阅读说明：本技术 基因目标区域的富集方法及体系 (Method and system for enriching gene target region ) 是由 郭志伟 李英辉 陈倩 胡荣君 于 2019-09-20 设计创作，主要内容包括：本发明首先提供了一种基因目标区域的富集方法,包括：(1)通过特异性探针扩增包含目标区域的片段化DNA,以提供捕获延伸产物,所述的特异性探针包括与所述片段化DNA的目标区域互补的序列,且其3’末端和5’末端核苷酸均被修饰；(2)向步骤(1)所提供的捕获延伸产物中加入连接酶,以提供连接产物。本发明其次提供了一种基因目标区域的富集体系,适用于本发明提供的基因目标区域的富集方法。本发明所提供的富集方法操作简单、结果可靠,应用于短片段DNA可以最大程度的减少原始分子尤其是稀有原始分子的损失,高效地富集目标分子。(The invention firstly provides an enrichment method of a gene target region, which comprises the following steps: (1) amplifying the fragmented DNA comprising the target region by a specific probe comprising a sequence complementary to the target region of the fragmented DNA and having both its 3 'and 5' terminal nucleotides modified to provide capture extension products; (2) adding a ligase to the captured extension product provided in step (1) to provide a ligation product. The invention further provides an enrichment system of the gene target region, which is suitable for the enrichment method of the gene target region provided by the invention. The enrichment method provided by the invention is simple to operate and reliable in result, and can reduce the loss of original molecules, particularly rare original molecules to the maximum extent and efficiently enrich target molecules when being applied to short-fragment DNA.)

1. A method of enriching for a gene target region comprising:

(1) amplifying the fragmented DNA comprising the target region by a specific probe to provide a capture extension product;

wherein the specific probe comprises sequences complementary and non-complementary to the target region of the fragmented DNA and both the 3 'and 5' terminal nucleotides of the specific probe are modified;

(2) adding a ligase to the captured extension product provided in step (1) to provide a ligation product comprising a circularized ligation product and a linear ligation product.

2. The method of enriching a target region of a gene according to claim 1,

in the step (1), the fragmented DNA comprises double-stranded DNA, single-stranded DNA and cDNA, and the length of the fragmented DNA is 25-200 bp;

and/or, the amplification system of the step (1) further comprises DNA polymerase and dNTP.

3. The method of enriching a target region of a gene according to claim 2,

the DNA polymerase has 3 '-5' exonuclease activity;

and/or, the dNTP is also coupled with a marker molecule, and the marker molecule is preferably biotin.

4. The method for enriching a gene target region according to claim 1, wherein the amplification system of step (1) further comprises:

an active substance for cleaving a 3' terminal modifying group of the specific probe after the specific probe binds to a target region of the fragmented DNA;

preferably, the active substance is a nuclease.

5. The method of enriching a target region of a gene according to claim 1,

the specific probe also comprises a universal sequence which can be recognized by a sequencing system;

and/or, the hydroxyl at position 3 of the 3' terminal nucleotide of the specific probe is substituted;

and/or, the methoxy group at position 2 of the 3' terminal nucleotide of the specific probe is substituted;

preferably, the substituent group at the 3' end of the specific probe is selected from a hydrogen atom, a C3Spacer group, a C6Spacer group, a phosphate group or an amino group;

and/or, the 3' end tail region of the specific probe comprises mismatched bases;

and/or, the hydroxyl at position 5 of the 5' terminal nucleotide of the specific probe is substituted;

preferably, the substituent group at the 5' end of the specific probe is selected from a phosphate group or an adenosine group.

6. The method of enriching a target region of a gene according to claim 1,

the ligation system in the step (2) comprises single-stranded ligase, and the single-stranded ligase is preferably T4RNA ligase or thermostable RNA ligase.

7. The method of enriching a target region of a gene according to claim 1,

(3) setting an enzyme cutting site in a sequence which is not complementary to the target region on the specific probe, and adding an endonuclease after the step (2) for cutting a cyclized ligation product in the ligation product provided in the step (2) at the enzyme cutting site to provide a linear ligation product;

preferably, the enzyme cleavage site is uracil and the endonuclease is ssDNA endonuclease or USER enzyme.

8. The method of enriching a gene target region according to claim 7, further comprising:

(4) after the step (2), PCR amplifying the ligation product provided in the step (2);

preferably, in the step (4), the PCR amplification primer has a sequence complementary to the specific probe and is not complementary to the sequence of the target region;

more preferably, the sequence complementary to the specific probe is a sequencing universal sequence; and/or the presence of a gas in the gas,

(5) after the step (3), PCR amplifying the ligation products provided in the step (2) and the step (3);

preferably, in the step (5), the PCR amplification primer has a sequence complementary to a sequence on both sides of the enzyme cutting site;

more preferably, the sequence complementary to the sequences on both sides of the restriction enzyme cutting site is a sequencing universal sequence;

and/or, product purification is performed after any of the steps (1) to (5).

9. The method of enriching a gene target region according to claim 8, further comprising:

(6) detecting the amplification product provided in step (4) or (5) by using a detection primer 1, a detection primer 2 and a probe 3, wherein at least one of the detection primer 1, the detection primer 2 and the probe 3 comprises a gene specific sequence;

preferably, the detection primer 1 comprises a gene specific sequence, and the detection primer 2 and the probe 3 comprise a universal sequence;

and/or, the detection primer 2 and/or the probe 3 also comprise a gene-specific sequence;

preferably, the probe 3 comprises a labeling molecule, and the sequence of the probe 3 is not complementary to the detection primer 1 or 2.

10. The method for enriching a target region of a gene according to claim 1, wherein the method for enriching a target region of a gene is applied to nucleic acid detection.

11. A system for enriching a fragmented DNA target region comprising a specific probe and a ligase suitable for use in a method of enriching a gene target region according to any one of claims 1 to 10.

12. The system for enriching fragmented DNA target regions according to claim 11,

also comprises one or more of the following components: dNTP coupled with a marker molecule, DNA polymerase, nuclease and endonuclease;

and/or, further comprising PCR amplification primers having a sequence complementary to at least part of the universal sequence of the specific probe;

and/or, at least one of the detection primer 1, the detection primer 2 and the probe 3 contains a gene specific sequence.

Technical Field

The invention relates to the field of biotechnology, in particular to a method and a system for enriching a gene target region.

Background

Gene sequencing technology has been around half a century since the seventies of the last century, and the advent of PCR technology in 1985 has driven the development of the entire field of molecular biology. The next generation sequencing technology (NGS) has the advantages of accuracy, sensitivity and high throughput, and the application range thereof is continuously expanded with the continuous reduction of the sequencing cost, but the application thereof is also restricted by the step of library construction which is diversified in demand and time-consuming and labor-consuming. In the process of banking clinical samples such as plasma samples, the existing forms of DNA are usually short fragments, damaged, single-stranded or partially double-stranded, and the existing PCR technology cannot capture and enrich DNA in the existing forms, especially DNA with fragments smaller than 200 bp.

For the enrichment of tiny DNA fragments, the prior art still mainly uses the traditional PCR library construction method, or firstly adds a joint connection and then amplifies, such as a hybridization capture method. However, for the former, because double-ended primers are needed, the length of a fragment suitable for amplification is greatly limited, and the problems that the preference in amplification causes high heterogeneity of products and the accumulated error of exponential amplification causes inaccurate results of subsequent sequencing exist; in the latter case, although the requirement for the length of the fragment to be enriched is not as high as that of PCR, ligation reaction is required first, and the ligation efficiency is usually only 20% to 50%, resulting in low capture efficiency and also in the problem that rare molecules are easily lost due to the difficulty in ligation. To solve the problem of the banking of NGS, recently developed technologies also include such as molecular inversion probes, multiplex PCR, and the like. Compared with the hybridization capture technology, the molecular inversion probe has better specificity, but the design of the pocket-shaped probe is complex, and the molecular inversion probe is not suitable for the enrichment of tiny DNA fragments. The multiplex PCR technology is suitable for large-scale samples and is most widely applied, but the primer design requirement is extremely high, the uniformity of an amplicon is poor, or the uniformity of an amplification product is good, but the requirement on the concentration of an initial sample is high, and the multiplex PCR technology is also not suitable for the enrichment of a tiny DNA fragment with low initial concentration. These prior art constructions typically require double-ended primers, necessitating the introduction of purification steps to remove adaptor dimer contamination, which results in the loss of information for small fragments of double-stranded DNA, damaged double-stranded DNA, and single-stranded DNA molecules. However, it is precisely these forms of DNA that exist in some genomic regions where transcription is active. In summary, the present invention provides a method for efficiently enriching a fragmented DNA target region, which overcomes the bottleneck of ligation efficiency on enrichment effect, inhibits the generation of non-target ligation products, captures rare molecules to the maximum extent and maintains the homogeneity of the products, and is a main technical problem to be solved by the present invention. In the application with application number 2019100024085, the applicant has previously established a technical scheme for achieving enrichment by first linearly amplifying a target region with a specific probe and then connecting a linker, and the main application direction is nucleic acid detection based on second-generation sequencing. The present invention will address the problem of targeted enrichment of fragmented DNA with another approach.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, it is an object of the present invention to provide a method for enriching a target region of a gene, which solves the problems of the prior art.

To achieve the above and other related objects, the present invention provides, in a first aspect, a method for enriching a target region of a gene, comprising:

(1) amplifying the fragmented DNA comprising the target region by a specific probe to provide a capture extension product;

the specific probe comprises sequences complementary and non-complementary to the target region of the fragmented DNA, and both the 3 'and 5' terminal nucleotides of the specific probe are modified;

(2) adding a ligase to the captured extension product provided in step (1) to provide a ligation product comprising a circularized ligation product and a linear ligation product.

Another objective of the invention is to provide a system for enriching fragmented DNA target regions, which comprises specific probes and ligase suitable for the gene target region enrichment method provided by the invention.

Drawings

FIG. 1 is a schematic flow chart of a method for enriching a target region according to an embodiment of the present invention.

FIG. 2 is a schematic representation of a molecule constructed in an example of the invention.

Detailed Description

The present inventors have conducted extensive exploratory studies and have provided a method for enriching a gene target region, which is simple to operate and reliable in result, and particularly has a good effect of enriching a short fragment of nucleic acid.

In a first aspect, the present invention provides a method for enriching a gene target region, comprising:

(1) amplifying fragmented DNA comprising a target region by means of a specific probe to provide captured extension products, the specific probe comprising both sequences complementary to the target region of the fragmented DNA and non-complementary to the target region, and the specific probe having both its 3 'and 5' terminal nucleotides modified to prevent ligation of the specific probe at its 3 'end, primarily self-ligation at its 5' end, prior to binding to the template;

(2) adding a ligase to the captured extension product provided in step (1) to provide a ligation product. The ligation product of this step comprises two parts, one is a circularized ligation product formed by intramolecular ligation of the captured extension product, and the other is a linear ligation product formed by ligation between the captured extension product and the specific probe molecule. Both circularized and linear ligation products contain the region of interest to be amplified and are suitable for library construction of the molecule of interest.

In the gene target region enrichment method provided by the invention, the fragmented DNA containing the target region is amplified through the specific probe, and the number of the fragmented DNA containing the target region can be one or more. Generally, the specific probes are in one-to-one correspondence with fragmented DNA containing the target region, i.e., the number of specific probes in the reaction system may be one or more. Extension as used herein refers to a complete linear pre-amplification step of a sample, including a cycle of single or multiple denaturation, annealing, and extension steps. In a preferred embodiment, the step is performed in multiple cycles, such as 2-100, 2-10, 10-20, 20-30, 30-40, 40-60, 60-80, 80-100 cycles, to effectively increase the number of molecules comprising the target region.

In the method for enriching the target region of the gene, the fragmented DNA may be double-stranded DNA, single-stranded DNA, cDNA, etc., which may be usually obtained by reverse transcription of RNA. For double-stranded DNA, a specific probe may include a sequence complementary to a target region on one strand of the fragmented DNA, as well as a sequence complementary to a target region on one strand of the fragmented DNA. Therefore, the enrichment method of the present invention is also suitable for fragmenting RNA, and one skilled in the art can reverse transcribe RNA into cDNA and perform subsequent operations by the enrichment method provided by the present invention. The length of the fragmented DNA can be 25-200 bps, 25-40 bps, 40-60 bps, 60-80 bps, 80-100 bps, 100-120 bps, 120-140 bps, 140-160 bps, 160-180 bps, or 180-200 bps.

In the method for enriching the target region of the gene, the amplification system in the step (1) may include a specific probe, a DNA polymerase and dntps. The reaction for amplifying the fragmented DNA containing the target region by the specific probe may be generally performed in the presence of a DNA polymerase. After the probe with the 3' end blocked and modified is combined with the template under the action of high-fidelity polymerase, the blocking group is cut off, and the probe is activated, so that the target sequence can be effectively extended. The DNA polymerase may have 3' -5 ' exonuclease activity to cleave off substituents at the 3' end of the probe after binding to the template, allowing the probe to extend along the template, and may preferably be a high fidelity DNA polymerase for further enhancing the amplification efficiency and purity of the product. The DNA polymerase may also be a conventional DNA polymerase, i.e.only polymerase activity, without 3 '-5' exonuclease activity. In this case, the amplification system of step (1) further comprises an active substance, wherein the active substance can be used for excising the 3' terminal modification group of the specific probe after binding to the target region, so that the active substance can be combined with a DNA polymerase (e.g., a common DNA polymerase) in the capture extension system to improve the efficiency of the linear amplification system, and the active substance is preferably a nuclease. The reaction for amplifying the fragmented DNA comprising the target region by the specific probe may be typically performed in the presence of dntps, which may typically be dntps coupled with label molecules, which may include but are not limited to biotin and the like, and dtnps, which may include but are not limited to dCTP, dATP and the like. The dntps may also be coupled with a label molecule, which may be biotin or the like, which may typically be used for purification of the captured extension product.

Enrichment method of gene target regionIn the method, the specific probe comprises a sequence complementary to the target region of the fragmented DNA, so that specific amplification of the target region of the fragmented DNA can be achieved, and a person skilled in the art can select a suitable target region of the fragmented DNA and design a suitable complementary sequence according to the target region of the fragmented DNA, the complementary sequence being generally close to the 3' end of the probe. The specific probe will typically also include sequences that are not complementary to the fragmented DNA, so that introduction of cleavage sites and universal sequences can be achieved, the non-complementary sequences typically being near the 5' end of the probe. The specific probe can be a specific probe with a modified 3 'terminal nucleotide, and is used for preventing the 3' terminal of the specific probe from carrying out a connection reaction with other groups, so that the self-connection of free probes or the connection of other non-target molecules can be avoided. One skilled in the art can select an appropriate modifying group to effect modification of the 3' end of a specific probe, for example, the modified group can replace a native group (e.g., hydroxyl, methoxy, etc.) on the nucleotide at the 3' end of a specific probe to prevent ligation at the 3' end of the specific probe, and the modifying group used can generally be a blocking group. After the probe is combined with a target region on the template through a complementary sequence, the modifying group at the 3' end can be removed by enzyme digestion, so that the probe is activated, and the target sequence can be effectively extended. The 3' terminal modifying group of the specific probe may be, but is not limited to, a hydrogen atom, a C3Spacer group, a C6Spacer group, a phosphate group (PO)₄) Amino (NH)₂) And the like. The selection of different substituent groups has obvious difference on the capture effect of the probe, and in a preferred embodiment of the invention, the effect obtained by replacing the hydroxyl at the 3' end of the probe with C3Spacer is optimal, and the method has obvious advantages compared with other substituent groups. The specific probes also include universal sequences that are typically recognized by a sequencing system so that subsequently provided ligation products can be sequenced by the sequencing system, e.g., for an Ion Torrent sequencing system, the universal sequences can be the reverse complements of the corresponding P1 and a sequences. The application of the sequence capable of being identified by the sequencing system makes the invention complete the text after the library is builtThe library can be sequenced by a high throughput sequencing platform to provide information needed for various subsequent studies and clinical applications. In a preferred embodiment of the present invention, the base of the tail region of the 3' end of the specific probe may comprise a mismatch, and the mismatch may be the last base of the 3' end of the probe or a base close to the 3' end; the mismatch may be one base or a plurality of bases. The mismatch does not affect the specificity and the binding efficiency of the probe, and is more beneficial to improving the cutting efficiency and the fidelity of high-fidelity DNA polymerase.

In the method for enriching a target region of a gene, the step (1) may further include purifying the captured extension product. The capture extension product can be purified by a suitable method selected by those skilled in the art, for example, the purification method of the capture extension product can be silica gel column purification, heating treatment, magnetic bead purification, etc. In a specific embodiment of the present invention, the captured extension product can be purified with respect to dNTP-coupled labeled molecules, and the purification process can be performed using avidin or streptavidin-coated magnetic beads, or the like.

The method for enriching a gene target region provided by the invention can further comprise the following steps: adding a ligase to the captured extension product provided in step (1) to provide a ligation product. The ligase is a single-stranded ligase, preferably T4RNA ligase or thermostable RNA ligase. The ligation product may be a circularized ligation product formed by intramolecular ligation of the capture extension product, or a linear ligation product formed by ligation between the capture extension product and a specific probe molecule.

In the method for enriching the gene target region, the specific probe can be a single-chain structure with modified 5 ' terminal nucleotide and at the reaction temperature of the step (2), so that a covalent bond can be generated by capturing the 3' terminal hydroxyl of the extension product and the modification group of the specific probe or the 5 ' terminal of the extension product under the catalysis of single-chain ligase to obtain the connection product. In one embodiment of the present invention, the 5 'terminal nucleotide (for example, the 5-hydroxyl group of the 5' terminal nucleotide) of the specific probe is substituted with a phosphate group to form a phosphorylation modification, and the 3 'hydroxyl group of the captured extension product is covalently bonded to the phosphorylated 5' terminal under catalysis of single-stranded ligase to obtain a ligation product. In this case, the specific probe has two states, one is a specific probe having a phosphorylated 5' end which binds to the upper template to form a captured extension product, and the other is a free specific probe. So the corresponding connection reaction can be two cases, namely the 3 'end hydroxyl of the captured extension product is connected with the 5' end phosphate group of the capture extension product, and the end to end connection in the molecule forms a ring; it is also possible to ligate the free probe to the 5 'phosphate group at the 3' hydroxyl end of the captured extension product, the ligation product remaining a linear product. In another embodiment of the invention, the 5 ' terminal nucleotide of the specific probe (e.g. the hydroxyl group at the 5 position of the 5 ' terminal nucleotide, which may be specifically) is substituted with an adenosine group to form an adenylation modification, the capture extension product may also be linked intramolecularly under the catalysis of a 5 ' App DNA/RNA thermostable ligase, or the 5 ' terminal adenosine group of the free probe may be linked to the 3' end of the capture extension product. In another embodiment of the invention, the 5 'terminal nucleotide of the specific probe (e.g., the hydroxyl group at the 5-position of the 5' terminal nucleotide in particular) is substituted with a phosphate group to form a phosphorylated modification, and the 5 'end of the specific probe can undergo a ligation reaction with the 3' end of the captured extension product catalyzed by thermostable RNA ligase to also generate both intramolecular and intermolecular linear ligation products. The specific probe can also be a partial double-stranded structure with a cohesive end at the 5 ' end region, and the cohesive end at the 5 ' end region has a single-stranded property, so that the 5 ' end can be modified by the method and connected with the capture extension product under the catalysis of a proper single-stranded ligase. In the method for enriching a target region of a gene, the step (2) may further include purifying the ligation product. The ligation product may be purified by a method selected by one skilled in the art, for example, the ligation product may be purified by a method including, but not limited to, silica gel column purification, heat treatment, magnetic bead purification, etc.

The method for enriching the gene target region provided by the invention can further comprise the following steps: (3) in the sequence of the specific probe that is not complementary to the target region, an enzyme cutting site may be generally set in the sequence near the 5' end, and an endonuclease is added after the step (2) to cut the circularized ligation product in the ligation product formed in the step (2) at the enzyme cutting site, so that the circularized ligation product is converted into a linear ligation product, which is beneficial to the subsequent PCR amplification and PCR detection reaction. In a specific embodiment of the invention, the enzyme cutting site is uracil U substituted for original thymine T, and the endonuclease is USER enzyme or SSDNA endonuclease. In another embodiment of the invention, a restriction enzyme site U is located near the 5' end of the specific probe, and after the capture extension products are connected end to form a circularization product, the USER enzyme or SSDNA endonuclease cleaves the circularization product at the restriction enzyme site U to form a linear ligation product, and then a PCR amplification primer can be used to achieve exponential amplification by combining a complementary sequence with the linear ligation product after restriction enzyme.

The method for enriching the gene target region provided by the invention can further comprise the following steps: (4) and (3) PCR amplifying the connection product provided by the step (2). The PCR amplification primers have a sequence complementary to the specific probe, which complementary sequence is not complementary to the sequence of the target region. The method for enriching the gene target region provided by the invention can further comprise the following steps: (5) after the step (3), PCR amplifying the ligation products provided in the steps (2) and (3), wherein the PCR amplification primer has a sequence complementary to the sequence on both sides of the enzyme cutting site; preferably, the sequence on both sides of the restriction enzyme site is a sequencing universal sequence, and the ligation product can be amplified by PCR (polymerase chain reaction) through DNA polymerase and PCR amplification primers, and the product of the DNA containing the target region can be further enriched by amplifying the ligation product. The ligation products provided in step (2) or step (3) may be amplified by one skilled in the art by selecting an appropriate method and system, for example, in one embodiment of the invention, PCR amplification primers having sequences complementary to and reverse to the sequence of unbound template near the 5' end of the specific probe may be amplified together with circularized and linear ligation products generated in step (2). In another embodiment of the invention, a restriction enzyme site U is arranged near the 5' end of the specific probe, and primers before and after PCR amplification are respectively complementary with sequences on two sides of the restriction enzyme site U on the specific probe and are reversely complementary. After the USER enzyme or ssDNA endonuclease cleaves the circularized ligation product at the cleavage site U to form a linear ligation product, the PCR amplification primers can be combined with both ends of the cleaved linear ligation product through complementary sequences to achieve exponential amplification, as shown in fig. 1. In the method for enriching a target region of a gene, the step (4) or (5) may further include purifying the amplification product. The method of the present invention may be used to purify the amplification product, and may include, but is not limited to, silica gel column purification, heat treatment, magnetic bead purification, etc.

The gene target region enrichment method provided by the invention can further comprise the following steps: (6) and (3) detecting the ligation product provided in the step (4) or (5) by using a detection primer 1, a detection primer 2 and a probe 3, and rapidly providing a detection result of the target region by means of non-secondary sequencing but PCR detection. At least one of the detection primer 1, the detection primer 2 and the probe 3 comprises a gene specific sequence, that is, for different gene target regions, the combination of the related specific sequences of the three primers/probes can be monospecific, bispecific or trispecific. In one embodiment of the invention, the detection primer 1 comprises a gene specific sequence, the detection primer 2 and the probe 3 comprise a universal sequence, or the detection primer 1 and the detection primer 2 comprise a gene specific sequence; or the detection primer 1 and the probe 3 comprise gene-specific sequences, or the detection primer 1, the detection primer 2 and the probe 3 comprise gene-specific sequences. Probe 3 may also comprise a labeling molecule, which may be, for example, a fluorescent molecule, and the sequence of probe 3 is not complementary to detection primer 1 or 2.

The enrichment method of the gene target region provided by the invention can be used for nucleic acid detection. Methods for further detection by amplified ligation products are known to those skilled in the art. The method for enriching the target region can be applied to PCR-based gene sequence detection, for example, the target region comprises: the site of sequence variation may be, more specifically, a single-base mutation site region, a base deletion site region, a base insertion site region, a fusion mutation site region, an epigenetic variation or gene-specific sequence, or the like. In one embodiment of the invention, the enrichment method of the gene target region provided by the invention is applied to the detection of EGFR SNP-Q787 site mutation and male specific SRY gene, and obtains ideal effect.

In a second aspect, the present invention provides a system for enriching a fragmented DNA target region, comprising a specific probe and a ligase suitable for the method for enriching a gene target region provided in the first aspect of the present invention. The ligase may be thermostable RNA ligase, T4RNA ligase, or 5' App DNA/RNA thermostable ligase. The structure of the specific probe has been described in detail in the first aspect of the present invention and will not be described herein.

The system provided by the invention can also comprise one or more of the following components: dNTP coupled with a marker molecule, DNA polymerase, nuclease, endonuclease and the like. In this case, dNTP may be dCTP or dATP. The coupled marker molecule may be biotin. The DNA polymerase may be a DNA polymerase having 3 '-5' exonuclease activity, preferably a high fidelity DNA polymerase. The nuclease has 3 '-5' exonuclease activity. The endonuclease may be a USER enzyme or an ssDNA endonuclease.

The system provided by the invention can also comprise a PCR amplification primer, the sequence of which is usually matched with the general sequence of the specific probe and the sequences at two sides of the cyclization enzyme cutting site, and specifically can be a sequence which is complementary with the sequences at two sides of the cyclization enzyme cutting site and is at least partially complementary with the general sequence of the specific probe.

The system provided by the invention can also comprise a detection primer 1, a detection primer 2 and a probe 3 for PCR detection, wherein at least one of the three primers contains a gene specific sequence, and specifically can be a primer/probe combination with single specificity, dual specificity or tri specificity. In a preferred embodiment, only detection primer 1 comprises a gene-specific sequence. It is applied to the scenes of non-secondary sequencing but PCR detection.

In a preferred embodiment of the present invention, after library construction by the method or system of the present invention, the library molecules comprise the following sequences in structural order: a 5 'terminal sequencing universal sequence, a gene specific probe sequence, an enriched target region sequence, and a 3' terminal sequencing universal sequence, as shown in FIG. 2. Wherein the enriched target region contains sequence information of sample DNA before enrichment, and is characterized in that: the position of the 5' end on the genome is fixed and is determined by a specific probe; while the 3' end position is not fixed and is determined by the initial DNA fragmentation state of the library. Thus, the position of the 3' end of the sequence on the genome can also serve as a molecular tag in the data analysis after enrichment.

The invention has the advantages that firstly, the target areas of all fragmented DNA samples are pre-amplified by means of capture extension before connection, so that the loss or omission of original target molecules, especially small fragments and rare molecules, caused by insufficient ligase connection efficiency in the connection stage is avoided; the extension reaction in the pre-amplification stage belongs to linear amplification, the preference of PCR amplification is avoided, errors caused by PCR amplification can not be accumulated, and compared with the conventional PCR library building technology, the product uniformity is good; secondly, in the pre-amplification stage, only one single-stranded probe with the length of about 30bp needs to be designed for each target gene, so that the difficulty of designing double-ended primers for short fragments such as cfDNA is avoided, the library building success rate is improved, and the library building convenience is also improved; the 3' end of the probe is closed, so that the non-target connection between the probe and the outside of the template can be blocked, the background noise caused by the free probe is effectively reduced, and the dNTP coupled with the labeled molecule and a purification system thereof are assisted, so that the purity of a target product is further improved, and the DNA molecule of the sample reaches the highest conversion rate; and thirdly, after the 5 ' end of the specific probe is modified, the specific probe can be well connected to the 3' end of the extension product under the catalysis of single-chain ligase but can not be connected to the 3' end of the free probe which is subjected to closed modification, so that the self-connection and misconnection of the free probe are avoided, and the proportion of the target connection product in a final product is improved. Finally, the enrichment method of the present invention, compared to the enrichment method disclosed in the applicant's application No. 2019100024085, eliminates linker DNA, provides more convenience in raw material preparation, has a lower probability of producing undesired products by mutual interference between different components, and further reduces costs. In conclusion, the enrichment method and the system of the invention have simple operation and reliable result, can reduce the loss of original molecules, especially rare molecules to the maximum extent when being used for DNA with the fragment length of less than 200bp, enrich target molecules with the highest efficiency, have high fidelity, specificity and sensitivity, and can detect rare mutant molecules with the mutation rate as low as 0.01 percent.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.