CN103898199B - A kind of high-throughput nucleic acid analysis method and application thereof - Google Patents

Abstract

The present invention relates to a high-throughput gene analysis method and its application. Specifically, it includes the step of: for each target nucleic acid fragment for n types of target nucleic acid fragments to be analyzed, providing different binding regions that bind to the target nucleic acid fragments At least 2 specific probes, each of the specific probes has a specific binding region and a general sequence region, and the sequence of the specific binding region is complementary to the sequence of the binding region of the target nucleic acid fragment, and the general sequence region The sequence corresponds to the sequencing primer sequence of the high-throughput single-molecule or single-molecule amplification cluster sequencing platform, wherein n is a positive integer ≥ 40; the nucleic acid sample containing the target nucleic acid fragment to be analyzed is hybridized with the probe, and Ligate the probes to obtain a mixture of probe ligation products; use the sequencing primers to sequence and analyze the probe ligation product mixture or its amplification products, thereby realizing the quantitative analysis of high-throughput target gene fragments Purpose.

Description

A high-throughput nucleic acid analysis method and its application

technical field

The invention belongs to the field of biotechnology and molecular diagnosis, in particular, the invention relates to a high-throughput nucleic acid analysis method and its application.

Background technique

Gene is the material basis of heredity, and it is a specific nucleotide sequence with genetic information on DNA or RNA molecules. Except for some viruses whose genetic material is RNA, the genetic material of almost all non-viral organisms is DNA. Different species have their specific gene sequences, so by detecting the gene sequences in a sample, the biological species in the sample can be judged.

In the process of life, genes are transcribed into mRNA through DNA, and then use mRNA as a template to translate biologically active protein molecules, thereby expressing the genetic information stored in the DNA sequence. By analyzing the amount of each mRNA in different tissues and combining the differences in physiological functions of different tissues, the function of genes can be understood. Therefore, gene expression analysis is one of the most basic research methods for molecular biology to study gene function.

Gene expression is coordinated by a variety of regulatory factors, among which DNA methylation is one of the important ways to regulate gene expression. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the interaction between DNA and proteins, thereby achieving the purpose of controlling gene expression. In most cases, methylation mainly occurs on the 5th carbon atom of the cytosine ring of the cytosine nucleotide in the CpG sequence.

In addition, errors may occur during gene replication to produce "mutations", such mutations include point mutations, large deletions/duplications (called copy number polymorphisms, CNVs), gene inversions, or gene translocations. Some mutations will seriously affect the function of key genes and cause diseases. Due to selection, although the frequency of such mutations in the population is very low, a considerable part of the mutations do not seriously affect gene functions or the affected genes do not cause survival for individuals. They will remain in the population and become a genetic polymorphism in the population due to random drift and frequency changes of the founder effect. The polymorphism with single base or oligobase change is called Single nucleotide polymorphisms (SNPs), and deletions or duplications of large segments are called copy number polymorphisms (CNPs). Genetic polymorphism and gene mutation analysis are the most common genetic analysis methods for studying gene function and the pathogenic mechanism of genetic diseases.

Therefore, gene identification, gene expression analysis, DNA methylation analysis, mutation screening, SNP typing, CNP typing and CNV detection are important molecular genetic research methods, and are also widely used in clinical molecular diagnosis. Because of the importance of these genetic assays, scientists and engineers have developed multiple assays for each assay.

Early detection methods focused on the analysis of limited fragments of interest. Use PCR amplification to identify the target gene, or use real-time fluorescent quantitative PCR to identify gene expression level, virus content, gene copy number and methylation level. Common DNA methylation analysis is mainly for methylation sequencing or methylation-specific PCR analysis of DNA after sulfurous acid treatment. Mutation screening mainly uses PCR amplification and Sanger sequencing, and then obtains the mutation status by comparing the sequencing sequence with the reference sequence. There are also many methods for SNP detection, such as TaqMan probe allele detection technology, restriction endonuclease reaction (RFLP), high-resolution melting curve reaction, single base extension technology (time-of-flight mass spectrometry platform, MultiplexSNaPshot), High-temperature ligase detection technology (LDR, SNPscan), etc. Medium and small throughput CNV detection methods mainly include real-time quantitative PCR, FISH, multiplex ligation probe amplification (MLPA), multiplex fluorescent competitive PCR (AccuCopy), etc. The above method is highly flexible, but the biggest defect is that the throughput is too low, and it is powerless for research projects or diagnostic needs that need to detect a large number of gene loci.

Microarray chip (Microarray) is characterized by high-density probe arrays. These microarrays are "printed" with a large number of DNA probes with known partial sequences. Using the principle of molecular hybridization, various processed fluorescently labeled samples are combined with the microarray The probes are hybridized, and then washed to remove non-specific hybridization signals. Finally, a scanner is used for fluorescence detection, and the signal amount related to the target gene is confirmed according to the intensity of the fluorescence signal and the position of the array where the fluorescence signal is located. The chip can simultaneously analyze tens of thousands or even millions of gene fragments or polymorphic sites, and is widely used in species identification, expression profile analysis, high-throughput SNP analysis, genome-wide methylation level analysis, and global DNA analysis. Genome copy number analysis and more. The biggest advantage of microarray chips is high throughput, which can analyze gene changes at the whole genome level, but its disadvantages are that due to the ubiquitous non-specific hybridization, the accuracy of quantification is poor, and expensive hybridization and scanning instruments are required. High and the custom chip takes a long time and is expensive, and it is impossible to detect unknown genes.

The emergence of second-generation sequencing technology has brought about a revolutionary change in the field of genetic testing. The main principle of the second-generation sequencing technology is sequencing after single-molecule PCR amplification on the chip, such as MiSeq, GAIIx, and Hiseq2000 sequencers from Illumina, Ion PGM and Solid sequencers from ABI, and 454 GSFLX sequencers from Roche. The second-generation sequencing technology can realize the sequencing of millions or even hundreds of millions of single-molecule amplification products at the same time. It is widely used in genome resequencing to quickly identify disease-causing genes, transcriptome analysis, methylation profile, microRNA identification, Genome-wide protein-DNA interaction research and genome sequencing of new species, etc.

A new generation of single-molecule direct sequencing technology is also under rapid research and development, and the main representative companies are PacificBiosciences and Helicos. The biggest advantage of this high-throughput sequencing technology is that the throughput is very high, and it can simultaneously identify and quantify known or unknown genes, so the specificity and efficiency are very high. But there are also some shortcomings, mainly compared with conventional sequencing, the accuracy of next-generation sequencing is slightly worse, mutations introduced by single-molecule amplification will affect the final result analysis, and this technology platform is suitable for the entire genome or transcription For detection and analysis of a target region or a group of genes, it is necessary to enrich the target gene segment in the sample in advance. The enrichment methods currently used include multiplex PCR and microfluidic digital PCR for limited gene regions, while methods for a large number of gene regions mainly use high-density probe sequences covering the target region to perform solid-phase or liquid-phase hybridization with samples. enrichment of target regions. These enrichment techniques are mainly used for mutation detection of candidate genes, but because these enrichment processes eliminate the proportional relationship between the product and the amount of the original template to a certain extent, the quantitative analysis of the enriched candidate gene fragments cannot be accurately realized, such as Expression and copy number analysis.

Therefore, there is still a lack of effective detection methods for gene detection in this field, especially gene identification, gene expression analysis, DNA methylation analysis, mutation screening, SNP typing, CNP typing and CNV detection, so there is an urgent need Development of an efficient method for high-throughput genetic analysis.

Contents of the invention

The main purpose of the present invention is to provide a high-throughput gene analysis method and its application.

In a first aspect of the present invention, a high-throughput nucleic acid analysis method is provided, comprising the steps of:

(1) For the n kinds of target nucleic acid fragments to be analyzed, for each target nucleic acid fragment, at least 2 specific probes that bind to different binding regions of the target nucleic acid fragments are provided, and each of the specific probes has a specific binding region and a universal sequence region, and the sequence of the specific binding region is complementary to the sequence of the binding region of the target nucleic acid fragment, and the sequence of the universal sequence region corresponds to the sequence of the sequencing primer, wherein n is a positive integer ≥ 40;

(2) hybridize the nucleic acid sample containing the target nucleic acid fragment to be analyzed with the probe described in step (1), and connect the probes to obtain a mixture of probe ligation products, wherein 3 of each probe ligation product Both ' and 5' ends are the general sequence regions whose sequences correspond to the sequences of the sequencing primers;

(3) Sequencing and/or analyzing the mixture of probe ligation products in step (2), so as to obtain the information of the target nucleic acid.

In another preferred example, the sequencing primer is a sequencing primer of a high-throughput single-molecule or single-molecule amplification cluster sequencing platform.

In another preferred example, n is a positive integer ≥ 100, preferably: a positive integer selected from 1000-10000.

In another preferred example, the sequence of the universal sequence region corresponds to the sequencing primer sequence means: the sequence of the universal sequence region is completely identical or at least 8 bp identical to the sequence of the sequencing primer, or the sequence of the universal sequence region is completely complementary to the sequence of the sequencing primer or Complementary at least 8bp.

In another preferred example, the specific probe also has one or more characteristics selected from the following group:

(1) The length of the specific probe is ≤100bp, preferably 30-70bp, more preferably 40-50bp.

(2) The length of the specific binding region of the specific probe is ≤50 bp, preferably 15-35 bp, more preferably 20-25 bp.

(3) The length of the general sequence region of the specific probe is ≥ 8 bp, preferably 15-35 bp, more preferably 20-25 bp.

(4) The sequence of the universal sequence region of the specific probe also corresponds to the sequence of the amplification primer;

(5) The specific probe includes a tag sequence.

In another preferred example, the tag sequence is a sequence (preferably 3-30, more preferably 6-9) of specific bases, which is used to distinguish probe ligation products from different sample sources.

In another preferred example, the two probes corresponding to each target nucleic acid fragment are: a 5' end probe and a 3' end probe. The binding region at the 'end is complementary, and the probe at the 3' end is complementary to the binding region at the 5' end of the target nucleic acid fragment to be analyzed.

In another preferred example, the structure of the 5' end probe or the 3' end probe is shown in formula I:

5'-A-L-B-3'

Formula I

In Formula I,

A represents the general sequence region;

B represents the specific binding region;

L represents the nucleic acid linking sequence between A and B;

Among them, the positions of A and B can be interchanged.

In another preferred example, said L is 0 bases.

In another preferred example, the connection relationship between the 5' end probe and the 3' end probe is selected from one or more of the following groups:

(a) The 5' end probe and the 3' end probe are adjacent probes: after the 5' end probe and the 3' end probe hybridize with the target nucleic acid fragment to be analyzed, the distance between the two is 0 Bases are connected under the action of ligase to obtain probe ligation products;

(b) The distance between the 5' end probe and the 3' end probe is 1-500 bases: after the 5' end probe and the 3' end probe are hybridized with the target nucleic acid fragment to be analyzed, the DNA polymerase Gap polymerization and ligation under the action of ligase to obtain probe ligation products;

(c) In addition to the 5' end probe and the 3' end probe, the hybridization system also includes probe 3, which is adjacent to the 5' end probe and the 3' end probe respectively, and the three probes After hybridizing with the target nucleic acid fragment to be analyzed, it is ligated under the action of ligase, so as to obtain the probe ligation product.

In another preferred example, the length of the probe 3 is 1-500bp, preferably 15-35bp, more preferably 20-25bp.

In another preferred embodiment, the 5' end of the 3' end probe described in (a) is phosphorylated.

In another preferred example, the 3' end of the 3' end probe and the 5' end of the 5' end probe described in (a) are modified and protected against exonucleases.

In another preferred example, the anti-exonuclease modification is a thio modification.

In another preferred example, in (b), the distance between the 5' end probe and the 3' end probe is preferably 1-10 bases.

In another preferred example, in (b), the DNA polymerase has no 5'-3' exonuclease activity.

In another preferred example, a step is further included between step (2) and step (3): amplifying the probe ligation product obtained in step (2).

In another preferred embodiment, in step (3), the mixture of probe ligation products obtained in step (2) is directly sequenced using a high-throughput single-molecule or single-molecule amplification cluster sequencing platform; or the probe The amplification products of the mixture of ligated products are sequenced using a high-throughput single molecule or single molecule amplification cluster sequencing platform.

In another preferred embodiment, in step (3), the mixture of probe ligation products or its amplification products are sequenced and analyzed by third-generation sequencing technology or second-generation sequencing technology.

In another preferred example, in step (3), the information of obtaining the target nucleic acid refers to one or more information selected from the following group: SNP typing information, DNA methylation information, mutation screening information , CNP typing information, CNV information, pathogenic microorganism gene information, genetic information of transgenic animal and plant products, gene expression level.

In the second aspect of the present invention, a high-throughput SNP typing method is provided, comprising the steps of: using the method described in the first aspect to perform sequencing and SNP analysis on the mixture of probe ligation products derived from the sample to be tested, Obtain the SNP typing information of the target nucleic acid.

In another preference, the high-throughput SNP typing method includes the steps of:

(1) For the n kinds of target nucleic acid fragments to be analyzed, for each target nucleic acid fragment, 3 specific probes that bind to different binding regions of the target nucleic acid fragment are provided: 2 5' end probes and 1 3 ' end probe, the 5' end probe is an allele-specific probe, and the last base corresponds to the corresponding allele base, and the 3' end probe is a shared probe, wherein n is a positive integer ≥ 40;

(2) hybridize the nucleic acid sample containing the target nucleic acid fragment to be analyzed with the probe described in step (1), and connect the probes to obtain a mixture of probe ligation products, wherein 3 of each probe ligation product Both ' and 5' ends are the general sequence regions whose sequences correspond to the sequences of the sequencing primers;

(3) Sequencing and analyzing the mixture of probe ligation products in step (2) by using the sequencing primers to obtain the SNP typing information of the target nucleic acid.

In the third aspect of the present invention, a method for detecting CNV is provided, comprising the steps of: using the method described in the first aspect to perform sequencing and CNV analysis on the mixture of probe ligation products derived from the sample to be tested to obtain the target nucleic acid CNV information.

In another preference, the method for detecting CNV includes the steps of:

(1) Design specific probes for each target gene fragment (preferably design 2 probes, 1 5' end probe and 1 3' end probe);

(2) Denaturing-annealing-ligating the ligation probes of all target gene fragments with the DNA template (preferably performing multiple denaturation-annealing-ligation cycles);

(3) The ligation product is amplified by PCR or directly digested with nuclease without amplification, and the amplified products of different samples are mixed for next-generation high-throughput chip sequencing;

(4) Sequencing data analysis to obtain the target gene copy number of the sample.

In the fourth aspect of the present invention, a high-throughput methylation analysis method is provided, comprising the steps of: using the method described in the first aspect to sequence and methylate the mixture of probe ligation products derived from the sample to be tested. Methylation analysis to obtain the methylation information of the target nucleic acid.

In another preferred example, the high-throughput methylation analysis method includes the steps of: treating the genomic DNA with a methylation-sensitive restriction endonuclease, designing probes for the cutting point, and using the method described in claim 1 The amount of uncut genomic DNA was detected by the method described above.

In another preferred example, the high-throughput methylation analysis method includes the steps of: performing sulfite treatment on genomic DNA, designing methylation-specific probes and non-methylation-specific probes for target gene segments, By detecting the amount of the ligation product of the two probes, the methylation level of the target gene segment is obtained.

In the fifth aspect of the present invention, a method for detecting gene expression is provided, comprising the step of: using the method described in the first aspect for detection.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

The following drawings are used to illustrate specific embodiments of the present invention, but not to limit the scope of the present invention defined by the claims.

Figure 1 shows the technical idea 1 of high-throughput measurement in a specific embodiment of the present invention.

Fig. 2 shows the technical idea 2 of high-throughput measurement in a specific embodiment of the present invention.

Figure 3 shows the workflow for high-throughput SNP typing based on high-throughput ligation product detection technology based on single-molecule direct or post-amplification sequencing.

Figure 4 shows the workflow for high-throughput CNV detection based on high-throughput ligation product detection technology based on single-molecule direct or post-amplification sequencing.

Figure 5 shows the flow of high-throughput ligation product detection technology based on single-molecule direct or post-amplification sequencing for high-throughput target gene mutation screening.

Figure 6 shows the workflow of high-throughput ligation product detection technology based on single-molecule direct or post-amplification sequencing for high-throughput candidate gene expression analysis.

Figure 7 shows the workflow of high-throughput ligation product detection technology based on single-molecule direct or post-amplification sequencing for high-throughput gene methylation level analysis.

FIG. 8 shows the detection results of the exon deletion and duplication of the DMD gene in Example 2.

detailed description

After extensive and in-depth research, the inventors first utilized the high specificity of the multiple ligation probe amplification technology and the good preservation of the quantitative information of the target fragments, and used the next-generation high-throughput sequencing technology platform to amplify the ligation probes. The products are identified and quantified by sequencing, so as to realize the quantitative analysis of high-throughput target gene fragments. The present invention has been accomplished on this basis.

Specifically, it includes the step of: for each target nucleic acid fragment to be analyzed, at least 2 specific probes that bind to different binding regions of the target nucleic acid fragment are provided, and each of the specific probes It has a specific binding region and a universal sequence region, and the sequence of the specific binding region is complementary to the sequence of the binding region of the target nucleic acid fragment, and the sequence of the universal sequence region corresponds to the sequence of the sequencing primer, wherein n is ≥ 40 positive Integer; the nucleic acid sample containing the target nucleic acid fragment to be analyzed is hybridized with the probe, and the probe is ligated to obtain a mixture of probe ligation products, wherein the 3' and 5' ends of each probe ligation product are both It is a universal sequence region whose sequence corresponds to the sequencing primer sequence; using the sequencing primer, the mixture of probe ligation products is sequenced and analyzed, thereby realizing quantitative analysis of high-throughput target gene fragments.

Multiplex Ligation Probe Amplification (MLPA)

Multiple ligation probe amplification is a technology that can accurately detect the number of target gene fragment molecules. Its basic process includes hybridization between the probe and the target nucleic acid sequence, followed by ligation, PCR amplification, product capillary electrophoresis and data collection, and analysis software Analyze the collected data and draw conclusions.

The MLPA probe is an oligonucleotide fragment including a primer sequence and a specific sequence. In the MLPA reaction, both hybridize to the target sequence, after which the two-part probe is ligated using ligase. The ligation reaction is highly specific. Only when the two probes are fully hybridized to the target sequence, that is, the target sequence is completely complementary to the probe-specific sequence, can the ligase link the two probes into a complete nucleic acid single strand; otherwise, if the target The sequence is not completely complementary to the probe sequence, even if there is only one base difference, it will lead to incomplete hybridization, so that the ligation reaction cannot be carried out or the ligation efficiency is greatly reduced.

After the ligation reaction is completed, a pair of universal primers are used to amplify the connected probes. The length of the amplification product of each probe is unique, ranging from 100 to 480 base pairs, and then separated and amplified by capillary electrophoresis. The product is analyzed by special software and a conclusion is drawn.

Only when the ligation reaction is completed, can the subsequent PCR amplification be carried out, and the amplification peak of the corresponding probe can be collected. Deletion, decrease or increase. Therefore, according to the change of the amplification peak, it can be judged whether there is an abnormal copy number or a point mutation in the target sequence.

The advantage of multiple ligation probe amplification technology is that the specificity of probe ligation is very high, so the analysis of multiple target gene fragments can be realized in one system at the same time, and there is a direct proportional relationship between the amount of ligation products and the amount of the original template, At the same time, because the ligation products of different gene fragments are amplified with universal primers, the amount of the amplified product well retains the information of the amount of the original template, and this method can detect the amount of the target gene of the original template through terminal analysis of the ligated PCR product.

Multiplex ligation probe amplification has been applied to research in many fields, including chromosomal aneuploidy changes, SNPs, point mutations, gene rearrangements of chromosomal subtelomeres, and the detection of common childhood genetic diseases.

The main disadvantages of this method are: 1. The ligation products usually have different lengths, and a pair of general-purpose PCR fluorescent primers are used to amplify, and the amplification amount of different sites is determined by electrophoresis technology according to the different lengths of the fluorescently labeled PCR products. This greatly limits the number of detection sites in a reaction system, and can only detect 40-50 nucleotide sequences at the same time, and the throughput is low; 2. The ligation probe sequence is usually very long (>100bp) and cannot be directly synthesized. It can be prepared by M13 clones, which is relatively cumbersome; 3. The ligation probe sequence is very long, and the length difference between the ligation probes and ligation products at different sites can reach hundreds of bases, so the ligation efficiency and amplification between different sites There will be large differences and fluctuations in efficiency, which will affect the detection accuracy.

High-throughput genetic analysis methods

The invention provides a high-throughput gene analysis method. The technical idea of this method is as follows:

Idea 1 (Figure 1): Take the analysis of two target gene fragments (F1 and F2) as an example, including the following steps:

1. Design specific DNA probes for target nucleic acid fragments. There are three optional methods for probe design:

The first method is to design two adjacent probes (probe 1 and probe 2) for each target fragment, one is the 5' end probe (i.e. probe 1) and the other is the 3' end probe (i.e. Probe 2). The first half of the 5’-end probe sequence (a of probe 1) is the general sequence consistent with the subsequent PCR amplification primers, while the second half (b1 of probe 1) is a specific sequence that hybridizes with the target nucleic acid fragment. The 5' end of the 3' end probe is modified by phosphorylation, the first half (b1 of probe 2) is the specific sequence that hybridizes with the target nucleic acid fragment, and the second half (a of probe 2) is the primer phase for subsequent PCR amplification. Consistent universal sequence. After the two probes hybridize with the template DNA, they are ligated under the action of ligase.

The second method also designs two probes (probe 1 and probe 2), the structure of the probe is the same as the method 1, but there is a distance of several to tens of bases between the two probes (the distance is optional 1-500bp, preferably 1-10bp), after the probe is hybridized to the template DNA, it is extended under the action of a polymerase without 5'->3' exonuclease activity, and the gap between the two probes is filled , and ligated under the action of ligase.

The third method is to design 3 pairs of probes (probe 1, probe 2 and probe 3), the structure of the 5' end and 3' end probes (probe 1 and probe 2) is the same as the first method, but this There are dozens to hundreds of bases distance (preferably 20-25bp) between the two probes, the 5' end of the middle probe (probe 3) is phosphorylated, just in line with the 5' end and 3' end The gaps of the probes are matched, and the three probes are ligated under the action of ligase after hybridization with the template DNA. In order to increase the amount of ligation products, it is preferable to use a high-temperature thermostable polymerase such as TaqDNA ligase to perform multiple cycles of denaturation-annealing-ligation.

2. Use a pair of PCR primers that match the amplification primers or sequencing primers of the next-generation sequencing platform to amplify the ligation product to obtain the target gene fragment containing the complete specific sequence.

Preferably, the PCR primer has a tag sequence (ie index) with a length of several to tens of bases, and the ligation products of different samples can be amplified with PCR primers with different tag sequences, so that the amplified products of different samples They can be mixed together, and the sequencing sequences can be classified into different samples according to the tag sequence in the subsequent sequencing data.

3. Use the next-generation high-throughput chip sequencing platform to perform single-molecule amplification sequencing or direct single-molecule sequencing of the amplification products of the ligation probe;

4. Analyze the sequencing data to realize the sample classification of the sequencing sequence, the classification of the gene locus and the quantification of the corresponding connection products of each gene fragment.

First, the sequence obtained by sequencing is assigned to the corresponding sample according to the tag sequence, and then assigned to the ligation product of the corresponding gene fragment according to the base composition of each sequence, and the number of sequencing sequences of each ligation product can be estimated to estimate the gene Relative amounts of fragment ligation products.

Idea 2 (Figure 2): Take the analysis of two target gene fragments (F1 and F2) as an example, including the following steps:

1. Design specific DNA probes for target nucleic acid fragments. There are three optional methods for probe design:

The first approach is to design two adjacent probes (Probe 1 and Probe 2), one at the 5’ end (Probe 1) and the other at the 3’ end (Probe 2). The first half of the 5'-end probe sequence is a universal sequence that matches the amplification primer or sequencing primer of the next-generation sequencing platform, while the second half is a specific sequence that hybridizes with the target nucleic acid fragment, and the 5'-end of the 3'-end probe is phosphated The first half is a specific sequence that hybridizes with the target nucleic acid fragment, the second half is a universal sequence that matches the amplification primer or sequencing primer of the next-generation sequencing platform, and a few bases at the 5' end of the 5' end probe are modified. Thio-modification or other protective group modification to prevent exonuclease degradation, a few bases at the 3' end of the 3'-end probe are modified by thio-modification or other protective groups to prevent exo-accurate degradation. After the probes hybridize to the template DNA, they are ligated under the action of ligase.

The second method also designs two probes, the probe structure is the same as method one, but there are several to tens of base distances between the two probes (the distance can be 1-500bp, preferably 1-10bp )), after the probe is hybridized to the template DNA, it is extended under the action of a polymerase without 5'->3' exonuclease activity, the gap between the two probes is filled, and then ligated under the action of ligase.

The third method is to design 3 pairs of probes, the structure of the 5' end and the 3' end probe is the same as the method one, but there are tens to hundreds of base distances between the two probes (preferably 20- 25bp), the 5' end of the middle probe is phosphorylated, just matching the gap between the 5' end and the 3' end probe. Usually, a tag sequence of several to tens of bases is added to the probe at the 5' end or 3' end, and the ligation products of different samples have different tag sequences, so that the ligation products of different samples can be mixed together , in the subsequent sequencing data, the sequencing sequence can be classified into different samples according to the tag sequence. After the three probes are hybridized with the template DNA, they are ligated under the action of ligase. In order to increase the amount of the ligated product, a high-temperature heat-resistant polymerase such as TaqDNA ligase can be used for multiple cycles of denaturation-refolding-ligation.

2. The ligation reaction product is digested with various exonucleases such as exonuclease I (exonuclease I), exonuclease III (exonuclease III) and lambda exonuclease (lamda exonuclease). The single-stranded or double-stranded DNA of the ligation product is removed and then purified (removing all nucleic acid sequences of the non-ligated product, the step of PCR amplification of the ligated product is not required, and the sequencing results can more truly reflect the information of the ligated product).

3. The non-amplified ligation products are directly subjected to single-molecule amplification sequencing or direct single-molecule sequencing using the next-generation high-throughput chip sequencing platform.

4. Analyze the sequencing data to realize the sample classification of the sequencing sequence, the classification of the gene locus, and the quantification of the connection products corresponding to each gene fragment: first, the sequence obtained by sequencing is assigned to the corresponding sample according to the tag sequence, and then according to each The base composition of the sequence is assigned to the ligation product of the corresponding gene fragment, and the relative amount of the ligation product of the gene fragment can be estimated by counting the number of sequenced sequences of each ligation product.

Primer

As used herein, the term "primer" refers to a general term for oligonucleotides capable of complementary pairing with a template and synthesizing a DNA chain complementary to the template under the action of DNA polymerase. Primers can be natural RNA, DNA, or any form of natural nucleotides, and even non-natural nucleotides such as LNA or ZNA.

A primer is "substantially" (or "essentially") complementary to a particular sequence on one strand of the template. A primer must be sufficiently complementary to one strand of the template to initiate extension, but the sequence of the primer does not have to be perfectly complementary to that of the template. For example, adding a sequence that is not complementary to the template at the 5' end of a primer that is complementary to the template at the 3' end, such a primer is still approximately complementary to the template. As long as there is a sufficiently long primer that can fully combine with the template, non-completely complementary primers can also form a primer-template complex with the template, thereby performing amplification.

In the present invention, primers include (but not limited to): degenerate primers, sequencing primers, linker primers and the like. Those skilled in the art can use conventional methods to design and optimize primers.

High-throughput sequencing

The "resequencing" of the genome enables humans to discover abnormal changes in disease-related genes as early as possible, which is helpful for in-depth research on the diagnosis and treatment of individual diseases.

Those skilled in the art can generally use three second-generation sequencing platforms for high-throughput sequencing: 454FLX (Roche Company), Solexa Genome Analyzer (Illumina Company), and SOLID from Applied Biosystems Company, etc. The common feature of these platforms is extremely high sequencing throughput. Compared with the 96-channel capillary sequencing of traditional sequencing, high-throughput sequencing can read 400,000 to 3 billion sequences in one experiment. Depending on the platform, the read length ranges from 25bp to 450bp, so different sequencing platforms can read bases ranging from 1G to 300G in one experiment.

Solexa high-throughput sequencing includes two steps: DNA cluster formation and on-machine sequencing: the mixture of PCR amplification products is hybridized with the sequencing probes immobilized on the solid-phase carrier, and solid-phase bridge PCR amplification is performed to form sequencing clusters; The sequencing clusters are sequenced by "sequencing-by-synthesis" to obtain the nucleotide sequence of the disease-associated nucleic acid molecule in the sample.

DNA clusters are formed by using a flow cell with a layer of single-stranded primers attached to the surface, and DNA fragments in a single-stranded state are immobilized on the chip through the adapter sequence and the primers on the surface of the chip through the principle of complementary base pairing. On the surface, through the amplification reaction, the immobilized single-stranded DNA becomes double-stranded DNA, and the double-stranded DNA is denatured again to become a single-strand, one end of which is anchored on the sequencing chip, and the other end is randomly complementary to another nearby primer to be anchored. Form a "bridge"; on the sequencing chip, there are tens of millions of DNA single molecules undergoing the above reactions at the same time; The double-strand is denatured into a single-strand, which becomes a bridge again, and the template called the next round of amplification continues to amplify; after 30 rounds of amplification, each single molecule is amplified by 1000 times, which is called monoclonal DNA clusters.

DNA clusters are synthesized and sequenced on the Solexa sequencer. In the sequencing reaction, the four bases are labeled with different fluorescence, and each base end is blocked by a protective base. Only one base can be added in a single reaction. After scanning , after reading the color of this reaction, the protection group is removed, and the next reaction can continue, and so on, and the precise sequence of the base is obtained. In the Solexa Multiplexed Sequencing (MultiplexedSequencing) process, the Index (label or barcode) is used to distinguish samples, and after the routine sequencing is completed, an additional 7 cycles of sequencing are performed on the Index part. Through the identification of the Index, it can be sequenced in one sequencing lane Distinguish up to 1000+ different samples.

application

The invention also provides the application of the high-throughput gene analysis method.

SNP typing

By using the method of the present invention to detect SNPs, hundreds or even thousands of SNP sites can be detected in each reaction. In a specific embodiment, the steps are as follows (Figure 3):

1. Preferably design 3 probes for each SNP site, 2 5'-end allele-specific probes and 1 3'-end common probe, the last base of each allele-specific probe The base corresponds to the corresponding allelic base. In order to increase the specificity of connection, change the base at one of the penultimate 2-4 positions of the probe to introduce additional mismatches to increase the specificity of connection;

2. Denature-anneal-ligate the ligation probes of all SNP sites with the DNA template. In order to increase the amount of ligation products, multiple denaturation-annealing-ligation cycles can be performed;

3. Amplify the ligation product by PCR, or directly digest and purify it with accounting enzymes without amplification, and perform next-generation high-throughput chip sequencing after mixing the amplification products of different samples;

4. Sequencing data analysis, genotype interpretation is performed according to the ratio of the two allelic connection products, or in the case of non-specific connection, the data of the number of two connection products of multiple samples can be taken for cluster analysis (it is expected that there will be 3 clustering area, corresponding to the three genotypes), and genotype interpretation is performed according to the clustering results.

CNV detection

By using the method of the invention to detect CNV, hundreds or even thousands of target gene fragments can be detected in each reaction. In a specific embodiment, the steps are as follows (Figure 4):

1. Each reaction system contains at least one reference gene fragment, which is a gene fragment that is considered to have no copy number polymorphism in the detected species population, and is used to correct the sampling differences of different samples;

2. For each target gene or reference gene fragment, preferably design 2 probes, 1 5' end probe and 1 3' end probe;

3. Denature-refold-connect the ligation probes of all target genes or reference gene fragments with the DNA template. In order to increase the amount of ligation products, multiple cycles of denaturation-refolding-connection can be performed;

4. The ligation product is amplified by PCR or directly digested with nuclease without amplification, and the amplified products of different samples are mixed for next-generation high-throughput chip sequencing;

5. Sequencing data analysis: Divide the detected quantity of the ligation product corresponding to each target gene by the detected quantity of the ligated product of the reference gene fragment to obtain the correction value R as shown in the figure N _T1 /N _R1 , and then divide the R value by the reference sample After the R value, the correction value RR is obtained. If there is more than one reference gene, a RR value is calculated for each reference gene fragment, and then the median is taken as the relative copy value of the target gene, and the value is multiplied by the reference gene. The copy number of the sample is the copy number of the target gene obtained in the sample, as shown in the figure CN _T1 .

Target gene mutation screening

Use the method of the present invention to screen the mutation of the target gene (Figure 5). In a specific embodiment, the steps are as follows: Since the DNA template corresponding to the ligation probe has a significant mutation, the ligation efficiency will be seriously reduced, and a high-density platform is designed for the target region. Spread the probes, and use the detection steps and data analysis methods of CNV detection to obtain the copy number of each probe region. For the probe region whose copy number deviates from the normal value, it can be used as a candidate region for mutation sites, which can be detected by conventional sequencing. verify.

Multiple candidate gene expression level analysis

Using the method of the present invention to analyze the expression levels of multiple candidate genes ( FIG. 6 ), each reaction can detect the expression levels of hundreds or even thousands of target genes. In a specific embodiment, the steps are as follows: multiple probes can be designed for each gene, the expression ratio of different splice bodies can be distinguished, and the cDNA obtained by reverse transcription or directly using RNA as a template is used for probe ligation, After the ligation products were amplified, the next-generation high-throughput chip sequencing was performed. The sequencing results were analyzed, and the number of ligation products in the target region of each gene was corrected by multiple reference genes, and the median was taken as the relative expression level of the gene, which was used for the differential analysis of the gene expression level among different samples.

High-throughput methylation analysis

Using the method of the present invention to analyze the methylation levels, each reaction can detect the methylation levels of hundreds or even thousands of CpG islands. In a specific embodiment, the method is as follows (Figure 7):

One method is to treat the genomic DNA with a methylation-sensitive restriction endonuclease, and design a probe for the cutting point to detect the amount of uncut genomic DNA; the other method is to treat the genomic DNA with sulfite Finally, methylation-specific probes and non-methylation-specific probes were designed for the target gene segment, and the methylation level of the target gene segment was estimated by detecting the ligation products of the two probes.

The probe ligation products were subjected to next-generation high-throughput chip sequencing to obtain the amount of each probe ligation product. In the first method, all methylated or hemimethylated regions in the genome need to be selected as reference DNA fragments, and samples that have not been treated with restriction endonucleases are selected as reference samples. In the second method, it is necessary to select a reference DNA sample whose methylation ratio in all target gene regions is known. The preparation of this sample can be done by using the whole gene amplification product and the whole genome after methylation modification. The amplification products are mixed in a certain ratio, usually 1:1 to obtain a reference sample with a 50% methylation ratio.

Identification of pathogenic microorganisms or transgenic animals and plants

By using the method of the invention to identify pathogenic microorganisms or transgenic animals and plants, hundreds or even thousands of species-specific gene fragments can be detected in each reaction.

Multiple specific probes are designed for each microorganism or transgenic gene, and probes are also designed for incorporated reference gene fragments. Probe ligation products were subjected to next-generation high-throughput chip sequencing. For the amount of each probe ligation product, the type of pathogenic microorganisms and the type of transgenic crops contained in the detection sample are confirmed after being corrected by incorporating the reference gene fragment.

The main advantages of the present invention are:

(1) One reaction can detect tens of thousands of gene fragment information at the same time, and the detection throughput is improved; it is applied on a non-proprietary detection platform without additional equipment investment, and one detection reaction can complete thousands of gene fragments at the same time Therefore, the detection cost of a single gene fragment is greatly reduced; the detection system can be quickly established for any target gene fragment that needs to be detected, and the application is flexible:

(2) Compared with the traditional chip hybridization, the present invention uses sequencing to identify the ligation products, and digital counting for quantification, without the influence of non-specific hybridization and detection background, thus greatly improving the accuracy;

(3) The lengths of all ligated products in the present invention are relatively consistent, and the difference in amplification efficiency between different fragments is relatively small when using universal primers for amplification. Compared with capillary electrophoresis that uses different lengths to distinguish ligated products, in this technology, The ratio of each ligation product in the amplification product is more likely to be consistent with the ratio before amplification;

(4) The ligation products are treated and purified by various exonucleases and then directly sequenced by high-throughput chips without PCR amplification, which reduces the correlation ratio of each ligation product introduced due to the difference in PCR amplification efficiency of different ligation products deviation;

(5) The sequence identification and digital counting quantitative method of single-molecule amplification product sequencing are adopted, which greatly improves the sensitivity.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

Example 1

Detect the typing of 48 SNP sites

Design junction probes for 48 SNP sites, design 3 probes for each site, 2 5'-end allele-specific probes and 1 3'-end consensus sequence, the first half of the 5'-end probe A general PCR sequence compatible with the next-generation sequencing platform of Illumina is added in part, and another general PCR sequence compatible with the next-generation sequencing platform of Illumina is added to the second half of the 5' end probe. The probe was ligated under the action of TaqDNA ligase under the condition of good pairing with the template, and the ligation product was amplified using general PCR primers compatible with the illumina next-generation sequencing platform. Different samples were amplified with general primers with different tag sequences, and then After uniform mixing and purification, 1x72 sequencing was performed on the Illumina GAIIx sequencer. After the Sequencing reads are read out by software, different sample sources are distinguished according to the tag sequence, and then each Sequencing read source is determined which ligation product is associated with, and the READS statistics are performed on each ligation product. Genotype interpretation was performed based on the ratio of the number of Sequencing reads of the two allele-specific junction products.

experiment process:

The sample comes from the whole blood sample of a normal individual in Shanghai Ruijin Hospital. The whole blood sample is extracted with phenol chloroform to extract DNA and then dissolved with 1XTE.

Take 100-200ng of DNA, dilute to 10μl with 1xTE, incubate at 98°C for 5 minutes, and place on ice immediately;

Configure the probe mixture (ProbeMix) with 1xTE, 0.005 μM for each probe;

Configure 2xLigation Premix, 10μl: 2μl 10*Taq ligase buffer, 1μl 40U/μl TaqLigase, 1μl ProbeMix, 6μl ddH ₂ O;

Add 10μl 2xLigation Premix to the denatured 10μl DNA sample, shake slightly to mix;

Carry out the ligation reaction with the following procedure: 4× (95°C for 30s, 58°C for 4h), after the ligation reaction is completed, immediately put it on ice for later use or store it below -20°C for later use;

Configure PCR primer mixtures Pmix1, Pmix2 and Pmix3, which are composed of NGMPCRF and NGMPCRR001, NGMPCRF and NGMPCRR002, NGMPCRF and NGMPCRR003 respectively, and the concentration of each primer is 2 μM;

Take 1 μl of the ligation product as a template for PCR reaction, the reaction system is 20 μl, including 2 μl 10x PCRbuffer, 2 μl 2.5mM dNTP mix, 2 μl Pmix1 for S1 (or Pmix2 for S2, or Pmix3 for S3), 1 μl Ligation product, 0.2 μl 5U/μl Taq DNA polymerase, 12.8μl Milli-Q water; the PCR program is: 95°C 5min; 8x (94°C 20s, 54°C 40s, 72°C 1min); 26x (94°C 20s, 68°C 1.5min); hold at 4°C ;

Electrophoresis was used to detect the amplification efficiency, and then the three PCR products were evenly mixed according to the product concentration, and the fragments between 100bp and 150bp were purified by electrophoresis and slicing using QIAquick Gel Extraction Kit;

Estimate the number of molecules after OD quantification of the purified product, then mix with other project samples and perform bridge amplification on the chip according to the requirements of TruSeqSRCluster Kit v2;

The amplified product was sequenced at 1x72+7 in Illumina GAIIX with TruSeq SBS Kit v5, the instrument control and data collection were performed using Genome Analyzer Data Collection Software SCS2.8, and the recipe selected for sequencing was GA2-PEM_MP_72+7Cycle_v<#>;

Divide the sequenced reads into different samples according to the tag sequence, and then compare them with the expected ligation product libraries; identify each read as an allele ligation product, and calculate the number of each allelic ligation product;

The genotype of the site is determined according to the ratio of the number of sequencing reads of the two junction products at each site and the proportion distribution of different samples: if the junction specificity is very strong, a certain allele junction product is more than 10 times or 1/2 that of the other Below 10, it can usually be directly judged as the homozygous dominant Allele. If not, it can be compared in multiple samples to see if there is a clustering phenomenon (for example, divided into 3 categories, corresponding to 3 genotypes).

The general primer sequences used in this example are as follows:

NGMPCRF (SEQ ID NO: 1)

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACNGMPCRR001 (SEQ ID NO: 2)

CAAGCAGAAGACGGCATACGAGATAAACTTGTGACTGGAGTTCAGACGTG

NGMPCRR002 (SEQ ID NO: 3)

CAAGCAGAAGACGGCATACGAGATTCCGGTGTGACTGGAGTTCAGACGTG

NGMPCRR003 (SEQ ID NO: 4)

CAAGCAGAAGACGGCATACGAGATCCAACTGTGACTGGAGTTCAGACGTG

The results of SNP sites and genotype calling (genotype interpretation) sequencing depth of the three samples are shown in Table 1.

Table 1

The results showed that by comparison with the previous sequencing results, except for rs2231926, all other 47 SNP sites were accurately typed in the 3 samples. The mistyping of locus rs2231926 is mainly due to the non-specific linkage of the G-specific probe, but if more samples are typed, this type of mistyping can be obtained by the cluster analysis of the amount of the two allelic junction products avoid.

Example 2

Detection of exon deletion duplications in the DMD gene

The basic principle is shown in Figure 4, 141 probes are designed for each sample, 129 of which are distributed on the 79 exons of the DMD gene, 6 reference gene probes, and 6 sex chromosome sex identification probes (3 located on X chromosomes, 3 on the Y chromosome). Two probes are designed for each site, one 5' end probe and one 3' end probe. For the specific sequence that hybridizes with the target nucleic acid fragment, the 5' end of the 3' end probe is modified by phosphorylation, the first half is the specific sequence that hybridizes with the target nucleic acid fragment, and the second half is the general sequence consistent with the subsequent PCR amplification primers . The probes were ligated under the action of Taq DNA ligase under the condition of good pairing with the template, and the ligation products were amplified with universal PCR primers compatible with the Illumina next-generation sequencing platform. Different samples were amplified with universal primers with different tag sequences, and then evenly mixed and purified on the IlluminaGAIIx sequencer for 1x72+7 sequencing. Sequence data for subsequent analysis.

Sample preparation: 2ml whole blood was drawn from 2 patients with pseudohypertrophic muscular dystrophy (P1, P2), 1 female carrier (P3) and 1 normal sample (P4), and the whole blood was extracted by traditional phenol chloroform method DNA was used in subsequent experiments.

The general primers NGMPCRF, NGMPCRR001, NGMPCRR002 and NGMPCRR003 used in this example are the same as in Example 1, and the sequence of NGMPCRR004 is as follows (SEQ ID NO: 5):

CAAGCAGAAGACGGCATACGAGAT AATTAG GTGACTGGAGTTCAGACGTG. AATTAG is the tag sequence used when sequencing with the Illumina next-generation sequencer, and is used to distinguish the sequencing data of different samples.

Experimental procedure: Take 100-200ng of DNA, dilute it to 10μl with 1xTE, incubate at 98°C for 5 minutes, and place it on ice immediately; use 1xTE to configure the probe mixture (ProbeMix), each probe 0.005μM; configure 2xLigationPremix, 10μl: 2μl10 *Taq ligase buffer, 1μl 40U/μl Taq DNA ligase, 1μl ProbeMix, 6μl sterile water; add 10μl 2xLigation Premix to the denatured 10μl DNA sample, shake slightly to mix; use the following procedure for ligation reaction: 4×( 95°C for 30s, 58°C for 4h), immediately after the ligation reaction, put it on ice for later use or store it below -20°C for later use; prepare the PCR primer mixture Pmix1, Pmix2, Pmix3 and Pmix4, respectively by NGMPCRF and NGMPCRR001, NGMPCRF and NGMPCRR002, NGMPCRF and NGMPCRR00, NGMPCRF and NGMPCRR004, the concentration of each primer is 2 μM; take 1 μl of the ligation product as a template for PCR reaction, the reaction system is 20 μl, including 2 μl 10x PCR buffer, 2 μl 2.5mM dNTP mix, 2 μl Pmix1 for P1 (or Pmix2 for P2, Pmix3 for P3, Pmix4 for P4), 1 μl ligation product, 0.2 μl 5U/μl Taq DNA polymerase, 12.8 μl sterile water; the PCR program is: 95°C 5min; 8x (94°C 20s, 54°C 40s , 72°C 1min); 26x (94°C 20s, 68°C 1.5min); hold at 4°C; 2% agarose electrophoresis to detect the amplification efficiency, then mix the 4 PCR products evenly according to the product concentration, and use QIAquick for electrophoresis separation and slicing GelExtraction Kit purifies fragments between 100bp-150bp; the purified product OD is quantified to estimate the number of molecules, and then mixed with other project samples to carry out bridge amplification on the chip according to the requirements of TruSeq SR Cluster Kit v2; the amplified product is used for TruSeq SBS Kit v5 was sequenced at Illumina GAIIX at 1x72+7. GenomeAnalyzer Data Collection Software SCS2.8 was used for instrument control and data collection. The recipe selected for sequencing was GA2-PEM_MP_72+7Cycle_v<#>; after the sequencing sequence was read out by the software, it was distinguished according to the tag sequence sample source, then determine which ligation product each sequence was derived from, and Sequencing depth statistics were performed for each ligation product. Divide the detection quantity of the ligation product corresponding to each target gene by the detection quantity of the reference gene fragment ligation product to obtain the first correction value (R), and then divide the R value by the R value of the reference sample to obtain the second correction value Value (RR), a RR value is calculated for each reference gene segment, there are 6 RR values in total, and then the median is taken, because the reference sample is a normal male individual, the DMD gene and the gene segments on the X and Y chromosomes The copy number is all 1, so the median is the copy number of the corresponding gene segment of the test sample.

Results: The detection results of the sequencing depth and copy number of each target gene fragment ligation product of the 4 samples are shown in Table 2.

Table 2

The test results are shown in Figure 8: Figure 8.1 is a male individual with a deletion of exon 18-41 of the DMD gene; Figure 8.2 is a male individual with a duplication of exon 63-67 of the DMD gene; Figure 8.3 is a carrier of a deletion of exon 50 of the DMD gene of female individuals.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (18)

1. A high-throughput nucleic acid analysis method for non-diagnostic purposes, characterized in that, comprising steps: (1) For the n kinds of target nucleic acid fragments to be analyzed, for each target nucleic acid fragment, at least 2 specific probes that bind to different binding regions of the target nucleic acid fragments are provided, and each of the specific probes has a specific binding region and a universal sequence region, and the sequence of the specific binding region is complementary to the sequence of the binding region of the target nucleic acid fragment, and the sequence of the universal sequence region corresponds to the sequence of the sequencing primer, wherein n is a positive integer ≥ 40; (2) hybridize the nucleic acid sample containing the target nucleic acid fragment to be analyzed with the probe described in step (1), and connect the probes to obtain a mixture of probe ligation products, wherein 3 of each probe ligation product Both ' and 5' ends are the general sequence regions whose sequences correspond to the sequences of the sequencing primers; (3) Sequencing and/or analyzing the mixture of probe ligation products in step (2), so as to obtain the information of the target nucleic acid; Wherein, the two probes corresponding to each target nucleic acid fragment are: a 5' end probe and a 3' end probe, and the 5' end probe can be complementary to the binding region located at the 3' end of the target nucleic acid fragment to be analyzed , the 3' end probe can be complementary to the binding region located at the 5' end of the target nucleic acid fragment to be analyzed, and the 3' end of the 3' end probe and the 5' end of the 5' end probe carry out anti- Modification and protection of exonucleases; And, in the step (2), multiple cycles of denaturation-annealing-ligation are performed. 2. The method of claim 1, wherein the specific probe also has one or more characteristics selected from the group consisting of: (1) The length of the specific probe is ≤100bp; (2) The length of the specific binding region of the specific probe is ≤50bp; (3) The length of the general sequence region of the specific probe is ≥ 8 bp; (4) The sequence of the universal sequence region of the specific probe also corresponds to the amplification primer sequence; (5) The specific probe includes a tag sequence. 3. The method according to claim 2, characterized in that the length of the specific probe is 30-70bp. 4. The method according to claim 2, characterized in that the length of the specific probe is 40-50bp. 5. The method according to claim 2, characterized in that the length of the specific binding region of the specific probe is 15-35 bp. 6. The method according to claim 2, characterized in that the length of the specific binding region of the specific probe is 20-25 bp. 7. The method according to claim 2, characterized in that the length of the general sequence region of the specific probe is 15-35 bp. 8. The method according to claim 2, characterized in that the length of the general sequence region of the specific probe is 20-25 bp. 9. The method of claim 1, wherein the 5' end of the 3' end probe is phosphorylated. 10. method as claimed in claim 9, is characterized in that, the structure of described 5 ' end probe or 3 ' end probe is as shown in formula I: 5'-A-L-B-3' Formula I In Formula I, A represents the general sequence region; B represents the specific binding region; L represents the nucleic acid linking sequence between A and B; Among them, the positions of A and B can be interchanged. 11. The method according to claim 9 or 10, wherein the connection relationship between the 5' end probe and the 3' end probe is selected from one of the following groups: (a) The 5' end probe and the 3' end probe are adjacent probes: after the 5' end probe and the 3' end probe hybridize with the target nucleic acid fragment to be analyzed, the distance between the two is 0 Bases are connected under the action of ligase to obtain probe ligation products; (b) The distance between the 5' end probe and the 3' end probe is 1-500 bases: after the 5' end probe and the 3' end probe are hybridized with the target nucleic acid fragment to be analyzed, the DNA polymerase Gap polymerization and ligation under the action of ligase to obtain probe ligation products; (c) In addition to the 5' end probe and the 3' end probe, the hybridization system also includes probe 3, which is adjacent to the 5' end probe and the 3' end probe respectively, and the three probes After hybridizing with the target nucleic acid fragment to be analyzed, it is ligated under the action of ligase, so as to obtain the probe ligation product. 12 . The method according to claim 1 , further comprising a step between step (2) and step (3): amplifying the probe ligation product obtained in step (2). 13 . 13. The method according to claim 1, characterized in that, in step (3), the mixture of probe ligation products or its amplified products is sequenced and analyzed using third-generation sequencing technology or second-generation sequencing technology . 14. The method according to claim 1, characterized in that, in step (3), the information of obtaining the target nucleic acid refers to one or more information optionally selected from the following group: DNA methylation information, Mutation screening information, genetic information of pathogenic microorganisms, genetic information of transgenic animal and plant products, and gene expression levels. 15. The method according to claim 1, characterized in that, in step (3), the information of obtaining the target nucleic acid refers to one or more information optionally selected from the following group: CNP typing information, and CNV information. 16. The method of claim 1, wherein n is a positive integer ≥ 100. 17. A method for detecting CNV for non-diagnostic purposes, characterized in that it comprises the step of: using the method according to claim 1 to perform sequencing and CNV analysis on the mixture of probe ligation products derived from the sample to be tested to obtain the target nucleic acid CNV information. 18. A high-throughput methylation analysis method for non-diagnostic purposes, characterized in that it comprises the step of: using the method of claim 1 to sequence and methylate the mixture of probe ligation products derived from the sample to be tested. Methylation analysis to obtain the methylation information of the target nucleic acid.