymerases use existing DNA or RNA as templates to synthesize RNA. In the methods of the present invention, the RNA polymerase may be a naturally occurring RNA polymerase, or a variant or fragment of a natural enzyme having the above-mentioned activities.

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

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

As used herein, a sequence in a detection probe that is capable of specifically hybridizing to a specified region of the nucleic acid molecule is also referred to as a "targeting sequence" or "target-specific sequence", the terms "targeting sequence" and "target" "Specific sequence" refers to a sequence capable of selectively/specifically hybridizing or annealing to a target nucleic acid sequence under conditions that permit hybridization, annealing, or amplification of the nucleic acid, which comprises a sequence complementary to the target nucleic acid sequence. In this application, the terms "targeting sequence" and "target-specific sequence" have the same meaning and are used interchangeably. It is readily understood that a targeting sequence or target-specific sequence is specific for a target nucleic acid sequence. In other words, a targeting sequence or target-specific sequence only hybridizes or anneals to a specific target nucleic acid sequence, and not to other nucleic acid sequences, under conditions that allow nucleic acid hybridization, annealing, or amplification.

As used herein, the term "complementary" means that two nucleic acid sequences are capable of forming hydrogen bonds between each other according to the principles of base pairing (Waston-Crick principle), and thereby forming duplexes. In this application, the term "complementary" includes "substantially complementary" and "completely complementary". As used herein, the term "completely complementary" means that every base in one nucleic acid sequence is capable of pairing with bases in another nucleic acid strand without mismatches or gaps. As used herein, the term "substantially complementary" means that a majority of bases in one nucleic acid sequence are capable of pairing with bases in the other nucleic acid strand, which allows for mismatches or gaps (eg, one or mismatches or gaps of several nucleotides). Typically, two nucleic acid sequences that are "complementary" (eg, substantially complementary or fully complementary) will selectively/specifically hybridize or anneal under conditions that allow nucleic acid hybridization, annealing, or amplification, and form a duplex . Accordingly, the term "non-complementary" means that two nucleic acid sequences cannot hybridize or anneal under conditions that permit hybridization, annealing, or amplification of the nucleic acids to form a duplex. As used herein, the term "not perfectly complementary" means that bases in one nucleic acid sequence cannot perfectly pair with bases in another nucleic acid strand, at least one mismatch or gap exists.

As used herein, the terms "hybridization" and "annealing" mean the process by which complementary single-stranded nucleic acid molecules form a double-stranded nucleic acid. In this application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Typically, two nucleic acid sequences that are completely complementary or substantially complementary can hybridize or anneal. The complementarity required for hybridization or annealing of two nucleic acid sequences depends on the hybridization conditions used, in particular 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 (polymerase chain reaction) that amplifies a target nucleic acid using a nucleic acid polymerase and primers.

As used herein, the term "detection probe" refers to an oligonucleotide labeled with a reporter group and a quencher group. When the probe is not hybridized to other sequences, the quencher group is positioned to absorb the signal of the quenched reporter group (eg, the quencher group is located adjacent to the reporter group), thereby absorbing or quenching the reporter group signal sent. In this case, the probe does not emit a signal. Further, when the probe is hybridized to its complementary sequence, the quencher group is located in a position that cannot absorb or quench the signal of the reporter group (eg, the quencher group is located away from the reporter group), so that it cannot absorb or quench the signal of the reporter group. Quench the signal from the reporter group. 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 the analysis of the presence or identity of a double-stranded nucleic acid molecule by determining the melting curve of the double-stranded nucleic acid molecule. method, 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 this 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, melting curve analysis can be performed by using a self-quenching probe labeled with a reporter group and a quencher group. Briefly, at ambient temperature, probes are capable of forming duplexes with their complementary sequences through base pairing. In this case, the reporter group (such as a fluorophore) and the quencher group on the probe are separated from each other, and the quencher group cannot absorb the signal (such as a fluorescent signal) emitted by the reporter group. At this time, it is possible to detect The strongest signal (eg fluorescent signal). As the temperature increases, the two strands of the duplex begin to dissociate (ie, the probe gradually dissociates from its complementary sequence), and the dissociated probe assumes a single-stranded free coil state. In this case, the reporter group (eg, fluorophore) and the quencher group on the dissociated probe are in close proximity to each other, whereby the signal (eg, fluorescence signal) emitted by the reporter group (eg, fluorophore) is absorbed by the quenching group. Therefore, as the temperature increases, the detected signal (eg, the fluorescence signal) gradually becomes weaker. When the two strands of the duplex are completely dissociated, all probes are in a single-stranded free coil state. In this case, all the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) on the probe is absorbed by the quencher group. Thus, the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) is substantially undetectable. Therefore, by detecting the signal (such as a fluorescent signal) emitted by the duplex containing the probe during the heating or cooling process, the hybridization and dissociation process of the probe and its complementary sequence can be observed, and the signal intensity changes with the temperature change. changing curve. Further, by derivation analysis of the obtained curve, a curve with the change rate of the signal intensity as the ordinate and the temperature as the abscissa (ie, the melting curve of the duplex) can be obtained. The peak in the melting curve is the melting peak, and the corresponding temperature is the melting point (T _m ) of the duplex. In general, the more closely the probe matches the complementary sequence (eg, fewer bases are mismatched and more bases are paired), the higher the _Tm of the duplex. Thus, from the _Tm of the duplex, the presence and identity of the sequence complementary to the probe in the duplex can be determined. Herein, the terms "melting peak", "melting point" and " _Tm " have the same meaning and are used interchangeably.

In certain preferred embodiments of the present application, melting curve analysis can be performed by using detection probes labeled with reporter and quencher groups. The detection principle is the same as above.

In order to solve the above problems, the present application uses detection probes to perform melting curve analysis on amplification products, thereby realizing the detection of nucleic acid molecule amplification products.

Accordingly, in a first aspect, the application provides a method for 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 group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and the detection probe emits a signal when hybridized to its complementary sequence that is different from the signal emitted when it is not hybridized to its complementary sequence; under conditions that allow nucleic acid hybridization or annealing Under the following conditions, the detection probe can specifically hybridize to a designated region of the nucleic acid molecule;

(c) using the polymerase and primer set to amplify the nucleic acid molecule to obtain an amplification product, and using the detection probe to perform melting curve analysis on the amplification product;

(d) Judging the length of the amplified product according to the result of 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 the amplification, a detection probe is added to the in the product of step (b), and performing melting curve analysis; or, in step (b), mixing the nucleic acid molecule with the polymerase, the primer set and the detection probe, and performing amplification , and then, after the end of amplification, perform melting curve analysis.

In certain embodiments, the method further comprises, prior to step (c), providing deoxynucleoside triphosphates (dNTPs), water, a solution containing an ion (eg, 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 eukaryotes (eg, animals, plants, fungi), prokaryotes (eg, bacteria, actinomycetes), viruses, bacteriophages, 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, Thermis flavus, Thermococcus literalis, Thermus antranildanii,Thermus caldophllus,Thermus chliarophilus,Thermus flavus,Thermus igniterrae,Thermus lacteus,Thermus oshimai,Thermus ruber,Thermus rubens,Thermus scotoductus,Thermus silvanus,Thermus thermophllus,Thermotoga maritima,Thermotoga neapolitana,Thermosipho africanus,Thermococcus litoralis,Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifex aeoolieus.

In certain embodiments, the polymerase is a DNA polymerase selected from the group consisting of 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 including 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 reverse primer each independently comprise or consist of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

In certain embodiments, the detection probe has a nucleotide sequence that is complementary (eg, fully complementary) to a specified region of the nucleic acid molecule. In certain embodiments, the designated region is a designated distance from the region to which the primer hybridizes (eg, 100nt, 200nt, 300nt, 400nt, 500nt, 800nt, 1000nt, 1500nt, 2000nt, 3000nt, 4000nt, 5000nt, or other designation the distance).

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

In certain embodiments, at least two detection probes (eg, a first detection probe and a second detection probe) are provided for each nucleic acid molecule's amplification product, wherein the first detection probe is capable of interacting with The first region of the nucleic acid molecule is hybridized, and the distance between the first region and the region where the forward primer is hybridized is the first distance, and the second detection probe can hybridize with the second region of the nucleic acid molecule, and the second region is hybridized with the forward primer. The distance of the area is the second distance, 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. According to whether there is a corresponding melting peak and/or melting point (T _m ) in the melting curve analysis, it can be determined whether there is an amplification product complementary to the corresponding detection probe. increase product. 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 the melting curve analysis, it can be determined that the length of the amplified product is greater than the first distance and less than the second distance. When the amplification of the nucleic acid molecule is completed and the melting curve analysis is performed to detect the first melting peak corresponding to the duplex formed by the first detection probe, the first melting peak corresponding to the duplex formed by the second detection probe is also detected. When there are two melting peaks, it can be determined that the length of the amplified 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 permit hybridization or annealing of the nucleic acid. In certain embodiments, each detection probe independently has a nucleotide sequence that is complementary (eg, fully complementary) to a different designated region of the nucleic acid molecule.

In certain embodiments, in step (d) of the method, the length of the amplification product is determined according to whether there is a corresponding melting peak and/or the size of the melting point (T _m ) in the melting curve analysis.

In the present application, according to the specific sequences of the detection probes, the melting points (T _m values) of different duplexes formed by combining different detection probes with their complementary sequences can be calculated in advance. Therefore, according to whether there is a corresponding melting peak and/or melting point (T _m ) in the melting curve analysis, it can be determined whether there is an amplification product complementary to the corresponding detection probe, and then, according to the specific hybridization of the detection probe region, the length of the amplified product can be determined.

In certain embodiments, the detection probes are designed such that the duplexes formed by each detection probe have different _Tm values from each other. Therefore, by detecting the presence or absence of melting peaks of different duplex melting points (T _m values) in the melting curve analysis, it is possible to determine whether there is an amplification product complementary to the corresponding detection probe, and then determine the length of the amplification product . In such embodiments, different detection probes can be labeled with the same or different reporter groups (eg, fluorophores).

In certain embodiments, detection probes are designed such that different detection probes are labeled with different reporter groups (eg, fluorophores). The different reporter groups can be detected in different detection channels. Thus, by detecting the presence or absence of melting peaks in each detection channel in the melting curve analysis, it is possible to determine whether there is an amplification product complementary to the corresponding detection probe, and then determine the length of the amplification product. In such embodiments, the duplexes formed by different detection probes may have the same or different _Tm values.

In certain embodiments, detection probes are designed such that duplexes formed by different detection probes have different melting points ( _Tm values), and different detection probes are labeled with different reporter groups (eg, fluorophores) . In such an embodiment, by detecting the presence or absence of various melting peaks of each detection channel in the melting curve analysis, it can be determined whether the amplification product complementary to the corresponding detection probe exists, and then the length of the amplification product can be determined.

In certain embodiments, each detection probe has a different melting point ( _Tm ) between the double-stranded hybrid formed by the amplification product of the nucleic acid molecule. In certain embodiments, the melting point ( _Tm ) between the detection probe and the double-stranded hybrid formed by the amplification product of the nucleic acid molecule differs by 1°C (eg, 1°C, 2°C, 3°C) )above.

In certain embodiments, the detection probes each independently comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleosides acid (eg, peptide nucleic acid (PNA) or locked nucleic acid), or any combination thereof.

In certain embodiments, the detection probes are each independently 15-1000nt in length, eg, 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, 900-1000nt.

In certain embodiments, the detection probes each independently have a 3'-OH terminus; alternatively, the 3'-terminus of the detection probes is blocked; Add a chemical moiety (eg, biotin or alkyl) to the 3'-OH of the acid by removing the 3'-OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a double deoxynucleotides, thereby blocking the 3'-end of the detection probe.

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

In certain embodiments, 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 labeled at their 3' end or downstream The reporter group is labeled and the quencher group is labeled at the 5' end or upstream.

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

In certain embodiments, the reporter groups in the detection probe are each independently a fluorophore (eg, 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 quenching group is a molecule or group capable of absorbing/quenching the fluorescence (eg, DABCYL, BHQ (eg, BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA).

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 groups.

In certain embodiments, the detection probes are each independently resistant to nuclease activity (eg, 5' nuclease activity, eg, 5' to 3' exonuclease activity); eg, the detection probes The backbone of the needle contains modifications that resist nuclease activity, such as phosphorothioate linkages, alkyl phosphotriester linkages, aryl phosphotriester linkages, alkyl phosphonate linkages, arylphosphonate linkages, hydrophosphates bond, alkyl phosphoramidate bond, aryl phosphoramidate bond, 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O- Butyl modification, and 1-(4'-thio-PD-ribofuranosyl) modification.

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.

beneficial effect

The detection method of the present application is different from the traditional detection method in the past, and the length of the amplification product of one or more nucleic acid molecules can be detected by using melting curve analysis. The method of the present application can simultaneously determine the lengths of amplification products of multiple nucleic acid molecules. Moreover, the operation is simple and the steps are simple, and the reaction time and the detection time are saved at the same time.

The 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 used to illustrate the present invention, rather than 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.

Description of drawings

Figure 1 shows the results of melting curve analysis using detection probe 1 to detect the length of fragment 1; wherein, the solid black line represents the detection result of fragment 1, and the solid gray line represents the detection result of the negative control.

Figure 2 shows the results of melting curve analysis using detection probe 2 to detect the length of fragment 2; wherein, the solid black line represents the detection result of fragment 2, and the solid gray line represents the detection result of the negative control.

Figure 3 shows the results of melting curve analysis using detection probe 3 to detect the length of fragment 3; wherein, the solid black line represents the detection result of fragment 3, and the solid gray line represents the detection result of the negative control.

Figure 4 shows the results of melting curve analysis using detection probe 4 to detect the length of fragment 4; wherein, the solid black line represents the detection result of fragment 4, and the solid gray line represents the detection result of the negative control.

Figure 5 shows the results of melting curve analysis using detection probes 1-4 to detect the length of fragment 5.

Figure 6 shows the results of melting curve analysis using detection probes 1-4 to detect the length of fragment 6.

Figure 7 shows the results of melting curve analysis using detection probes 1-4 to detect the length of fragment 7.

Figure 8 shows the results of melting curve analysis using detection probes 1-4 to detect the length of Fragment 8.

Detailed ways

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

Unless otherwise indicated, the experiments and methods 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, Fritsch and Maniatis, " MOLECULAR CLONING: A LABORATORY MANUAL, 2nd editor (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al.) Editor, (1987)); "METHODS IN ENZYMOLOGY" series (Academic Publishing Company): "PCR 2: A PRACTICAL APPROACH" (M.J. MacPherson), B.D. Edited by B.D. Hames and G.R. Taylor (1995)).

In addition, if the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market. Those skilled in the art appreciate that the examples describe the invention by way of example 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, four nucleic acid fragments of known length were pre-constructed, and the method of the present invention was applied to detect the length of the constructed DNA fragments to verify the feasibility of the method of the present invention.

1. Construction of DNA Fragments of Different Lengths

In this example, λ DNA (purchased from Life technologies (Shanghai)) was used as the template to construct fragments of different lengths. The PCR method and corresponding primers are used for amplification, wherein the length of the amplified fragment 1 is 2024 bp, the length of the amplified fragment 2 is 3036 bp, the length of the amplified fragment 3 is 4026 bp, and the length of the amplified fragment 4 is is 5044bp, and the primers used are shown in Table 1. The enzyme used for PCR amplification was 2×TaKaRa Taq ^™ HS Perfect Mix (purchased from TaKaRa). 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

reactive components	Volume (μL)
λDNA template	5
2xTaKaRa Taq TM HS Perfect Mix	25
50 μM λDNA-F	0.2
50 μM λDNA-2024-R	0.2
RNase Free Water	19.6

Table 3. Reaction system for PCR of fragment 2

reactive components	Volume (μL)
λDNA template	5
2xTaKaRa Taq TM HS Perfect Mix	25
50 μM λDNA-F	0.2
50 μM λDNA-3036-R	0.2
RNase Free Water	19.6

Table 4. Reaction system for PCR of fragment 3

reactive components	Volume (μL)
λDNA template	5
2xTaKaRa Taq TM HS Perfect Mix	25
50 μM λDNA-F	0.2
50 μM λDNA-4026-R	0.2

RNase Free Water

19.6

Table 5. Reaction system for PCR of fragment 4

reactive components	Volume (μL)
λDNA template	5
2xTaKaRa Taq TM HS Perfect Mix	25
50 μM λDNA-F	0.2
50 μM λDNA-5044-R	0.2
RNase Free Water	19.6

Table 6. Reaction program for PCR

According to the nucleotide sequence of the product amplified by the primers, probes 1-4 are designed to bind to the amplified product, wherein probe 1 can bind to the 1152nt to 1189nt nucleotide sequence of the amplified product, and probe 2 Can bind to the 2108 nt to 2134 of the nucleotide sequence of the amplification product, probe 3 can bind to the 3137 nt to 3167 nt of the nucleotide sequence of the amplification product, and probe 4 can bind to the 4246 nt to 4274 nt of the nucleotide sequence of the amplification product .

Using detection probes 1-4, the lengths of fragments 1-4 were determined by melting curve analysis, respectively. In short, after denaturing the amplification product obtained above at high temperature, the probe is hybridized with it at a lower temperature, and the double-strand formed by the probe and the amplification product is calculated by gradually increasing the temperature and detecting its fluorescence signal. The melting temperature (T _m ) corresponding to the body is used to indicate whether there is a corresponding detection target by the presence or absence of a melting peak at the corresponding temperature of the probe. The probes used in this example are shown in Table 7, and the fluorescent quantitative PCR reaction program is shown in Table 8. The detection system used was 25 μL: 2.5 μL of 10x PCR buffer, 1.5 μL of MgCl ₂ (25 mM), 2 μL of 5 μM probe (probe 1-4), 12.5 μL of amplified fragment (fragment 1-4), 6.5 μL of RNase Free Water. Wherein, the formula of 10× PCR buffer is: (NH ₄ ) ₂ SO ₄ 21.142 g, Tris 81.164 g, Tween-20 1.0 mL, pH 8.8.

Table 7. Sequences of probes

Table 8. Reaction procedure for length detection

The measurement results of the lengths of fragments 1-4 are shown in Figures 1 to 4, respectively. The solid gray line is the negative control, and the solid black line is the nucleic acid fragment detected. The results prove that the length of the nucleic acid fragment can be detected by the method of the present invention, and the detection result is accurate.

Example 2

In this example, λDNA (purchased from Life technologies (Shanghai)) was used as the template, and after the polymerase synthesis reaction was completed, the method of the present invention was applied to detect the length of the synthesized product. The λDNA was amplified by the PCR method and the primers shown in Table 9, and four amplifications with different product lengths were carried out, wherein the length of fragment 5 was 1463 bp, the length of fragment 6 was 2875 bp, and the length of fragment 7 was 3362 bp , the length of fragment 8 is 4528bp. 2xTaKaRa Taq ^™ HS Perfect Mix (purchased from TaKaRa) was used for PCR amplification, and the specific reaction systems are shown in Table 10 to Table 13.

Table 9. Primers and sequences used

Table 10. Reaction system for fragment 5

reactive components	Volume (μL)
λDNA template	5
2xTaKaRa Taq TM HS Perfect Mix	25
50 μM λDNA-F	0.2
λDNA-fragment 5-R	0.2
RNase Free Water	19.6

Table 11. Reaction system for fragment 6

reactive components	Volume (μL)
λDNA template	5
2xTaKaRa Taq TM HS Perfect Mix	25
50 μM λDNA-F	0.2
λDNA-fragment 6-R	0.2

RNase Free Water

19.6

Table 12. Reaction system for fragment 7

reactive components	Volume (μL)
λDNA template	5
2xTaKaRa Taq TM HS Perfect Mix	25
50 μM λDNA-F	0.2
λDNA-fragment 7-R	0.2
RNase Free Water	19.6

Table 13. Reaction system for fragment 8

reactive components	Volume (μL)
λDNA template	5
2xTaKaRa Taq TM HS Perfect Mix	25
50 μM λDNA-F	0.2
λDNA-fragment 8-R	0.2
RNase Free Water	19.6

According to the probes and methods described in Example 1, the assays were carried out respectively, and the detection system used in this example is shown in Table 14. Wherein, the formula of 10x PCR buffer is: (NH ₄ ) ₂ SO ₄ 21.142 g, Tris 81.164 g, Tween-20 1.0 mL, pH 8.8.

Table 14. Detection system

reactive components	Volume (μL)
10x PCR buffer	2.5
MgCl 2 (25mM)	1.5
5 μM Probe 1 (1152-1189)	2
5 μM Probe 2 (2108-2134)	2
5 μM Probe 3 (3137-3167)	2
5 μM Probe 4 (4246-4274)	2
Amplified fragment to be tested	12.5
RNase Free Water	6.5

The measurement results of the lengths of fragments 5-8 are shown in Figures 5 to 8, respectively. The results showed that fragment 5 could bind to probe 1, but not to the rest of the probes, proving that the length of fragment 5 ranged from 1189 nt to 2108 nt; fragment 6 could bind to probe 1 and probe 2, but not to the rest of the probes. It is proved that the length of fragment 6 ranges from 2134nt to 3137nt; fragment 7 can bind to probes 1, 2, and 3, but not probe 4, which proves that the length of fragment 7 ranges from 3167nt to 4246nt, and fragment 8 can bind to probe 4. Pins 1-4 bind, demonstrating that fragment 8 has a length range of 4274 nt and above. The above results prove that the method of the present invention can simultaneously determine the lengths of amplification products of multiple nucleic acid molecules, and the process is simple and fast. Moreover, the results of the multiple fragment length analysis were consistent with the designed fragment lengths, indicating that the results were reliable and stable.

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 group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and the detection probe emits a signal when hybridized to its complementary sequence that is different from the signal emitted when it is not hybridized to its complementary sequence; under conditions that allow nucleic acid hybridization or annealing Under the following conditions, the detection probe can specifically hybridize to a designated region of the nucleic acid molecule;

   (c) using the polymerase and primer set to amplify the nucleic acid molecule to obtain an amplification product, and using the detection probe to perform melting curve analysis on the amplification product;

   (d) Judging the length of the amplified product according to the result of 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 the amplification is completed, the detection probe is added to the step in the product of (b), and performing melting curve analysis; or, in step (b), mixing the nucleic acid molecule with the polymerase, the primer set and the detection probe, and performing amplification, Then, after the end of the amplification, a melting curve analysis was performed.
3. The method of claim 1 or 2, in step (d) of the method, the length of the amplification product is determined according to whether there is a corresponding melting peak and/or melting point (T _m ) in the melting curve analysis.
4. The method of any one of claims 1-3, further comprising, prior to step (c), providing deoxynucleoside triphosphates (dNTPs), water, a solution containing ions (eg Mg ²⁺ ), a single Stranded DNA binding proteins, 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 following:

   (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 eukaryotes (eg, animals, plants, fungi), prokaryotes (eg, bacteria, actinomycetes), viruses, bacteriophages, or any combination thereof.
6. The method of any one of claims 1-5, wherein the polymerase has one or more technical characteristics selected from the group consisting of:

   (1) the polymerase is selected from DNA polymerase, 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, Thermis flavus, Thermococcus literalis, Thermus antranildanii, Thermus caldophllus ,Thermus chliarophilus,Thermus flavus,Thermus igniterrae,Thermus lacteus,Thermus oshimai,Thermus ruber,Thermus rubens,Thermus scotoductus,Thermus silvanus,Thermus thermophllus,Thermotoga maritima,Thermotoga neapolitana,Thermosipho africanus,Thermococcus litoralis,Thermococcus barossi,Thermococcus gorgonarius,Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifex aeoolieus;

   (3) The polymerase is a DNA polymerase, and the DNA polymerase is 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, and the DNA polymerase 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 set comprising at least one forward primer and at least one reverse primer.
8. The method of claim 7, wherein the forward primer and the reverse primer each independently comprise or consist of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof composition.
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 nucleic acid molecule amplification product (for example, one, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detection probes).
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 complementary (eg, fully complementary) to a specified region of the nucleic acid molecule. In certain embodiments, the designated region is a designated distance from the region to which the primer hybridizes (eg, 100nt, 200nt, 300nt, 400nt, 500nt, 800nt, 1000nt, 1500nt, 2000nt, 3000nt, 4000nt, 5000nt, or other designation the distance);

    (2) Each detection probe and the double-stranded hybrid formed by the amplification product of the nucleic acid molecule have different melting points ( _Tm ); preferably, the amplification product of the detection probe and the nucleic acid molecule has different melting points (Tm). The melting point (T _m ) between the double-stranded hybrids formed by the amplification products differs by more than 1°C (for example, 1°C, 2°C, 3°C);

    (3) The detection probes each independently comprise or consist of naturally occurring nucleotides (such as deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (such as peptides) nucleic acid (PNA) or locked nucleic acid), or any combination thereof;

    (4) The lengths of the detection probes are each independently 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, 900-1000nt;

    (5) The detection probes each independently have a 3'-OH end; alternatively, the 3'-end of the detection probe is blocked; The addition of a chemical moiety (eg, biotin or alkyl) to the -OH by removing the 3'-OH of the last nucleotide of the detection probe, or replacing the last nucleotide with a dideoxynucleotide , thereby blocking the 3'-end of the detection probe;

    (6) in step (d), the product of step (c) is gradually heated or cooled and the signal emitted by the reporter group on each detection probe is monitored in real time, so as to obtain the signal of each reporter group a curve of signal intensity as a function of temperature; then, the curve is derived to obtain the melting curve of the product of step (d);

    (7) the reporting group and the quenching group are separated by a distance of 10-80nt or longer;

    (8) The reporter groups in the detection probe are each independently a fluorescent group (for example, 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 (eg DABCYL, BHQ (eg BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA);

    (9) Each of the detection probes independently has the same or different reporter groups; preferably, each of the detection probes independently has the same or different quenching groups;

    (10) The detection probes are each independently resistant to nuclease activity (eg, 5' nuclease activity, eg, 5' to 3' exonuclease activity); eg, the backbone of the detection probe Contains modifications that resist nuclease activity, such as phosphorothioate linkages, alkyl phosphotriester linkages, aryl phosphotriester linkages, alkylphosphonate linkages, arylphosphonate linkages, hydrophosphonate linkages, alkyl phosphoramidate bond, aryl phosphoramidate bond, 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification, and 1-(4'-thio-PD-ribofuranosyl) modification;

    (11) The detection probes are each independently linear, or have a hairpin structure;

    (12) Each of the detection probes is independently labeled with a reporter group at its 5' end or upstream and labeled with a quencher group at its 3' end or downstream, or labeled with a reporter group at its 3' end or downstream And a quencher group is labeled at the 5' end or upstream.

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