CN101280338A

CN101280338A - Nucleic acid amplification method for detecting polymorphism of nucleic acid

Info

Publication number: CN101280338A
Application number: CNA2007100931272A
Authority: CN
Inventors: 黄庆; 府伟灵
Original assignee: First Affiliated Hospital of TMMU
Current assignee: First Affiliated Hospital of TMMU
Priority date: 2007-12-13
Filing date: 2007-12-13
Publication date: 2008-10-08
Anticipated expiration: 2027-12-13
Also published as: CN101280338B

Abstract

The invention provided a nucleic acid amplification method for detecting nucleic acid polymorphyism. The method is to utilize the probe the basic structure of which is shown in the figure to be interbred with a target molecule, and a base interstice exists in the formed probe-target molecule crossbred, then the interstice in the probe-target molecule crossbred is filled through polymerization-connection reaction, in order to ensure the two monomers to connected into a single chain to be the template used for the next polymerase chain reaction amplification, finally, the analyzing on nucleotide polymorphyism of the target molecule is performed through detecting the polymerase chain reaction amplification product. The method has the advantages that the template used for the next polymerase chain reaction amplification can be obtained only through polymerization-connection reaction, therefore the steps, such as restriction enzyme or extension, are omitted, and the detecting efficiency is improved.

Description

Nucleic acid amplification method for detecting nucleic acid polymorphism

Technical Field

The present invention relates to a nucleic acid amplification method, particularly to a nucleic acid amplification method for detecting nucleic acid polymorphisms.

Background

The detection of Single Nucleotide Polymorphisms (SNPs) and nucleic acid polymorphisms such as gene mutations is generally performed by using a multiplex Polymerase Chain Reaction (PCR) technique, which is based on the principle of simultaneously amplifying a plurality of target molecules in the same reaction tube, not only greatly increasing the analysis throughput, but also saving the amount of target molecule nucleic acid templates in a sample to be detected, but most of the current multiplex PCR methods are limited to simultaneously amplifying 5-10 target molecule fragments, and the main reason is that n pairs of different primers, i.e., 2n primers, must be added in n-multiplex PCR, which makes the conditions of PCR amplification reaction difficult to control, and in addition, primer dimerization and non-specific amplification are easily generated due to the interaction between primers. Therefore, the bottleneck of the multiplex PCR technology is how to reduce the usage amount of primers while improving the amplification multiplexing, so that the multiplex PCR becomes a real high-throughput nucleic acid analysis technology platform.

The universal primer is one of the important methods for improving the multiplex PCR strategy. In the design of multiplex PCR, the design of universal primers is usually performed in such a way that they are present in the target nucleic acid template or primary PCR amplification product to be amplified, for example, the multiplex PCR technology based on golden gate, MIP and SNPlex, so that the universal primers can be used to amplify the target sequence with high throughput, greatly increasing the multiplicity of multiplex PCR.

The probe structures of MIP (Hardenbol P, Banner J, Jain M et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat Biotechnol. 2003; 21 (6): 673-: 1. hybridizing a sequence complementary to the target gene in the probe with the target gene; 2. filling in the corresponding base of SNP site in the probe under the action of DNA polymerase, and connecting the two sequences by ligase; 3. cutting the enzyme cutting site of the primer sequence part in the probe by using restriction endonuclease to linearize the probe; 4. adding a primer into the digested probe as a template to perform PCR amplification; 5. detecting the amplified product, and judging the sequence of the SNP locus of the target gene according to the base added in the step 2. Both of the two techniques need to utilize enzyme digestion to linearize the probe hybridized with the target gene to be used as a template for subsequent PCR amplification, so that the operation becomes complicated, the detection efficiency is reduced, and even the detection accuracy is possibly influenced.

GoldenGate technology (Shen R, Fan JB, Campbell D et al, high-through SNP mutagenesis on undivided target array. Mutat Res.2005; 573 (1-2): 70-82) is characterized in that a probe is composed of two oligonucleotide strands, each strand comprises two parts complementary to a primer sequence and a target gene sequence, wherein one strand also comprises a tag sequence, and the end of the part complementary to the target gene sequence corresponds to the SNP site of the target gene, when the two probes are hybridized with the target gene, if the end of the part complementary to the target gene sequence in one of the probes is complementary to the SNP site of the target gene, the target gene is extended to the junction of the other probe by the template under the action of DNA polymerase, the two fragments are connected by ligase, then a primer is added and PCR is carried out by using the connected sequence as the template, finally, amplification products are detected, the SNP site sequence can be judged from the terminal base corresponding to the SNP site of the target gene in the probe. Although this method does not require the enzyme digestion method to linearize the PCR template, it does not require the extension of one of the probes to achieve the ligation of the two probes, thereby obtaining the template for the subsequent PCR.

Disclosure of Invention

The present invention aims to improve the detection efficiency of a nucleic acid amplification method for detecting a nucleic acid polymorphism.

The technical scheme for solving the technical problems is as follows:

a nucleic acid amplification method for detecting a nucleotide polymorphism, comprising the steps of:

(1) designing and preparing an LDPP probe, wherein the LDPP probe comprises an upstream oligonucleotide monomer and a downstream oligonucleotide monomer; wherein, the 5 '-end and the 3' -end of the upstream oligonucleotide monomer are respectively a universal sequence region and a complementary region, and the 5 '-end and the 3' -end of the downstream oligonucleotide monomer are respectively a complementary region and a universal sequence region; the upstream and downstream oligonucleotide monomers are complementary with the corresponding sequence of the same single-stranded nucleic acid of the target molecule in a head-to-tail adjacent mode, and in the LDPP probe-target molecule hybrid, only a gap of one base exists between the head and the tail of the upstream and downstream oligonucleotide monomers (the structure of the LDPP probe-target molecule hybrid of the invention is shown in figure 1, P1 and P2 are general sequence regions of the probe, C1 and C2 are complementary regions of the probe, and the sequence in the figure is only used for explaining the position relation of the upstream and downstream oligonucleotide monomers in the hybrid and is not the exact sequence of the probe or the target gene of the invention);

(2) setting 1-4 reaction systems, adding a target gene and at least one LDPP probe into each reaction system, and then carrying out polymerization-ligation reaction in the presence of deoxyribonucleotide triphosphates, DNA polymerase and DNA ligase, wherein the deoxyribonucleotide triphosphates in each reaction system are respectively one of dATP, dTTP, dCTP and dGTP;

(3) taking the reaction product obtained in the step (2) as a reaction template, and adding a universal primer corresponding to the LDPP probe to perform PCR amplification; one of the universal primers is the same as the sequence of the universal sequence region of one oligonucleotide monomer in the probe, and the other universal primer is complementary with the sequence of the universal sequence region of the other oligonucleotide monomer in the probe;

(4) and (4) detecting the amplification product obtained in the step (3), and performing qualitative and/or quantitative analysis according to the existence and abundance of the amplification product in each reaction system.

The LDPP probe of the method of the invention can also comprise an upstream complementary oligonucleotide monomer and a downstream complementary oligonucleotide monomer, said upstream complementary oligonucleotide monomer being complementary to part or all of the other nucleic acid sequence outside the region of complementarity of the upstream oligonucleotide monomer, and the downstream complementary oligonucleotide monomer being complementary to part or all of the other nucleic acid sequence outside the region of complementarity of the downstream oligonucleotide monomer, thereby improving the effectiveness and specificity of the complementary hybridization of the LDPP probe and the target molecule (as shown in FIG. 2, LDPP-1 and LDPP-2 are respectively an upstream oligonucleotide monomer and a downstream oligonucleotide monomer, LDPP-3 and LDPP-4 are respectively an upstream complementary oligonucleotide monomer and a downstream complementary oligonucleotide monomer; the sequence in the figure is only used for explaining the position relation of the upstream oligonucleotide monomer and the downstream oligonucleotide monomer in the hybrid and is not the exact sequence of the probe or the target gene of the invention).

The upstream complementary oligonucleotide monomer and/or the downstream complementary oligonucleotide monomer of the LDPP probe can be designed into a structure with one or more of the following characteristics:

(1) the universal sequence region and the complementary region in the upstream oligonucleotide monomer and/or the downstream oligonucleotide monomer are directly linked as shown in FIG. 3A: p1 and P2 are the universal sequence regions of the upstream and downstream oligonucleotide monomers, respectively, C1 and C2 are the complementary regions of the upstream and downstream oligonucleotide monomers, respectively; or

(2) A spacer sequence region exists between the universal sequence region and the complementary region in the upstream oligonucleotide monomer and/or the downstream oligonucleotide monomer, the spacer sequence region has universality, the minimum length of the sequence is one base, the maximum length is unlimited, the spacer sequences can be the same or similar to each other, but do not cross-hybridize with the universal primer, and the spacer sequence has no homology or lower homology with the species genome nucleic acid sequence of the object to be detected, as shown in figure 3C, S in the probe is the spacer sequence region; or

(3) A tag sequence region exists between the universal sequence region and the complementary region in the upstream oligonucleotide monomer and/or the downstream oligonucleotide monomer, the tag sequence region has self specificity, oligonucleotides with tag sequences of 10-40 mer length do not cross hybridize with each other and with the universal primer, and the tag sequence has no homology or low homology with the species genome nucleic acid sequence of the object to be detected, as shown in FIG. 3B, T in the probe is the tag sequence region; or

(4) An enzyme cutting site exists between the universal sequence region and the complementary region in the upstream oligonucleotide monomer and/or the downstream oligonucleotide monomer, as shown in FIG. 3B, and X in the probe is the enzyme cutting site.

The method can also design LDPP probes targeting different target molecules into fragments with different lengths, so that the target molecules can be qualitatively and/or quantitatively detected directly through the difference of the lengths and/or abundances of PCR amplification products, and the aim of simultaneously detecting a plurality of different target molecules is fulfilled. When the target molecule is RNA or mRNA, the complementary region of the LDPP probe of the invention can also be Oligo d (T), and the length of the Oligo d (T) is 8-40 mers.

Part or all of the bases of the LDPP probe can be base analogues or base modifications, and artificial mismatched bases can be introduced into the complementary regions of two target molecules in the LDPP probe and are natural A, T, C, G four bases or analogues thereof.

In order to facilitate detection of amplification products, modification or labeling of a phosphate group, a hydroxyl group or other similar group may be performed at the 5 '-and/or 3' -end of one or more monomers of the upstream oligonucleotide monomer, the downstream oligonucleotide monomer, the upstream complementary oligonucleotide monomer and the downstream complementary oligonucleotide monomer of the LDPP probe of the present invention; one or more than two of the four deoxyribonucleotides triphosphates added in step (2) can also be labeled with a fluorescent molecule, a luminescent group, a rare element or non-fluorescent chromophore, biotin, a digoxigenin molecule, an isotope, other similar luminescent molecules, or a labeled molecule suitable for separation or identification.

The polymerization-linking reaction of step (2) of the process of the present invention comprises the following four processes: denaturation, renaturation, polymerization and ligation. Wherein,

modification: the target molecule may be pre-denatured or denatured after addition of a series of reagents to render the target molecule single-stranded; the denaturation temperature is usually 92-99 ℃, the duration is 1 sec-10 min, the preferred temperature is 94-96 ℃, and the denaturation time is 2-5 min.

Renaturation: putting the whole polymerization-ligation reaction system at a certain temperature to ensure that the complementary regions of the LDPP probe are respectively complementarily hybridized with target molecules to form an LDPP probe-target molecule hybrid; the temperature is usually 25-72 ℃ and the duration is 30 sec-30 min; the temperature is preferably 45-65 ℃ and the duration time is 5-15 min.

③ polymerization-ligation: when the only deoxyribonucleotide triphosphate (e.g., dATP) present in the reaction system matches or is complementary to a single base (e.g., base T) at the head-to-tail gap between the upstream and downstream oligonucleotide monomers in the LDPP probe-target hybrid at an appropriate temperature, then under the action of the DNA polymerase, the DNA polymerase extends the 3 '-terminal sequence of the upstream oligonucleotide monomer in the LDPP probe-target hybrid and fills the 5' -terminal of the downstream oligonucleotide monomer in the 5 '→ 3' direction. The DNA polymerase described herein does not possess strand displacement activity and therefore, after the strand has been extended to the 5 ' -end of the downstream oligonucleotide monomer, the DNA polymerase falls off the nucleic acid template, whereupon the DNA ligase blocks the gap between the 3 ' -and 5 ' -ends of the upstream and downstream oligonucleotide monomers of the LDPP probe, converting the LDPP probe into a single, intact oligonucleotide-single stranded molecule. At this stage, extension and ligation coexist, and the single-stranded DNA molecule formed has a universal sequence that binds to the universal primer, and the single-stranded DNA molecule can be PCR-amplified using the universal primer corresponding to the universal sequence. When the LDPP probe shown in FIG. 3B is used, the single-stranded DNA molecule formed by the polymerization-ligation reaction contains a tag sequence region; when the LDPP probe shown in FIG. 3C is used, the single-stranded DNA molecule formed by the poly-ligation reaction contains a spacer region.

The DNA polymerase and DNA ligase (or other types of nucleic acid polymerases and nucleic acid ligases or other enzymes having both ligase and polymerase activity) in the polymerization-ligation reaction of the present invention may be cold or warm, or heat resistant. In addition to one kind of DNA ligase (or other types of nucleic acid ligase, or other enzymes having both ligase and polymerase activities), a mixture of 2 or more kinds of DNA ligase (or other types of nucleic acid ligase, or other enzymes having both ligase and polymerase activities) may be added to the reaction system. In addition to a DNA polymerase (or other types of nucleic acid polymerases, or other enzymes having both ligase and polymerase activities), a mixture of 2 or more DNA polymerases (or other types of nucleic acid polymerases, or other enzymes having both ligase and polymerase activities) may be added to the reaction system.

The polymerization-ligation reaction of step (2) of the method of the present invention may be carried out in multiple cycles, i.e., in four steps of denaturation, renaturation, extension, and ligation, which are repeated multiple times, to improve the efficiency of the polymerization-ligation reaction. The polymerization-ligation reaction product of step (2) may also be treated with one or more exonucleases to maintain the integrity of only the LDPP probe that has been ligated into a single entity, reducing the complexity of the polymerization-ligation reaction product.

The target molecule or the sample to be detected in step (2) of the method of the present invention refers to various nucleic acid molecules, including DNA, RNA, polynucleotide analogs of RNA or DNA, oligonucleotide analogs of RNA or DNA, nucleic acid products prepared by Polymerase Chain Reaction (PCR) or other techniques, mRNA, cDNA, nucleic acids modified by various methods including bisulfite modification, nucleic acid products treated by various restriction endonucleases including methylation-sensitive restriction endonucleases (MSRE), such as HpaII and HhaI, or other similar nucleic acid fragments.

One or more of the four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP) used in step (2) of the method of the invention are labeled with a fluorescent molecule, a luminescent group, a rare element or non-fluorescent chromophore, biotin, a digoxigenin molecule, an isotope, or other similar luminescent molecules or labeling or modifying molecules suitable for isolating or identifying PCR amplification products.

The polymerization-ligation reaction in step (2) of the method of the present invention can be carried out in 1, 2, 3 or 4 reaction tubes, respectively, but only one of the four deoxyribonucleotides triphosphates (dATP, dCTP, dGTP, dTTP) is used in each reaction tube, and the other reaction components and conditions in each reaction tube are the same, for example, when the polymerization-ligation reaction is carried out in 4 reaction tubes, dATP, dCTP, dGTP and dTTP are used in 4 reaction tubes, respectively, and the other reaction components and conditions in 4 reaction tubes are the same.

The step (3) of the method of the invention is to use the product obtained in the step (2) as a template and use a universal primer to carry out PCR amplification, wherein,

firstly, processing a polymerization-linking reaction product: after an upstream oligonucleotide monomer and a downstream oligonucleotide monomer in the LDPP probe-target molecule hybrid form a complete oligonucleotide single-stranded DNA molecule through polymerization-ligation reaction, treating a polymerization-ligation reaction product by adopting one or more exonucleases or nucleases or other enzymes, so that the complete single-stranded DNA molecule formed by only the LDPP probe in the polymerization-ligation reaction product still keeps the integrity; or collecting the polymerization-ligation reaction product using a capture elution system; or directly performing PCR amplification without treatment.

PCR amplification: the product of the polymerization-ligation reaction can be used as a PCR amplification template after the treatment steps or can be directly used as the PCR amplification template without treatment. And carrying out PCR amplification on the polymerization-ligation reaction product by using a pair of universal primers corresponding to the universal sequence of the LDPP probe in a PCR amplification system. The PCR reaction system mainly comprises: a poly-ligation reaction product, a pair of universal primers, four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP), a thermostable DNA polymerase and a suitable buffer system therefor. The use of universal primers ensures that each polymerization-ligation reaction product can be amplified equivalently.

The PCR amplification in the step (3) of the method can be carried out in 1-4 reaction systems, and the reaction templates of the PCR amplification are corresponding products obtained in the step (2); it is also possible to carry out the reaction in 1 reaction system, wherein the reaction template is a mixture of products obtained from each reaction system in the step (2).

The universal primer used in step (3) of the method of the present invention may be labeled with a fluorescent molecule, a luminescent group, a rare element or a non-fluorescent chromophore, biotin, a digoxigenin molecule, an isotope, or other similar luminescent molecules or labeling or modifying molecules suitable for separating or identifying PCR amplification products. All or part of the bases of the universal primer can be base analogues or base modifications.

One or more of the four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP) used in step (3) of the method of the invention are labeled with a fluorescent molecule, a luminescent group, a rare element or non-fluorescent chromophore, biotin, a digoxigenin molecule, an isotope, or other similar luminescent molecules or labeling or modifying molecules suitable for separating or identifying PCR amplification products; or dUTP labeled with the above-mentioned labeling molecule may be used in place of dTTP in part or in whole.

The PCR amplification product obtained in the step (3) of the method can be amplified for the second time or multiple times by using a universal primer, the reaction can be carried out in 1-4 reaction systems, and the reaction templates are the PCR amplification products obtained in the step (3); it is also possible to carry out the reaction in 1 reaction system, wherein the reaction template is a mixture of products obtained from each reaction system in the step (3).

The method for detecting the PCR amplification product in the step (4) of the method can be realized by the following modes: the amplified product is detected directly with agarose gel electrophoresis, polyacrylamide electrophoresis, capillary electrophoresis or other similar electrophoresis technology.

A detection system based on the length of the PCR amplification product: when the LDPP probes used in the reaction system targeting each target molecule have different lengths and the PCR amplification products corresponding to each target molecule have different lengths, the target molecule can be quantitatively and/or qualitatively detected using a detection system based on the lengths of the PCR products. Detection systems based on the length of the PCR amplification product include agarose gel electrophoresis, polyacrylamide electrophoresis, capillary electrophoresis, and other similar electrophoresis techniques.

Thirdly, a detection system based on the PCR amplification product label sequence: when the LDPP probes used in the reaction system and targeting different target molecules have no length difference or have small length difference but have self-specific label sequences, the target molecules can be detected quantitatively and/or qualitatively by detecting the label sequences in PCR products directly. The detection system based on the label sequence in the PCR amplification product comprises a universal chip, other similar arrays or microbead technology and the like which are fixed on the surface of a solid phase substrate, wherein the universal chip, the other similar arrays or microbead technology and the like are the same as or complementary to the label sequence in each target molecule PCR amplification product, and oligonucleotide fragments which are the same as or complementary to the label sequence in each target molecule PCR amplification product are fixed on single detection points of the universal chip, the other similar arrays or the microbead technology and the like, and after the PCR amplification products are hybridized with the universal chip, the target molecules can be detected quantitatively and/or qualitatively through the existence and the intensity values of hybrid points.

A detection system based on real-time fluorescence PCR or real-time fluorescence quantitative PCR: the target molecule is quantitatively and/or qualitatively detected by adopting a sequence which is the same as or complementary to the LDPP probe sequence, or adopting a sequence which is the same as or complementary to the target molecule sequence in a polymerization-ligation reaction product or a PCR amplification product as a target sequence and adopting a real-time fluorescent PCR or real-time fluorescent quantitative PCR technology based on a TaqMan probe, a molecular beacon or other similar technologies.

After the LDPP probe and the target molecule form an LDPP probe-target molecule hybrid, the upstream and downstream oligonucleotide monomers of the LDPP probe are complementary in head-to-tail adjacency to the corresponding sequences of the same single-stranded nucleic acid of the target molecule, in which case the upstream and downstream oligonucleotide monomers are separated by only one base between the head and tail of the head-to-tail, then filling the gap of 1 base between the 5 '-and 3' -ends of the upstream and downstream oligonucleotide monomers of the LDPP probe in the LDPP probe-target molecule hybrid by using DNA polymerase with high polymerization capability, then the gap is closed by DNA ligase, thus forming a template required by the next step of PCR amplification of the universal primer, the detection method does not need to be obtained through steps of restriction enzyme treatment or further extension and the like, so that the detection efficiency is improved, and the detection accuracy is also improved. When the polymerization-ligation reaction is performed in 4 reaction tubes and only one type of deoxyribonucleotide triphosphate is used in each reaction tube, the PCR amplified fragment corresponding to the polymerization-ligation reaction product is detected to determine whether the PCR amplified fragment is positive, so that the base of the target molecule corresponding to the deoxyribonucleotide triphosphate in the gap can be analyzed, and the high-throughput detection and analysis of nucleic acid polymorphisms such as SNP and gene mutation can be realized.

In the present invention, the following words/terms have the following meanings, unless otherwise specified:

the term "nucleic acid" as used herein refers to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), polynucleotide analogs of RNA or DNA, oligonucleotide analogs of RNA or DNA, nucleic acid products produced by Polymerase Chain Reaction (PCR) or other techniques, mRNA, cDNA, modified nucleic acids (e.g., bisulfite modified genomic DNA), nucleic acid products digested or digested with various restriction enzymes or other enzymes (e.g., digestion products of methylation-sensitive restriction endonucleases (MSRE)), and other similar nucleic acid fragments.

The "oligonucleotide" of the invention refers to a small molecule nucleic acid, which is formed by connecting (polymerizing) nucleotide residues (fragments) through phosphodiester bonds, has a molecular weight between that of nucleic acid and nucleotide, and is inclined to nucleotide. The present invention is not limited to the number of nucleotide residues.

The "monomer" of the present invention refers to a single-stranded oligonucleotide.

The term "target molecule" as used herein refers to a substance, including, but not limited to, nucleic acids, which is detected directly or indirectly by the method of the present invention.

The term "analyte" as used herein refers to any sample or analyte that contains or may contain a target molecule.

The term "qualitative detection" as used herein refers to the direct or indirect detection of the presence of a target molecule, or the direct or indirect detection of the presence of a target molecule in an analyte.

The term "quantitatively detecting" as used herein refers to directly or indirectly detecting the concentration of a target molecule, or directly or indirectly detecting the concentration of a target molecule in an analyte, for example, detecting the copy number of a target molecule in an analyte.

As used herein, "complementary region" refers to the region of an oligonucleotide sequence in a probe that is complementary to a target molecule.

As used herein, the term "universal sequence region" refers to the region of the oligonucleotide sequence of a probe corresponding to a universal primer.

The "tag sequence region" refers to a specific oligonucleotide sequence region in each LDPP probe, has self specificity, oligonucleotides with tag sequences of 10-40 mer length do not cross-hybridize with each other and with the universal primer, and the tag sequences have no homology or low homology with the species genome nucleic acid sequences of the object to be detected.

The "spacer sequence region" as used herein refers to a region of oligonucleotide sequence that is identical or similar to the region of oligonucleotide sequence present in a plurality of LDPP probes, and can vary in length among different LDPP probes, with a minimum length of one base, without strict restriction on the maximum length, spacer sequences that are identical or similar to each other but do not cross-hybridize with the universal primers, and the spacer sequences that are not or less homologous to the species genomic nucleic acid sequence of the test object.

The "restriction site region" as used herein refers to a recognition site for a specific restriction enzyme artificially designed in the LDPP probe.

The term "LDPP probe-target hybrid" as used herein refers to an LDPP probe in which upstream and downstream oligonucleotide monomers are complementarily hybridized head-to-tail adjacent to the corresponding sequence of a single-stranded nucleic acid of a target molecule, and there is only one base gap between the heads and tails of the upstream and downstream oligonucleotide monomers of the LDPP probe.

The solid phase matrix comprises glass sheets, silicon sheets, ceramic sheets, plastics, nitrocellulose, nylon membranes or rubber and the like which are treated by various methods.

As used herein, "immobilization" refers to the attachment of an oligonucleotide probe or target molecule to the surface of a solid substrate by physical adsorption and/or chemical coupling.

The term "physical adsorption" as used herein means that the oligonucleotide probe or target molecule is attached to and immobilized on the surface of the solid matrix via a secondary bond (e.g., an ionic bond), or the oligonucleotide probe or target molecule is directly or constantly adsorbed onto the surface of the solid matrix via a non-covalent bond, or the oligonucleotide probe or target molecule is immobilized via an electrostatic interaction between the phosphate anions in the metastable oligonucleotide probe and the positively charged modified layer on the surface of the solid matrix.

The "chemical coupling" in the present invention is to make the oligonucleotide probe or target molecule interact with the active group on the surface of the solid phase matrix by forming a covalent bond (such as amide bond, ester bond, ether bond, etc.), so as to fix the oligonucleotide probe or target molecule on the surface of the solid phase matrix, for example: the surface of the solid phase substrate is first activated to introduce various desired reactive groups such as amino, carboxyl, thiol, hydroxyl, halogen (including fluorine, chlorine, bromine, iodine, etc.), etc., or to derivatize nucleotides to carry appropriate functional genes, and then the oligonucleotide probes or target molecules are immobilized to the surface of the solid phase substrate by contact with bifunctional reagents or coupling activators, such as Glutaraldehyde (GA), p-nitrophenyl chloroformate (NPC), Maleimide (MA), diisothiocyanate, etc.

Drawings

FIG. 1 is the schematic diagram of the design and construction of LDPP probe.

FIG. 2 is a schematic diagram of the design principle of the upstream complementary oligonucleotide monomer and the downstream complementary oligonucleotide monomer of the LDPP probe.

FIG. 3 is a schematic diagram of the design principle of LDPP probes with various structures.

FIG. 4 is a schematic diagram of the principle of multiplex SNP detection based on the difference in length of LDPP probes.

FIG. 5 is a schematic diagram of the principle of high-throughput SNP detection of a universal biochip based on the label difference of LDPP probes.

Detailed Description

The process of the invention will be further illustrated by the following specific examples, but the process of the invention is not limited to the following limited examples. Various changes or modifications of the invention can be made by those skilled in the art based on the content of the specification and the actual needs, and these equivalents also fall within the scope of the claims appended to the present application.

The first embodiment is as follows: the multiple SNP detection method based on the LDPP probe is shown in figure 4, and the specific implementation steps are as follows:

(1) constructing an LDPP probe:

as shown in fig. 4, the LDPP probe is composed of a pair of oligonucleotide monomers (as shown in fig. 4A, the sequences in the figure are only for illustrating the position relationship of the upstream and downstream oligonucleotide monomers in the hybrid, and are not the exact sequences of the probe or target gene of the present invention), wherein the nucleic acid sequence of the 5 '-to 3' -end of the upstream oligonucleotide monomer is the universal sequence region, the spacer sequence region and the complementary region, and the nucleic acid sequence of the 5 '-to 3' -end of the downstream oligonucleotide monomer is the complementary region and the universal sequence region; in the LDPP probe-target hybrid, the upstream and downstream oligonucleotide monomers of the LDPP probe are complementary to the corresponding sequences of the same single-stranded nucleic acid of the target molecule in a head-to-tail adjacent manner, there is only one base gap between the head and tail of the complementary hybridization region between the upstream and downstream oligonucleotide monomers, and the gap corresponds well to the base where the SNP site is located (as shown in fig. 4A); since the length of the spacer sequence of the upstream oligonucleotide monomer targeting different SNP sites differs by 5nt or more (as shown in FIG. 4C), the PCR amplification products corresponding to different SNP sites can be directly determined by the length of the PCR product.

(2) Polymerization-linking reaction:

the poly-ligation reactions were performed in 4 reaction tubes (as shown in FIG. 4B), each using only one of the four deoxyribonucleotides triphosphates (dATP. dTTP, dCTP and dGTP) (dXTP in FIG. 4B), and the remaining reaction components were the same. Therefore, since only one deoxyribonucleotide triphosphate is used in each reaction tube, the polymerization-ligation reaction product of each reaction tube represents whether a corresponding base exists at a SNP site, and at the same time, since 4 reaction tubes take into account the possible bases existing at each SNP site, it is suitable for the detection of all SNP sites.

(3) Universal primer PCR amplification

And (3) respectively carrying out PCR amplification on the polymerization-ligation reaction products in the 4 reaction tubes in the step (2) in the 4 reaction tubes, wherein the upstream primer (or the downstream primer) in the universal primer in each reaction tube is labeled with fluorescent molecules with different colors, and the rest reaction components are the same. Since only one deoxyribonucleotide triphosphate (dXTP in FIG. 4B) is used in each polymerization-ligation reaction tube, and the fluorescence color of the universal primer label in each reaction tube is different in the amplification at this step, it can be judged whether a base corresponding to the added deoxyribonucleotide triphosphate exists at the SNP site by the fluorescence color; in addition, since the length of the spacer sequence of the upstream oligonucleotide monomer of the LDPP probe targeting different SNP sites is different, the length of the PCR amplification product of different SNP sites is also different, therefore, which SNP site is detected can be finally judged according to the length of the PCR amplification product, and the specific existence condition of the base of the SNP site can be judged according to the fluorescence color of the PCR amplification product with the specific length.

(4) Detection of PCR amplification product

Mixing the PCR amplification products in the 4 reaction tubes in the step (3) into one reaction tube, performing capillary electrophoresis on the mixed solution of the PCR amplification products in a capillary electrophoresis mode, and finally judging the type and the base condition of the SNP site according to the size of the fragment and the fluorescence color of the fragment (as shown in FIG. 4D).

Example two: general biochip high-throughput SNP detection method based on LDPP probe

As shown in fig. 5, the specific implementation steps are as follows:

(1) construction of LDPP Probe

The LDPP probe consists of a pair of oligonucleotide monomers (shown in figure 5A), wherein the nucleic acid sequence from 5 '-end to 3' -end of the upstream oligonucleotide monomer is a universal sequence region, a label sequence region, a restriction enzyme site region and a complementary region in sequence, the restriction enzyme site region contains a specific restriction enzyme site (X in figure 5A), and the nucleic acid sequence from 5 '-end to 3' -end of the downstream oligonucleotide monomer is a complementary region and a universal sequence region in sequence; in the LDPP probe-target hybrid, the upstream and downstream oligonucleotide monomers of the LDPP probe are complementary to the same single-stranded nucleic acid sequence of the target molecule in a head-to-tail adjacent manner, and there is only one base gap between the head and tail of the complementary hybridization region for the upstream and downstream oligonucleotide monomers, and the gap corresponds well to the base where the SNP is located (fig. 5A); the tag sequences of the upstream oligonucleotide monomers targeting different SNP sites have self-specificity, so that PCR amplification products corresponding to different SNP sites can be judged directly through the tag sequences of the PCR amplification products.

(2) Polymerization-linking reaction:

the poly-ligation reactions were performed in 4 reaction tubes (as shown in FIG. 5B), each using only one of the four deoxyribonucleotides triphosphates (including dATP, dTTP, dCTP, and dGTP) (dXTP in FIG. 5B), and the remaining reaction components were the same. Since only one deoxyribonucleotide triphosphate is used in each reaction tube, the polymerization-ligation reaction product of each reaction tube represents whether a corresponding base exists at a SNP site, and at the same time, since 4 reaction tubes take into account the possible bases existing at each SNP site, it is suitable for the detection of all SNP sites.

(3) Universal primer PCR amplification

And (3) performing PCR amplification on the polymerization-ligation reaction products in the 4 reaction tubes in the step (2) in the 4 reaction tubes respectively, wherein the upstream primer (or the downstream primer) in the universal primers in each reaction tube is labeled with fluorescent molecules with different colors, and the rest reaction components are the same (as shown in FIG. 5B). Since only one deoxyribonucleotide triphosphate (dXTP in FIG. 4B) is used in each polymerization-ligation reaction tube, and the fluorescence color of the universal primer label in each reaction tube is different in the amplification at this step, it can be judged whether a base corresponding to the added deoxyribonucleotide triphosphate exists at the SNP site by the fluorescence color; in addition, because the tag sequences of the upstream oligonucleotide monomers of the LDPP probes targeting different SNP sites have self-specificity, the detected SNP sites can be finally judged according to the tag sequences of PCR amplification products, and the specific existence condition of the bases of the SNP sites can be judged according to the fluorescence color of the PCR amplification products of the specific tag sequences. (4) Detection of PCR amplification product

Mixing PCR amplification products in the 4 reaction tubes in the step (3) into one reaction tube, and detecting by adopting a universal biochip, wherein the specific steps are as follows (as shown in FIG. 5C):

immobilizing oligonucleotides complementary to the tag sequence in the LDPP probe on the surface of the solid phase matrix by physical adsorption and/or chemical coupling, wherein part or all of the sequence in the oligonucleotide probe is complementary to the tag sequence in the LDPP probe.

Mixing the PCR amplification products in the 4 reaction tubes in the step (3) into one reaction tube, and treating the PCR amplification products by using restriction enzymes corresponding to specific restriction enzyme recognition sites (X in the figure 5A) in the enzyme cutting site region of the oligonucleotide monomer at the upstream of the LDPP probe.

Hybridizing the enzyme digestion product obtained in the step II with the chip prepared in the step I, and then judging the type and base distribution condition of the SNP according to the type of the label sequence and the fluorescence color and signal intensity of the hybrid point.

Claims

1. A nucleic acid amplification method for detecting a nucleic acid polymorphism, comprising the steps of:

(1) designing and preparing an LDPP probe, wherein the LDPP probe comprises an upstream oligonucleotide monomer and a downstream oligonucleotide monomer; wherein, the 5 '-end and the 3' -end of the upstream oligonucleotide monomer are respectively a universal sequence region and a complementary region, and the 5 '-end and the 3' -end of the downstream oligonucleotide monomer are respectively a complementary region and a universal sequence region; the upstream and downstream oligonucleotide monomers are complementary in head-to-tail adjacency to the corresponding sequences of the same single nucleic acid strand of the target molecule, and in the LDPP probe-target molecule hybrid, there is only a one-base gap between the head-to-tail of the upstream and downstream oligonucleotide monomers;

(2) setting 1-4 reaction systems, adding a target molecule and at least one LDPP probe into each reaction system, and then carrying out polymerization-ligation reaction in the presence of deoxyribonucleotide triphosphates, DNA polymerase and DNA ligase, wherein the deoxyribonucleotide triphosphates in each reaction system are respectively one of dATP, dTTP, dCTP and dGTP;

2. The method of claim 1, wherein said LDPP probe further comprises an upstream complementary oligonucleotide monomer complementary to part or all of the other nucleic acid sequence except the complementary region of the upstream oligonucleotide monomer and a downstream complementary oligonucleotide monomer complementary to part or all of the other nucleic acid sequence except the complementary region of the downstream oligonucleotide monomer.

3. The method of claim 1, wherein the universal sequence region and the complementary region of the upstream oligonucleotide monomer and/or the downstream oligonucleotide monomer of the LDPP probe are directly linked.

4. The method of claim 1, wherein a spacer sequence region is present between the universal sequence region and the complementary region in the upstream oligonucleotide monomer and/or the downstream oligonucleotide monomer in the LDPP probe, the spacer sequence region has universality, the minimum length of the sequence is one base, the maximum length is unlimited, the spacer sequences can be the same or similar to each other, but have no cross hybridization with the universal primer, and the spacer sequence has no homology or low homology with the species genome nucleic acid sequence of the object to be detected.

5. The method as claimed in claim 1, wherein a tag sequence region exists between the universal sequence region and the complementary region in the upstream oligonucleotide monomer and/or the downstream oligonucleotide monomer in the LDPP probe, the tag sequence region has self-specificity, oligonucleotides with tag sequences of 10-40 mer length do not cross-hybridize with each other and with the universal primer, and the tag sequences have no homology or low homology with the species genome nucleic acid sequence of the substance to be detected.

6. The method of claim 1, wherein said LDPP probe comprises a cleavage site between said universal sequence region and said complementary region in said upstream oligonucleotide monomer and/or said downstream oligonucleotide monomer.

7. The method of claim 1, wherein LDPP probes targeting different target molecules are designed to have different lengths.

8. The method of claim 1, wherein when the target molecule is RNA or mRNA, the complementary region of the LDPP probe is also Oligo d (T) which is 8-40 mer in length.

9. The method of claim 1, wherein the one or more monomers selected from the group consisting of the upstream oligonucleotide monomer, the downstream oligonucleotide monomer, the upstream complementary oligonucleotide monomer and the downstream complementary oligonucleotide monomer of the LDPP probe are modified or labeled at the 5 '-and/or 3' -end with a phosphate group, a hydroxyl group or the like.

10. The method of claim 1, wherein some or all of the bases of the LDPP probe are base analogues or base modifications.

11. The method of claim 1, wherein the two target molecule complementary regions of the LDPP probe incorporate artificial mismatched bases comprising A, T, C, G four bases or analogues thereof, which are naturally occurring.

12. The method according to claim 1, wherein one or more of the four deoxyribonucleotides triphosphates added in step (2) and/or step (3) are labeled with a fluorescent molecule, a luminescent group, a rare element or non-fluorescent chromophore, biotin, a digoxigenin molecule, an isotope, other similar luminescent molecules, or a labeled molecule suitable for separation or identification.

13. The method according to claim 1, wherein the number of the reaction systems in the step (2) is 4, and the deoxyribonucleotide triphosphates used in each reaction system are dATP, dTTP, dCTP and dGTP, respectively.

14. The method according to claim 1, wherein the upstream primer or the downstream primer of the universal primer used in step (3) is labeled with a fluorescent molecule, a luminescent group, a rare element or non-fluorescent chromophore, biotin, a digoxigenin molecule, an isotope, or other similar luminescent molecules or labeling molecules suitable for separating or identifying PCR amplification products.

15. The method according to claim 1, wherein the method for detecting the amplification product in step (4) is:

(1) detecting the PCR amplification product by agarose gel electrophoresis, polyacrylamide electrophoresis, capillary electrophoresis and other similar electrophoresis techniques; or

(2) Detecting PCR amplification products by using a universal chip, other similar arrays or a microbead technology, wherein the universal chip, the other similar arrays or the microbead technology is fixed with oligonucleotides which are the same as and/or complementary to the label sequence of the LDPP probe on the surface; or

(3) Detecting the polymerization-ligation reaction product by using TaqMan probe, molecular beacon or other similar real-time fluorescent PCR or real-time fluorescent quantitative PCR technology; or

(4) The PCR amplification product is detected by TaqMan probe, molecular beacon or other similar real-time fluorescent PCR or real-time fluorescent quantitative PCR technology.