Method and kit for enriching target nucleic acid

Abstract

The invention discloses a method and a kit for enriching target nucleic acid, and the method comprises the following steps: processing a sample nucleic acid to obtain a partial double-chain product containing a gap, binding oligonucleotide to the gap and extending the oligonucleotide, and connecting the oligonucleotide extending product with an adjacent chain to perform separation and enrichment. The oligonucleotide is connected with any chain of the target nucleic acid through a covalent bond, so that the combination stability of the oligonucleotide and the target nucleic acid is effectively improved, a denaturation process is not involved, and the method has the characteristics of high controllability and flexible scheme; the method provided by the invention can realize enrichment of long-fragment target nucleic acid, is suitable for enrichment of target nucleic acid containing a complex structure region, and has important significance for enrichment of target nucleic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method and a kit for enriching target nucleic acid. Background Art

[0002] Targeted enrichment of specific nucleic acids of interest is an important molecular biological operation that can specifically separate the target nucleic acid from the sample and accumulate it in quantity. Enrichment technology can be further combined with other detection methods to identify the enriched products, and is widely used in gene research, gene function research, variation / polymorphism analysis, genetic disease diagnosis, drug development, crop breeding and other fields.

[0003] Hybridization capture is one of the main technologies for enriching target nucleic acids. Conventionally, an oligonucleotide (i.e., probe) that is complementary to the target nucleic acid is designed to anneal with the sample nucleic acid that has been denatured into a single strand, and the specific binding of the oligonucleotide to the target nucleic acid is achieved through hydrogen bonds formed by complementary base pairing. Biotin labels are usually added to the oligonucleotides, and combined with magnetic beads modified with streptavidin or with various molecules modified with streptavidin anchored on the surface of the matrix to achieve capture and enrichment of the target nucleic acid. Compared with PCR amplification, another main technology for enriching target nucleic acids, hybridization capture technology can more realistically reflect the original state of the target nucleic acid and has a better enrichment effect on regions with complex structures and regions containing structural variations.

[0004] Therefore, capturing longer and more complete target nucleic acid fragments is more conducive to exerting the technical advantages of hybrid capture. In conventional hybrid capture, in order to ensure the capture quality, it is recommended to randomly break the sample nucleic acid into 150-350bp fragments before participating in the capture reaction, and, according to the sequence and structural characteristics of the target nucleic acid fragment, the length of the oligonucleotide used varies from 80 to 120nt. In order to achieve the capture of longer fragments, two main methods are currently adopted. One method is to design longer oligonucleotides to form more hydrogen bonds through base complementary pairing to enhance the binding force between oligonucleotides and target nucleic acids, thereby achieving the capture of long fragments. This method has the following problems. First, the longer the oligonucleotide, the higher the synthesis difficulty and the higher the synthesis cost. In the process of oligonucleotide synthesis, the current mainstream column synthesis has a maximum synthesis efficiency of 99%. Taking the synthesis of 100 base-length oligonucleotides as an example, the theoretical success rate is only 36.6%, that is, about 63.4% of the initial synthesis products are incompletely synthesized and need to be removed by purification. Second, longer oligonucleotides will lead to more nonspecific binding. Theoretically, only 10-20 consecutive bases of complementary pairing are required for the oligonucleotide to bind to the sample nucleic acid. If the oligonucleotide is long, the possibility of binding to non-target nucleic acids by segment is greatly increased, resulting in non-specific capture. Third, longer oligonucleotides are more likely to form secondary structures, reducing the binding efficiency with the target nucleic acid chain. Another way is to increase the density of oligonucleotides and use multiple oligonucleotides to achieve the capture of a target nucleic acid fragment. Its essence is to form more hydrogen bonds through base complementary pairing to enhance the binding force between the oligonucleotide and the target nucleic acid. In some cases, in order to ensure the capture effect, each oligonucleotide covers the target nucleic acid in a tile-like manner. Moreover, as the number of oligonucleotides increases, its cost will also increase accordingly. Therefore, how to achieve efficient enrichment of long and complete fragments of target nucleic acids is an urgent problem to be solved. Summary of the invention

[0005] In view of this, one of the purposes of the present invention is to provide a method for enriching target nucleic acids, and the method can achieve efficient enrichment of long-fragment target nucleic acids.

[0006] A method for enriching target nucleic acid, comprising the following steps:

[0007] Processing the sample nucleic acid into a partially double-stranded product containing a gap, and then binding an oligonucleotide to the gap of the partially double-stranded product; wherein the 3' portion of the oligonucleotide is consistent with the partial gap sequence;

[0008] Extending the oligonucleotide to obtain an extension product, wherein the extension product covers the 3' end of the oligonucleotide to the 3' end of the gap, and then connecting the extension product to the 3' side adjacent strand of the extension product by a ligase and / or a ligase active fragment;

[0009] Finally, oligonucleotides are used to enrich the target nucleic acid.

[0010] The target nucleic acid described in the present invention is a double-stranded nucleic acid, which is the smallest unit captured by the oligonucleotide. In the enrichment method provided by the present invention, there is no specific restriction on the number of target nucleic acids. When there are multiple target nucleic acids, they can be multiple arranged continuously, multiple arranged non-continuously, or a combination of various situations. For example, when the method of the present invention is used to enrich long fragments or even ultra-long fragments of nucleic acid, the nucleic acid fragment can be divided into multiple continuous, relatively short, equal or unequal length target nucleic acids, thereby achieving enrichment. For another example, multiple target nucleic acids located on different chromosomes are enriched. For another example, for different fragments located on different chromosomes, a part of the fragments can be used as a separate plurality of target nucleic acids, and another part of the fragments can be divided into multiple continuous, relatively short, equal or unequal length target nucleic acids for enrichment.

[0011] In the partially double-stranded product of the present invention, for a certain target nucleic acid, the number of gaps can be one or more; when there are multiple gaps, the gaps can be located on the same strand of the target nucleic acid or on different strands of the target nucleic acid. The gaps described in the present invention specifically refer to regions where one or more consecutive nucleotides are missing.

[0012] It is understood that, for a target nucleic acid strand (i.e., any strand of the target nucleic acid and located on the same strand as the gap), its relative position to the gap includes the following situations:

[0013] ① The gap is completely located inside the target nucleic acid chain;

[0014] ② The gap is completely located outside the 5' end of the target nucleic acid chain;

[0015] ③ The gap portion is located inside the target nucleic acid chain and the other portion is located outside the 5' end of the target nucleic acid chain;

[0016] ④ The gap portion is located inside the target nucleic acid chain and the other portion is located outside the 3' end of the target nucleic acid chain;

[0017] ⑤ The gap is completely located outside the 3' end of the target nucleic acid chain.

[0018] Preferably, in the above-mentioned enrichment method, at least one gap in the partial double-stranded product is not located outside the 3' end of the target nucleic acid chain, that is, at least one gap in the partial double-stranded product does not belong to the above-mentioned situation ⑤, so that the gap can be used to effectively capture and enrich the target nucleic acid. This preferred embodiment can also be expressed as: at least one gap in the partial double-stranded product belongs to any of the above-mentioned situations ①, ②, ③, and ④ for the binding of oligonucleotides.

[0019] Preferably, in the above-mentioned enrichment method, the partially double-stranded product is prepared by:

[0020] Processing sample nucleic acid to generate nicks (Nick);

[0021] Then, a nuclease and / or an exonuclease active fragment is used to perform single-stranded cleavage from the nick to form a gap, thereby obtaining a partial double-stranded product.

[0022] In the above preferred enrichment method, the exonuclease and / or exonuclease active fragment acts only on a single strand, starting from the nick, cleaving the phosphodiester bond between adjacent nucleotides to release a single nucleotide.

[0023] In the above preferred enrichment method, the cutting direction of the exonuclease and / or exonuclease active fragment used can be 5' to 3', or 3' to 5', or a combination of enzymes and / or enzyme active fragments having two cutting directions. Moreover, in the specific operation, enzymes and / or their active fragments with different exonuclease activities, enzymes and / or their active fragments with different enzyme cutting speeds can be selected, and the formation of the gap and the length of the gap can be controlled by controlling the enzyme cutting reaction environment and the enzyme cutting reaction duration.

[0024] Therefore, the number of gaps and their relative positions to the target nucleic acid are mainly controlled by the three factors of the incision position, the exonuclease and / or the exonuclease active fragment, and the enzyme digestion reaction conditions. Based on this, it can be understood that when the incision is located outside the 5' end of any strand of the target nucleic acid, the resulting gap may be spaced from the 5' end of the target nucleic acid chain, may be adjacent to the 5' end of the target nucleic acid chain, and may also include a partial sequence within the target nucleic acid chain; when the incision is located inside any strand of the target nucleic acid, the resulting gap may be completely located inside the target nucleic acid, or may include a partial sequence outside the 5' end or the 3' end of the target nucleic acid.

[0025] In addition, when the incision is located inside any strand of the target nucleic acid, no double-strand break of the nucleic acid is generated at the incision; when the incision is located outside the 5' end of any strand of the target nucleic acid, a double-strand break may or may not be generated at the incision. At this time, in order to ensure the integrity of the target nucleic acid, the nuclease and / or nuclease active fragment used has no recognition site inside the target nucleic acid.

[0026] It should be noted that in the above-mentioned preferred enrichment method, the exonuclease and / or exonuclease active fragment can not only perform single-strand cutting from the incision, but also perform single-strand cutting from the break; the break is caused by the incision produced by the endonuclease cutting at the same or similar position of the two chains, but also by random physical breaks caused during the operation. In order to avoid the influence of physical breaks on this scheme, in specific operations, the integrity of the sample nucleic acid can be improved by optimizing the separation, extraction and / or purification methods of the sample nucleic acid, reducing physical breaks, so as to further improve the enrichment effect of the scheme of the present invention on long fragments.

[0027] In the partially double-stranded product of the present invention, for a certain target nucleic acid, although there may be one or more gaps that fall into the situations of ①, ②, ③, and ④, it should be noted that not all gaps need to be bound to oligonucleotides. In actual operation, those skilled in the art can first determine the preset gaps that need to be bound to oligonucleotides according to the target nucleic acid, then process the sample nucleic acid to generate incisions at the preset positions, and then process the incisions into preset gaps.

[0028] More preferably, the present invention can process the sample nucleic acid by any one of a) to f) or a combination of several methods to generate a cut at a preset position:

[0029] a) treating the sample nucleic acid with one or more endonucleases A and / or endonuclease A active fragments, wherein the endonuclease A is a nicking enzyme or a variant of a nicking enzyme;

[0030] b) treating the sample nucleic acid with one or more endonucleases B and / or endonuclease B active fragments, wherein the endonuclease B is an endonuclease guided by a guide nucleic acid or a variant of such an enzyme;

[0031] c) treating the sample nucleic acid with one or more endonucleases C and / or endonuclease C active fragments, wherein the endonuclease C is a restriction endonuclease or a variant of a restriction endonuclease;

[0032] d) treating the sample nucleic acid with one or more endonucleases D and / or endonuclease D active fragments, wherein the endonuclease D is a transcription activator-like effector nuclease or a variant of such an enzyme;

[0033] e) treating the sample nucleic acid with one or more endonucleases E and / or endonuclease E active fragments, wherein the endonuclease E is a zinc finger nuclease or a variant of a zinc finger nuclease;

[0034] f) treating the sample nucleic acid with one or more endonucleases F and / or endonuclease F active fragments, wherein the endonuclease F is a recombinant protein-dependent endonuclease or a variant thereof.

[0035] In the above-mentioned methods of generating incisions, variants of the involved endonucleases include, but are not limited to, mutants obtained by changing the nucleic acid sequence or amino acid sequence of the wild-type protein, fusion proteins comprising endonuclease domains, and proteins with one or more chemical modifications. Chemical modifications include, but are not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, addition of protective groups or blocking groups, proteolytic cleavage, and connection with cell ligands or any other molecules.

[0036] In the above method a), the nucleic acid nicking enzyme or its variant recognizes a fixed nucleic acid sequence (i.e., the cleavage site). For one cleavage site, under the action of the nucleic acid nicking enzyme or its variant, the nucleic acid double strand only breaks one phosphodiester bond on one strand, producing an incision. Usually, the cleavage sites of different nucleic acid nicking enzymes or their variants are not adjacent, and no nucleic acid double strand breaks are produced. The cleavage effect of the nucleic acid nicking enzyme or its variant can be estimated by sequence analysis. Nt.BstNBI, Nb.BtsI, and Nb.BsrDI are common nucleic acid nicking enzymes.

[0037] In the above-mentioned mode b), a guide nucleic acid-guided endonuclease or its variant is used, which needs to be assembled with the guide nucleic acid to form a complex to exert biological functions. There is no restriction on the assembly process. It can be carried out in an independent system, and then the assembled complex is put into the reaction system for producing the incision; or the guide nucleic acid, the guide nucleic acid-guided endonuclease or its variant can be directly added to the reaction system for producing the incision, and self-assembled in the reaction system. The guide nucleic acid-guided endonuclease has an endonuclease domain, which acts on the phosphodiester bond between adjacent nucleotides inside the nucleic acid chain, resulting in the cleavage of the phosphodiester bond and the formation of an incision. Generally, the number of endonuclease domains determines the effect of its cutting. If it contains only one endonuclease domain, it usually cuts only once on one chain to form an incision; if it contains two endonuclease domains, it usually cuts once on each of the two chains, and the cutting positions are the same or adjacent, forming two incisions and causing the break of the double-stranded nucleic acid, and the break can be a flat end or a single-stranded protruding end. Cas protein family members, Ago protein family members, and Fanzor protein family members are reported guide nucleic acid-guided endonucleases. Taking the widely used Cas9 protein as an example, it contains two endonuclease domains, which cut the single strand of nucleic acid complementary to the guide nucleic acid and the single strand of nucleic acid non-complementary to the guide nucleic acid, respectively, to produce double-strand breaks in the nucleic acid.

[0038] The present invention may use a guide nucleic acid-guided endonuclease or a variant thereof. In specific cases, the guide nucleic acid-guided endonuclease or a variant thereof may differ from the wild type in terms of the optimal reaction temperature, the number of endonuclease domains contained, and the activity strength of the endonuclease domains contained.

[0039] The present invention does not limit the number and function of the endonuclease domains of the guide nucleic acid-guided endonuclease. For example, a guide nucleic acid-guided endonuclease that cuts only on a single strand and does not produce a double-strand break in the nucleic acid can be used, and a guide nucleic acid-guided endonuclease that cuts on a double strand of the nucleic acid and produces a double-strand break in the nucleic acid can also be used.

[0040] The guide nucleic acid comprises a nucleic acid sequence that is complementary or substantially complementary to the region near the cleavage site. With the help of the complementary nucleic acid sequence, the guide nucleic acid-guided endonuclease binds and cuts at a preset position. Different types of guide nucleic acid-guided endonucleases require different types of guide nucleic acids, such as DNA guide nucleic acids, RNA guide nucleic acids, and can be further subdivided. Taking the Cas protein as an example, it requires RNA guide nucleic acid, some Cas proteins use crRNA alone as a guide nucleic acid, and some Cas proteins require a combination of crRNA and tracrRNA as a guide nucleic acid. For the latter, crRNA and tracrRNA can be fused together by artificial design to form sgRNA (single guide RNA) to perform the function.

[0041] The present invention does not limit the type and structure of the guide nucleic acid, as long as it matches the endonuclease guided by the guide nucleic acid used. The present invention can also use derivatives of the guide nucleic acid, which may include mutants obtained by changing the nucleic acid sequence, and nucleic acids with one or more chemical modifications. Chemical modifications include, but are not limited to, acetylation, phosphorylation, methylation, deletion of one or more original chemical groups, and covalent attachment of any other type of molecule to the guide nucleic acid.

[0042] In addition, the present invention does not limit the number of complexes. For example, a guide nucleic acid can be assembled with a guide nucleic acid-guided nuclease or a variant thereof to obtain a complex to cut a preset cleavage site; for another example, a guide nucleic acid can be assembled with a guide nucleic acid-guided nuclease or a variant thereof to obtain a complex, the guide nucleic acid is complementary to the same conserved sequence contained in multiple target nucleic acids, and cuts multiple preset cleavage sites; for another example, multiple guide nucleic acids can be assembled with guide nucleic acid-guided nuclease or a variant thereof to obtain multiple complexes to achieve cutting of multiple preset cleavage sites.

[0043] In the above method c), the restriction endonuclease or its variant recognizes a fixed nucleic acid sequence (i.e., the restriction site), generates at least one set of cuts on the double-stranded nucleic acid, and each set of cuts includes two cuts located at the same or adjacent positions on the double-stranded nucleic acid, which will cause the break of the double-stranded nucleic acid. The restriction endonuclease and / or restriction endonuclease active fragment has a known or artificially designed recognition site, and the restriction effect can be preset by sequence analysis to achieve fixed-point cutting, replacing the random interruption in conventional hybridization capture.

[0044] In the above method d), the transcription activator-like effector nuclease (TALEN) or its variant comprises a TALE nucleic acid binding domain and a nuclease endonuclease domain that recognizes and binds to a specific nucleic acid sequence. The binding of the nucleic acid binding domain at a preset position can be achieved by artificial design, and the nuclease endonuclease domain is pulled to cut at the preset position to produce a phosphodiester bond break. Generally, the number of nuclease endonuclease domains determines the effect of its cutting. For example, the most commonly used FokI nuclease endonuclease domain needs to form a dimer to have biological activity, that is, the formed TANLEN contains two nuclease endonuclease domains, so it will cut on the two nucleic acid chains to produce incisions, resulting in the breakage of the nucleic acid chain. Similar to method b), there is no limit to the number of TALEN, which will not be repeated here.

[0045] In the above method e), the zinc finger nuclease (Zinc-finger nuclease, ZFN) or its variant comprises a zinc finger protein nucleic acid binding domain and a nuclease endonuclease domain that recognizes and binds to a specific nucleic acid sequence. The binding of the nucleic acid binding domain at a preset position can be achieved by artificial design, and the nuclease endonuclease domain is pulled to cut at the preset position to produce a phosphodiester bond break. Generally, the number of nuclease endonuclease domains determines the effect of its cutting. For example, the most commonly used FokI nuclease endonuclease domain needs to form a dimer to have biological activity, that is, the formed ZFN contains two nuclease endonuclease domains, so it will cut on the two nucleic acid chains to produce incisions, resulting in the breakage of the nucleic acid chain. Similar to method b), there is no limit to the number of TALENs, which will not be repeated here.

[0046] In the above method f), a recombinant protein-dependent nuclease (Rec-dependent endonuclease) or its variant can also achieve cutting of the preset position of the sample nucleic acid. Single-stranded nucleotides complementary to the preset position can be artificially designed, combined with the sample nucleic acid under the action of the recombinant protease, and the double strands of the sample nucleic acid are cut under the action of the nuclease to cause a break. For example, a commonly used recombinant protein-dependent nuclease is Ref, which relies on the recombinant protein RecA to perform its function.

[0047] Preferably, in the above enrichment method, before the sample nucleic acid is nicked, the sample nucleic acid is first pretreated for dephosphorylation, and after the nick is generated, a nuclease and / or an exonuclease active fragment that is selective for phosphorylated ends is used to perform single-stranded cleavage from the nick to form a gap.

[0048] More preferably, in the above preferred enrichment method, the exonuclease selective for phosphorylated ends is lambda exonuclease. Lambda exonuclease is a 5' to 3' exonuclease that specifically acts on DNA and can selectively digest double-stranded DNA phosphorylated at the 5' end along the 5' to 3' direction, and has low enzymatic activity on DNA that is not phosphorylated at the 5' end.

[0049] The oligonucleotide of the present invention specifically includes a 5' portion and a 3' portion, wherein the 5' portion is a sequence close to the 5' end of the oligonucleotide, and the 3' portion is a sequence close to the 3' end of the oligonucleotide.

[0050] In the enrichment method provided by the present invention, there is no specific restriction on the number of oligonucleotides. Only one oligonucleotide can be used to enrich one target nucleic acid. One oligonucleotide can also be used to enrich multiple target nucleic acids containing the same conserved sequence. Multiple oligonucleotides can also be used to enrich multiple target nucleic acids.

[0051] In the above-mentioned enrichment method, it is only required that the 3' portion of the oligonucleotide is consistent with the sequence of a partial region in the gap, so that the 3' portion of the oligonucleotide can bind to the partial double-stranded product through base complementary pairing; it is understandable that the partial region can be any region in the gap, so the oligonucleotide can bind to any position in the gap (such as the 5' end position or the 3' end position or any position in the middle); for the 5' portion of the oligonucleotide, it can be consistent with the partial sequence in the gap, or it can be inconsistent. In actual operation, the combination of the oligonucleotide and the partial double-stranded product is usually achieved by controlling the reaction temperature. More specifically, the reaction temperature is set according to the theoretical annealing temperature of the oligonucleotide to achieve annealing and binding of the oligonucleotide and the partial double-stranded product.

[0052] Preferably, in the above enrichment method, the 5' portion of the oligonucleotide is identical to a portion of the sequence in the gap. In this case, the entire oligonucleotide may bind to the gap, not just the 3' portion.

[0053] More preferably, in the above preferred enrichment method, the 5' end of the oligonucleotide is bound to the 5' end of the gap. This scheme is particularly suitable for the two situations where the gap is completely located inside the target nucleic acid chain (case ①) or the gap is partially located inside any chain of the target nucleic acid and the other part is located outside the 3' end of the target nucleic acid chain (case ④), which can effectively ensure the integrity of the target nucleic acid.

[0054] More preferably, after the 5' end of the oligonucleotide is bound to the 5' end of the gap, a ligase and / or a ligase active fragment is used to catalyze the 5' end phosphate of the oligonucleotide and the 3' end hydroxyl of the 5' side adjacent chain to form a phosphodiester bond, and then the oligonucleotide is extended and connected. This solution can further improve the binding stability of the oligonucleotide and the target nucleic acid.

[0055] In the above-mentioned enrichment method, a polymerase and / or a polymerase active fragment can be used to start from the nucleotide complementary to the partial double-stranded product at the 3' end of the oligonucleotide and extend along the gap to obtain an extended product. It is understandable that for the gap of the unbound oligonucleotide, under the action of the polymerase and / or the polymerase active fragment, if there is a nucleic acid chain 5' upstream of the gap, the 3' end of the nucleic acid chain can also be used as the amplification starting position and extend along the gap, and the extension can fill the gap in whole or in part, and the extension will not have a negative impact on the enrichment of the target nucleic acid.

[0056] The polymerase and / or polymerase active fragment have polymerization activity. The polymerase and / or polymerase active fragment may also have other activities. For example, the polymerase and / or polymerase active fragment may have 3'-5' exonuclease activity, which can be used to remove mismatched nucleotides in the extension process to improve fidelity. For another example, the polymerase and / or polymerase active fragment may have 5'-3' exonuclease activity, and after extending to the 3' end of the gap, the downstream chain of the 3' gap can continue to be cut and extended.

[0057] In the above-mentioned enrichment method, the extension product includes a sequence covering the 3' end of the oligonucleotide to the 3' end of the gap. The present invention fills the single-stranded region between the 3' end of the oligonucleotide and the 3' end of the gap through an extension reaction. If the oligonucleotide is bound to any position in the middle of the gap, then the partial region of the gap can be filled by the extension reaction; if the 5' end of the oligonucleotide is bound to the 5' end of the gap, then the entire gap can be filled by the extension reaction. In specific operations, enzymes and / or their active fragments with different polymerase activities, enzymes and / or their active fragments with different extension speeds can be selected, and the gap can be filled by controlling the extension reaction environment and the extension reaction duration.

[0058] In addition, it can be understood that when the polymerase and / or the polymerase active fragment do not have 5'-3' exonuclease activity, the extension product includes the oligonucleotide and a partial sequence covering the 3' end of the oligonucleotide to the 3' end of the gap; when the polymerase and / or the polymerase active fragment has 5'-3' exonuclease activity, the extension product may include the oligonucleotide, a sequence covering the 3' end of the oligonucleotide to the 3' end of the gap and the synthesized 3' downstream chain of the gap.

[0059] In the above enrichment method, ligase and / or ligase active fragment is used to catalyze the formation of phosphodiester bond between the hydroxyl group at the 3' end of the extension product and the phosphate group at the 5' end of the 3' side adjacent chain of the extension product to form a complete chain.

[0060] Preferably, in the above-mentioned enrichment method, artificial sequences and / or modifications are added to the oligonucleotide. The artificial sequence includes, but is not limited to: a barcode sequence for distinguishing the nucleic acid sources of different samples, and a linker sequence for adapting to downstream detection methods. The modification includes, but is not limited to: biotinylation, acetylation, phosphorylation, methylation, deletion of one or more original chemical groups, and covalent attachment of any other type of molecule to the oligonucleotide. Specifically, the oligonucleotide may be added with only an artificial sequence, only a modification, or both an artificial sequence and a modification.

[0061] More preferably, in the above preferred enrichment method, the oligonucleotide is added with biotin modification and / or thio modification.

[0062] When the oligonucleotide is modified with biotin, the oligonucleotide can be combined with magnetic beads modified with avidin or with various molecules modified with avidin anchored on the surface of the matrix to achieve separation of the connection product. The prior art shows that three types of avidin, streptavidin, avidin, and neutrophil interleukin, have strong affinity with biotin and can be used to separate the connection product of the present invention.

[0063] When oligonucleotides are modified with thiolation, the oligonucleotides can be protected from the action of 5'-3' exonucleases.

[0064] More preferably, in the above preferred enrichment method, uracil (U) is added to the oligonucleotide, and when to be separated, USER enzyme is used to separate the target nucleic acid from the solid phase carrier, thereby releasing the target nucleic acid.

[0065] More preferably, a linker sequence is added to the 5' end of the oligonucleotide to facilitate direct sequencing of the enriched product.

[0066] Preferably, in the above enrichment method, it also includes:

[0067] When the oligonucleotide is used to enrich the target nucleic acid, the single strand complementary to the oligonucleotide is digested with an exonuclease, and only the single strand of the target nucleic acid connected to the oligonucleotide is enriched.

[0068] Preferably, in the above-mentioned enrichment method, the following steps are also included:

[0069] The enriched product is amplified or sequenced. For example, the amount of the enriched product can be further increased by PCR amplification technology, wherein the PCR method can be a conventional three-step method, or other methods such as isothermal amplification and whole genome amplification. For another example, the enriched product can be sequenced and detected by first-generation sequencing, second-generation sequencing, third-generation sequencing, etc.

[0070] In addition, in the above-mentioned enrichment method, in addition to the biotin-avidin method, a method similar to a gene chip can be used to separate the target nucleic acid, that is, a nucleic acid probe that is partially complementary to the oligonucleotide chain is fixed on the surface of the matrix, and then the connection product is separated by molecular hybridization. Furthermore, the matrix surface can be modified with APTES-PDITC or APTES-glutaraldehyde for the fixation of the nucleic acid probe.

[0071] In the enrichment method provided by the present invention, the sample nucleic acid can be genomic DNA, plasmid DNA, DNA fragments, RNA, DNA and / or RNA analogs, and can also be modified derivatives of the above nucleic acids. Among them, genomic DNA includes nuclear genome and organelle genome; DNA fragments can be obtained by various technical means, such as physical methods and enzyme cutting methods to break genomic DNA to obtain DNA fragments, and further, the DNA fragments obtained by breaking can be used to build a library to obtain a DNA fragment library.

[0072] The sources of the sample nucleic acid include, but are not limited to, animals (human and other animals), plants, bacteria, fungi, viruses, etc. The sample nucleic acid can be separated, extracted and / or purified by methods known in the art, such as enzymatic hydrolysis, SDS method, CTAB method, phenol extraction method, guanidine isothiocyanate method or a combination thereof for nucleic acid separation and / or extraction, and magnetic bead method, permeable membrane method, ethanol or isopropanol precipitation method or a combination thereof for nucleic acid purification. In addition, commercially available reagents or kits can also be used to separate, extract and / or purify nucleic acids.

[0073] In the enrichment method provided by the present invention, there is no specific restriction on the function of the target nucleic acid. For example, the target nucleic acid may be part or all of the structural genes, regulatory genes, and other genes without translation products. For example, the target nucleic acid may contain one or more variant sites, and the variant sites include but are not limited to sites and polymorphic sites that cause phenotypic changes in the organism. The variation includes but is not limited to: single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP), single nucleotide site variation (Single Nucleotide Variant, SNV), insertion-deletion mutation (Indel), simple sequence repeat marker (Simple Sequence Repeat, SSR), short tandem repeat (Short Tandem Repeat, STR), microsatellite DNA (Microsatellite DNA), microsatellite instability (Microsatellite Instability, MSI), copy number variation (Copy Number Variation, CNV), copy number polymorphism (Copy Number Polymorphism, CNP), fusion gene (Fusion Gene), structural variation (Structural Variation, SV).

[0074] In the enrichment method provided by the present invention, the enzyme and enzymatic activity fragments to which each step is related are intended to clearly provide enzyme molecules with the same function in the reaction system, and the number and combination of enzymes and enzymatic activity fragments are not limited. For the enzyme and enzymatic activity fragments of a certain function, for example, one or more enzymes can be used, one or more enzymatic activity fragments can also be used, a combination of an enzyme and another enzymatic activity fragment can also be used, a combination of multiple enzymes and the active fragments of other enzymes can also be used, a combination of an enzyme and the active fragments of other multiple enzymes can also be used, and a combination of multiple enzymes and the active fragments of other multiple enzymes can also be used. In some embodiments, variants of enzymes and / or enzymatic activity fragments can be used, such as enzymes and / or enzymatic activity fragments with one or more modifications and / or one or more mutation sites, and / or recombined with other proteins, fused.

[0075] Based on the above method for enriching target nucleic acid, a second object of the present invention is to provide a kit for enriching target nucleic acid.

[0076] Preferably, the kit comprises at least: an endonuclease and / or an endonuclease active fragment for generating a nick on the sample nucleic acid, an exonuclease and / or an exonuclease active fragment for performing single-strand cleavage at the nick and forming a gap, a polymerase and / or a polymerase active fragment for extending an oligonucleotide to obtain an extension product, and a ligase and / or a ligase active fragment for connecting the extension product to its adjacent strand.

[0077] More preferably, the kit may also include other reagents and materials involved in each step, such as oligonucleotides of specific sequences, buffer, dNTPs, magnetic beads, etc.

[0078] Compared with the prior art, the present invention has the following advantages:

[0079] (1) The binding of oligonucleotide to target nucleic acid is stable. The existing technologies all achieve the binding of oligonucleotide to target nucleic acid based on complementary base pairing, the essence of which is the formation of hydrogen bonds. Generally speaking, hydrogen bonds are much weaker than covalent bonds. In the present invention, the oligonucleotide is connected to any strand of the target nucleic acid through a covalent bond (i.e., a phosphodiester bond), and its stability is much higher than that of hydrogen bonds.

[0080] (2) The structure of the enriched product is stable. In the prior art, only the limited region where the oligonucleotide binds to the target nucleic acid is a double-stranded structure. The target nucleic acid obtained in the present invention is a stable long double-stranded structure, that is, it contains any strand of the target nucleic acid and its complementary strand. The stable double-stranded structure ensures the integrity of the target nucleic acid in the subsequent separation step.

[0081] (3) Based on the first two advantages, the present invention can achieve the enrichment of long-fragment target nucleic acids. The length of the enriched product is related to the nick interval on the side of the target nucleic acid or inside the target nucleic acid, and the enrichment of ultra-long-fragment target nucleic acids can be achieved by increasing the nick interval.

[0082] (4) The enrichment process does not involve a denaturation process, and can truly reflect the original state of the target nucleic acid. The prior art requires denaturation of the target nucleic acid to form a single strand in order to achieve complementary pairing with the oligonucleotide. In the present invention, a gap for the target nucleic acid is cleverly created by combining endonucleases and exonucleases, providing a position for the binding of the oligonucleotide.

[0083] (5) Strong controllability. In practical applications, physical random interruption is usually used to fragment the sample nucleic acid before hybridization capture, and the interruption position cannot be accurately controlled. The present invention uses an endonuclease with a fixed recognition site to process the sample nucleic acid, and the cutting site can be artificially designed and controlled. In addition, combined with the use of an exonuclease, the oligonucleotide binding position can be artificially designed and controlled.

[0084] (6) Flexible technical solution: The present invention can generate a gap for binding oligonucleotide at a fixed point on any strand of the target nucleic acid, and can be designed according to actual needs.

[0085] (7) Based on the above advantages, the present invention is also applicable to the enrichment of target nucleic acids containing complex structural regions. For example, oligonucleotide binding sites can be obtained by enzyme cleavage outside the complex structural region, and a ligation product containing the complex structural region can be obtained by subsequent extension and ligation, thereby ultimately achieving the enrichment purpose.

[0086] (8) Short time consumption. The present invention can complete the process from processing sample nucleic acid to stably combining oligonucleotides with target nucleic acid within 4 hours at the fastest. In the prior art, the hybridization of oligonucleotides with sample nucleic acid alone requires several hours or even overnight reaction. Compared with the two, the present invention has obvious advantages.

[0087] (9) The enriched product can be used in a variety of ways. Unlike the prior art, which can only release the enriched product complementary to the oligonucleotide by denaturation, the enriched product obtained in the present invention is a double-stranded structure, which can be used directly, or only the complementary strand of any strand of the target nucleic acid released by denaturation can be used, or only any strand of the target nucleic acid connected to the oligonucleotide can be used. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 This is a schematic diagram of a specific implementation mode 1 of the method for enriching target nucleic acid provided by the present invention.

[0089] Figure 2 This is a schematic diagram of a second specific implementation mode of the method for enriching target nucleic acid provided by the present invention.

[0090] Figure 3 This is a schematic diagram of a third specific implementation mode of the method for enriching target nucleic acid provided by the present invention.

[0091] Figure 4 A schematic diagram of a fourth specific implementation mode of the method for enriching target nucleic acid provided by the present invention.

[0092] Figure 5 This is a schematic diagram of a fifth specific implementation mode of the method for enriching target nucleic acid provided by the present invention.

[0093] Figure 6 It is a detection gel image of the enrichment product in Examples 1 and 2, wherein Flank1-1, Flank1-2, Flank1-3, Flank1-4, CK1, and CK2 are the names of the primer pairs used, lanes 1 to 3 are three technical repeats in Example 1, lanes 4 to 6 are three technical repeats in Example 2, ck- is a no-template negative control, and ck+ is a sample nucleic acid positive control.

[0094] Figure 7 It is a detection gel image of the enrichment product in Example 4, wherein Flank2-1, Flank2-2, Flank2-3, Flank2-4, Flank2-5, Flank2-6, CK1, and CK2 are the names of the primer pairs used, and lanes 1 to 4 represent four concentration gradients of oligonucleotide input, which are 44 nM, 4.4 nM, 0.44 nM, and 0.044 nM, respectively. ck- is a no-template negative control, and ck+ is a sample nucleic acid positive control. DETAILED DESCRIPTION

[0095] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0096] The present invention provides a method for enriching target nucleic acid, comprising the following steps:

[0097] The sample nucleic acid is processed into a partially double-stranded product containing a gap, and then an oligonucleotide is bound to the gap of the partially double-stranded product; wherein the 3' portion of the oligonucleotide is consistent with the partial gap sequence, and at least one of the gaps is not located outside the 3' end of the target nucleic acid chain; the target nucleic acid chain is any chain of the target nucleic acid and is located on the same chain as the gap;

[0098] Extending the oligonucleotide to obtain an extension product, the extension product covers the 3' end of the oligonucleotide to the 3' end of the gap, and then connecting the extension product to the 3' side adjacent strand of the extension product by a ligase and / or a ligase active fragment;

[0099] The oligonucleotides are used to enrich target nucleic acids.

[0100] The gap is formed by: treating the sample nucleic acid to generate a nick; and then using an exonuclease and / or an exonuclease active fragment to perform single-stranded cutting from the nick to form a gap.

[0101] Based on the method for enriching target nucleic acid provided by the present invention, the present invention further illustrates several specific embodiments in conjunction with the accompanying drawings:

[0102] (1) Method 1. Figure 1 As shown, a nuclease is used to generate only one cut outside the 5' end of any strand of the target nucleic acid, and the target nucleic acid strand is the part filled with slashes. The specific process is as follows:

[0103] First, use an endonuclease or its active fragment to Figure 1 -A arrow position is cut by enzyme, and specifically, endonuclease A, B, D, E or a combination thereof can be used. The incision produced by enzyme cutting is as follows Figure 1 As shown in triangle -B, the nick is located outside the 5' end of the target nucleic acid chain.

[0104] Then use 3'-5' exonuclease to cut the single strand from the 3' end and the nick of the double-stranded nucleic acid to generate a gap and obtain a partial double-stranded product ( Figure 1 -C). Wherein, region 1 is the gap produced by cutting from the nick, and regions 2 and 3 are the gaps produced by cutting from the 3' end.

[0105] Gap 1 was used for oligonucleotide binding. Figure 1 -D shows an oligonucleotide (shown by a thick black line) that binds to a portion of the double-stranded product. The 3' portion of the oligonucleotide is consistent with the sequence of the partial gap, so that the oligonucleotide binds to the 5' end of the gap, and the 5' portion of the oligonucleotide is inconsistent with the sequence of the gap and will not bind to the gap.

[0106] Further extension along the 5'-3' direction under the action of polymerase ( Figure 1 -D arrow), and the extension product ( Figure 1 -E is shown in black thick lines). The extension product fills the entire gap and has a nick on its 3' side adjacent strand ( Figure 1 -E triangle). The extension of the other gaps (areas 2 and 3) is not shown.

[0107] After ligation with ligase, a phosphodiester bond is generated at the incision to form a complete nucleic acid chain ( Figure 1 -F), to obtain the ligation product.

[0108] Since the oligonucleotide contains a 5' part that is inconsistent with the gap sequence, it cannot form a complete nucleic acid chain with the 5' side chain by ligase. The ligation product includes any chain of the complete target nucleic acid connected to the oligonucleotide and its complementary chain. The enrichment of the target nucleic acid can be achieved by enriching the ligation product.

[0109] (2) Method 2. Figure 2 As shown, a nuclease is used to generate multiple cuts on both sides of the target nucleic acid, and the cuts are located on different single strands. The target nucleic acid strand is the portion filled with diagonal lines.

[0110] First, use an endonuclease or its active fragment to Figure 2 -A arrow position is cut by enzyme, and specifically, endonuclease A, B, D, E or a combination thereof can be used. The incision produced by enzyme cutting is as follows Figure 2 -B triangle, where the cut indicated by the black filled triangle is located outside the 5' end of the target nucleic acid chain. The two cuts are not adjacent, so no double-strand break of the nucleic acid will occur.

[0111] Then use 5'-3' exonuclease to cut the single strand from the 5' end and the incision of the double-stranded nucleic acid to generate a gap and obtain a partial double-stranded product ( Figure 2 -C). Region 1 is the gap generated by cutting from the 5' end of the physical break, and region 2 is the gap generated by cutting from the incision. Regions 1 and 2 are the normal situation after exonuclease cutting, that is, there is a spacer sequence between the two. Region 3 is the gap generated by cutting from the 5' end of the physical break and from the incision. Due to the special situation that the incision is close to the 5' end, a gap is formed after exonuclease cutting.

[0112] Gap 2 was used for oligonucleotide binding. Figure 2 -D shows an oligonucleotide (shown by a thick black line) that binds to a portion of the double-stranded product. The 3' portion of the oligonucleotide is consistent with the sequence of the partial gap, so that the oligonucleotide binds to the middle position of the gap, and the 5' portion of the oligonucleotide is inconsistent with the sequence of the gap, so it will not bind to the gap.

[0113] Under the action of polymerase, the oligonucleotide is extended in the 5'-3' direction ( Figure 2 -D arrow), and the extension product ( Figure 2 -E is shown by a thick black line). The extension product fills part of the gap and has a nick on its 3' side adjacent strand (as shown in Figure 2 -E triangle).

[0114] After ligation with ligase, phosphodiester bonds are generated at the cut to form a complete nucleic acid chain (such as Figure 2 -F), and obtain a ligation product. The ligation product includes any complete target nucleic acid strand and its complementary strand connected to the oligonucleotide, and the enrichment of the target nucleic acid can be achieved by enriching the ligation product.

[0115] Obviously, in this method, an oligonucleotide having a sequence completely identical to that of the gap portion can also be used to achieve the same technical effect.

[0116] In addition, for the complementary strand of the target nucleic acid strand, Figure 2 -B The cut indicated by the white filled triangle is located outside its 5' end. Figure 2 The gap in region 3 of -C can also be used for oligonucleotide binding and can be enriched using the same method, which will not be described in detail here.

[0117] (3) Method 3. Figure 3 As shown, a nuclease is used to generate multiple cuts on both sides of the target nucleic acid, and the cuts are located on the same single strand. The target nucleic acid strand is the portion filled with diagonal lines.

[0118] Use of endonucleases or their active fragments Figure 3-A arrow position is cut by enzyme, and specifically, endonuclease A, B, D, E or a combination thereof can be used. The incision produced by enzyme cutting is as follows Figure 3 -B, wherein the nick indicated by the black filled triangle is located at the 5' end side of the target nucleic acid chain.

[0119] Then use 5'-3' exonuclease to cut the single strand from the 5' end and the incision of the double-stranded nucleic acid to generate a gap and obtain a partial double-stranded product ( Figure 3 -C). Regions 1 and 4 are gaps generated by cutting from the 5' end of the physical break, and regions 2 and 3 are gaps generated by cutting from the incision. Regions 1 and 2 are the normal situation after exonuclease cutting, that is, there is a spacer sequence between the two. At gap 2, because the incision is close to the 5' end of the target nucleic acid chain, part of the target nucleic acid chain is removed during exonuclease cutting.

[0120] Gap 2 was used for oligonucleotide binding. Figure 3 -D is the oligonucleotide (shown by the thick black line) binding to the partial double-stranded product. The oligonucleotide has the same sequence as the gap and binds to the middle region of the gap.

[0121] Under the action of polymerase, the oligonucleotide is extended along the 5'-3' direction (indicated by the arrow in the figure) to obtain the extension product ( Figure 3 -E is shown by a thick black line). The extension product fills part of the gap and contains part of the target nucleic acid chain sequence, and there is a nick on its 3' side adjacent chain ( Figure 3 -E triangle). The extension of the other gap (area 3) is not shown.

[0122] After ligation with ligase, a phosphodiester bond is generated at the incision to form a complete nucleic acid chain ( Figure 3 -F), and obtain the ligation product. The ligation product includes any complete target nucleic acid strand connected to the oligonucleotide and its complementary strand, and the enrichment of the target nucleic acid can be achieved by enriching the ligation product.

[0123] In this embodiment, the oligonucleotide binds to the middle region of the gap, and there is a gap with the 5' side chain, which is not adjacent, and cannot form a complete nucleic acid chain through ligase. Of course, in this embodiment, an oligonucleotide whose only 3' portion is consistent with the sequence of the gap portion (i.e., refer to the situation in embodiment one or two) can also be used to achieve the same technical effect.

[0124] (4) Method 4. Figure 4 As shown, a nick is produced inside any strand of the target nucleic acid using a nuclease, and the target nucleic acid strand is the portion filled with diagonal lines.

[0125] Use of endonucleases or their active fragments Figure 4-A arrow position is cut by enzyme, and specifically, endonuclease A, B, D, E or a combination thereof can be used. The incision produced by enzyme cutting is as follows Figure 4 -B triangle.

[0126] Then use exonuclease to cut the single strand to create a gap and obtain a partial double-stranded product. Figure 4 -C1, cleavage is performed from the nick and the 5' end of the physical break using a 5'-3' exonuclease. Figure 4 -C2, 3'-5' exonuclease is used to cut from the incision and the 3' end of the physical break. In practical applications, it can be used alone as Figure 4 -C1 as shown, used alone as Figure 4 - Method shown in C2, combined use Figure 4 -C1 and Figure 4 - The gap is produced in the manner shown in C2.

[0127] Taking the situation shown in 4-C1 as an example, the oligonucleotide ( Figure 4 -D (shown by thick black lines) binds to part of the double-stranded product, such as Figure 4 -D. Since the exonuclease cuts off part of the target nucleic acid chain at gap 1, the oligonucleotide is completely consistent with the gap and binds to the 5' end of the gap, with a cut between the 5' side adjacent chain ( Figure 4 -D triangle).

[0128] The oligonucleotide is extended in the 5'-3' direction by the polymerase ( Figure 4 -D arrow), and the extension product ( Figure 4 -E is shown in black thick lines). The extension product fills the entire gap and there is a nick on the 3' side adjacent strand ( Figure 4 -E) as indicated by the black filled triangle.

[0129] After ligation with ligase, phosphodiester bonds are generated at both cuts to form a complete nucleic acid chain ( Figure 4 -F), to obtain a ligation product. Preferably, in this embodiment, before the oligonucleotide is extended, a ligase may be used to Figure 4 -D triangle cut to connect; after obtaining the extension product, use ligase to Figure 4 -E The cutouts shown by the black filled triangles are connected.

[0130] The ligation products obtained in this embodiment include any complete target nucleic acid chain connected to the oligonucleotide and its complementary chain. The enrichment of the target nucleic acid can be achieved by enriching the ligation products.

[0131] It should be noted that, in this embodiment, the extension product needs to be connected to the adjacent chains on both sides in order to achieve the enrichment of the complete target nucleic acid.

[0132] (5) Method 5. Figure 5 As shown, a nuclease is used to generate multiple cuts on the outside of the double-stranded target nucleic acid and generate a break in the nucleic acid chain. The target nucleic acid chain is the portion filled with diagonal lines.

[0133] Use nuclease or its active fragment to cut at the same or adjacent position of each nucleic acid single strand to produce a group of cuts, such as Figure 5 -A1 or 5-A2, the endonuclease performs enzymatic cleavage at the position indicated by the arrow, and specifically, endonuclease B, C, D, E, F or a combination thereof can be used. Figure 5 -A1 is to cut only on the 5' outer side of any strand of the target nucleic acid, forming a set of incisions. Figure 5 -A2 is to perform enzyme cleavage on both the 5' and 3' outer sides of any strand of the target nucleic acid, forming two sets of cuts. Obviously, Figure 5 -A1 method causes the sample nucleic acid to break at one point. Figure 5 -A2 method causes the sample nucleic acid to break at two locations.

[0134] Taking the case shown in A1 as an example, the product after enzyme cleavage is as follows Figure 5 -B. Cutting is performed using a 5'-3' exonuclease to form a gap from the nick break and the physical break at the 5' end. The partial double-stranded product obtained is as shown in Figure 5 -C. In which, at position 1, the exonuclease cuts off part of the target nucleic acid chain.

[0135] like Figure 5 -D, an oligonucleotide (shown by a thick black line) is bound to the partially double-stranded product. The 3' portion of the oligonucleotide is consistent with the sequence of the partial gap, so that the oligonucleotide is bound to the middle region of the gap.

[0136] Under the action of polymerase, the oligonucleotide is extended along the 5'-3' direction ( Figure 5 -D arrow), and the extension product ( Figure 5 -E is shown by a thick black line). The extension product fills part of the gap, including part of the target nucleic acid sequence, and has a nick on the 3' side adjacent chain ( Figure 5 -E triangle).

[0137] After ligation with ligase, a phosphodiester bond is generated at the incision to form a complete nucleic acid chain ( Figure 5 -F), and obtain the ligation product. The ligation product includes any complete target nucleic acid strand connected to the oligonucleotide and its complementary strand, and the enrichment of the target nucleic acid can be achieved by enriching the ligation product.

[0138] It is understandable that in this embodiment, an oligonucleotide that is completely consistent with a partial sequence of the gap (ie, refer to the situation in the third or fourth embodiment) can also be used to achieve the same technical effect.

[0139] In the above cases, some ligation products are combined with part of the non-target chain (as shown by the gray dotted line in each figure). This part can be removed by increasing the elution intensity and number during the separation of the ligation products.

[0140] In addition, multiple endonuclease cleavage sites can be designed for multiple target nucleic acids at the same time, and multiple gaps can be further formed by exonucleases, so that multiple oligonucleotides can be annealed, extended, and connected, and finally multiple connection products are obtained. By separating the connection products, enriched products of multiple target nucleic acids are obtained.

[0141] In order to verify the method of the present invention, further verification and demonstration are performed through the following several examples.

[0142] Unless otherwise specified, the examples were prepared according to conventional experimental conditions or the conditions recommended by the manufacturer's instructions. All reagents and materials used were commercially available unless otherwise specified.

[0143] Example 1

[0144] In this example, the corn genome extracted by CTAB method was used as the sample nucleic acid. The specific enrichment process is as follows:

[0145] (1) The sample nucleic acid is cleaved using a complex formed by the commercial cas9 enzyme and the guide nucleic acid sgRNA.

[0146] The preparation method of the complex is as follows: add 1 μl cas9 enzyme (1 μM), 2 μl sgRNA (1 μM) and 3 μl 10×Buffer, add water to 30 μl, and then react at 25°C for 10 minutes; wherein the sequence of sgRNA is:

[0147] 5'-CCUAAGAAUAGCAAACAGCUUGA-3' (SEQ ID NO. 1).

[0148] 20 μl of sample nucleic acid was added to the prepared 30 μl complex product and reacted at 37° C. for 2 h. Using this complex, double-stranded cleavage between positions 21853525 and 21853526 (B73V5.0) of chromosome 1 can be achieved.

[0149] (2) Use T5 exonuclease to digest the cas9-sgRNA complex digestion product.

[0150] Add 6 μl 10× Buffer and 0.2 μl T5 exonuclease (10 U / μl) to the 50 μl product obtained in step (1), add water to 60 μl, and react at 50°C for 30 min.

[0151] (3) Synthesis and annealing of oligonucleotides.

[0152] A biotin-modified oligonucleotide was synthesized, and its sequence (SEQ ID NO.2) is:

[0153] 5'- AggaatcTGTACTAGCGAACCACCTC CTTAGGGGACAGGCAAGTGATATGTTATATT-3'.

[0154] The 5' end of the underlined oligonucleotide is inconsistent with the gap sequence, the bases in italics are biotin-modified, and the bases in lowercase are thio-modified (thio-modification can prevent the action of 5'-3' nuclease on the oligonucleotide); the 3' end of the non-underlined oligonucleotide is consistent with the partial sequence of the target gap.

[0155] The oligonucleotide was annealed with the partially double-stranded product obtained in step (2), and the reaction system was: 60 μl T5 exonuclease digestion product, 0.5 μl oligonucleotide (2 μM), 7 μl 10×Buffer, and water was added to 70 μl. The reaction was carried out at 65° C. for 10 min.

[0156] (4) Oligonucleotide extension and 3' end ligation reaction.

[0157] The reaction system was: 70 μl annealing product, 8 μl 10× Buffer, 0.5 μl 20× DNA polymerase, 0.5 μl Lagse, 1 μl 25 mM dNTPs. The reaction was carried out at 60°C for 30 min.

[0158] In this example, oligonucleotide extension and 3' end ligation are placed in the same reaction system to directly obtain the ligation product.

[0159] (5) Use streptavidin affinity magnetic beads to separate the ligation products.

[0160] Use 10μl streptavidin affinity magnetic beads, wash twice, resuspend with 10μl buffer, add to 50μl ligation product, mix, and incubate at room temperature for 30min. According to the operation manual, perform multiple adsorption and elution, and finally resuspend the magnetic beads with 50μl TE to obtain the enriched product.

[0161] The enriched products were detected as follows:

[0162] Primers were designed at different distances downstream of the 3' annealing region of the oligonucleotide, and PCR amplification was performed using the enriched product as a template to verify the length of the enriched product. In addition, PCR is highly sensitive and obvious products will be detected if a non-specific capture negative control is present.

[0163] The PCR reaction system was: 1.5 μl of enriched product, 2 μl of 10× Buffer, 1 μl of 20× DNA polymerase, 1 μl of 25 mM dNTPs, 0.5 μl of each of upstream and downstream primers (10 μM), and water was added to 20 μl.

[0164] The PCR program was: 95°C, 15 min; 95°C, 30 s, 55°C, 30 s, 72°C, 30 s, 35 cycles; 72°C, 3 min.

[0165] The sample nucleic acid was used as the positive control sample, and the sample without template was used as the negative control sample. At the same time, a pair of primers 5' upstream of the oligonucleotide annealing region and two pairs of primers in the non-target nucleic acid region on the corn genome were designed as negative controls. Because cas9 causes double-strand breaks at the preset position, according to the design principle, only downstream nucleic acid fragments bound by the oligonucleotide can be enriched, so the upstream primer pair cannot be amplified. The details of each primer are shown in Table 1 below, where the distance from the 3' downstream of the oligonucleotide is represented by a positive number, and the distance from the 5' upstream is represented by a negative number.

[0166] Table 1

[0167]

[0168] The PCR products were detected by agarose gel electrophoresis. Figure 6 As shown (lanes numbered 1, 2, 3, CK-, CK+). The results showed that the target nucleic acid downstream of the oligonucleotide 3' was enriched as expected, and the fragment 8.3kb downstream of the oligonucleotide 3' was enriched. In addition, the primer pairs of Flank1-4, CK1, and CK2 did not amplify, indicating that the enrichment can be achieved as designed and the enrichment specificity is good.

[0169] Example 2

[0170] Take the enriched product in Example 1, digest the complementary strand with exonuclease, and only retain the target nucleic acid strand connected to the oligonucleotide. The specific process is as follows:

[0171] (1) The λ exonuclease used has high activity on phosphorylated 5' ends, so phosphorylation is performed first to facilitate the action of the exonuclease. The phosphorylation reaction system is: 10μl enriched product, 1.5μl 10× Buffer, 0.5μl Polynucleotide Kinase (10U / μl), add water to 15μl. React at 37℃ for 1h.

[0172] (2) Using λ exonuclease and RecJ f Add 4.5 μl 10× Buffer, 0.5 μl λ exonuclease (5 U / μl) and 0.5 μl RecJ to the phosphorylated product in the previous step. f Nuclease exonuclease (30U / μl) was added to 45μl of water. After reacting at 37℃ for 3h, it was inactivated at 65℃ for 20min. Since the 5' end of the oligonucleotide was thiolated (see Example 1), lambda exonuclease and RecJ f Exonucleases only act on the complementary strand, starting from the 5' end.

[0173] Only the target nucleic acid strands linked to the oligonucleotides can be subsequently processed, which can reduce the non-target nucleic acids enriched due to non-specific binding of the oligonucleotides.

[0174] The same primers, reaction system and reaction procedure as in Example 1 were used to detect the enzyme digestion product obtained in step (2). The PCR product was detected by agarose gel electrophoresis. The results are as follows Figure 6 As shown (lanes numbered 4, 5, 6, CK-, CK+). The results show that the target nucleic acid 3' downstream of the oligonucleotide can still be enriched as expected, and the length of the enriched fragment is consistent with that in Example 1.

[0175] Example 3

[0176] This example uses the same sample nucleic acid as in Example 1, except that:

[0177] (1) In this example, the sample nucleic acid was dephosphorylated before using the cas9-sgRNA complex for enzyme digestion. The dephosphorylation system was: 70 μl genomic DNA (300 ng / μl), 10 μl 10× Buffer, 8 μl SAP (1 U / μl), and water was added to 100 μl. The reaction was carried out at 25°C overnight and inactivated at 65°C for 10 min.

[0178] After dephosphorylation, the 5' end produced by physical cleavage does not have a phosphate group. After the sample nucleic acid is dephosphorylated, the cas9-sgRNA complex is digested in the same manner as in Example 1.

[0179] (2) In this example, lambda exonuclease was used to enzymatically digest the cas9-sgRNA complex cleavage product.

[0180] The enzyme digestion system is: 50 μl product, 2 μl 10× Buffer, 0.5 μl λ exonuclease (5 U / μl), add water to 60 μl, and react at 37°C for 40 min.

[0181] The 5' end produced by physical cleavage does not have a phosphate group after dephosphorylation, and the 5' end produced by cas9-sgRNA complex digestion has a phosphate group. Taking advantage of the difference in the activity of λ nuclease on phosphorylated 5' ends and unphosphorylated 5' ends, in this step, exonuclease digestion is preferentially performed on the designed cutting position to produce a gap.

[0182] The other operation steps are the same as those in Example 1, including oligonucleotide annealing, extension, ligation, separation of ligation products, and detection of enriched products.

[0183] The test results in this example show that the target nucleic acid downstream of the oligonucleotide 3' can be enriched as expected. In addition, when the enrichment product is detected, after the PCR cycle number is increased to 48, it can be clearly observed that the non-specific amplification of the sample nucleic acid after dephosphorylation treatment is significantly weakened, indicating that dephosphorylation treatment improves the specificity of capture.

[0184] Example 4

[0185] This example uses the same sample nucleic acid as in Example 1, and the enrichment process is as follows:

[0186] (1) Use the nuclease Nb.BbvCI to digest the sample nucleic acid.

[0187] The reaction system was: 80 μl sample nucleic acid (500 ng / μl), 10 μl 10× Buffer, 4 μl Nb.BbvCI (10 U / μl), and water was added to 100 μl. After overnight reaction at 37°C, the reaction was inactivated at 80°C for 20 min.

[0188] The cleavage between positions 21843767 and 21843768 of chromosome 1 was selected as the target incision, and the adjacent Nb.BbvCI recognition site (B73 V5.0) was located about 5 kb upstream of the 5' strand and about 7.2 kb downstream of the 3' strand of the reverse complementary strand. According to the design principle, the adjacent restriction site does not affect the enrichment effect.

[0189] (2) Using T5 exonuclease to digest the cleavage product of the nuclease in step (1).

[0190] The reaction system is: 20 μl of the Nb.BbvCI digestion product obtained in step (1), 4 μl of 10× Buffer, 0.5 μl of T5 exonuclease (10 U / μl), add water to 40 μl, and react at 50°C for 1 hour.

[0191] (3) Synthesis and annealing of oligonucleotides.

[0192] A biotin-modified oligonucleotide was synthesized and phosphorylated. The oligonucleotide sequence is:

[0193] 5'-TGAGGCTACAGTGAAGAC T AGCTGGAGATT-3' (SEQ ID NO.15),

[0194] The underlined T is modified with biotin. The oligonucleotide is designed to be consistent with the partial sequence of the target gap and can anneal to the 5' end of the target gap.

[0195] The phosphorylation system of oligonucleotide is: 10μl oligonucleotide (10μM), 5μl 10×Buffer, 5μl Polynucleotide Kinase (10U / μl), add water to 50μl. React at 37℃ for 1h. The 5' end of the phosphorylated oligonucleotide has a phosphate group, which can form a phosphodiester bond with the 3' end hydroxyl group of another nucleic acid chain under the action of ligase.

[0196] The oligonucleotides were annealed with some double-stranded products and connected to the 5' end. The reaction system was: 40μl T5 exonuclease digestion product, 1μl phosphorylated oligonucleotide, 5μl 10×Buffer, 0.2μl Lagse, and water was added to 50μl. The reaction was carried out at 65℃ for 3h. Among them, the oligonucleotide input amount was designed into 4 concentration gradients, and the final concentrations were 44nM, 4.4nM, 0.44nM, and 0.044nM respectively.

[0197] Through the above reaction, the oligonucleotide can be annealed with part of the double-stranded product and connected with the adjacent strand on the 5' side after annealing. This can avoid the extension of the adjacent strand on the 5' side of the gap after adding the polymerase, so that the gap is filled, resulting in the inability of the oligonucleotide to anneal at the designed site.

[0198] (4) Oligonucleotide extension reaction and ligation.

[0199] The reaction system is: 50μl annealing product, 6μl 10× Buffer, 3μl 20× DNA polymerase, 1μl 25mM dNTPs. Reaction at 50℃ for 1h. Since the annealing product already contains ligase, the extension product can be connected to the 3' side adjacent chain in this system to obtain a ligation product.

[0200] (5) Use streptavidin affinity magnetic beads to separate the ligation products.

[0201] Use 10μl streptavidin affinity magnetic beads, wash twice, resuspend with 10μl buffer, add to 60μl ligation product, mix, and incubate at room temperature for 30min. According to the operation manual, perform multiple adsorption and elution, and finally resuspend the magnetic beads with 20μl TE to obtain the enriched product.

[0202] The enriched products were detected as follows:

[0203] Primers were designed at different distances upstream and downstream of the oligonucleotide annealing region, and PCR amplification was performed using the enriched product as a template to verify the length of the enriched product.

[0204] The PCR reaction system was: 1.5 μl of enriched product, 2 μl of 10× Buffer, 1 μl of 20× DNA polymerase, 1 μl of 25 mM dNTPs, 0.5 μl of each of upstream and downstream primers (10 μM), and water was added to 20 μl.

[0205] The PCR program was: 95°C, 15 min; 95°C, 30 s, 55°C, 30 s, 72°C, 30 s, 35 cycles, 72°C, 3 min.

[0206] The sample nucleic acid was used as the positive control sample, and the sample without template was used as the negative control sample. At the same time, two pairs of primers for non-target nucleic acid regions on the corn genome were designed as negative controls. The details of each primer are shown in Table 2 below, where the distance to the 3' downstream of the oligonucleotide is represented by a positive number, and the distance to the 5' upstream is represented by a negative number.

[0207] Table 2

[0208]

[0209] The PCR products were detected by agarose gel electrophoresis. Figure 7 As shown. The results showed that the target nucleic acids on both sides of the oligonucleotide were enriched as expected, and the fragments were enriched to 4124bp upstream of the 5' and 11kb downstream of the 3', and the length of the enriched fragments was not less than 15kb. In addition, there was no amplification of the CK1 and CK2 primer pairs, indicating that the enrichment specificity was good. The restriction site at about 7.2kb downstream of 3' was confirmed to have no effect on the enrichment effect. In addition, the increase in the amount of oligonucleotide input was beneficial to the enrichment of long fragments.

[0210] In summary, the method of the present invention can achieve efficient enrichment of long-fragment target nucleic acids.

[0211] The above implementation cases are only for illustrating the technical solutions and features of the present invention, and their purpose is to enable people familiar with the technology to implement them better. They cannot be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention are within the protection scope of the present invention, and those not described in detail are prior art.

Claims

1. A method for enriching a target nucleic acid, characterized in that: The following steps are involved: Processing the sample nucleic acid into a partially double-stranded product containing a gap, and then binding an oligonucleotide to the gap of the partially double-stranded product; wherein the 3' portion of the oligonucleotide is consistent with the partial gap sequence; Extending the oligonucleotide to obtain an extension product, the extension product covers the 3' end of the oligonucleotide to the 3' end of the gap, and then connecting the extension product to the 3' side adjacent strand of the extension product by a ligase and / or a ligase active fragment; The oligonucleotides are used to enrich target nucleic acids.

2. The method for enriching target nucleic acid according to claim 1, characterized in that: The gap to which the oligonucleotide binds is not located outside the 3' end of the target nucleic acid chain, and the target nucleic acid chain is any chain of the target nucleic acid and is located on the same chain as the gap.

3. The method for enriching target nucleic acid according to claim 1 or 2, characterized in that: The partially double-stranded product is obtained by the following method: treating the sample nucleic acid to produce nicks; Then, a nuclease and / or an exonuclease active fragment is used to perform single-stranded cleavage from the nick to form a gap, thereby obtaining a partial double-stranded product.

4. The method for enriching target nucleic acid according to claim 3, characterized in that: The method for processing the nucleic acid sample to generate a nick is any one or a combination of the following: a) treating the sample nucleic acid with one or more endonucleases A and / or endonuclease A active fragments, wherein the endonuclease A is a nicking enzyme or a variant thereof; b) treating the sample nucleic acid with one or more endonucleases B and / or endonuclease B active fragments, wherein the endonuclease B is an endonuclease guided by a guide nucleic acid or a variant thereof; c) treating the sample nucleic acid with one or more endonucleases C and / or endonuclease C active fragments, wherein the endonuclease C is a restriction endonuclease or a variant thereof; d) treating the sample nucleic acid with one or more endonucleases D and / or endonuclease D active fragments, wherein the endonuclease D is a transcription activator-like effector nuclease or a variant thereof; e) treating the sample nucleic acid with one or more endonucleases E and / or endonuclease E active fragments, wherein the endonuclease E is a zinc finger nuclease or a variant thereof; f) treating the sample nucleic acid with one or more endonucleases F and / or endonuclease F active fragments, wherein the endonuclease F is a recombinant protein-dependent endonuclease or a variant thereof.

5. The method for enriching target nucleic acid according to claim 3, characterized in that: The sample nucleic acid is pretreated with dephosphorylation before the incision is made, and after the incision is made, a nuclease and / or an exonuclease active fragment that is selective for the phosphorylated end is used to perform single-stranded cutting from the incision to form a gap.

6. The method for enriching target nucleic acid according to claim 1, characterized in that: The 5' portion of the oligonucleotide also corresponds to a portion of the sequence of the gap.

7. The method for enriching target nucleic acid according to claim 6, characterized in that: The 5' end of the oligonucleotide binds to the 5' end of the gap.

8. The method for enriching target nucleic acid according to claim 7, characterized in that: Prior to extending the oligonucleotide, the oligonucleotide is ligated to the 5' adjacent strand of the oligonucleotide by a ligase and / or a ligase active fragment.

9. The method for enriching target nucleic acid according to claim 1, characterized in that: Artificial sequences and / or modifications are added to the oligonucleotides.

10. The method for enriching target nucleic acid according to claim 9, characterized in that: The oligonucleotides are added with biotin modification and / or thio modification.

11. The method for enriching target nucleic acid according to claim 9, characterized in that: The oligonucleotides have uracil added to them.

12. The method for enriching target nucleic acid according to claim 9, characterized in that: A linker sequence is added to the 5' end of the oligonucleotide.

13. The method for enriching target nucleic acid according to claim 1, characterized in that: When the oligonucleotide is used to enrich the target nucleic acid, a single strand complementary to the oligonucleotide is digested with an exonuclease, and only the single strand of the target nucleic acid connected to the oligonucleotide is enriched.

14. A kit, characterized in that: The kit is used to enrich target nucleic acid, and the kit comprises: An endonuclease and / or an endonuclease active fragment for generating nicks on the sample nucleic acid; An exonuclease and / or exonuclease active fragment for performing single-strand cleavage at the nick and forming a gap; A polymerase and / or a polymerase-active fragment for extending an oligonucleotide to obtain an extension product; and a ligase and / or a ligase-active fragment for ligating the extension product to its adjacent strand.

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