阅读说明：本技术 基因目标区域富集方法及试剂盒 (Gene target region enrichment method and kit ) 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：本发明涉及生物技术领域,特别是涉及一种基因目标区域富集方法及试剂盒。本发明提供一种基因目标区域富集方法,包括：(1)通过特异性探针扩增包括目标区域的片段化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,所述特异性探针的3’末端核苷酸被修饰,用于阻止所述特异性探针的3’末端发生连接反应；(2)将步骤(1)所提供的捕获延伸产物的3’末端连接接头DNA,以提供连接产物。本发明所提供的富集方法操作简单、结果可靠,针对片段长度较短的DNA使用,可以最大程度的减少原始分子尤其是稀有分子的损失,最高效地富集目标分子。(The invention relates to the technical field of biology, in particular to a gene target region enrichment method and a kit. The invention provides a gene target region enrichment method, 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 to provide capture extension products, the specific probe having a3 'terminal nucleotide modified to prevent ligation at the 3' terminal of the specific probe; (2) ligating the 3' end of the captured extension product provided in step (1) with a linker DNA to provide a ligation product. 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 molecules to the greatest extent and enrich target molecules most efficiently by aiming at DNA with shorter fragment length.)

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

(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 to provide capture extension products, the specific probe having a3 'terminal nucleotide modified to prevent ligation at the 3' terminal of the specific probe;

(2) ligating the 3' end of the captured extension product provided in step (1) to a linker DNA to provide a ligation product.

2. The method for enriching a gene target region according to claim 1, wherein 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 to 200 bp;

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

3. The method for enriching a target region of a gene according to claim 2, wherein 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 target region of a gene according to claim 1, wherein the amplification system of step (1) further comprises an active substance for cleaving the 3' -terminal modifying group of the specific probe after binding to the target region, the active substance preferably being a nuclease;

and/or, the specific probe further comprises a universal sequence capable of being 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 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.

5. The method for enriching a gene target region according to claim 1, wherein the ligation system in the step (2) comprises a single-stranded ligase, preferably T4RNA ligase or thermostable RNA ligase;

and/or, the 5 'terminal nucleotide of the linker DNA is modified and has a single-stranded structure at the reaction temperature of step (2), preferably, the 5' terminal nucleotide of the linker DNA is substituted with a phosphate group or an adenosine group;

and/or the joint DNA is a partial double-stranded structure with a cohesive end at the 5' end region;

and/or the adaptor DNA comprises one or more of a combination of universal sequence, sample tag sequence and molecular tag sequence which can be recognized by a sequencing system.

6. The method of claim 1, wherein the step (1) further comprises purifying the capture extension product, preferably by a method selected from the group consisting of magnetic bead purification;

and/or, in the step (2), the method further comprises purifying the ligation product, wherein the purification method of the ligation product is preferably selected from silica gel column purification and/or heating treatment.

7. The method of claim 1, further comprising:

(3) amplifying the ligation product provided in step (2), preferably, in step (3), the PCR amplification primers have a sequence that matches the universal sequence of the specific probe and/or the universal sequence of the adaptor DNA.

8. The method of claim 7, further comprising:

(4) sequencing the amplified ligation product to provide a sequencing result of the target region.

9. The method of enriching for a gene target region according to claim 1, further comprising:

(5) detecting the ligation product provided in step (2) with 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 of claim 1, wherein the method is used for gene detection.

11. A kit for enriching a fragmented DNA target region, comprising a specific probe and linker DNA suitable for use in the method for enriching a gene target region according to any one of claims 1 to 9.

12. The kit of claim 11, further comprising one or more of: RNA ligase, dNTP coupled with a marker molecule, DNA polymerase and nuclease;

and/or further comprising a forward primer and a reverse primer having a sequence that is at least partially complementary to the universal sequence of the specific probe and the universal sequence of the adaptor DNA.

13. The kit of claim 12,

also comprises a detection primer 1, a detection primer 2 and a probe 3, wherein at least one of the three contains a gene specific sequence.

Technical Field

The invention relates to the technical field of biology, in particular to a gene target region enrichment method and a kit.

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 the method of adding a joint and then amplifying, such as the hybrid 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.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present invention aims 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 a method for enriching a target region of a gene, comprising:

(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 to provide capture extension products, the specific probe having a3 'terminal nucleotide modified to prevent ligation at the 3' terminal of the specific probe;

(2) ligating the 3' end of the captured extension product provided in step (1) to a linker DNA to provide a ligation product.

In another aspect, the present invention provides a kit for enriching a fragmented DNA target region, comprising a specific probe and linker DNA suitable for the gene target region enrichment method.

Drawings

FIG. 1 is a schematic flow chart of the enrichment process for a target region in an embodiment of the present invention.

FIG. 2 is a schematic diagram of library molecules constructed in accordance with an embodiment of the present invention.

FIG. 3 is a schematic diagram of the construction of the calibrator in the embodiment of the present invention.

FIG. 4 is a schematic flow chart of a monospecific detection system according to an embodiment of the present invention.

Detailed Description

The inventors of the present invention have made extensive exploratory studies to provide 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, thereby completing the present invention.

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

(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 to provide capture extension products, the specific probe having a3 'terminal nucleotide modified to prevent ligation at the 3' terminal of the specific probe;

(2) ligating the 3' end of the captured extension product provided in step (1) to a linker DNA to provide a ligation product.

The method for enriching a gene target region provided by the invention can comprise the following steps: the fragmented DNA comprising the target region is amplified by specific probes to provide capture extension products. In reaction, the specific probe can be extended based on the Capture of the complementary sequence of the target region to obtain a Capture-Extension Product (CEP).

In the gene target region enrichment method, in a reaction for amplifying fragmented DNA including a target region by a specific probe, the number of fragmented DNA including the target region may be one or more. Generally, the specific probes are in one-to-one correspondence with fragmented DNA including 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 the entire pre-amplification step of a sample, including cycles 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 a target region of a gene, the fragmented DNA may be double-stranded DNA, single-stranded DNA, cDNA, and the like, and the cDNA may be usually obtained by reverse transcription from RNA. For double-stranded DNA, a specific probe may comprise 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 of amplifying fragmented DNA including a target region by a specific probe can be usually performed in the presence of DNA polymerase, and after the 3' end-blocked modified probe is combined with a template under the action of high fidelity polymerase, the blocking group is cleaved, 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.one which correspondingly does not have 3 '-5' exonuclease activity). 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 the target region, and can also be combined with a DNA polymerase (for example, a common DNA polymerase) in the capture extension system to improve the efficiency of the amplification system, and the active substance is preferably a nuclease. The reaction for amplifying the fragmented DNA including the target region by the specific probe may be generally performed in the presence of dntps, which may be generally dntps coupled with label molecules, which may be, but are not limited to, biotin and the like, and dtnps, which may be, 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 be typically used for purification of captured extension products.

In the gene target region enrichment 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. 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 a free probe or the connection of other non-target molecules can be avoided, for example, the non-target molecules can be joint DNA and the like. 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, etc.) on the nucleotide at the 3' end of a specific probe to prevent a ligation reaction from occurring 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 a universal sequence that is 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 sequence can be the corresponding P1 sequence. In a preferred embodiment of the present invention, the specific probe has a 3' end tail regionThe base of the domain may comprise a mismatch, which may be the last base at the 3 'end of the probe, or a base near 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 gene target region, the step (1) may further include purifying the captured extension product. The capture extension product can be purified by a suitable method selected by one skilled in the art, for example, the purification method of the capture extension product can include magnetic bead purification, etc. In one embodiment of the present invention, the captured extension product can be purified by using a labeled molecule, and the purification process can be performed by using a solid phase purification method using magnetic beads coated with avidin or streptavidin, or the like.

The method for enriching a gene target region provided by the invention can further comprise the following steps: ligating the 3' end of the captured extension product provided in step (1) to a linker DNA to provide a ligation product.

In the method for enriching a target region of a gene, the ligation system for ligating the 3' end of the captured extension product to the adaptor DNA may include a single-stranded ligase for ligating the 5 ' end of the adaptor DNA to the 3' end of the captured extension product, and the single-stranded ligase is preferably T4RNA ligase, thermostable RNA ligase, or the like.

In the gene target region enrichment method, the linker DNA may be a single-stranded structure in which the 5 ' terminal nucleotide is modified and which is at the reaction temperature in step (2), so that a covalent bond may be generated by capturing the 3' terminal hydroxyl group of the extension product and the modifying group at the terminal of the linker DNA5 ' under the catalysis of a single-stranded ligase to obtain a ligation 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 linker DNA is substituted with a phosphate group to form a phosphorylation modification, and the 3 'hydroxyl group of the captured extension product and the phosphorylated 5' terminal of the linker DNA form a covalent bond under catalysis of a single-stranded ligase to obtain a ligation product. In another embodiment of the invention, the 5 ' terminal nucleotide of the linker DNA (e.g., the 5-hydroxyl group of the 5 ' terminal nucleotide, which may be specifically) is substituted with an adenosine group to form an adenylation modification, and the 5 ' end of the linker DNA can undergo a ligation reaction with the 3' end of the captured extension product catalyzed by a5 ' App DNA/RNA thermostable ligase. In another embodiment of the invention, the 5 'terminal nucleotide of the linker DNA (e.g., the 5-hydroxyl group of the 5' terminal nucleotide, which may be specifically) is substituted with a phosphate group to form a phosphorylated modification, and the 5 'end of the linker DNA may undergo a ligation reaction with the 3' end of the captured extension product catalyzed by a thermostable RNA ligase. The adaptor DNA may also be a partially double-stranded structure with a cohesive end in the 5 ' end region, and the cohesive end in the 5 ' end region has a single-stranded nature, so that the 5 ' end can be modified by the method described above and can be ligated to the capture extension product under catalysis of a suitable single-stranded ligase. The linker DNA may also include a combination of one or more of universal sequences, sample tag sequences, molecular tag sequences, and the like, which are typically recognized by a sequencing system to allow sequencing of the ligation products prepared. For example, for the Ion Torrent sequencing system, the universal sequence may be an a sequence, and the sample tag sequence may be a barcode, a molecular tag sequence corresponding thereto, and the like. The introduction of the barcode is beneficial to distinguishing samples from different sources in subsequent biological analysis, so that the sequencing of a plurality of samples can be simultaneously carried out in a single reaction to improve the flux. The introduction of the molecular tag is beneficial to identifying different molecules in subsequent biological analysis so as to further identify the variation generated in subsequent amplification. The use of these sequences, which can be identified by a sequencing system, allows the library of the invention to be sequenced via a high throughput sequencing platform to provide information needed for a variety of subsequent research and clinical applications.

In a preferred embodiment of the present invention, the hydroxyl group at the 3 'end of the linker DNA is also substituted with other groups to prevent ligation of the 3' end of the linker DNA, thereby avoiding self-ligation or ligation of other non-target molecules, such as free probes from step (1).

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 those skilled in the art, for example, the method for purifying the ligation product may include, but is not limited to, silica gel column purification, heat treatment, and the like.

The gene target region enrichment method provided by the invention can be used for gene detection. Methods for further gene detection by amplified ligation products will be known to those skilled in the art. In one embodiment of the present invention, the method for enriching the target region can be applied to high-throughput sequencing. In another embodiment of the present invention, the method for enriching a target region of the present invention can be applied to 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, or the like. In another embodiment of the present invention, the method for enriching the target region of the present invention can be applied to the detection of the genetic modification status, for example, the target region includes a region where a methylation site may exist, and for example, the DNA fragment is treated with bisulfite.

The gene target region enrichment method provided by the invention can further comprise the following steps: (3) amplifying the ligation product provided in step (2). The ligation products can be amplified by PCR, typically by a DNA polymerase and PCR amplification primers, and by amplifying the ligation products, the products of the DNA containing the target region can be further enriched. The skilled person can select suitable methods and systems for amplifying the ligation product provided in step (2), for example, PCR amplification primers having a sequence at least partially complementary to the universal sequence of the specific probe and/or the universal sequence of the adaptor DNA.

The gene target region enrichment method provided by the invention can further comprise the following steps: (4) sequencing the amplified ligation product to provide a sequencing result of the target region. After PCR amplification is carried out on the connection product, sequencing can be carried out on the amplification product by identifying the universal sequence in the amplification product, and a sequencing result of the target region is obtained. The person skilled in the art can select a suitable method and system to sequence the amplified ligation products, for example, sequencing the P1 sequence and the a sequence based on Ion Torrent platform to obtain the sequencing result of the target region. It should be noted that the enrichment method of the present invention can make the ligation product obtained in step (2) include any sequencing universal sequence, such as Ion Torrent sequencing platform, Illumina sequencing platform, or other sequencing platform universal sequences.

The gene target region enrichment method provided by the invention can further comprise the following steps: (5) and (3) detecting the ligation product provided in the step (2) 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. In some embodiments, probe 3 may further comprise a labeling molecule, such as a fluorescent molecule, and the sequence of probe 3 is not complementary to detection primer 1 or 2.

In a second aspect, the present invention provides a kit for enriching a fragmented DNA target region, comprising a specific probe and linker DNA suitable for use in the method for enriching a gene target region provided in the first aspect of the present invention. The structures of the specific probe and the linker DNA have been described in detail in the first aspect of the present invention, and are not described herein in detail.

The kit provided by the invention can also comprise one or more of the following components: RNA ligase, dNTP coupled with a labeled molecule, DNA polymerase, nuclease and the like. Wherein, the RNA ligase can be thermostable RNA ligase, T4RNA ligase, or 5' App DNA/RNA thermostable ligase; dNTP may be dCTP or dATP. The DNA polymerase can be DNA polymerase with 3 '-5' exonuclease activity, and preferably, the DNA polymerase is high-fidelity polymerase.

The kit provided by the invention can also comprise a forward primer and a reverse primer for PCR amplification, which are usually matched with the universal sequence of the specific probe and the universal sequence of the joint DNA, and particularly can have sequences at least partially complementary with the universal sequence of the specific probe and the universal sequence of the joint DNA.

The kit 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. Preferably, only detection primer 1 contains a gene-specific sequence, and the detection scheme refers to FIG. 4.

In a preferred embodiment of the present invention, after library construction by the method or kit of the present invention, the library molecules (e.g., the ligation products provided in step (2), the structure of which can be shown in fig. 2) structurally comprise the following sequences in sequence: a5 'terminal sequencing universal sequence, a gene specific probe sequence, an enriched target region sequence, a linker sequence, a barcode, a molecular tag sequence and a 3' terminal sequencing universal sequence, which are shown in figure 2. Wherein the enriched target region contains sequence information of the sample DNA before enrichment, and the partial sequence 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 molecular label can be used for distinguishing different molecules, and the sensitivity and the accuracy of detection are improved.

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 and the subsequent sequencing result is more accurate; 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 and shorter fragments such as ctDNA is avoided, the library construction success rate is improved, and the library construction 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 theoretical highest conversion rate; and thirdly, after the 5 'end of the joint DNA is modified, the joint DNA can be well connected to the 3' end of the extension product under the catalysis of single-stranded ligase but cannot be connected to the 3 'end of the specific probe subjected to blocking modification, and meanwhile, the 3' end of the joint is blocked to avoid self-connection and misconnection of the joint, so that the proportion of the target connection product in a final product is improved. Finally, a sequencing universal sequence, a sample tag sequence and a molecular tag sequence are set in the process of enriching a target area or on a probe and a joint, so that the aims of direct sequencing and data analysis after library building are fulfilled; or the single, double or triple specific primer/probe system of the invention is used for rapid detection of the target region. In conclusion, the enrichment method and the kit of the invention have simple operation and reliable result, can reduce the loss of original molecules, especially rare molecules to the maximum extent and enrich target molecules with the highest efficiency by aiming at the DNA with the fragment length of less than 200 bp; the enrichment method and the kit have extremely high fidelity, and the base errors generated in the sequencing process can be effectively corrected by a subsequent letter generation means, so that the highest sequencing accuracy rate which can be theoretically reached is obtained.

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.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; methodsin Enzymology, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.