WO2021051665A1 - 基因目标区域的富集方法及体系 - Google Patents

基因目标区域的富集方法及体系 Download PDF

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WO2021051665A1
WO2021051665A1 PCT/CN2019/123892 CN2019123892W WO2021051665A1 WO 2021051665 A1 WO2021051665 A1 WO 2021051665A1 CN 2019123892 W CN2019123892 W CN 2019123892W WO 2021051665 A1 WO2021051665 A1 WO 2021051665A1
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probe
target region
sequence
gene
specific
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PCT/CN2019/123892
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English (en)
French (fr)
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郭志伟
李英辉
陈倩
胡荣君
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上海臻迪基因科技有限公司
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Priority to EP19946206.0A priority Critical patent/EP4032986A4/en
Priority to US17/760,939 priority patent/US20220325318A1/en
Publication of WO2021051665A1 publication Critical patent/WO2021051665A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/532—Closed or circular

Definitions

  • the present invention relates to the field of biotechnology, in particular to a method and system for enriching gene target regions.
  • NGS Next-generation sequencing technology
  • the cost of sequencing continues to decrease, its application range continues to expand, but its application is also subject to the diversified demand and time-consuming and laborious library construction step Constraints.
  • the DNA is usually short fragments, damaged, single-stranded or partially double-stranded.
  • the existing PCR technology cannot achieve good capture and enrichment.
  • the prior art still mainly uses the traditional PCR library construction method, or first add adaptor ligation and then amplify, such as hybrid capture method.
  • the length of fragments suitable for amplification is greatly limited, and the preference in amplification leads to high inhomogeneity of products, and the accumulation of errors in exponential amplification leads to subsequent sequencing The result is inaccurate; for the latter, although the length of the fragment to be enriched is not as high as PCR, it needs to be ligated first, and the ligation efficiency is usually only 20% to 50%, resulting in low capture efficiency.
  • Difficulty in connection and easy loss of rare molecules are also.
  • molecular inversion probes In order to solve the problem of NGS library construction, recently developed technologies include molecular inversion probes, multiplex PCR and so on. Compared with hybrid capture technology, molecular inversion probe has better specificity, but its pocket probe design is complicated, and it is not suitable for the enrichment of tiny DNA fragments. Multiplex PCR technology is suitable for large-scale samples and is the most widely used, but either requires extremely high primer design and poor amplicon uniformity, or the amplification product has good uniformity but requires high starting sample concentration, and it is also not suitable for starting Enrichment of small DNA fragments with low concentration. These existing technologies usually require double-ended primers when constructing libraries.
  • the applicant has established a technical solution to first linearly amplify the target region with a specific probe and then connect the adapter to achieve enrichment.
  • the main application direction is nucleic acid detection based on second-generation sequencing.
  • the present invention uses another idea to solve the problem of targeted enrichment of fragmented DNA.
  • an object of the present invention is to provide a method for enriching a gene target region to solve the problems in the prior art.
  • the present invention first provides a method for enriching gene target regions, including:
  • the specific probe includes complementary and non-complementary sequences to the target region of the fragmented DNA, and the 3'end and 5'end nucleotides of the specific probe are both modified;
  • a ligase is added to the capture extension product provided in step (1) to provide a ligation product, and the ligation product includes a circularized ligation product and a linear ligation product.
  • Another object of the present invention is to provide a system for enriching a target region of fragmented DNA, which includes a specific probe and a ligase suitable for the method for enriching a target region of a gene provided by the present invention.
  • Fig. 1 is a schematic flowchart of a method for enriching a target area in an embodiment of the present invention.
  • Figure 2 is a schematic diagram of a molecule constructed in an embodiment of the present invention.
  • the method for enriching the gene target region is simple in operation and reliable in results, especially for short-segment nucleic acids, which has a good enrichment effect.
  • the present invention has been completed on this basis.
  • the first aspect of the present invention provides a method for enriching a gene target region, including:
  • the specific probe includes both complementary to the target region of the fragmented DNA and non-compliance with the target region. Complementary sequence, and both the 3'end and 5'end nucleotides of the specific probe are modified to prevent the 3'end of the specific probe from being ligated to the template before the specific probe binds to the template, mainly with itself Self-ligation reaction at the 5'end;
  • step (2) Add ligase to the capture extension product provided in step (1) to provide the ligation product.
  • the ligation product in this step contains two parts, one is the circular connection product formed by the intramolecular connection of the capture extension product, and the other is the linear connection product formed by the intermolecular connection between the capture extension product and the specific probe. Both the circular ligation product and the linear ligation product contain the target region to be amplified, and both are suitable for library construction of target molecules.
  • the fragmented DNA containing the target region is amplified by a specific probe, and there may be one or more fragmented DNA containing the target region.
  • the specific probe and the fragmented DNA containing the target region that is, the number of specific probes in the reaction system can be one or more.
  • the extension described in this step refers to a complete linear pre-amplification step for the sample, including a single or multiple cycles of denaturation, annealing, and extension steps. In a preferred embodiment, this step is implemented in multiple cycles, such as 2-100, 2-10, 10-20, 20-30, 30-40, 40-60, 60-80, 80-100 cycles, effectively Increase the number of molecules containing the target area.
  • the fragmented DNA may be double-stranded DNA, single-stranded DNA, cDNA, etc., and the cDNA can usually be obtained by reverse transcription of RNA.
  • the specific probe may include a sequence complementary to the target region of one strand of the fragmented DNA. Therefore, the enrichment method of the present invention is also suitable for fragmenting RNA, and those skilled in the art can reverse transcription of RNA into cDNA and then perform subsequent operations by the enrichment method provided by the present invention.
  • the length of the fragmented DNA may be 25 to 200 bps, 25 to 40 bps, 40 to 60 bps, 60 to 80 bps, 80 to 100 bps, 100 to 120 bps, 120 to 140 bps, 140 to 160 bps, 160 to 180 bps, or 180 to 200 bps.
  • the amplification system of step (1) may include specific probes, DNA polymerase and dNTPs.
  • the reaction of amplifying fragmented DNA containing the target region by a specific probe can usually be carried out in the presence of a DNA polymerase. After the 3'-end blocking modified probe binds to the template under the action of the high-fidelity polymerase, the blocking group is removed and the probe is activated, which can effectively extend the target sequence.
  • the DNA polymerase may have 3'-5' exonuclease activity, so that the substitution group at the 3'end of the probe after binding to the template can be removed, so that the probe can extend beyond the template, preferably a high-fidelity DNA polymerase, Used to further improve the amplification efficiency and purity of the product.
  • the DNA polymerase can also be an ordinary DNA polymerase, that is, it has only polymerase activity and no 3'-5' exonuclease activity.
  • the amplification system of step (1) also includes an active substance, which can be used to remove the 3'end modification group of the specific probe after binding to the target region, so it can be used in
  • the capture extension system is combined with a DNA polymerase (for example, a common DNA polymerase) to improve the efficiency of the linear amplification system, and the active substance is preferably a nuclease.
  • the reaction to amplify the fragmented DNA containing the target region by a specific probe can usually be carried out in the presence of dNTPs, which can usually be dNTPs coupled to label molecules, and the coupled label molecules can include but not Limited to biotin and the like, the dTNP may include, but is not limited to, dCTP, dATP, and the like.
  • the dNTP may also be coupled with a labeling molecule, the labeling molecule may be biotin or the like, and the labeling molecule can be generally used for the purification of the captured extension product.
  • the specific probe includes a sequence complementary to the target region of the fragmented DNA, so that the specific amplification of the target region of the fragmented DNA can be realized.
  • the skilled person can select a suitable target region of fragmented DNA, and design a suitable complementary sequence according to the target region of fragmented DNA.
  • the complementary sequence is usually close to the 3'end of the probe.
  • the specific probe usually also includes a sequence that is not complementary to the fragmented DNA, so that restriction sites and universal sequences can be introduced, and the non-complementary sequence is usually close to the 5'end of the probe.
  • the specific probe may be a specific probe with a modified 3'end nucleotide, which is used to prevent the 3'end of the specific probe from ligating with other groups, so as to avoid free probes. Self-linking or linking to other non-purpose molecules.
  • a suitable modification group to modify the 3'end of the specific probe.
  • the modified group can replace the natural group on the 3'end nucleotide of the specific probe (e.g., Hydroxy, methoxy, etc.) in order to prevent the 3'end of the specific probe from ligating reaction, the modification group used can usually be a blocking group.
  • the 3'terminal modification group of the specific probe may include, but is not limited to, a hydrogen atom, a C3 Spacer group, a C6 Spacer group, a phosphate group (PO 4 ), an amino group (NH 2 ), and the like.
  • the selection of different substituent groups has obvious differences in the capture effect of the probe.
  • substituting the 3'terminal hydroxyl group of the probe with C3 Spacer has the best effect, which has the best effect compared with other substituent groups. obvious advantage.
  • the specific probe also includes a universal sequence, which can usually be recognized by the sequencing system, so that the ligation product provided subsequently can be sequenced by the sequencing system.
  • the universal sequence may be The corresponding P1 sequence and the reverse complement of the A sequence.
  • the application of the sequence that can be recognized by the sequencing system enables the library after the library construction of the present invention to be sequenced through the high-throughput sequencing platform to provide various information required for subsequent research and clinical applications.
  • the bases in the tail region of the 3'end of the specific probe may contain mismatches, which may be the last base at the 3'end of the probe, or may be close to the 3'end. Base; the mismatch can be one base or more than one base. This mismatch not only does not affect the specificity and binding efficiency of the probe, but is also more conducive to improving the cutting efficiency and fidelity of the high-fidelity DNA polymerase.
  • the step (1) may further include purifying and capturing the extension product.
  • the purification method of the captured extension product may include silica gel column purification, heat treatment, magnetic bead purification, and the like.
  • the capture extension product can be purified for the dNTP-coupled label molecule, and the purification process can use avidin or streptavidin-coated magnetic beads for purification.
  • the method for enriching the gene target region provided by the present invention may further include: adding a ligase to the capture extension product provided in step (1) to provide a ligation product.
  • the ligase is a single-stranded ligase, preferably T4 RNA ligase or thermostable RNA ligase or the like.
  • the ligation product may be a circular ligation product formed by the intramolecular connection of the capture extension product, or a linear ligation product formed by the intermolecular connection between the capture extension product and the specific probe molecule.
  • the specific probe may have a modified 5'terminal nucleotide and a single-stranded structure at the reaction temperature of step (2), so that it can be connected in a single-stranded manner.
  • a covalent bond is formed by capturing the 3'end hydroxyl group of the extension product with itself or the modification group of the 5'end of the specific probe to obtain the ligation product.
  • the 5'terminal nucleotide of the specific probe (for example, the 5'hydroxyl group of the 5'terminal nucleotide) is replaced by a phosphate group to form a phosphorylation modification
  • the 3'end hydroxyl group of the captured extension product forms a covalent bond with the phosphorylated 5'end to obtain the ligation product.
  • the specific probe has two states, one is a specific probe that binds to the template to form a phosphorylated 5'end that captures the extension product, and the other is a free specific probe.
  • the corresponding ligation reaction can also be in two situations, either by capturing the 3'end hydroxyl group of the extension product to connect to its own 5'end phosphate group, and then connecting the end to end of the molecule to form a ring; or it can be capturing the 3'end of the extension product.
  • the hydroxyl group is connected to the 5'end phosphate group of the free probe, and the connection product is still a linear product.
  • the 5'terminal nucleotide of the specific probe (for example, the 5-hydroxyl group of the 5'terminal nucleotide) is replaced by an adenosine group to form adenosine Acidification modification, under the catalysis of 5'App DNA/RNA thermostable ligase, the capture extension product can also be cyclically connected within the molecule, or the 5'end adenosine group of the free probe can be connected to the 3'end of the capture extension product Phase connection.
  • the 5'terminal nucleotide of the specific probe (for example, the 5'hydroxyl group of the 5'terminal nucleotide) is replaced by a phosphate group to form a phosphorylation modification
  • the 5'end of the specific probe can be ligated with the 3'end of the capture extension product, and two ligation products, intramolecular cyclization and intermolecular linearity, are also produced.
  • the specific probe can also be a partially double-stranded structure with a sticky end in the 5'end region, and the sticky end of the 5'end region has a single-stranded property, so that the 5'end can be processed by the method described above.
  • the step (2) may further include purifying the ligation product.
  • the purification method of the ligation product may include but not limited to silica gel column purification, heat treatment, magnetic bead purification, and the like.
  • the method for enriching the gene target region provided by the present invention may further include: (3) in the sequence where the specific probe is not complementary to the target region, usually the sequence near the 5'end may be provided with restriction enzyme digestion. Site, and after the step (2), an endonuclease is added for cutting the cyclization connection product in the ligation product formed in the step (2) at the restriction site to convert the cyclization connection product It is a linear connection product, which is conducive to the subsequent PCR amplification and PCR detection reaction.
  • the restriction site is uracil U substituted for prothymidine T
  • the endonuclease is USER enzyme or SSDNA endonuclease.
  • restriction site U there is a restriction site U near the 5'end of the specific probe.
  • the USER enzyme or SSDNA endonuclease is located at the restriction site U
  • the circularization product is cut to make it a linear ligation product, and then PCR amplification primers can be used to achieve exponential amplification by combining the complementary sequence with the digested linear ligation product.
  • the method for enriching the gene target region provided by the present invention may further include: (4) the ligation product provided in the PCR amplification step (2).
  • the PCR amplification primer has a sequence complementary to the specific probe, and the complementary sequence is not complementary to the sequence of the target region.
  • the method for enriching the gene target region provided by the present invention may further include: (5) after the step (3), PCR amplifies the ligation products provided in the steps (2) and (3), and the PCR
  • the amplification primer has a sequence complementary to the sequence on both sides of the restriction site; preferably, the sequence on both sides of the restriction site is a universal sequence for sequencing.
  • the product can usually be ligated by a DNA polymerase and PCR amplification primer pair.
  • Carry out PCR amplification, by amplifying the ligation product, the product containing the DNA of the target region can be further enriched.
  • PCR amplification primers have the same specificity as described above.
  • the complementary and reverse complementary sequences in the sequence near the 5'end of the probe that are not bound to the template can be amplified together with the circularization and linear ligation products produced in step (2).
  • the primers before and after PCR amplification are respectively complementary and reverse to the sequences on both sides of the restriction site U on the specific probe.
  • PCR amplification primers can combine the complementary sequence with the two ends of the digested linear ligation product to achieve index Amplification, as shown in Figure 1.
  • the step (4) or (5) may further include purifying the amplified product.
  • a suitable method to purify the amplified product for example, it can include but not limited to silica gel column purification, heat treatment, magnetic bead purification, and the like.
  • the method for enriching the gene target region provided by the present invention may further include: (6) using detection primer 1, detection primer 2 and probe 3 to detect the ligation product provided in step (4) or (5) for non-second-generation sequencing Instead, the PCR detection method quickly provides the detection result of the target area.
  • At least one of the detection primer 1, the detection primer 2 and the probe 3 contains a gene-specific sequence, that is, for different gene target regions, the combination of the three primers/probes with specific sequences can be It is monospecific, bispecific or trispecific.
  • the detection primer 1 contains a gene-specific sequence
  • the detection primer 2 and the probe 3 contain a universal sequence
  • the detection primer 1 and the detection primer 2 contain a gene-specific sequence
  • the detection primer 1 and the probe 3 contain a gene-specific sequence
  • the needle 3 contains a gene-specific sequence, or the detection primer 1, the detection primer 2 and the probe 3 all contain a gene-specific sequence.
  • the probe 3 may also include a labeling molecule, such as a fluorescent molecule, and the sequence of the probe 3 is not complementary to the detection primer 1 or 2.
  • the method for enriching gene target regions provided by the present invention can be used for nucleic acid detection.
  • the method for further detection by the amplified ligation product is known to those skilled in the art.
  • the method for enriching the target region of the present invention can be applied to PCR-based gene sequence detection.
  • the target region includes: the site where the sequence is changed, more specifically, it can be the single-base mutation site region, base deletion Site region, base insertion site region, fusion mutation site region, epigenetic variation or gene-specific sequence, etc.
  • the method for enriching the gene target region provided by the present invention is applied to the detection of EGFR SNP-Q787 site mutation and male-specific SRY gene, and has achieved ideal results.
  • the second aspect of the present invention provides a system for enriching a target region of fragmented DNA, which includes a specific probe and a ligase suitable for the method for enriching a target region of a gene provided in the first aspect of the present invention.
  • the ligase can be a thermostable RNA ligase, T4 RNA ligase, or 5'App DNA/RNA thermostable ligase, etc.
  • the structure of the specific probe has been described in detail in the first aspect of the present invention, and will not be repeated here.
  • the system provided by the present invention may also include one or more of the following components: dNTPs, DNA polymerases, nucleases, endonucleases, etc. coupled to the labeled molecules.
  • dTNP can be dCTP or dATP.
  • the coupled labeling molecule can be biotin.
  • the DNA polymerase may be a DNA polymerase having 3'-5' exonuclease activity, preferably a high-fidelity DNA polymerase.
  • the nuclease has 3'-5' exonuclease activity.
  • the endonuclease can be USER enzyme or ssDNA endonuclease.
  • the system provided by the present invention may also include PCR amplification primers, whose sequence is usually matched with the general sequence of the specific probe and the sequence on both sides of the cyclase cutting site, and specifically may be matched with the cyclase site.
  • the sequences on both sides of the cleavage site are complementary and are at least partially complementary to the general sequence of the specific probe.
  • the system provided by the present invention may also include detection primer 1, detection primer 2 and probe 3 for PCR detection. At least one of the three contains a gene-specific sequence, and the specific ones may be monospecific or bispecific. Sexual or tri-specific primer/probe combinations. In a preferred embodiment, only the detection primer 1 contains a gene-specific sequence. It is applied to scenarios where PCR detection is not second-generation sequencing.
  • the library molecules sequentially contain the following sequences in structure: 5'end sequencing universal sequence, gene-specific probe sequence, and enriched target Regional sequence, 3'end sequencing general sequence, as shown in Figure 2.
  • the enriched target region contains the sequence information of the sample DNA before enrichment. Its characteristics are: the position of the 5'end on the genome is fixed and determined by the specific probe; and the position of the 3'end is not fixed, which is determined by the specific probe. The initial DNA fragmentation status of the library is determined. Therefore, in the data analysis after enrichment, the position of the 3'end of the sequence on the genome can also serve as a molecular tag.
  • the beneficial effects of the present invention are that, firstly, the target regions of all fragmented DNA samples are pre-amplified by means of capture and extension before ligation, so as to avoid the loss of the original target molecules due to insufficient ligase ligation efficiency during the ligation stage. Or missed detection, especially small fragments and rare molecules; the extension reaction in the pre-amplification stage is linear amplification, and there is no preference for PCR amplification, and it will not accumulate errors introduced by PCR amplification. It is compatible with conventional PCR library building technology.
  • the product has good uniformity; secondly, in the pre-amplification stage, only a single-stranded probe with a length of about 30 bp is designed for each target gene, which avoids the difficulty of designing double-ended primers for short fragments such as cfDNA, and improves
  • the success rate of library construction also improves the convenience of library construction; and blocking the 3'end of the probe can block the non-intended connection between the probe and the template, effectively reducing the background noise caused by the free probe, and the coupling has
  • the dNTP of the labeled molecule and its purification system further improve the purity of the target product and achieve the highest conversion rate of the sample DNA molecule; again, after the 5'end of the specific probe is modified, it can be well connected under the catalysis of single-stranded ligase.
  • the enrichment method of the present invention eliminates the need for linker DNA, makes the preparation of raw materials more convenient, and interferes with each other to produce non-target products. The odds are lower and the cost is further reduced.
  • the enrichment method and system of the present invention are simple in operation and reliable in results. They can be used for DNA with a fragment length of less than 200bp, which can minimize the loss of original molecules, especially rare molecules, and enrich the target molecules most efficiently. , And its fidelity, specificity and sensitivity are high, and it can detect rare mutant molecules with a mutation rate as low as 0.01%.
  • the probes in this example all contain universal sequences (underlined). All oligonucleotides are synthesized by Shanghai Bailige Biological Co., Ltd. After the probe was purified twice by HPLC, the purity was over 99%.
  • the specific sequences of the probes of SEQ ID NO. 1-11 in the table are all directed to the 20th exon region of the EGFR gene.
  • probe 6 SEQ ID NO.6 3'terminal nucleoside is replaced with a hydrogen atom, which becomes a dideoxynucleotide
  • probe 8 SEQ ID NO.8 3'terminal nucleoside
  • the 2-position hydrogen atom of the acid is replaced by a methoxy group
  • the 5-position hydroxyl group of the 5'-terminal nucleotide of probe9 SEQ ID NO.9 is replaced by adenosine
  • the deoxygenation near the 3'-end in the probe10 SEQ ID NO.10) sequence Ribonucleotide G is replaced with ribonucleotide rG
  • probe10 and probe11 and the last base of SEQ ID NO.10, SEQ ID NO.11 are all mismatched bases.
  • UF and UR are the front and back primers used for PCR amplification of circularized products
  • F1 is the front primer used to detect the 20th exon region of the EGFR gene
  • RX1 and MX1 are the back primer and MGB probe used to detect the 20th exon region of the EGFR gene, respectively.
  • RNA extraction kit and RNA Clean Kit for purification of single-stranded amplification products were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • rTaq DNA polymerase, KOD plus high-fidelity DNA polymerase, and reaction master mix (QPK-101) for PCR detection were purchased from TOYOBO, Japan;
  • RNAse H2 thermostable nuclease was purchased from IDT, USA.
  • APG ssDNA ligase and APG Enchant high-fidelity DNA polymerase were purchased from Shanghai Yiming Biotechnology Co., Ltd.
  • RNA Clean Kit to purify the single-stranded amplification product, remove the unextended probe, and elute with 30 ⁇ L of elution buffer.
  • the PCR detection system for linear amplification products is shown in Table 2, and the PCR program for detecting linear amplification is shown in Table 3.
  • step 3 select part of the linear amplification product obtained in step 2 and the control sample QC for circularization reactions according to Table 8, Table 9 and Table 10 respectively.
  • the library product was detected using the detection system and conditions in Table 11 and Table 12.
  • the four parameters of linear amplification factor, circularization connection efficiency, molecular conversion rate and library expansion factor are used to evaluate the technical effect of the method and system of the present invention on the amplification and connection of the target region.
  • the specific calculation method is as follows:
  • Linear amplification rate output copy number of target gene after linear amplification/input copy number of target gene before linear amplification
  • Multiplier of library expansion copy number after library expansion/circulation product output copy number ⁇ 100%
  • the linear amplification reaction system and conditions involved in Table 15 are shown in Tables 4 to 7.
  • the high-fidelity polymerase reaction system uses APG Enchant high-fidelity DNA polymerase, which has 3'-5' exonuclease activity , While the rTaq enzyme used in the ordinary polymerase reaction system has 5'-3' exonuclease activity but not 3'-5' exonuclease activity.
  • thermostable nuclease to the ordinary polymerase reaction system, we can also cut off the modified closed 3'end, achieving a technical effect similar to the use of high-fidelity DNA polymerase.
  • thermostable nuclease is used in combination with the common polymerase rTaq, which can also activate the probe modified by the ribonucleotide group, which enhances the applicability of the enrichment method of the present invention; 3) different substitution groups
  • C3 has obvious differences in linear amplification effect. Substituting the probe's 3'end hydroxyl group with C3 Spacer has an ideal effect, which has obvious advantages compared with other substituent groups.
  • the data starting from Result 2 in this example is mainly for the result data obtained by substituting the 3'terminal hydroxyl group with C3 Spacer probe. 4) When there is a mismatch near the 3'end of the probe modified by the 3'end nucleotide blocking (Probe 11), it has no significant effect on the linear amplification reaction driven by the high-fidelity polymerase with proofreading function, or it is helpful .
  • step 2 for the linear amplification reaction system and conditions involved in Table 16, and refer to step 3 for the detection method of linear amplification efficiency.
  • test results show that whether it is a high-fidelity polymerase system or an ordinary polymerase plus nuclease system, the increase in the number of cycles can effectively increase the linear amplification rate of the 3'-end blocked probe.
  • step 4 for the cyclization reaction system involved in Table 17, and refer to step 5 for the method for detecting the ligation efficiency.
  • the collection method can be completely non-destructive from the original molecule to the library molecule equipped with sequencing universal sequences at both ends; 2) After the reaction of the unmodified probe system, only a very small amount of target circularized molecules can be detected; 3) Probes Only after the 5'end nucleotide is phosphorylated and modified, can it realize self-cyclization under the action of APG ssDNA ligase; 4) For probes that are blocked and modified by ribonucleotides near the 3'end, use ordinary polymerase The system with nuclease can also achieve a higher yield of target circularized molecules.
  • the results show that the target circularized molecule of the closed probe can be better amplified.
  • the library expansion factor reaches about 100 times, which is significantly better than unblocked probes.
  • the blocking modification of the 3'-end nucleotide hydroxyl group of the probe is very important. It can prevent the probe from connecting and greatly improve the ligation efficiency of the target molecule.
  • the probe after the template is bound by the DNA polymerase to remove the modified group. Perform multiple cycles of linear amplification.
  • the selection of thermostable APG ssDNA ligase, the purification treatment after circularization, and the amplification with universal primers after circularization can also play a role in improving the purity of the library product and the detection sensitivity.
  • the probes in this example also contain universal sequences. After the probe is purified twice by HPLC, the purity of more than 99% is guaranteed.
  • probe 12 SEQ ID NO.18
  • a base T is replaced with U in the general sequence part.
  • the rest of the detection primers and probes are the same as in Example 1.
  • the APG ssDNA endonuclease was purchased from Shanghai Yiming Biotechnology Co., Ltd., and the USER enzyme was purchased from NEB in the United States, and the rest were the same as in Example 1.
  • the sample preparation, quantitative detection of the sample, linear amplification and circularization connection are the same as in Example 1.
  • the initial template is 20000 copies, and the linear amplification uses APG Enchant high-fidelity polymerase, 80 cycles, circularization APG ssDNA ligase is used for ligation.
  • APG ssDNA endonuclease can specifically cleave single-stranded DNA containing 5'GGCC 3'in the sequence. After digestion, the digested product was purified with RNA Clean Kit and eluted with 35 ⁇ L of elution buffer.
  • step 6 in Example 1 the eluted product obtained in step 4 after the circularization was subjected to PCR amplification according to the reaction system in Table 13 and the procedure in Table 14.
  • the library products were detected using the detection systems and conditions in Table 11 and Table 12 above.
  • Example 1 Similar to Example 1, this example also uses the four parameters of linear amplification factor, circularized molecule yield, molecular conversion rate and library expansion factor to evaluate the technical effect of this method on the amplification and connection of the target region.
  • the specific calculation method See Example 1.
  • This embodiment evaluates the detection ability of rare molecules and rare variant molecules of small fragments after linear amplification and circularization of real samples are used to build a library using the enrichment method and system of the present invention.
  • oligonucleotide sequence used in this example is shown in Table 23:
  • the specific sequence of probe 13 is directed to the SRY gene on sex chromosome Y.
  • FX1 (SEQ ID NO.20) is a pre-primer used to specifically detect SNP-Q787 (2361 G>A) in the 20th exon region of EGFR gene.
  • RX1 and MX1 paired to form a specific detection SNP-Q787 PCR detection system.
  • FX2, RX2 and MX2 (SEQ ID NO. 21-23) are the front primer, back primer and MGB probe used to detect the amplification product of the SRY probe, respectively.
  • UM (SEQ ID NO.24) is an MGB probe used to detect universal sequences.
  • the free DNA extraction kit was purchased from Shanghai Zhendi Gene Technology Co., Ltd., and the others were the same as in Examples 1 and 2.
  • the first-generation sequencing method was used to detect the status of SNP-Q787. Male cfDNA samples homozygous for SNP were spiked into female wild-type cfDNA samples at a mass ratio of 1%, 0.1%, 0.03%, and 0.01%. At the same time, unincorporated female wild-type cfDNA samples were used as blank controls (QC ).
  • Use 50ng (about 15000 copies, 15ul in total) as the total starting template for a single sample for subsequent targeted library building. Two replicate holes are set for each gradient, and finally the average value of the multiple hole results is used as the test result of this kind of sample.
  • APG ssDNA endonuclease Use APG ssDNA endonuclease to target the sample to build a library.
  • the probes are probe4 and probe13, and the concentration is 50nM.
  • the number of linear amplification cycles is 80.
  • APG ssDNA ligase is used for circularization, and 12 cycles are amplified after digestion and purification, and then purified with AMPureXP magnetic beads to obtain 20 uL library products.
  • Step 2 qPCR detection with different specific primer/probe combinations
  • Three-specific PCR detection system was performed on the original sample and the target library sample, and then the dual-specific and single-specific PCR detection was performed on the target library sample. Take the average CT value of the test sample, use the average CT value of the QC sample as the test background, and use the difference ⁇ CT between the two as the evaluation standard. When ⁇ CT ⁇ 2.5, it is considered to have a significant difference.
  • the three-specific, bi-specific, and mono-specific detection systems for the Q787Q site of exon 20 of the EGFR gene are shown in Tables 24-26, respectively.
  • the SRY gene tri-specific, dual-specific, and mono-specific detection systems are shown in Table 27-29, respectively.
  • the qPCR detection procedure is shown in Table 30.
  • the experimental results show that the closed probe is very important to the circular enrichment method of the present invention. It can significantly improve the detection rate of rare molecules in cfDNA, and can increase the detection rate of library molecules by more than 10 times.
  • the detection of original samples The rate is generally 0.1%, and the detection rate of library samples can reach 0.01%.
  • This example compares the traditional gene detection scheme with three-specific system, and the dual-specific system and the single-specific two-primer/probe system.
  • the mono-specific system and the bi-specific system have a larger ⁇ CT value and a higher effective resolution than the three-specific system.
  • the detection sensitivity of different specific primer/probe systems is high, and the three systems can reach 0.01%.
  • the blocking modification of the 3 hydroxyl group in the 3'-end nucleotide of the probe is very important, which can prevent the probe from self-linking and greatly improve the efficiency of cyclization.
  • the selection of APG ssDNA ligase, the digestion treatment after circularization, and the amplification with universal primers after circularization can also play a role in improving the purity and detection sensitivity of the library product, respectively/together constitute the invention Technical solutions.

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Abstract

本发明提供了一种基因目标区域的富集方法，包括：（1）通过特异性探针扩增包含目标区域的片段化DNA，以提供捕获延伸产物，所述的特异性探针包括与所述片段化DNA的目标区域互补的序列，且其3'末端和5'末端核苷酸均被修饰；（2）向步骤（1）所提供的捕获延伸产物中加入连接酶，以提供连接产物。本发明还提供了一种基因目标区域的富集体系，适用于本发明提供的基因目标区域的富集方法。

Description

基因目标区域的富集方法及体系 技术领域
本发明涉及生物技术领域，特别是涉及一种基因目标区域的富集方法及体系。
背景技术
基因测序技术自上世纪七十年代问世以来已经历了近半个世纪，1985年PCR技术的横空出世推动了整个分子生物学领域的发展。下一代测序技术(NGS)具有准确、灵敏、通量高的优点，随着测序成本的不断降低，其应用范围不断扩大，但其应用也受到了需求多样化且费时费力的文库构建这一步骤的制约。在对临床样本比如血浆样本的建库过程中，DNA的存在形式通常是短片段的，受损的，单链或部分双链的，对于这些存在形式的尤其是片段小于200bp的DNA来说，现有的PCR技术并不能做到很好的捕获和富集。
对于微小DNA片段的富集，现有技术仍主要使用传统PCR建库方法，或先加接头连接再扩增比如杂交捕获法。但对于前者来说，由于需要双端引物，极大限制了适合扩增的片段的长度，且存在扩增中的偏好性导致产物的高不均一性，以及指数扩增累积的错误导致后续测序结果不准确的问题；而对于后者，虽然对待富集片段长度的要求没有PCR高，但需要先进行连接反应，而连接的效率通常只有20％～50％，导致捕获效率低下，还存在因连接困难容易丢失稀有分子的问题。为了解决NGS的建库问题，近期发展起来的技术还包括诸如分子倒置探针、多重PCR等。分子倒置探针与杂交捕获技术相比，特异性较好，但其口袋状探针设计复杂，也不适用于微小DNA片段的富集。多重PCR技术适合大规模样本，应用最为广泛，但要么引物设计要求极高且扩增子均一性差，要么扩增产物均一性较好但对起始样本浓度要求很高，同样不适用于起始浓度低的微小DNA片段的富集。这些现有技术构建文库时通常需要双端引物，为了去除接头二聚体污染必然引入纯化步骤，这导致小片段的双链DNA、受损伤的双链DNA和单链DNA分子的信息丢失。然而，在一些转录活跃的基因组区域恰恰是这些存在形式的DNA。综上所述，现有技术对片段化的，尤其是200bp以下的微小DNA进行富集的需求严重未能满足，而找到高效富集片段化DNA目标区域的方法，突破连接效率对富集效果的瓶颈，抑制非目的连接产物的产生，最大程度的捕获稀有分子并保持产物的均一性，是本发明要解决的主要技术问题。申请人在申请号为2019100024085的申请中，已先行确立了先用特异性探针线性扩增目标区域再连接接头实现富集的技术方案，主要的应用方向是基于二代测序的核酸检测。本发明将用另一思路解决片段化DNA靶向富集的问题。
发明内容
鉴于以上所述现有技术的缺点，本发明的一个目的在于提供一种基因目标区域的富集方 法，用于解决现有技术中的问题。
为实现上述目的及其他相关目的，本发明首先提供一种基因目标区域的富集方法，包括：
(1)通过特异性探针扩增包含目标区域的片段化DNA，以提供捕获延伸产物；
所述特异性探针包括与所述片段化DNA的目标区域互补和不互补的序列，且所述特异性探针的3’末端和5’末端核苷酸均被修饰；
(2)向步骤(1)所提供的捕获延伸产物中加入连接酶，以提供连接产物，所述连接产物包括环化连接产物和线性连接产物。
本发明另一个目的在于提供一种用于富集片段化DNA目标区域的体系，包括适用于本发明提供的基因目标区域富集方法的特异性探针和连接酶。
附图说明
图1是本发明实施例中对于目标区域富集方法的流程示意图。
图2是本发明实施例中构建的分子的示意图。
具体实施方式
本发明发明人经过大量探索性研究，提供了一种基因目标区域的富集方法，所述基因目标区域的富集方法操作简单、结果可靠，尤其对于短片段核酸具有良好的富集效果，在此基础上完成了本发明。
本发明第一方面提供一种基因目标区域的富集方法，包括：
(1)通过特异性探针扩增包含目标区域的片段化DNA，以提供捕获延伸产物，所述特异性探针既包括与所述片段化DNA的目标区域互补也包括与所述目标区域不互补的序列，且所述特异性探针的3’末端和5’末端核苷酸均被修饰，用于阻止所述特异性探针结合模板前其3’末端发生连接反应，主要是和自身5’末端发生的自连反应；
(2)将步骤(1)所提供的捕获延伸产物中加入连接酶，以提供连接产物。这一步的连接产物包含两部分，其一是捕获延伸产物分子内连接形成的环化连接产物，其二是捕获延伸产物与所述特异性探针分子间连接形成的线性连接产物。不论是环化连接产物或是线性连接产物都包含需要扩增的目标区域，均适用于目标分子的文库构建。
本发明所提供的基因目标区富集方法中，通过特异性探针扩增包含目标区域的片段化DNA，包含目标区域的片段化DNA可以是一个，也可以是多个。通常来说，特异性探针与包含目标区域的片段化DNA是一一对应的，即反应体系中特异性探针的数量可以是一种，也可以多种。此步骤所述的延伸指的是对样本完整的线性预预扩增步骤，包括单轮或多轮变性、退火、延伸步骤的循环。优选的实施例中，此步骤实施多轮循环，比如2～100、2～10、10～20、 20～30、30～40、40～60、60～80、80～100个循环，有效地增加包含目标区域的分子数。
所述基因目标区域的富集方法中，所述片段化DNA可以是双链DNA、单链DNA和cDNA等，所述cDNA通常可以由RNA反转录获得。对于双链DNA来说，特异性探针可以包括与所述片段化DNA其中一链的目标区域互补的序列，也包括。所以本发明的富集方法也适用于片段化RNA，本领域技术人员可以将RNA反转录成cDNA即可通过本发明所提供的富集方法进行后续操作。所述片段化DNA的长度可以是25～200bps、25～40bps、40～60bps、60～80bps、80～100bps、100～120bps、120～140bps、140～160bps、160～180bps、或180～200bps。
所述基因目标区域的富集方法中，所述步骤(1)的扩增体系中可以包括特异性探针、DNA聚合酶和dNTP。通过特异性探针扩增包含目标区域的片段化DNA的反应通常可以在DNA聚合酶存在的条件下进行。3'端封闭修饰的探针在高保真聚合酶的作用下结合模板后，封闭基团被切除，探针被活化，从而可以对目标序列进行有效延伸。所述DNA聚合酶可以具有3’-5’外切酶活性，从而可以切除结合模板后的探针3’端的取代基团，使探针能够延模板延伸，优选可以为高保真DNA聚合酶，用于进一步提高产物的扩增效率和纯度。所述DNA聚合酶也可以是普通的DNA聚合酶，即只有聚合酶活性，没有3’-5’外切酶活性。这种情况下，所述步骤(1)的扩增体系中还包括活性物质，所述活性物质可以用于切除结合目标区域后所述特异性探针的3’末端修饰基团，故可以在捕获延伸体系中与DNA聚合酶(例如，普通的DNA聚合酶)组合，以提升线性扩增体系的效率，所述活性物质优选为核酸酶。通过特异性探针扩增包含目标区域的片段化DNA的反应通常可以在dNTP存在的条件下进行，所述dNTP通常可以是耦联标记分子的dNTP，所述耦联标记分子可以是包括但不限于生物素等，所述dTNP可以是包括但不限于dCTP、dATP等。所述dNTP还可以耦联有标记分子，所述标记分子可以是生物素等，所述标记分子通常可以用于捕获延伸产物的纯化。
所述基因目标区域的富集方法中，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，从而可以实现对片段化DNA的目标区域的特异性扩增，本领域技术人员可选择合适的片段化DNA的目标区域，并根据片段化DNA的目标区域，设计合适的互补的序列，互补的序列通常靠近探针3’末端。所述的特异性探针通常还包括与所述片段化DNA不互补的序列，从而可以实现酶切位点和通用序列的引入，不互补的序列通常靠近探针5’末端。所述特异性探针可以是3’末端核苷酸被修饰的特异性探针，用于阻止所述特异性探针的3’末端与其他基团发生连接反应，从而可以避免游离探针的自连或连接其它非目的分子。本领域技术人员可选择合适的修饰基团以实现特异性探针3’末端的修饰，例如，所修饰的基团可以取代特异性探针3’末端核苷酸上的天然基团(例如，羟基、甲氧基等)，以阻止所述特异性探 针3’端发生连接反应，所使用的修饰基团通常可以是封闭基团。探针通过互补序列与模板上的目标区域结合后，3’端的修饰基团可以被酶切除，使得探针被活化，从而可以对目标序列进行有效延伸。特异性探针的3’末端修饰基团可以是包括但不限于氢原子、C3 Spacer基团、C6Spacer基团、磷酸基团(PO 4)、氨基(NH 2)等。不同取代基团的选择对于探针的捕获效果有明显差异，在本发明一优选实施例中，将探针3’端羟基取代为C3 Spacer得到的效果最佳，与其它取代基团相比具有明显优势。所述特异性探针还包括通用序列，所述通用序列通常能被测序系统识别，从而可以将后续提供的连接产物通过测序系统进行测序，例如，对于Ion Torrent测序系统，所述通用序列可以是对应的P1序列和A序列的反向互补序列。这种能被测序系统识别的序列的应用，使本发明完成建库后的文库能够通过高通量测序平台进行测序，以提供各种后续研究和临床应用所需要的信息。在本发明一优选实施方式中，所述特异性探针3’端尾部区域的碱基可以包含错配，错配的可以是探针3’端最后一个碱基，也可以是接近3’端的碱基；错配的可以是一个碱基，也可以是复数个。该错配不仅不影响探针的特异性和结合效率，还更有利于提高高保真DNA聚合酶的切割效率和保真度。
所述基因目标区域的富集方法中，所述步骤(1)中，还可以包括纯化捕获延伸产物。本领域技术人员可选择合适的方法对捕获延伸产物进行纯化，例如，捕获延伸产物的纯化方法可以是包括硅胶柱纯化、加热处理、磁珠纯化等。在本发明一具体实施例中，可以针对dNTP耦联的标记分子对捕获延伸产物进行纯化，纯化过程可以使用亲和素或者链霉亲和素包被的磁珠纯化等。
本发明所提供的基因目标区富集方法中，还可以包括：向步骤(1)所提供的捕获延伸产物中加入连接酶，以提供连接产物。所述连接酶是单链连接酶，优选为T4 RNA连接酶或热稳定性RNA连接酶等。所述连接产物可以是包括捕获延伸产物分子内连接形成的环化连接产物，也可以是捕获延伸产物与特异性探针分子间连接形成的线性连接产物。
所述基因目标区域的富集方法中，所述特异性探针可以是5’末端核苷酸被修饰的、且在步骤(2)的反应温度下为单链结构，从而可以在单链连接酶的催化下，通过捕获延伸产物的3’端羟基与其自身或特异性探针的5’末端的修饰基团生成共价键，得到连接产物。在本发明一具体实施方式中，特异性探针的5’末端核苷酸(例如，具体可以为的5’末端核苷酸的5位羟基)被磷酸基团取代，从而形成磷酸化修饰，在单链连接酶的催化下，捕获延伸产物的3’端羟基与与磷酸化的5’末端生成共价键，得到连接产物。此时特异性探针有两种状态，一种是结合上模板形成了捕获延伸产物的具有磷酸化的5’末端的特异性探针，另一种游离的特异性探针。所以对应的连接反应也可以是两种情况，既可以是捕获延伸产物的3’端羟基连接自 身的5’端磷酸基团，分子内首尾连接成环；也可以是捕获延伸产物的3’端羟基连接游离探针的5’端磷酸基团，连接产物仍然是线性产物。在本发明另一具体实施方式中，特异性探针的5’末端核苷酸(例如，具体可以为的5’末端核苷酸的5位羟基)被腺苷基团取代，从而形成腺苷酸化修饰，在5′App DNA/RNA热稳定连接酶的催化下，捕获延伸产物也可以分子内环化连接，或者游离探针的5’末端腺苷基团可以与捕获延伸产物的3’端相连接。在本发明另一具体实施方式中，特异性探针的5’末端核苷酸(例如，具体可以为5’末端核苷酸的5位羟基)被磷酸基团取代，从而形成磷酸化修饰，在热稳定RNA连接酶的催化下，特异性探针的5’末端可以与捕获延伸产物的3’端发生连接反应，也产生分子内环化的和分子间线性的两种连接产物。所述特异性探针也可以是5’端区域具有黏性末端的部分双链结构，其5’端区域的黏性末端具有单链性质，从而可以通过如上所述的方法对5’端进行修饰，在合适的单链连接酶催化下，与捕获延伸产物连接。所述基因目标区域的富集方法中，所述步骤(2)还可以包括纯化连接产物。本领域技术人员可选择合适的方法对连接产物进行纯化，例如，连接产物的纯化方法可以是包括但不限于硅胶柱纯化、加热处理、磁珠纯化等。
本发明所提供的基因目标区域的富集方法，还可以包括：(3)在所述特异性探针与所述目标区域不互补的序列中，通常可以是靠近5’端的序列中设置酶切位点，并在所述步骤(2)之后加入内切酶，用于在所述酶切位点切割所述步骤(2)形成的连接产物中的环化连接产物，使环化连接产物转变为线性连接产物，有利于后续的PCR扩增和PCR检测反应的进行。在本发明一具体实施方式中，所述酶切位点是替换了原胸腺嘧啶T的尿嘧啶U，所述内切酶是USER酶或SSDNA内切酶。在本发明另一具体实施例中，特异性探针5’端附近有酶切位点U，当捕获延伸产物首尾相连形成环化产物后，USER酶或SSDNA内切酶在酶切位点U切开环化产物使之成为线性连接产物，后续可以使用PCR扩增引物通过互补序列结合上酶切后的线性连接产物实现指数扩增。
本发明所提供的基因目标区域的富集方法，还可以包括：(4)PCR扩增步骤(2)提供的连接产物。所述PCR扩增引物具有与所述特异性探针互补的序列，所述互补的序列与所述目标区域的序列不互补。本发明所提供的基因目标区域的富集方法，还可以包括：(5)在所述步骤(3)后，PCR扩增所述步骤(2)和(3)提供的连接产物，所述PCR扩增引物具有与所述酶切位点两侧的序列互补的序列；优选的，所述酶切位点两侧的序列为测序通用序列通常可以通过DNA聚合酶和PCR扩增引物对连接产物进行PCR扩增，通过扩增连接产物，可以进一步富集含有目标区域的DNA的产物。本领域技术人员可选择合适的方法和体系对步骤(2)或步骤(3)所提供的连接产物进行扩增，例如，在本发明一实施例中，PCR扩增 引物具有与所述特异性探针5’端附近未结合模板的序列中互补和反向互补的序列，可以将或步骤(2)产生的环化和线性连接产物一起扩增。在本发明另一实施例中，特异性探针5’端附近有酶切位点U，PCR扩增的前后引物分别与特异性探针上的酶切位点U两侧的序列互补和反向互补。当USER酶或ssDNA内切酶在酶切位点U切开环化连接产物使之成为线性连接产物后，PCR扩增引物可通过互补序列结合上酶切后的线性连接产物的两端实现指数扩增，如图1所示。所述基因目标区域的富集方法中，所述步骤(4)或(5)还可以包括纯化扩增产物。本领域技术人员可选择合适的方法对扩增产物进行纯化，例如可以是包括但不限于硅胶柱纯化、加热处理、磁珠纯化等。
本发明所提供的基因目标区域富集方法，还可以包括：(6)用检测引物1、检测引物2和探针3检测步骤(4)或(5)提供的连接产物，以非二代测序而是PCR检测的手段快速提供目标区域的检测结果。所述的检测引物1、检测引物2和探针3中，至少其一包含基因特异性序列，也即是说，对于不同的基因目标区域，三种引物/探针有关特异性序列的组合可以是单特异性、双特异性或三特异性。在本发明一个实施例中，所述检测引物1包含基因特异性序列，检测引物2和探针3包含通用序列，或检测引物1和检测引物2包含基因特异性序列；或检测引物1和探针3包含基因特异性序列，或检测引物1、检测引物2和探针3均包含基因特异性序列。探针3还可以包含标记分子，比如可以是荧光分子，且探针3的序列与检测引物1或2不互补。
本发明所提供的基因目标区域的富集方法，可以用于核酸检测。通过扩增后的连接产物进一步进行检测的方法对于本领域技术人员来说是已知的。本发明目标区域富集的方法可以应用于基于PCR的基因序列检测中，例如，所述的目标区域包括：序列发生变异的位点，更具体可以是单碱基突变位点区域，碱基缺失位点区域，碱基插入位点区域，融合突变位点区域，表观遗传变异或基因特异性序列等。在本发明一实施例中，本发明所提供的基因目标区域的富集方法应用于EGFR SNP-Q787位点突变和男性特有SRY基因的检测，取得了理想的效果。
本发明第二方面提供一种用于富集片段化DNA目标区域的体系，包括适用于本发明第一方面所提供的基因目标区域的富集方法的特异性探针和连接酶。其中，连接酶可以是热稳定性RNA连接酶、T4 RNA连接酶、或5′App DNA/RNA热稳定连接酶等。所述特异性探针的结构在本发明第一方面已经详细描述，在此不作赘述。
本发明所提供的体系中，还可以包括以下组分的一种或多种：耦联标记分子的dNTP、DNA聚合酶、核酸酶、内切酶等。其中，dTNP可以是dCTP，也可以是dATP。耦联的标记分子可以是生物素。所述DNA聚合酶可以是具有3’-5’外切酶活性的DNA聚合酶，优选高保 真DNA聚合酶。所述核酸酶具有3’-5’外切酶活性。所述内切酶可以是USER酶或者ssDNA内切酶。
本发明所提供的体系中，还可以包括PCR扩增引物，其序列通常与特异性探针的通用序列和环化酶切位点两侧的序列相配合，具体的可以是与位于环化酶切位点两侧的序列互补，并与所述特异性探针的通用序列至少部分互补的序列。
本发明所提供的体系中，还可以包括用于PCR检测的检测引物1、检测引物2和探针3，三者中至少其一含有基因特异性序列，具体的可以是单特异性、双特异性或三特异性的引物/探针组合。优选的实施例中，仅检测引物1包含基因特异性序列。其应用于非二代测序而是PCR检测的场景。
在本发明一优选实施例中，通过本发明的方法或体系进行文库构建后，文库分子在结构上依次包含以下序列：5’端测序通用序列、基因特异性探针序列、富集到的目标区域序列、3’端测序通用序列，如图2所示。其中富集到的目标区域包含富集前样本DNA的序列信息，其特点是：5’端在基因组上的位置是固定的，由特异性探针决定；而3’端位置不固定，由建库初始的DNA片段化状态决定。因此，在富集后的数据分析中，该序列的3’端在基因组上的位置也可以起到分子标签的作用。
本发明的有益效果在于，首先，在连接前先对所有片段化DNA样本的目标区域通过捕获延伸的手段进行预扩增，避免在连接阶段因连接酶连接效率不足所致的原始目标分子的损失或漏检，特别是小片段和稀有分子；预扩增阶段的延伸反应属于线性扩增，没有PCR扩增的偏好性，也不会累积PCR扩增所引入的错误，与常规PCR建库技术相比，产物均一性好；其次，预扩增阶段，只需为每个目标基因设计一条长约30bp的单链探针，避开了为短片段如cfDNA设计双端引物的困难，既提高了建库成功率还提升了建库便利性；而封闭探针3’端，可以阻断探针与模板之外的非目的连接，有效降低游离探针引起的背景噪声，辅以耦联有标记分子的dNTP及其纯化系统，进一步提高目的产物纯度，使样本DNA分子达到最高转化率；再次，将特异性探针的5’端修饰后，在单链连接酶催化下能很好地连接到延伸产物的3’端但不会连接到游离探针经过封闭修饰的的3’端，避免游离探针的自连和错连，提高目的连接产物在终产物中的比例。最后，本发明的富集方法与申请人在申请号为2019100024085中公开的富集方法相比，省去了接头DNA，原料准备上更具便利性，不同组分之间相互干扰生成非目的产物的几率更低，且进一步降低了成本。综上所述，本发明的富集方法及体系，操作简单，结果可靠，针对片段长度小于200bp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，且其保真性、特异性和灵敏度高，可检出突 变率低至0.01％的稀有突变分子。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
实施例1
实施例中所用寡聚核苷酸如表1所示：
表1
Figure PCTCN2019123892-appb-000001
Figure PCTCN2019123892-appb-000002
此实施例中的探针都包含通用序列(下划线部分)。所有寡聚核苷酸由上海百力格生物有限公司合成。探针经HPLC两次纯化后，达到99％以上的纯度。表中SEQ ID NO.1-11的探针的特异性序列都针对EGFR基因的第20外显子区域。
各探针5’端核苷酸与3’端核苷酸的修饰如表1中所示。其中，probe 6(SEQ ID NO.6)3’端核苷酸的3位羟基被替换为氢原子，变成了双脱氧核苷酸；probe 8(SEQ ID NO.8)3’端核苷酸的2位氢原子被甲氧基取代；probe9(SEQ ID NO.9)5’端核苷酸的5位羟基被腺苷替代；probe10(SEQ ID NO.10)序列中靠近3’端的脱氧核糖核苷酸G被替换成核糖核苷酸rG；probe10和probe11和(SEQ ID NO.10，SEQ ID NO.11)的最后一个碱基均为错配碱基。
UF和UR(SEQ ID NO12，SEQ ID NO.13)是用于PCR扩增环化产物的前后引物，F1(SEQ ID NO.17)是用于检测EGFR基因第20外显子区域的前引物，RX1和MX1(SEQ IDNO.19和SEQ ID NO.20)分别是用于检测EGFR基因第20外显子区域的后引物和MGB探针。
主要试剂与材料
细胞DNA提取试剂盒和用于纯化单链扩增产物的RNA Clean Kit试剂盒购自天根生化科技(北京)有限公司。rTaq DNA聚合酶、KOD plus高保真DNA聚合酶和用于PCR检测的反应预混液(QPK-101)购自日本TOYOBO公司；RNAse H2热稳定性核酸酶购自美国IDT公司。APG ssDNA连接酶、APG Enchant高保真DNA聚合酶购自上海羿鸣生物科技有限公司。USER酶、Q5高保真DNA聚合酶、T4 RNA连接酶与5′App DNA/RNA热稳定连接酶购自美国NEB公司；Agencourt AMPure磁珠购自美国Beckman公司；相关过程中定量检测使用的校准品按照REF序列由上海百力格生物技术有限公司合成，按照一定浓度梯度配置于20％的甘油TE缓冲液中。
实验方法
步骤1、样本准备
使用细胞DNA提取试剂盒提取健康人志愿者血细胞样本中gDNA，超声打断成平均长度为150bp左右的片段，使用qubit定量后备用。使用去离子水作为对照样本NC。
步骤2、样本的定量检测
按照表2配制样本的PCR定量检测体系，并用表3的扩增条件，使用针对EGFR20外显子区域的检测体系定量检测样本的拷贝数浓度。
Figure PCTCN2019123892-appb-000003
Figure PCTCN2019123892-appb-000004
步骤3、线性扩增
3.1按表4、表5、表6分别配制线性扩增反应体系。线性扩增的PCR程序如表7所示。
Figure PCTCN2019123892-appb-000005
Figure PCTCN2019123892-appb-000006
Figure PCTCN2019123892-appb-000007
Figure PCTCN2019123892-appb-000008
3.2线性扩增程序完成后，用RNA Clean Kit试剂盒对单链扩增产物进行纯化，去除未延伸的探针，用30μL洗脱缓冲液洗脱。
3.3线性扩增效率检测
线性扩增产物的PCR检测体系如表2，检测线性扩增的PCR程序如表3所示。
步骤4、环化连接
根据步骤3的结果，选择步骤2得到的部分线性扩增产物与对照样本QC分别按表8、表9和表10进行环化反应。
Figure PCTCN2019123892-appb-000009
Figure PCTCN2019123892-appb-000010
Figure PCTCN2019123892-appb-000011
Figure PCTCN2019123892-appb-000012
步骤5、环化产物得率检测
使用TE缓冲液将环化产物稀释10倍，按照表11配制PCR检测体系对步骤4的中的目标环化产物进行得率检测，同时检测不加模板的对照样本NC。PCR程序如表12所示。使用校准品对目标环化产物分子进行定量，评价目标环化产物得率。
Figure PCTCN2019123892-appb-000013
Figure PCTCN2019123892-appb-000014
步骤6、扩增环化产物
按表13的反应体系和表14的程序对步骤4得到的环化产物进行PCR扩增。
Figure PCTCN2019123892-appb-000015
Figure PCTCN2019123892-appb-000016
Figure PCTCN2019123892-appb-000017
反应结束后，取80μL AMPure XP磁珠对扩增后文库进行纯化，参照操作说明，最终将纯化产物于20μL洗脱缓冲液(10 mM Tris-HCl，0.1mM EDTA，pH8.0)中洗脱，在磁力架上取上清转移到新的微量离心管。至此，包含通用序列的靶向文库分子扩库完成。
将扩增后的文库产物用TE缓冲液稀释100倍后，使用表11和表12的检测体系和条件检测文库产物。
实验结果及分析
本实施例用线性扩增倍数、环化连接效率、分子转化率与扩库倍数这四个参数评价本发明的方法和体系对目标区域扩增和连接的技术效果。具体计算方式如下：
线性扩增倍率＝目的基因线性扩增后输出拷贝数/目的基因线性扩增前输入拷贝数
环化分子得率＝目的基因环化产物输出拷贝数/目的基因线性扩增产物输入拷贝数×100％
分子转化率＝目的基因环化产物输出拷贝数/扩增前输入拷贝数×100％
扩库倍数＝扩库纯化后拷贝数/环化产物输出拷贝数×100％
结果1、不同类型的聚合酶对不同修饰探针的线性扩增效率影响如表15所示
Figure PCTCN2019123892-appb-000018
Figure PCTCN2019123892-appb-000019
对于所有对照样本，均没有检测到信号。
表15中所涉及的线性扩增反应体系和条件参见表4至表7，其中，高保真聚合酶反应体系中使用的是APG Enchant高保真DNA聚合酶，具有3’-5’外切酶活性，而普通聚合酶反应体系中使用的rTaq酶，具有5’-3’外切酶活性，但不具有3’-5’外切酶活性。为了实现外切酶的功能，我们在普通聚合酶反应体系中添加了热稳定性核酸酶后，也能切除修饰后封闭的3’末端，达到与使用高保真DNA聚合酶类似的技术效果。
从测试结果可知：1)3'端核苷酸封闭修饰的探针在高保真聚合酶的作用下结合模板后，封闭基团被切除，探针被活化，从而可以对目标序列进行有效扩增；2)热稳定性核酸酶与普通聚合酶rTaq合用，对核糖核苷酸基团修饰的探针也能起到活化效果，增强了本发明富集方法的适用性；3)不同取代基团的选择对于线性扩增效果有明显差异，将探针3’端羟基取代为C3 Spacer得到的效果理想，与其它取代基团相比具有明显优势。本实施例从结果2开始的数据主要是针对将3’端羟基取代为C3 Spacer的探针得到的结果数据。4)3’端核苷酸封闭修饰的探针的3’端附近出现错配时(Probe 11)，对于具有proofreading功能的高保真聚合酶驱动的线性扩增反应没有显著影响，或还有帮助。
结果2、不同扩增循环数对线性扩增效率的影响如表16所示。
表16中所涉及的线性扩增反应体系和条件参见步骤2，线性扩增效率检测方法参见步骤3。
Figure PCTCN2019123892-appb-000020
测试结果表明，无论是高保真聚合酶体系还是普通聚合酶加核酸酶的体系，都可通过循环数的增加可以有效提高3’端封闭探针的线性扩增倍率。
结果3、封闭探针对环化结果的影响如表17所示
表17中所涉及的环化反应体系见步骤4，连接效率检测的方法见步骤5。
Figure PCTCN2019123892-appb-000021
Figure PCTCN2019123892-appb-000022
结果表明:1)使用将3’端核苷酸羟基替换为C3 Spacer的探针，能够获得理想的目标环化分子得率和分子转化率，后者可超过100％，表明用本发明的富集方法可以做到从原始分子到两端配置有测序通用序列的文库分子的完全无损；2)未经修饰的探针体系反应后，只能检出极少量目标环化分子；3)探针的5’端核苷酸只有被磷酸化修饰后，在APG ssDNA连接酶作用下，才能实现自身环化；4)对于3’端附近被核糖核苷酸封闭修饰的探针，使用普通聚合酶加核酸酶的体系，也能取得较高的目标环化分子得率。
结果4、不同5’端修饰的探针和不同连接酶对环化效率的影响如表18所示。
表18中所涉及的连接反应体系参见表8、表9和表10，环化效率检测方法参见步骤5。
Figure PCTCN2019123892-appb-000023
结果表明，使用5’端磷酸化修饰的封闭探针配合APG ssDNA连接酶环化效果理想，优于腺苷修饰的封闭探针配合其他连接酶的体系。
结果5、连接产物扩库效率检测如表19所示。
Figure PCTCN2019123892-appb-000024
表19中所涉及的扩库反应体系与程序参见步骤6。
结果表明，封闭探针的目标环化分子可以得到较好的扩增。扩库倍数达到100倍左右， 显著优于未封闭探针。
结论：
探针3'端核苷酸羟基的封闭修饰至关重要，可以防止探针自连，大幅提高目标分子的连接效率，而结合模板后的探针被DNA聚合酶切除修饰基团后还能进行多个循环的线性扩增。另外，热稳定性APG ssDNA连接酶的选用，环化后的纯化处理，以及环化后使用通用引物扩增也都能起到提高建库产物纯度和检测灵敏度的作用。
实施例2
实施例中所用寡聚核苷酸如表20所示：
表20.
Figure PCTCN2019123892-appb-000025
同实施例1，本实施例中的探针也包含通用序列。探针经HPLC两次纯化后，保证99％以上的纯度。
其中，probe 12(SEQ ID NO.18)两端的修饰与probe4相同，通用序列部分有一个碱基T被替代为U。其余检测引物与探针与实施例1相同。
主要试剂与材料
APG ssDNA内切酶购自上海羿鸣生物科技有限公司，USER酶购自美国NEB公司，其余与实施例1相同。
实验方法
本实施例的样本准备，样本的定量检测，线性扩增与环化连接均与实施例1相同，其中初始模板为20000拷贝，线性扩增选用APG Enchant高保真聚合酶，80个循环，环化连接选用APG ssDNA连接酶。
取环化产物加入USER酶或APG ssDNA内切酶，37度酶切1小时，并以未酶切的环化产物作为对照。APG ssDNA内切酶可以特异性酶切序列中包含5’GGCC 3’的单链DNA。酶切后用RNA Clean Kit纯化酶切产物，用35μL洗脱缓冲液洗脱。
取5μL洗脱产物使用TE缓冲液将洗脱产物稀释10倍，采用实施例1中表11 PCR检测体系检测目标环化产物得率，PCR程序如表12所示。
按照实施例1步骤6的方案，按表13的反应体系和表14的程序对步骤4得到的环化后洗脱产物进行PCR扩增。
反应结束后，取80μL AMPure XP磁珠对扩增后文库进行纯化，参照操作说明，最终将纯化产物于20μL洗脱缓冲液(10 mM TrisCl，0.1mM EDTA，pH8.0)中洗脱，在磁力架上取上清转移到新的微量离心管。至此，包含通用序列的靶向文库分子扩库完成。
扩增后的文库产物，TE缓冲液稀释100倍后，使用上文表11和表12检测体系和条件检测文库产物。
实验结果及分析
同实施例1，本实施例也用线性扩增倍数、环化分子得率、分子转化率与扩库倍数这四个参数以评价本方法对目标区域扩增和连接的技术效果，具体计算方法见实施例1。
结果2.1环化后酶切对检测的影响如表21所示。
Figure PCTCN2019123892-appb-000026
结果表明，环化产物在酶切之后，检测出的环化分子得率与分子转化率均有显著提升。
结果2.2环化后酶切对扩库效率的影响如表22所示。
Figure PCTCN2019123892-appb-000027
结果表明，环化产物在酶切之后，扩库效率得到了显著的提升。
实施例3
本实施例评估用本发明的富集方法和体系对真实样本进行线性扩增-环化建库后，针对微小片段的稀有分子和稀有变异分子的检出能力。
本实施例所用寡聚核苷酸序列如表23所示：
表23
Figure PCTCN2019123892-appb-000028
Figure PCTCN2019123892-appb-000029
其中探针13(SEQ ID NO.19)特异性序列针对性染色体Y上的SRY基因。FX1(SEQ ID NO.20)为用来特异检测EGFR基因第20外显子区域SNP-Q787(2361 G>A)的前引物，前实施例中RX1和MX1配对组成特异性检测SNP-Q787的PCR检测体系。FX2、RX2和MX2(SEQ ID NO.21-23)分别是用于检测SRY探针扩增产物的前引物，后引物和MGB探针。UM(SEQ ID NO.24)是用来检测通用序列的MGB探针。
主要试剂与材料
游离DNA提取试剂盒购自上海臻迪基因科技有限公司，其他同实施例1和2。
实验方法
步骤1、靶向文库制备
使用游离DNA提取试剂盒提取健康人血浆样本中的cfDNA，使用Qubit定量后备用。通过F1和RX1扩增EGFR-20外显子区域的片段。使用一代测序方法检测其中SNP-Q787的状态。将SNP纯合子的男性cfDNA样本，按照1％，0.1％，0.03％，0.01％的质量比掺入到女性野生型cfDNA样本中，同时以未掺入的女性野生型cfDNA样本作为空白对照(QC)。以50ng(约15000拷贝，共15ul)作为单个样本总起始模板量进行后续靶向建库。每种梯度设置两个复孔，最后以复孔结果的平均值作为该种样本的检测结果。
使用APG ssDNA内切酶对样本进行靶向建库。探针选用probe4和probe13，浓度为50nM。线性扩增循环数为80，环化使用APG ssDNA连接酶，酶切纯化后扩增12个循环，再用AMPureXP磁珠纯化后获得20 uL文库产物。
步骤2、不同特异性引物/探针组合的qPCR检测
对原始样本和靶向建库样本分别进行三特异性PCR检测体系检测，再对靶向建库样本进行双特异性、单特异性PCR检测。取检测样本的平均CT值，以QC样本的平均CT值为检测背景，以两者之间的差值ΔCT，作为评价标准。当ΔCT≥2.5时认为具备显著性差异。
EGFR基因20外显子Q787Q位点三特异性、双特异性、单特异性检测体系分别如表24-26所示。SRY基因三特异性、双特异性、单特异性检测体系分别如表27-29所示。qPCR检测程序如表30所示。
表24.Q787Q位点三特异性检测体系：
Figure PCTCN2019123892-appb-000030
Figure PCTCN2019123892-appb-000031
表25.Q787Q位点双特异性检测体系：
试剂 用量(μL) 终浓度
2×Taqman master Mix 10 1×
FX1(10μM) 0.6 300nM
UR(10μM) 0.6 300nM
MX1(10μM) 0.2 100nM
H2O 6.6 /
待测样本 2 /
Total 20 /
表26.Q787Q位点单特异性检测体系：
试剂 用量(μL) 终浓度
2×Taqman master Mix 10 1×
FX1(10μM) 0.6 300nM
UR(10μM) 0.6 300nM
UM(10μM) 0.2 100nM
H 2O 6.6 /
待测样本 2 /
Total 20 /
表27.SRY基因三特异性检测体系：
试剂 用量(μL) 终浓度
2×Taqman master Mix 10 1×
FX2(10μM) 0.6 300nM
RX2(10μM) 0.6 300nM
MX2(10μM) 0.2 100nM
H 2O 6.6 /
待测样本 2 /
Total 20 /
表28.SRY基因双特异性检测体系：
试剂 用量(μL) 终浓度
2×Taqman master Mix 10 1×
FX2(10μM) 0.6 300nM
UR(10μM) 0.6 300nM
MX2(10μM) 0.2 100nM
H 2O 6.6 /
待测样本 2 /
Total 20 /
表29.SRY基因单特异性检测体系：
试剂 用量(μL) 终浓度
2×Taqman master Mix 10 1×
FX2(10μM) 0.6 300nM
UR(10μM) 0.6 300nM
UM(10μM) 0.2 100nM
H 2O 6.6 /
待测样本 2 /
Total 20 /
表30.
Figure PCTCN2019123892-appb-000032
实验结果及分析
针对原始样本和靶向建库后的样本使用不同特异性体系检测EGFR SNP-Q787位点和SRY基因的检测结果分别如表31，32所示。
表31、不同掺入比下的EGFR SNP-Q787位点检测结果
Figure PCTCN2019123892-appb-000033
Figure PCTCN2019123892-appb-000034
表32.不同掺入比下的SRY基因检测结果
Figure PCTCN2019123892-appb-000035
结论：
实验结果表明，封闭探针对于本发明的环化富集方法至关重要，能够显著提高cfDNA中稀有分子的检出率，可将文库分子的检出率提升10倍以上，原始样本的检出率一般在0.1％，而文库样本的检出率可达到0.01％。
本实施例比较了传统基因三特异体系检测方案，和双特异性体系及单特异性两种引物/探针的体系。对单一碱基变异的Q787位点，单特异性体系和双特异性体系相比三特异性体系而言，ΔCT值更大，具有更高的有效分辨率。对于特异性基因SRY，不同特异性引物/探针体系的检测灵敏度都较高，三种体系都能达到0.01％。
综上所述，探针3'端核苷酸中3位羟基的封闭修饰至关重要，可以防止探针自连，大幅提高环化效率。结合上模板的探针被切除修饰基团后，还能进行多个循环的线性扩增，最大程度的降低了原始分子的损失。另外，APG ssDNA连接酶的选用，环化后的酶切处理，以及环化后使用通用引物扩增也都能起到提高建库产物纯度和检测灵敏度的作用，分别/共同组成了本发明的技术方案。

Claims (12)

  1. 一种基因目标区域的富集方法，包括：
    (1)通过特异性探针扩增包含目标区域的片段化DNA，以提供捕获延伸产物；
    其特征在于，所述特异性探针包括与所述片段化DNA的目标区域互补和不互补的序列，且所述特异性探针的3’末端和5’末端核苷酸均被修饰；
    (2)向步骤(1)所提供的捕获延伸产物中加入连接酶，以提供连接产物，所述连接产物包括环化连接产物和线性连接产物。
  2. 如权利要求1所述的基因目标区域的富集方法，其特征在于，
    所述步骤(1)中，所述片段化DNA包含双链DNA、单链DNA和cDNA，所述片段化DNA的长度为25～200bp；
    和/或，所述步骤(1)的扩增体系中还包括DNA聚合酶和dNTP。
  3. 如权利要求2所述的基因目标区域的富集方法，其特征在于，
    所述DNA聚合酶具有3’-5’外切酶活性；
    和/或，所述dNTP还偶联有标记分子，所述标记分子优选为生物素。
  4. 如权利要求1所述的基因目标区域的富集方法，其特征在于，所述步骤(1)的扩增体系中还包括：
    活性物质，所述活性物质在所述特异性探针结合所述片段化DNA的目标区域后，用于切除所述特异性探针的3’末端修饰基团；
    优选的，所述活性物质是核酸酶。
  5. 如权利要求1所述的基因目标区域的富集方法，其特征在于，
    所述特异性探针还包括能被测序系统识别的通用序列；
    和/或，所述特异性探针的3’末端核苷酸的3位羟基被取代；
    和/或，所述特异性探针的3’末端核苷酸的2位甲氧基被取代；
    优选的，所述特异性探针的3’末端的取代基团选自氢原子、C3 Spacer基团、C6 Spacer基团、磷酸基团或氨基基团；
    和/或，所述特异性探针3’端尾部区域包含错配碱基；
    和/或，所述特异性探针的5’末端核苷酸的5位羟基被取代；
    优选的，所述特异性探针的5’末端的取代基团选自磷酸基团或腺苷基团。
  6. 如权利要求1所述的基因目标区域的富集方法，其特征在于，
    所述步骤(2)的连接体系中包括单链连接酶，所述单链连接酶优选为T4 RNA连接酶或热稳定性RNA连接酶。
  7. 如权利要求1所述的基因目标区域的富集方法，其特征在于，
    (3)在所述特异性探针上与所述目标区域不互补的序列中设置酶切位点，并在步骤(2)后加入内切酶，用于在所述酶切位点切割步骤(2)提供的连接产物中的环化连接产物以提供线性连接产物；
    优选的，所述酶切位点是尿嘧啶，所述内切酶是ssDNA内切酶或USER酶。
  8. 如权利要求7所述的基因目标区域的富集方法，其特征在于，还包括：
    (4)在所述步骤(2)后，PCR扩增所述步骤(2)提供的连接产物；
    优选的，所述步骤(4)中，PCR扩增引物具有与所述特异性探针互补的序列，且与所述目标区域的序列不互补；
    更优选的，与所述特异性探针互补的序列为测序通用序列；和/或，
    (5)在所述步骤(3)后，PCR扩增所述步骤(2)和步骤(3)提供的连接产物；
    优选的，所述步骤(5)中，PCR扩增引物具有与所述酶切位点两侧的序列互补的序列；
    更优选的，与所述酶切位点两侧的序列互补的序列为测序通用序列；
    和/或，在在所述步骤(1)至(5)的任意步骤后进行产物纯化。
  9. 如权利要求8所述的基因目标区域的富集方法，其特征在于，还包括：
    (6)用检测引物1、检测引物2和探针3检测步骤(4)或(5)提供的扩增产物，其特征在于，所述检测引物1、检测引物2和探针3中，至少其一包含基因特异性序列；
    优选的，所述检测引物1包含基因特异性序列，所述检测引物2和探针3包含通用序列；
    和/或，所述检测引物2和/或探针3也包含基因特异性序列；
    优选的，所述探针3包含标记分子，且所述探针3的序列与检测引物1或2不互补。
  10. 如权利要求1所述的基因目标区域的富集方法，其特征在于，所述基因目标区域的富集方法应用于核酸检测。
  11. 一种用于富集片段化DNA目标区域的体系，包括适用于如权利要求1～10 任一权利要求所述的基因目标区域的富集方法的特异性探针和连接酶。
  12. 如权利要求11所述用于富集片段化DNA目标区域的体系，其特征在于，还包括以下组分的一种或多种：偶联标记分子的dNTP，DNA聚合酶，核酸酶，内切酶；
    和/或，还包括PCR扩增引物，所述扩增引物具有与所述特异性探针的通用序列中至少部分互补的序列；
    和/或，还包括检测引物1、检测引物2和探针3，三者中至少其一含有基因特异性序列。
PCT/CN2019/123892 2019-09-20 2019-12-09 基因目标区域的富集方法及体系 WO2021051665A1 (zh)

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