WO2013075313A1 - 一种检测病毒在待测样本中整合方式的探针及其制备方法和应用 - Google Patents

一种检测病毒在待测样本中整合方式的探针及其制备方法和应用 Download PDF

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WO2013075313A1
WO2013075313A1 PCT/CN2011/082855 CN2011082855W WO2013075313A1 WO 2013075313 A1 WO2013075313 A1 WO 2013075313A1 CN 2011082855 W CN2011082855 W CN 2011082855W WO 2013075313 A1 WO2013075313 A1 WO 2013075313A1
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probe
sample
nucleic acid
virus
genome
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PCT/CN2011/082855
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English (en)
French (fr)
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李伟阳
易赏
曾玺
陈盛培
杨焕明
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深圳华大基因科技有限公司
深圳华大基因研究院
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Priority to CN201180074786.6A priority Critical patent/CN103975075A/zh
Priority to PCT/CN2011/082855 priority patent/WO2013075313A1/zh
Priority to CN201280029759.1A priority patent/CN103797130B/zh
Priority to PCT/CN2012/077175 priority patent/WO2012175013A1/zh
Priority to TW101143949A priority patent/TW201321519A/zh
Priority to TW101143948A priority patent/TW201321566A/zh
Publication of WO2013075313A1 publication Critical patent/WO2013075313A1/zh

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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701Specific hybridization probes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701Specific hybridization probes
    • C12Q1/702Specific hybridization probes for retroviruses
    • C12Q1/703Viruses associated with AIDS
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701Specific hybridization probes
    • C12Q1/706Specific hybridization probes for hepatitis
    • C12Q1/707Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/16Primer sets for multiplex assays

Definitions

  • Probe for detecting integration mode of virus in sample to be tested, preparation method and application thereof
  • the present invention belongs to the field of biotechnology, and in particular, the present invention relates to a probe for detecting the manner of integration of a virus in a sample to be tested, a preparation method thereof and an application thereof. Background technique
  • Hepatitis B virus is a global chronic viral infection.
  • the hepatitis B virus infection rate in China is about 60%-70%; and the hepatitis B surface antigen carrying rate is about 7.18% of the total population.
  • about 93 million people in the country carry hepatitis B virus, of which about 30 million are hepatitis B patients.
  • Around the world about 45% of people live in high-risk areas of chronic HBV infection, 43% live in the middle-incidence area of chronic HBV infection, and another 12% live in low-risk areas of chronic HBV infection.
  • the risk of HBV infection is > 60%.
  • Most HBV infections are transmitted vertically through mother-infant; in areas where chronic HBV infection occurs, the risk of HBV infection is 20%-60%; In the low-risk areas of chronic HBV infection, the risk of HBV infection is ⁇ 20%.
  • the hepatitis B virus genome is a partially double-stranded circular double-stranded DNA molecule with different lengths of two single strands.
  • the HBV virus has multiple genotypes, and eight common genotypes are A-H. Type A is distributed in Northern Europe and Western Europe; B and C are distributed in Southeast Asia; Type D is distributed in Southern Europe, Indian; Type E is distributed in West Africa and South Africa; F, Type H is distributed in Central America, South America; Type G is distributed in France, United States.
  • HBV-DNA is integrated into the genome of liver cancer cells.
  • the monoclonal method was used to find that the HBV gene was integrated around the human genome site, and rearrangements and deletions occurred.
  • HBV carriers have turned into liver cancer, and the study found that there are three ways:
  • HBV integration into the genome causes chromosomal instability, resulting in multiple site deletions
  • the expressed viral protein can regulate the proliferation of liver cancer cells.
  • HCC hepatocellular carcinoma
  • HBV can be re-integrated on the basis of integration.
  • the translocation of chromosomes 17 and 18 at both ends of the HBV DNA integration site is sometimes detected, and chromosome 18 has at least a 1.3 kb base deletion at the integration site.
  • the human genome sequence has 5 bp and 19 bp deletions; in 3 cases, the human chromosomes on both sides of the HBV integration fragment were detected, although the same chromosome, but the direction Instead, a rearrangement took place.
  • the research methods for integrating the HBV genome into the host genome are mainly Southern Blot, probe isotope labeling, monoclonal sequencing, ALU-PCR, and oligonucleotide probes.
  • these methods mainly reflected in: 1.
  • the suppliers of conventional probe customization are mainly Agilent and imblegene, which are more expensive to customize, usually several hundred dollars/reaction; An attempt to integrate.
  • Another object of the present invention is to provide a method and application for the preparation of the probe.
  • a nucleic acid probe set comprising a plurality of probes, the probe set having the following characteristics: (1) having one or more biotinylated dNTPs per probe; and/or
  • the entire nucleic acid sequence of the probe set covers 70%-100% of the corresponding viral genome sequence.
  • the probe set has 1-20000 nucleic acid probes; preferably, the probe set has 1000-5000 nucleic acid probes; more preferably, the probe set has 2500 Nucleic acid probes.
  • the biotin-labeled dNTP has an abundance of 1:4 in the probe set.
  • the probes have a partial overlap between them.
  • the probe is 100-500 bp in length; preferably, the probe is 200-300 bp in length ; more preferably, the probe is 250 bp in length.
  • the probe is obtained by PCR amplification using a viral genome as a template, and preferably, the amplification template is a hepatitis B virus (HBV) genome, hepatitis C virus (HCV). a genome, an HIV (HIV) genome, a papillomavirus (HPV) genome, or a combination thereof; more preferably the sample is a type B HBV genome and/or a type C HBV genome.
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HPV papillomavirus
  • nucleic acid chip having a probe set immobilized on the second aspect of the invention.
  • nucleic acid probe set of the first aspect of the invention and the nucleic acid chip of the second aspect of the invention for detecting the manner in which the virus is integrated in the sample;
  • the integrated mode is selected from the group consisting of rearrangement, ectopic, insertion, replacement, or a combination thereof.
  • a method of preparing the nucleic acid probe of the first aspect of the invention comprising the steps of:
  • the dNTP of the PCR amplification system is a biotin-labeled dNTP, and a PCR amplification product with biotin labeling is obtained;
  • the sample of step (a) has the following characteristics:
  • the sample is a virus sample containing nucleic acid
  • the sample is virions, serum, blood, tissue samples, exfoliated cells, epithelial cells, or a combination thereof; and/or
  • the sample is selected from the group consisting of: hepatitis B virus (HBV), hepatitis C virus (HCV), HIV (HIV), papillomavirus (HPV), or a combination thereof; and/or
  • the sample is type B HBV and / or type C HBV.
  • step (b) has the following characteristics:
  • step (b) The amplification described in step (b) is to amplify the full length of the viral DNA in the sample.
  • the ratio of the labeled dNTP to the unlabeled dNTP in the step (b) is 1: 2-8; the preferred ratio is 1: 3-6; more preferably the ratio is 1:4.
  • the interruption in step (c) is interrupted by ultrasonication.
  • step (d) is further included: purifying and/or quantifying the probe obtained in the step (c).
  • the probe has a length of 100 to 500 bp; preferably, the probe has a length of 200 to 300 bp, and more preferably, the probe has a length of 250 bp.
  • a method of detecting a manner of gene integration of a virus in a sample to be tested comprising the steps of:
  • step (ii) performing library construction on the samples obtained in step (i);
  • step (iii) hybridizing the probe of the first aspect of the invention to the library obtained in step (ii) to capture a nucleic acid sequence associated with viral gene integration;
  • step (iv) amplifying the nucleic acid sequence captured in step (iii) to obtain an amplification product associated with viral integration
  • step (V) sequencing the amplification product obtained in step (iv) to obtain nucleic acid related to virus integration
  • step (i) has the following characteristics:
  • the sample to be tested is tissue, blood, exfoliated cells, epithelial cells; and/or
  • the sample to be tested is derived from a human or non-human mammal, preferably from a human; and/or
  • the sample to be tested is derived from an HBV infected person or a liver cancer patient.
  • step (iii) has the following characteristics:
  • the probe is a denatured single-stranded DNA; and/or
  • linker blocking molecule The sequence of the linker blocking molecule is set forth in SEQ ID NO: 7; and/or
  • the sequence of the tag blocking molecule is shown in SEQ ID NO: 8 and SEQ ID NO: 9.
  • step (V) the amplification product is hybridized with a sequencing probe immobilized on a solid phase carrier, and subjected to solid phase bridge PCR amplification to form a sequencing cluster;
  • the sequencing clusters were sequenced by the "Synthesis-Side Sequencing" method to obtain nucleic acid information related to the way of virus integration.
  • the library in the step (ii), is constructed as: end-repairing the interrupted genomic DNA, adding a linker, and amplifying the fragment having the linker, and obtaining the expanded with the linker
  • the addition mixture is the sample library.
  • the linker has the sequence set forth in SEQ ID ⁇ :1 and SEQ ID NO:2; and/or the constructed library has SEQ ID:3 and SEQ ID:4 The sequence of tags.
  • kit for use in the method of the fourth aspect of the invention comprising:
  • the kit further comprises a reagent selected from the group consisting of:
  • Figure 1 shows the results of electrophoresis after HBV genome-wide PCR amplification, with a genome length of approximately 3.2 ⁇ M.
  • Figure 2 shows the results of electrophoresis after breaking the full-length HBV product.
  • the size of the fragment was between 250 bp and 300 bp.
  • Figure 3 shows the results of two library fragment sizes after the establishment of the library, which are 271 bp and 876 bp, respectively.
  • Figure 4 shows the flow of the method for detecting the integration of HBV in the sample to be tested according to the present invention.
  • Fig. 5 is a view showing the flow of information analysis of the method for detecting the integration of HBV in the sample to be tested according to the present invention. detailed description
  • the present inventors have for the first time constructed a probe for detecting the manner in which a virus is integrated in a sample to be tested and an application thereof.
  • the probe set has the following characteristics:
  • the entire nucleic acid sequence of the probe set covers 70%-100% of the corresponding viral genome sequence.
  • the probe set has 1 to 20,000 nucleic acid probes; preferably, the probe set has 1000 to 5000 nucleic acid probes; more preferably, the probe set There are 2500 nucleic acid probes.
  • the probe set further has the following features:
  • the biotin-labeled dNTP has an abundance of 1:4 in the probe set;
  • the probe has a length of 100 to 500 bp; preferably, the probe has a length of 200 to 300 bp; more preferably, the probe has a length of 250 bp; and/or
  • the probe is obtained by PCR amplification using a viral genome as a template.
  • the amplification template is a hepatitis B virus (HBV) genome, a hepatitis C virus (HCV) genome, and AIDS.
  • the invention also provides a method for preparing the probe and an application thereof.
  • the present invention has been completed on this basis.
  • the term “containing” includes “comprise”, “consisting essentially of” and “consisting of.”
  • the terms “above” and “below” include the number, for example “80% or more” means ⁇ 80%, and “2% or less” means ⁇ 2%.
  • the term "abundance" refers to the proportion of biotin-labeled dNTPs in the entire probe set.
  • the abundance of the biotinylated dNTP in the probe set is 1:6-1:2; moreover, the abundance of the biotinylated dNTP in the probe set is 1: 4.
  • the term "primer” refers to a generic term for an oligonucleotide that is complementary to a template and which synthesizes a DNA strand complementary to a template in the action of DNA polymerase.
  • the primer may be natural RNA, DNA, or any form of natural nucleotide, and the primer may even be a non-natural nucleotide such as LNA or ZNA.
  • the primer is “substantially” (or “substantially") complementary to a particular sequence on a strand on the template.
  • the primer must be sufficiently complementary to a strand on the template to initiate extension, but the sequence of the primer does not have to be fully complementary to the sequence of the template. For example, a sequence that is not complementary to the template is added to the 5' end of the primer complementary to the template at a 3' end, such primers are still substantially complementary to the template. As long as there are sufficiently long primers to bind well to the template, the non-fully complementary primers can also form a primer-template complex with the template for amplification.
  • probe refers to a simple DNA or RNA molecule capable of detecting a complementary nucleic acid sequence.
  • the probe must be pure and not affected by other different sequence nucleic acids.
  • Typical probes are cloned DNA sequences or DNA obtained by PCR amplification, synthetic oligonucleotides or RNA obtained after cloning and cloning DNA sequences in vitro, and can also be used as probes.
  • the length of the probe may range from 20 to 500 mers, preferably from 50 to 300 mers, more preferably from 250 mers.
  • Probe design and synthesis methods are well known to those skilled in the art It is known that synthetic probes can be synthesized using artificial chemical synthesis or commercially available probes.
  • the present invention provides a probe for detecting the manner in which a virus is integrated in a sample to be tested, the probe set having the following characteristics:
  • the entire nucleic acid sequence of the probe set covers 70%-100% of the corresponding viral genome sequence.
  • the probe set has 1 to 20,000 nucleic acid probes; preferably, the probe set has 1000 to 5000 nucleic acid probes; more preferably, the probe set There are 2500 nucleic acid probes.
  • the probe set further has the following features:
  • the biotin-labeled dNTP has an abundance of 1:4 in the probe set;
  • the probe has a length of 100 to 500 bp; preferably, the probe has a length of 200 to 300 bp; more preferably, the probe has a length of 250 bp; and/or
  • the probe is obtained by PCR amplification using a viral genome as a template.
  • the amplification template is a hepatitis B virus (HBV) genome, a hepatitis C virus (HCV) genome, and AIDS.
  • the invention also provides a method of preparing the probe.
  • the method comprises the steps of:
  • the labeled dNTP is preferably a biotin-dNTP, and the labeled dNTP can be combined with a streptomycin affinity magnetic bead;
  • the probe of the present invention has a length of 100 to 500 bp, preferably, the probe has a length of 200 to 300 bp, and more preferably the probe has a length of 250 bp.
  • chip refers to the use of micromachining techniques to add to the substrate material of a chip. A variety of fine structures are produced, and the necessary biochemical substances are applied and surface-treated, and various probe molecules and surfaces are immobilized to obtain a material containing a large amount of probes.
  • a person skilled in the art can obtain a chip using a general method.
  • the first type is a light-guided in-situ synthesis method in which a photolithography process is combined with a photochemical synthesis method in a micromachining technique.
  • the second method is a chemical jet method in which a synthesized oligonucleotide probe is spot-sprayed onto a chip and immobilized to produce a DNA chip.
  • the third method is a contact dot coating method in which a DNA probe is applied to a chip by a precise movement of a high-speed precision robot to bring the pipetting head into contact with the glass chip.
  • the fourth method is to synthesize DNA probes on the chip in parallel using four piezoelectric nozzles each containing A, T, G, and C nucleosides.
  • the present invention provides a chip having a surface-immobilized manner for detecting viral gene integration.
  • the virus is HBV, preferably B-type HBV and C-type HBV.
  • the chip of the present invention is useful for detecting the manner of gene integration of a virus in a host to be tested.
  • DNA library preparation refers to the interruption of a fragment of interest in a genome to obtain a mixture of DNA fragments of a certain size.
  • Methods for preparing a sample library include, but are not limited to, steps: repairing the end of the disrupted genomic DNA to have a blunt end; adding a linker, amplifying the fragment having the linker, obtaining The mixture with the linker is a library.
  • the linker has the sequences set forth in SEQ ID ⁇ :1 and SEQ ID NO:2.
  • the constructed library has a tag sequence as set forth in SEQ ID NO: 3 and SEQ ID NO: 4.
  • the disrupted product, the end repair product, the linker product, and the enriched product can also be purified.
  • Purification conditions and parameters are well known to those skilled in the art, and it is within the ability of those skilled in the art to make certain changes or optimizations to the conditions of the reaction.
  • NGS Next-generation sequencing technology
  • the next-generation sequencing technology enables simultaneous sequencing of each base in a short period of time through the simultaneous parallel sequencing of millions of short DNA fragments, and the cost is greatly reduced.
  • NGS technology is used in many ways, such as genomics, transcriptomics, and appearance. Genomics, clinical diagnosis, etc.
  • Those skilled in the art can typically perform high throughput sequencing using a variety of second generation sequencing platforms including, but not limited to, Ileqia's Hiseq 2000, 454 FLX (oche), Solexa Genome Analyzer, and SOLID from Applied Biosystems.
  • the common feature of these platforms is the extremely high sequencing throughput. Compared to the 96 sequencing capillary sequencing of traditional sequencing, high-throughput sequencing can read 400,000 to 4 million sequences in one experiment. According to the platform, the read length is from 25bp. Up to 450 bp, so different sequencing platforms can read the number of bases ranging from 1G to 14G in one experiment.
  • Solexa high-throughput sequencing includes two steps: DNA cluster formation and on-machine sequencing: the mixture of PCR amplification products is hybridized with the immobilized sequencing probe immobilized on the solid phase carrier, and subjected to solid phase bridge PCR amplification to form a sequencing. Clusters; the sequencing clusters were sequenced by "edge synthesis-edge sequencing”.
  • the DNA cluster is formed by using a flow cell with a single-stranded primer attached to the surface, and the DNA fragment of the single-stranded state is immobilized on the chip by the principle that the linker sequence and the primer on the surface of the chip are complementary to each other by base complementation.
  • the fixed single-stranded DNA becomes double-stranded DNA
  • the double strand is denatured into a single strand, one end of which is anchored on the sequencing chip, and the other end is randomly and adjacent to another primer to be anchored, Forming a "bridge"; on the sequencing chip, there are tens of millions of DNA single molecules simultaneously reacting; forming a single-stranded bridge, using the surrounding primers as amplification primers, and amplifying again on the surface of the amplification chip to form a double
  • the strand, the double strand is denatured into a single strand, and becomes a bridge again.
  • the template called the next round of amplification continues to expand; after repeated rounds of 30 rounds of amplification, each single molecule is amplified 1000 times, called a single clone. DNA cluster.
  • DNA clusters were sequenced on a Solexa sequencer. During the sequencing reaction, the four bases were labeled with different fluorescence, and each base was blocked by a protected base. Only one base could be added to a single reaction. After reading the color of the reaction, the protection group is removed, and the next reaction can be continued. Thus, the exact sequence of the base is obtained.
  • INDEX label or BARCODE
  • an additional 7 cycles of sequencing for the INDEX portion can be performed. Up to 1 can be identified by INDEX. Twelve different samples were distinguished in the sequencing ramp.
  • the present invention also provides a method for detecting the manner of gene integration of a virus in a host cell.
  • the method mainly comprises the following steps: 1. HBV probe production
  • PCR is performed on the full length genome of HBV, wherein the labeled dNTP is introduced during the PCR process;
  • the labeled dNTP is biotin-dNTP, the ratio of biotin-dNTP to normal dNTP is 1:4, and the total concentration is 2.5 mM;
  • it is purified using 1.5 volumes of AMPURE magnetic beads and then purified using QIAGEN's 250MinElute PCR Purification Kit;
  • the sample may be from the plasma or tissue of a patient with HBV or liver cancer, and the library is constructed according to the Illumina Paired-End Sample Preparation Guide; the probe is mixed with the library to capture specific sample nucleic acid fragments.
  • the captured nucleic acid fragment is eluted
  • the purified PCR product was subjected to high-throughput sequencing, and the sequencing platform used ILLUMINA's HISEQ2000.
  • the sequencing length it was not limited to PEI101, and PEI91 could also be used.
  • the off-machine data is removed from the duplicates and the joints contaminated by the joints, and the basic information of the machine data is counted (for example: library length; reads length; number of reads; number of bases; repetition rate, etc.); respectively, intercepting two reads of PE
  • the 50 bp base forms a pair of 50 bp new reads
  • B PE50readsc uses the new PE50 reads using the soap alignment software ( -r 1 -V 2 ) with the human reference sequence (hgl9) and HBV various reference sequences, respectively.
  • Fig. 5 is a view showing the flow of information analysis of the method for detecting the integration of HBV in the sample to be tested according to the present invention.
  • the invention also provides a kit for detecting the manner of integration of a virus in a host, mainly comprising Lower component:
  • the kit further comprises a reagent selected from the group consisting of:
  • the price required to prepare the probe using the present invention is only 1% to 5% of the preparation price of the prior art
  • the invention has flexible form in the process of making the probe, can be designed according to its own needs, and the proportion and quantity of HBV are added in proportion;
  • the process of making the probe is simple, and the probe can be obtained by PCR and interrupted purification without synthesis;
  • the probe of the present invention is a double-stranded probe, which can reduce information errors and losses caused by PCR;
  • the detection method of the present invention can be used in combination with the database construction technology to enrich HBV fragments; and has wide applicability.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions.
  • NanoDrop 1000 Spectrophotometer Thermo Fisher Scientific Electrophoresis DYY-6C Beijing Liuyi Instrument Factory Nucleic Acid Concentrator 5305 Eppendorf Gel Imaging System Tanon Shanghai Tianneng Technology Co., Ltd.
  • the source of the sample is liver cancer tissue of the same patient, and the liver cancer tissue of the patient has full genome sequencing information.
  • the genomic DNA was interrupted by Covaris s2, the end was filled and repaired, the end was added with A, and the joint was added.
  • the joints used in the construction process were:
  • the fragment added to the adaptor was subjected to PCR to obtain a sample library, and the constructed library was provided with an INDEX label sequence.
  • the INDEX sequence is as follows:
  • the designed primers are:
  • PI SEQ ID NO: 5 : TTTTTCACCTCTGCCTAATCA
  • the PCR reaction system is shown in Table 2.
  • HBV virus genome (lng/ ⁇ ) 3 ⁇ 1
  • the PCR reaction was carried out on an AB-9700 PCR instrument, and the reaction procedure is shown in Table 3.
  • the PCR product was detected by 1% agarose gel electrophoresis and purified by using 1.2-1.5 volumes of AMPURE BEADS and dissolved in 80 ul of water. It was then purified using a 250MinElute PCR Purification Kit and dissolved in 60 ⁇ l of water. The 1% agarose gel electrophoresis assay detects whether the PCR product has been purified. The results are shown in Figure 1.
  • the DNA was completely transferred to Covaris interrupted tubules and supplemented with TE buffer to a total volume of 8 ( ⁇ l (Nanodrop detected a total of 5 g), Covaris S2 instrument (Gene Co., Ltd.) interrupted, hit See Table 4 for the breaking conditions.
  • the size of the fragment was detected by 2% agarose gel electrophoresis. The results showed that the fragment was at 250-300 bp (Fig.
  • the obtained fragmented product can be used as a probe for hybridization.
  • the fragment product was purified using a MinElute PCR Purification Kit, dissolved in 40 ⁇ l of buffer, and the concentration of the probe DNA was measured with a Nanodrop instrument so that the concentration of the probe was about 120 ⁇ 3 ⁇ 4/ ⁇ 1.
  • the resulting probe can be stored at -20 ° C or -80 ° C.
  • the probe must be denatured at 95 °C for 10 minutes before use, and then rapidly placed on ice to cool to form a single strand.
  • the amount of the library is lg
  • the amount of the probe is 600 ng (Nanodrop quantification)
  • add the linker blocking molecule the ratio of the amount of the blocking molecule to the amount of the library is lnmol: 1 ⁇ ⁇
  • tag blocking molecule and library The ratio of the amount is lnmol: ⁇ g.
  • the closed molecular sequence of the linker is:
  • Two reagents in the EZ hybridization system were separately added to the EP tube: 2 X SC Hybridiation Buffer hybridization buffer 7.5 ⁇ and 1 X SC Hybridiation Component A 3 ⁇ 1, then denatured at 95 ° C for 10 minutes, and the self-made probe was added to the above hybridization mixture. 600 ng, probe volume total 5 ⁇ 1. After shaking, mix and centrifuge at full speed for 10 seconds on a centrifuge, and transfer all the samples to a 200 ⁇ l tube.
  • the hybridization mixture was aspirated and added to the magnetic beads prepared in the previous step; the mixture was pipetted 10 times with a pipette; the tube was placed on a PCR machine and incubated at 47 ° C for 45 min (the vortex was shaken every 15 min 3 s to prevent precipitation of magnetic beads); After incubation for 45 min, the mixture was transferred from a 0.2 mL vial into a 1.5 mL EP tube.
  • PFX polymerase purchased from Invirtogen
  • PFX reaction buffer (10 X ) PFX reaction buffer (10 X )
  • dNTP 10 mM
  • SEQ ID NO: 10 AATGATACGGCGACCACCGAGATC:.
  • each sample was purified using 1.5 volumes of Ampme Beads, and the recovered PCR product was dissolved in 30 ⁇ l of ultrapure water and measured by Nanodrop 1000.
  • Example 6 PCR product sequencing
  • the purified PCR product was determined by 2100 Bioanalyzer (Agilent) to determine the size and insert size. See Figure 3 and Figure 4.
  • the purified product was 271 bp and 876 bp, respectively.
  • QPCR was accurately quantified and sequenced. In this example, the sequencing of the upper machine was carried out in accordance with the specifications of c-Bot and HISEQ2000Hiseq 2000 published by Illumina/Solexa.
  • the off-machine data is removed from the duplicates and the joints contaminated by the joints, and the basic information of the machine data (the length of the library; the length of the reads; the number of reads; the number of bases; the repetition rate) are counted; and the 50 bp of the two reads of the PE are respectively intercepted.
  • a pair of reads that are read to the human reference sequence and another to the HBV reference sequence are selected from the alignment results; such reads are likely to cross the HBV insertion site; statistics on this part of the reads ratio For information, find the insertion hotspot area in the human genome.
  • a kit for detecting the manner in which a virus is integrated in a sample to be tested including the following

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Abstract

本发明公开了一种检测病毒在待测样本中整合方式的探针及其制备方法和应用。具体地,所述探针至少具有以下一个特征:(1)每个探针上具有一个或多个生物素标记的dNTP;(2)生物素标记的dNTP在探针集中的丰度为1:6-1:2;(3)所述探针集的全部核酸序列覆盖对应病毒基因组序列的70%-100%。

Description

一种检测病毒在待测样本中整合方式的探针及其制备方法和应用 技术领域
本发明属于生物技术领域, 具体地, 本发明涉及一种检测病毒在待测样本 中整合方式的探针及其制备方法和应用。 背景技术
乙型肝炎病毒 (hepatitis B vims, HBV) 是一种全球性的慢性病毒感染性 疾病。 我国的乙肝病毒感染率约 60%-70%; 而乙肝表面抗原携带率约占总人口 的 7.18%, 以此计算, 全国约有 9300万人携带乙肝病毒, 其中乙肝患者大约有 3000万。 在全世界, 大约有 45%的人群生活在慢性 HBV感染的高发区, 43%的 人群生活在慢性 HBV感染的中发区, 另有 12%的人群生活在慢性 HBV感染低发 区。 在慢性 HBV感染的高发区, HBV感染的危险性 > 60%, 绝大多数 HBV感染 是通过母婴垂直传播; 在慢性 HBV感染中发地区, HBV感染的危险性为 20%— 60%; 在慢性 HBV感染的低发区, HBV感染的危险性 <20%。
在慢性 HBV感染的过程中, 一定比例的 HBV感染发展成肝硬化与肝细胞 癌, 还有一部分由于肝病变的恶化而发生肝衰竭。 因此, 在慢性 HBV感染的自 然过程中, 不少患者会因肝硬化、 肝细胞癌, 以及肝功能衰竭而导致死亡, 给 社会和家庭带来巨大的经济损失。
乙肝病毒基因组是一个有部分单链的环状双链 DNA分子, 两条单链长度不 同, HBV病毒具有多种基因型, 常见基因型有八种, 分别为 A-H。 A型分布于 北欧与西欧; B, C型分布于东南亚; D型分布于南欧, 印第安; E型分布于西 非与南非; F, H型分布中美, 南美; G型分布法国, 美国。
现今关于肝癌与 HBV基因组整合研究, 发现 HBV-DNA整合入肝癌细胞基 因组中。采用单克隆方法发现 HBV基因整合入人类基因组位点周围,发生重排、 缺失。 HBV携带者转变为肝癌, 研究发现有以下三个途径:
( 1 ) HBV整合入基因组引起染色体不稳定, 造成多位点缺失;
( 2) HBV片段插入特定位点引起基因插入缺失, 激活原癌基因, 例如细 胞增殖分化基因, 与端粒逆转录酶基因;
( 3 ) 表达的病毒蛋白可调控肝癌细胞增殖。
HBV病毒基因组整合入人类基因组破坏基因组稳定性引起非正常易位, DNA重排,删除,杂合丢失,以及激活抑制相关基因,从而诱导肝细胞癌(HCC ) 的发生。 现已知在 14号、 15号、 18号、 19号和 Y染色体上, HBV DNA的整合位 点附近有一些与癌症相关的基因, 如 BCL2L2基因与细胞凋亡相关, SMOCl、 Calmodulin-1基因与钙通道相关, FBLN5基因与血管生长相关, NOTCH3与细 胞信号转导途径相关。 HCC组织中可以存在多个整合位点, HBV在整合的基础 上还可以发生再次整合。 如在肝癌患者的肝组织中有时能检测到 HBV DNA整 合位点两端发生 17号和 18号染色体的易位, 同时 18号染色体在整合位点处有至 少 1.3 kb的碱基缺失。 通过对肝癌患者的肝组织 HBV整合的研究发现: 有的病 例中人基因组序列各有 5bp和 19bp的缺失; 在 3例病例中检测到 HBV整合片段两 侧的人染色体虽然是同一染色体, 但是方向相反, 发生了重排。
HBV基因组整合入宿主基因组的研究方法目前主要为 Southern Blot, 探针 同位素标记、 单克隆测序、 ALU-PCR、 寡聚核苷酸探针等。 目前这些方法存在 很多问题, 主要体现在: 1. 对于高通量测序来说常规探针定制的供应商主要为 Agilent和 imblegene, 定制价格较高, 一般为几百美元 /反应; 2. 尚未做过整合 方面的尝试。
目前本领域还没有一种经济简单快速的 HBV基因组整合入宿主基因组的 研究方法。 因此本领域迫切需要开发新的 HBV基因组整合入宿主基因组的检测 方法, 为研究 HBV以及肝脏相关疾病提供方便廉价的研究方法与手段。 发明内容
本发明的目的是提供一种用于检测病毒在待测样本中整合方式的探针。
本发明的另一目的是提供所述探针的制备方法和应用。
本发明的另一目的是提供一种检测病毒在待测样本中整合方式的方法。 在本发明的第一方面, 提供了一种核酸探针集, 包括多个探针, 所述探针集 具有以下特征: (1)每个探针上具有 1个或多个生物素标记的 dNTP; 和 /或
(2)生物素标记的 dNTP在探针集中的丰度为 1 :6-1 :2; 和 /或
(3)所述探针集的全部核酸序列覆盖对应病毒基因组序列的 70%-100%。
在另一优选例中, 所述探针集具有 1-20000个核酸探针; 较佳地, 所述探针 集具有 1000-5000个核酸探针; 更佳地, 所述探针集具有 2500个核酸探针。
在另一优选例中, 所述生物素标记的 dNTP在所述探针集中的丰度为 1 :4。 在另一优选例中, 所述探针之间具有部分重叠。
在另一优选例中, 所述探针长度为 100-500bp ; 较佳地, 所述探针长度为 200-300bp; 更佳地, 所述探针长度为 250bp。
在另一优选例中, 所述探针是以病毒基因组作为模板, PCR法扩增获得, 较佳 地, 所述扩增模板为为乙型肝炎病毒 (HBV) 基因组、 丙型肝炎病毒 (HCV) 基 因组、 艾滋病病毒 (HIV) 基因组、 乳头瘤病毒 (HPV) 基因组, 或其组合; 更佳 地所述样本为 B型 HBV基因组和 /或 C型 HBV基因组。
在本发明的第二方面,提供了一种表面固定有本发明第二方面所述探针集的 核酸芯片。
在本发明的第三方面,提供了本发明的第一方面所述核酸探针集和本发明的第 二方面所述核酸芯片的用途, 用于检测病毒在待样本中的整合方式; 较佳地, 所 述的整合方式选自下组: 重排、 异位、 插入、 替换, 或其组合。
在本发明的第四方面, 提供了一种制备本发明的第一方面所述核酸探针的方 法, 包括步骤:
a. 获得探针来源样本;
b. 对步骤(a)获得的样本进行 PCR扩增, PCR扩增体系的 dNTP为生物素标记 的 dNTP, 获得带有生物素标记的 PCR扩增产物;
c 对步骤 (b) 获得的生物素标记的 PCR扩增产物进行打断, 得到片段化的生 物素标记的 PCR扩增产物, 即为探针;
在另一优选例中, 步骤 (a) 所述样本具有以下特征:
样本为含有核酸的病毒样本; 和 /或
所述样本为病毒粒子、 血清、 血液、 组织样本、 脱落细胞, 上皮细胞, 或其 组合; 和 /或 所述样本选自下组: 乙型肝炎病毒 (HBV) 、 丙型肝炎病毒 (HCV) 、 艾滋 病病毒 (HIV) 、 乳头瘤病毒 (HPV) , 或其组合; 和 /或
所述样本为 B型 HBV和 /或 C型 HBV。
在另一优选例中, 步骤 (b) 具有以下特征:
步骤 (b) 所述的扩增为对样本中病毒 DNA全长进行扩增; 和 /或
步骤 (b) 所述标记的 dNTP为 biotin-dNTP, 且所述标记的 dNTP能够与链霉素 亲和磁珠结合; 和 /或
步骤(b)所述标记的 dNTP与非标记 dNTP的比例为 1 : 2-8; 优选比例为 1 : 3-6; 更优选比例为 1 : 4。
在另一优选例中, 步骤 (c) 所述打断为超声法打断。
在另一优选例中, 还包括步骤 (d) : 对步骤 (c) 获得的探针进行纯化和 /或 定量。
在另一优选例中, 所述探针的长度为 100-500bp; 较佳地, 所述探针的长度为 200-300bp, 更佳地, 所述探针的长度为 250bp。
在本发明的第五方面, 提供了一种检测病毒在待测样本中基因整合方式的方 法, 包括步骤:
(i) 获得待测样本;
(ii) 对步骤 (i) 获得的样本进行文库构建;
(iii) 将本发明第一方面所述的探针与步骤 (ii) 获得的文库进行杂交, 捕获 与病毒基因整合有关的核酸序列;
(iv) 对步骤 (iii) 捕获的核酸序列进行扩增, 获得与病毒整合有关的扩增产 物;
( V ) 对步骤 (iv ) 获得的扩增产物进行测序, 获得与病毒整合方式有关核酸
I Ή自、
在另一优选例中, 步骤 (i) 具有以下特征:
所述待测样本为组织、 血液、 脱落细胞, 上皮细胞; 和 /或
所述待测样本来源于人或非人哺乳动物, 较佳地来源于人; 和 /或
所述待测样本来源于 HBV感染者或肝癌患者。
在另一优选例中, 步骤 (iii) 具有以下特征: 所述探针为变性的单链 DNA; 和 /或
在杂交液中加入接头封闭分子和标签封闭分子; 和 /或
所述接头封闭分子的序列如 SEQ ID NO:7所示; 和 /或
所述标签封闭分子的序列如 SEQ ID NO:8和 SEQ ID NO:9所示。
在另一优选例中, 在步骤 (V ) 中, 将所述的扩增产物与固相载体上固定的测 序探针进行杂交, 进行固相桥式 PCR扩增, 形成测序簇; 然后对所述测序簇用 "边 合成-边测序"法进行测序, 从而得到与病毒整合方式有关核酸信息。
在另一优选例中, 在步骤 (ii) 中, 所述的文库构建为: 对打断的基因组 DNA 进行末端修复, 加入接头, 对具有接头的片段进行扩增, 获得的带有接头的扩增混 合物即为样本文库。
在另一优选例中,所述的接头具有如 SEQ ID ΝΟ:1和 SEQ ID NO:2所示的序列; 和 /或, 所构建的文库具有如 SEQ ID:3和 SEQ ID:4所示的标签序列。
在本发明的第六面,提供了一种可用于本发明第四方面所述方法的试剂盒,所 述试剂盒包括:
(1)第一容器以及位于容器内本发明第二方面所述的核酸芯片, 或本发明第 一方面所述的探针;
(2)第二容器以及位于容器内的用于构建样本文库的接头;
(3)第三容器以及位于容器内的接头封闭分子;
(4)第四容器以及位于容器内的标签封闭分子;
(5)检测说明书;
在另一优选例中, 所述试剂盒还包括选自下组的试剂:
用于进行 PCR扩增所需的试剂、
用于进行封闭反应所需的试剂、
用于进行杂交反应所需的试剂、
用于进行测序反应所需的试剂、
或其组合。 应理解,在本发明范围内中,本发明的上述各技术特征和在下文 (如实施例) 中具体描述的各技术特征之间都可以互相组合, 从而构成新的或优选的技术方 案。 限于篇幅, 在此不再一一累述。 附图说明
下列附图用于说明本发明的具体实施方案, 而不用于限定由权利要求书所 界定的本发明范围。
图 1显示了对 HBV全基因组 PCR扩增后的电泳检测结果, 基因组全长约为 3·2Κ。
图 2显示了对 HBV全长产物打断后电泳检测结果, 打断的片段大小集中于 250bp-300bp之间。
图 3显示了建库杂交后两种文库片断大小检测结果, 分别为 271bp和 876bp。 图 4显示了本发明检测 HBV在待测样本基因整合方式的流程。
图 5显示了本发明检测 HBV在待测样本基因整合方式的信息分析流程。 具体实施方式
本发明人经过广泛而深入的研究, 首次构建了一种用于检测病毒在待测样 本中整合方式的探针及其应用, 具体地, 所述探针集具有以下特征:
(1)每个探针上具有 1个或多个生物素标记的 dNTP; 和 /或
(2)生物素标记的 dNTP在探针集中的丰度为 1 :6-1 :2; 和 /或
(3)所述探针集的全部核酸序列覆盖对应病毒基因组序列的 70%-100%。
在本发明的一个优选例中, 所述探针集具有 1-20000个核酸探针; 较佳地, 所述探针集具有 1000-5000个核酸探针; 更佳地, 所述探针集具有 2500个核酸探 针。
在另一优选例中, 所述探针集还具有以下特征:
(i)所述生物素标记的 dNTP在所述探针集中的丰度为 1 :4; 和 /或
(ii)所述探针之间具有部分重叠; 和 /或
(iii)所述探针长度为 100-500bp; 较佳地, 所述探针长度为 200-300bp; 更佳地, 所述探针长度为 250bp; 和 /或
(iv)所述探针是以病毒基因组作为模板, PCR法扩增获得, 较佳地, 所述扩增 模板为为乙型肝炎病毒 (HBV) 基因组、 丙型肝炎病毒 (HCV) 基因组、 艾滋病 病毒 (HIV) 基因组、 乳头瘤病毒 (HPV) 基因组, 或其组合; 更佳地所述样本为 B型 HB V基因组和 /或 C型 HB V基因组。
本发明还提供了所述探针的制备方法及其应用。 在此基础上完成了本发明。 术语
如本文所用,术语"含有"包括"具有 (comprise)"、 "基本上由…构成"和 "由… 构成"。
如本文所用, 术语"以上"和"以下"包括本数, 例如" 80%以上"指≥80%, "2% 以下"指≤2%。
丰度
如本文所用, 术语 "丰度 "指的生物素标记的 dNTP数量在整个探针集中所 占的比例。 在本发明的一个优选例中, 生物素标记的 dNTP在探针集中的丰度为 1 :6-1 :2; 更加地, 生物素标记的 dNTP在所述探针集中的丰度为 1 :4。
引物
如本文所用, 术语 "引物"指的是能与模板互补配对, 在 DNA聚合酶的作 用合成与模板互补的 DNA链的寡聚核苷酸的总称。 引物可以是天然的 RNA、 DNA, 也可以是任何形式的天然核苷酸, 引物甚至可以是非天然的核苷酸如 LNA或 ZNA等。
引物"大致上 "(或 "基本上")与模板上一条链上的一个特殊的序列互补。 引 物必须与模板上的一条链充分互补才能开始延伸, 但引物的序列不必与模板的 序列完全互补。 比如, 在一个 3'端与模板互补的引物的 5'端加上一段与模板不 互补的序列, 这样的引物仍大致上与模板互补。 只要有足够长的引物能与模板 充分的结合, 非完全互补的引物也可以与模板形成引物-模板复合物, 从而进行 扩增。
探针及其制备方法
如本文所用, "探针 "一词是指能够检测互补核酸序列的简单 DNA或 RNA分 子。 探针必须是纯净的, 而且不受其他不同序列核酸的影响。 典型的探针是克 隆的 DNA序列或通过 PCR扩增获得的 DNA, 人工合成的寡核苷酸或从体外转录 克隆 DNA序列后获得的 RNA, 也可以作为探针。 探针长度可以从 20-500mer, 较佳地 50-300mer, 更佳地 250mer。探针设计和合成方法为本领域技术人员所熟 知可以使用人工化学合成法合成探针或使用市售探针。
本发明提供了一种用于检测病毒在待测样本中整合方式的探针, 所述探针 集具有以下特征:
(1)每个探针上具有 1个或多个生物素标记的 dNTP; 和 /或
(2)生物素标记的 dNTP在探针集中的丰度为 1 :6-1 :2; 和 /或
(3)所述探针集的全部核酸序列覆盖对应病毒基因组序列的 70%-100%。
在本发明的一个优选例中, 所述探针集具有 1-20000个核酸探针; 较佳地, 所述探针集具有 1000-5000个核酸探针; 更佳地, 所述探针集具有 2500个核酸探 针。
在另一优选例中, 所述探针集还具有以下特征:
(i)所述生物素标记的 dNTP在所述探针集中的丰度为 1 :4; 和 /或
(ii)所述探针之间具有部分重叠; 和 /或
(iii)所述探针长度为 100-500bp; 较佳地, 所述探针长度为 200-300bp; 更佳地, 所述探针长度为 250bp; 和 /或
(iv)所述探针是以病毒基因组作为模板, PCR法扩增获得, 较佳地, 所述扩增 模板为为乙型肝炎病毒 (HBV) 基因组、 丙型肝炎病毒 (HCV) 基因组、 艾滋病 病毒 (HIV) 基因组、 乳头瘤病毒 (HPV) 基因组, 或其组合; 更佳地所述样本为 B型 HB V基因组和 /或 C型 HB V基因组。
本发明还提供了一种所述探针的制备方法,在一个优选例中,所述方法包括步 骤:
a. 获得病毒样本;
b. 获得样本进行扩增, 扩增中引入标记的 dNTP, 获得标记的 PCR扩增产物, 标记的 dNTP优选为 biotin-dNTP, 标记的 dNTP能够与链霉素亲和磁珠结合;
c 对标记的 PCR扩增产物进行片段化, 即为探针;
d: 对获得的探针进行纯化和 /或定量。
本发明所述探针的长度为 100-500bp, 较佳地, 所述探针的长度为 200-300bp, 更佳地所述探针的长度为 250bp。
芯片
如本文所用, "芯片 "一词是指可以采用微加工技术在芯片的基底材料上加 工出各种微细结构, 施加必要的生物化学物质并进行表面处理, 将各种探针分 子与表面固定化, 制得含有大量探针的材料。
本领域技术人员可以使用通用的方法获得芯片。 DNA芯片制备方法通常有 4种。 第 1种是光引导原位合成法, 在微加工技术中用光刻工艺与光化学合成法 相结合。 第 2种方法是化学喷射法, 将合成好的寡核苷酸探针定点喷射到芯片 上并加以固定化来制作 DNA芯片。 第 3种方法是接触式点涂法, 通过高速精密 机械手的精确移动让移液头与玻璃芯片接触而将 DNA探针涂敷在芯片上。 第 4 种方法是使用 4支分别装有 A, T, G, C核苷的压电喷头在芯片上并行合成出 DNA 探针。
本发明提供了一种表面固定有用于检测病毒基因整合方式的芯片。 在一个 优选例中, 所述病毒为 HBV, 较佳地, 为 B型 HBV和 C型 HBV。 本发明的芯片 用于检测病毒在待测宿主中的基因整合方式。
DNA文库及其制备
如本文所用, "DNA文库制备"一词是指对基因组的目的片段进行打断, 获 得一组具有一定大小的 DNA片段混合物。
样本文库的制备方法为本领域技术人员所熟知, 包括 (但不局限于)步骤: 对打断的基因组 DNA末端修复, 使之具有平末端; 加入接头, 对具有接头的 片段进行扩增, 获得的带有接头的混合物为文库。
在一个优选例中,所述的接头具有如 SEQ ID ΝΟ:1和 SEQ ID NO:2所示的序列。 在一个优选例中, 所构建的文库具有如 SEQ ID NO:3和 SEQ ID NO:4所示的标 签序列。
在一个优选例中, 还可以对打断产物、 末端修复产物、 接头产物和富集产 物进行纯化。 纯化条件及参数为本领域技术人员所熟知, 对反应的条件进行一 定的变化或优化也在本领域技术人员能力范围之内。
高通量测序
新一代测序技术 (NGS)改变了以往对 DNA/RNA样本的分析模式, 几乎成为 所有研究领域中必不可少的研究工具。 新一代测序技术是通过对上百万条 DNA 短片段的同时的平行测序, 使得在短时间内就能够完成每个碱基的测序, 且成 本大幅度降低。 NGS技术在很多方面得到应用, 如基因组学、 转录组学、 表观 基因组学、 临床诊断等。
本领域技术人员通常可以采用多种第二代测序平台进行高通量测序: 包括 但不限于 illumina公司的 Hiseq2000, 454 FLX( oche公司)、 Solexa Genome Analyzer和 Applied Biosystems公司的 SOLID等。 这些平台共同的特点是极高的 测序通量, 相对于传统测序的 96道毛细管测序, 高通量测序一次实验可以读取 40万到 400万条序列, 根据平台的不同, 读取长度从 25bp到 450bp不等, 因此不 同的测序平台在一次实验中,可以读取 1G到 14G不等的碱基数。其中, Solexa 高 通量测序包括 DNA簇形成和上机测序两个步骤: PCR扩增产物的混合物与固相载 体上固定的测序探针进行杂交, 并进行固相桥式 PCR扩增, 形成测序簇; 对所述测 序簇用 "边合成 -边测序法"进行测序。
DNA簇的形成是使用表面连有一层单链引物 (primer)的测序芯片 (flow cell), 单链状态的 DNA片段通过接头序列与芯片表面的引物通过碱基互补配对的原 理被固定在芯片的表面, 通过扩增反应, 固定的单链 DNA变为双链 DNA, 双链 再次变性成为单链, 其一端锚定在测序芯片上, 另一端随机和附近的另一个引 物互补从而被锚定, 形成"桥"; 在测序芯片上同时有上千万个 DNA单分子发生 以上的反应; 形成的单链桥, 以周围的引物为扩增引物, 在扩增芯片的表面再 次扩增, 形成双链, 双链经变性成单链, 再次成为桥, 称为下一轮扩增的模板 继续扩增; 反复进行了 30轮扩增后, 每个单分子得到 1000倍扩增, 称为单克隆 的 DNA簇。
DNA簇在 Solexa测序仪上进行边合成边测序, 测序反应中, 四种碱基分别 标记不同的荧光,每个碱基末端被保护碱基封闭,单次反应只能加入一个碱基, 经过扫描, 读取该次反应的颜色后, 该保护集团被除去, 下一个反应可以继续 进行, 如此反复, 即得到碱基的精确序列。 在 Solexa多重测序 (Multiplexed Sequencing)过程中会使用 INDEX (标签 或 BARCODE)来区分样品, 并在常规 测序完成后, 针对 INDEX部分额外进行 7个循环的测序, 通过 INDEX的识别, 最多可以在 1条测序甬道中区分 12种不同的样品。
检测方法
本发明还提供了一种检测病毒在宿主细胞中基因整合方式的方法, 以 HBV 病毒为例, 在本发明的一个优选例中, 所述方法主要包括以下步骤: 1. HBV探针制作
对 HBV全长基因组进行 PCR, 其中 PCR过程中引入标记的 dNTP;
在一个优选例中, 所述标记 dNTP为 biotin-dNTP, biotin-dNTP与普通 dNTP 的比例是 1 : 4, 总浓度为 2.5mM;
2. 对 HBV全长产物进行纯化
在一个优选例中, 采用 1.5倍体积 AMPURE磁珠纯化, 后采用 QIAGEN的 250MinElute PCR Purification Kit纯化;
3. 对 HBV全长产物进行片段化
将 DNA转移至缓冲液中, 进行超声片段化;
4. 探针与样本杂交
样品来源可为 HBV患者或肝癌患者的血浆或组织, 根据 Illumina公司 Paired-End Sample Preparation Guide (操作流程)进行文库构建; 将探针与文库 混合, 捕获特异性的样本核酸片段。
5. 洗脱
将捕获到的核酸片段洗脱下来;
6. 将上述洗脱下来的 DNA进行 PCR扩增和纯化、 定量;
7. 上机测序系统及信息分析
将上述纯化后的 PCR产物经进行高通量测序, 测序平台采用 ILLUMINA公 司的 HISEQ2000, 对于测序长度不限于 PEI101 , 也可采用 PEI91。 将下机数据除 去重复和被接头污染的 reads,统计下机数据的基本信息(例如: 文库长度; reads 长度; reads条数; 碱基数; 重复率等) ; 分别截取 PE的两条 reads 前面的 50bp 碱基, 形成一对长为 50bp新 reads, B PE50readsc 将新的 PE50 reads运用 soap比 对软件( -r 1 -V 2 )分别与人的参考序列(hgl9)和 HBV各种参考序列进行比对, 从比对结果中挑选出一条 read比到人的参考序列并且另一条比对到 HBV参考序 列的一对 reads; 这样的 reads很有可能跨过 HBV插入的位点; 统计这部分 reads 比对信息, 找到在人类基因组的插入热点区域。 图 5显示了本发明检测 HBV在 待测样本基因整合方式的信息分析流程。
试剂盒
本发明还提供了一种用于检测病毒在宿主中整合方式的试剂盒, 主要包括以 下组分:
( 1 )第一容器以及位于容器内的检测芯片或探针集;
(2)第二容器以及位于容器内的用于构建样本文库的接头;
(3)第三容器以及位于容器内的接头封闭分子;
(4)第四容器以及位于容器内的标签封闭分子;
(5)检测说明书。
在另一优选例中, 所述试剂盒还包括选自下组的试剂:
用于进行 PCR扩增所需的试剂、 用于进行封闭反应所需的试剂、 用于进行 杂交反应所需的试剂、 用于进行测序反应所需的试剂、 或其组合。
本发明的主要优点包括:
1. 采用本发明制备探针所需的价格仅为现有技术制备价格的 1%-5%;
2. 本发明在制作探针过程中形式灵活, 可以按自身需要设计, 比例加入 HBV种类以及数量;
3. 制作探针过程简单, 通过 PCR以及打断纯化即可得到探针, 无须合成;
4. 本发明的探针为双链探针, 能够降低因 PCR 原因引起的信息错误及丢 失;
5. 本发明的检测方法可以与建库技术联合使用起到富集 HBV片段的作用; 具有广泛的适用性。 下面结合具体实施例, 进一步阐述本发明。 应理解, 这些实施例仅用于说 明本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方 法,通常按照常规条件如 Sambrook等人,分子克隆:实验室手册 (New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件, 或按照制造厂商所建议的 条件。
仪器和试剂
本方面用到的仪器和试剂见表 1。
Figure imgf000014_0001
Cycler/9700
Agilent 2100 2100 Bioanalyzer Agilent
NanoDrop 1000 Spectrophotometer Thermo Fisher Scientific 电泳仪 DYY-6C 北京六一仪器厂 核酸浓缩仪 5305 Eppendorf 凝胶成像系统 Tanon 上海天能科技有限公司
Covaris打碎仪 S-2 Covaris
Thermomixer Thermomixer comfort Eppendorf
Dynabeads M-280
Invitrogen
Strep tavidin磁珠
MinElute PC
28004 QIAGEN
Purification Kit
实施例 1 样本文库制备
1. 样本来源
样品的来源为同一患者的肝癌组织, 且此患者肝癌组织有全基因组测序信 息。
2. 样本文库制备
文库构建按照 Illumina公司的标准文库制备流程说明书(Paired-End Sample Preparation Guide) 进行构建, 具体方法如下:
采用 Covaris s2打断基因组 DNA, 末端补平修复, 末端加 A, 加入接头, 建 库过程所用的接头为:
SEQ ID NO: l 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' ;
SEQ ID NO:2 5,-TACACTCTTTCCCTACACGACGCTCTTCCGATCT-3,。 对加入接头的片段进行 PCR, 得到样本文库, 所构建的文库带有 INDEX标 签序列。 INDEX序列如下:
SEQ ID NO: 3
ACGTGTGCTCTTCCGATCT-3, SEQ ID NO: 4
5'-CAAGCAG
ACGTGTGCTCTTCCGATCT-3' c
将样本分别做成两种片段大小文库, 对构建文库片段进行片段大小检; 主带为 170bp左右和 800bp左右。 实施例 2 制备 HBV探针
1. 引物设计
本实施例中, 所设计的引物为:
PI ( SEQ ID NO:5 ) : TTTTTCACCTCTGCCTAATCA
P2 ( SEQ ID NO:6 ) : AAAAAGTTGCATGGTGCTGG
2. PCR反应体系
PCR反应体系见表 2。
表 2
LA Taq 0.25μ1
10 X LA buffer ( Mg+2.00mM ) 2.5μ1
dNTP (2.5mM) 4μ1
PI ( ΙΟμΜ) 1.5μ1
P2 ( ΙΟμΜ) 1.5μ1
HBV病毒基因组 (lng/μΐ) 3μ1
H2O 12.25μ1
总体积 25 μΐ
(注: dNTP中 biotin-dNTP与普通 dNTP的比例是 1 : 4, 总浓度为 2.5mM) 3. PCR反应条件
PCR反应在 AB-9700PCR仪上进行, 反应程序见表 3。
表 3
温度 时间 循环数
94 °C 3min 1
94 °C 30s 35 56 °C 50s
68 °C 150s
72 °C lOmin
4°C 保持
4. PCR纯化及电泳检测
反应结束后, 采用 1%琼脂糖凝胶电泳检测 PCR产物, 并用 1.2-1.5倍体积 AMPURE BEADS纯化,采用 80ul水溶解。然后采用 250MinElute PCR Purification Kit纯化, 采用 60μ1水溶解。 1%琼脂糖凝胶电泳检测 PCR产物是否已经纯化干净 检测结果见图 1。
结果表明, 扩增并纯化出了大小约 3.2K的 HBV的片段。
5. PCR产物片段化
纯化完成后,将 DNA全部转移至 Covaris打断小管并补加 TE缓冲液至总体积 为 8(^l(Nanodrop检测其总量为 5 g), Covaris S2仪器 (基因有限公司) 打断, 打断条件见表 4。
表 4
Figure imgf000017_0001
6. 片段化产物电泳检测
2%琼脂糖凝胶电泳检测片段的大小, 结果表明, 片段主带在 250-300bp (图
2) 。 所获得的片段化的产物即可用作杂交的探针。
7. 探针保存
使用 MinElute PCR Purification Kit纯化片段产物, 溶于 40μ1缓冲液中, 用 Nanodrop仪检测探针 DNA的浓度, 使得探针的浓度为 120ι¾/μ1左右。 得到的探 针可以保存在 -20°C或 -80°C。
实施例 3 HBV探针与样本文库进行杂交
1. 探针变性
探针使用前必须 95°C变性 10分钟, 然后迅速放于冰上冷却形成单链。 2. 选用已确定的整合文库, 文库用量为 l g, 探针用量为 600ng (Nanodrop 定量) , 加入接头封闭分子, 接头封闭分子的量与文库量的比值为 lnmol: 1μ§, 标签封闭分子与文库量的比值为 lnmol: ^g。
接头封闭分子序列为:
SEQ ID NO: 7
5'-AATGATA(
GCTCTTCCGATCT-3'
标签封闭分子序列为
SEQ ID NO: 8
S'-AAGCAGA,
CGTGTGCTCTTCCGATCT-3';
SEQ ID NO: 9
5'-AAGCAGA,
CGTGTGCTCTTCCGATCT-3'。
在一个 1.5mL的 EP管中加入 l g的待杂交文库, lnmol接头封闭分子, lnmol 标签封闭分子, 5 g Cot DNA。 盖好管盖, 用干净的 50ml注射器针在分装的 EP 管盖上戳一个孔, 然后置于 60°C旋蒸仪中蒸干。 使用新的离心管管盖替换戳孔 的管盖, 并做好标记。 EP管中分别加入 EZ杂交系统中的两种试剂: 2 X SC Hybridiation Buffer杂交缓冲液 7.5 μΐ和 1 X SC Hybridiation Component A 3 μ1,然 后 95°C变性 10分钟, 在上述杂交混合物加入自制探针 600ng, 探针体积共 5μ1。 震荡混匀后置于离心机上全速离心 10秒, 并将样品全部转移到 200μ1 ΡΟ小管 中。
杂交混合物中含有的成分见表 5。 文库蒸干混合物 l g (文库) +5 g (Cot DNA) + 2nmol封闭分子
2Χ SC Hybridiation Buffer 7.5μ1
SC Hybridiation Component A 3μ1
探针 600ng
总体积 15μ1 将 200μ1ΡΟ小管放置于 PCR仪上, 47°C条件下杂交 24h。 实施例 4 杂交后洗脱
1. 准备链霉亲和素磁珠 (Invitrogen M280)
提前从冰箱中拿出链霉亲和素磁珠; 漩涡震荡磁珠 lmin, 使其充分混匀; 在 1.5mL的 EP管中加入 ΙΟΟμΙ磁珠; 将 ΕΡ管置于磁力架上至液体澄清, 用移液器 小心地去除上清; 保持 ΕΡ管在磁力架上, 加入 200 μΐ (2倍体积) 的结合缓冲液 (购于 Agilent公司) ; 从磁力架上取下 EP管, 漩涡震荡 10s, 使其混匀; 将 EP 管重新放回磁力架至液体澄清, 用移液器小心地去除上清; 重复清洗两次; 用 100 μΐ 的 Agilent 结合缓冲液 r悬浮磁珠; 将其转入 0.2 ml的小管中; 用磁力架 结合磁珠 (将小管靠到磁力架上) , 直到液体澄清, 用移液器小心去除上清; 现在磁珠可以用来结合捕获的 DNA了。
2. 将捕获到的 DNA结合到链霉素磁珠上
将杂交混合物吸出来 , 加到上步准备好的磁珠中; 用移液器吹打 10次混 匀; 将小管放在 PCR仪上 47°C孵育 45 min (每隔 15 min拿出来漩涡震荡 3 s以防 止磁珠沉淀); 孵育 45 min后, 将混合物从 0.2mL的小管中转入 1.5 mL的 EP管中。
3. 洗涤结合了捕获 DNA的链霉亲和素磁珠
1 ) 将 EP管置于磁力架上至液体澄清, 用移液器小心地去除上清;
2 ) 力口 100 L 预热到 47°C的 I X清洗缓冲液 I;
3 ) 漩涡震荡 10 s, 使其混匀;
4 ) 将 EP管置于磁力架上至液体澄清, 用移液器小心地去除上清;
5) 从磁力架上取下 EP管, 加入 200 μ1 预热到 47°C的 1 X严谨清洗缓冲液, 用移液器吹打混匀 10次(该步操作应迅速以尽量使管中液体温度不低于 47°C );
6 ) 47°C孵育 5 min;
7 ) 重复步骤 5 ) -7) , 总共用 I X严谨清洗缓冲液洗两次;
8 ) 将 EP管置于磁力架上至液体澄清, 用移液器小心地去除上清;
9 ) 力 P200 L室温下放置的 I X清洗缓冲液 I (不用 47°C预热的) , 漩涡震荡 2 min, 使其混匀;
10) 将 EP管置于磁力架上至液体澄清, 用移液器小心地去除上清; 1 1 )力 P200 室温下放置的 l X Wash Buffer ll, 漩涡震荡 1 min, 使其混匀;
12) 将 EP管置于磁力架上至液体澄清, 用移液器小心的去除上清;
13 ) 力 P200 室温下放置的 I X Wash Buffer III, 漩涡震荡 30 s, 使其混匀;
14) 将 EP管置于磁力架上至液体澄清, 用移液器小心地去除上清;
15) 从磁力架上取下 EP管, 加入 76μ1超纯水 (不用将 DNA从磁珠上洗脱下 来, 可以直接进行 PCR, 取样 35μ1进行后面的 PCR反应) 。 实施例 5 PCR反应
预先从 -20°C保存的试剂盒中取出 PFX聚合酶 (购于 Invirtogen公司), PFX 反 应缓冲液(10 X ), dNTP(10mM) o 引物序列为:
PCR Flowcell-Primer F ( 1 Opm/μΐ)
SEQ ID NO: 10 AATGATACGGCGACCACCGAGATC:。
PCR Flowcell-Primer (lOpm/μΙ)
SEQ ID NO: 11 CAAGCAGAAGACGGCATACGA
在 PCR小管上, 每孔按照表格 6配置 PCR反应体系。
表 6
DNA 35μ1
PFX酶 Ιμΐ
PFX 反应缓冲液 (10*) 5μ1
MgSO4 2μ1
dNTP(lOmM) 2μ1
Flowcell-primer-F 1 (lOpm/μΙ) 2.5μ1
Flowcell-primer- 1 (lOpm/μΙ) 2.5ul
总体积 50ul
反应条件见表 7。
表 7
温度 时间 循环
94 °C 2min 1
94 °C 15s 14 58 °C 30s
72 °C 30s
72 °C 5min 1
4°C 保持
PCR结束后, 每个样品都用 1.5倍体积的 Ampme Beads纯化, 回收的 PCR产 物溶于 30μ1超纯水中, Nanodrop 1000测浓度。 实施例 6 PCR产物上机测序
上述纯化后的 PCR产物经 2100 Bioanalyzer (Agilent)确定大小及插入片段大小 见图 3和图 4, 纯化产物大小分别为 271bp和 876bp, QPCR精确定量后上机测序。 在本实施例中,上机测序按照 Illumina/Solexa官方公布的 c-Bot和 HISEQ2000Hiseq 2000说明书进行操作。 实施例 7信息分析
将下机数据除去重复和被接头污染的 reads, 统计下机数据的基本信息 (文 库长度; reads长度; reads条数; 碱基数; 重复率等) ; 分别截取 PE的两条 reads 前面的 50bp碱基, 形成一对长为 50bp新 reads, B PE50reads c 将新的 PE50 reads 运用 soap比对软件 ( -r 1 -V 2 )分别与人的参考序列 (hgl9)和 HBV各种参考序 列进行比对, 从比对结果中挑选出一条 read比到人的参考序列并且另一条比对 到 HBV参考序列的一对 reads; 这样的 reads很有可能跨过 HBV插入的位点; 统计 这部分 reads比对信息, 找到在人类基因组的插入热点区域。
杂交结果见表 8。
表 8
Figure imgf000021_0001
Chrl9:30315005 , Chrl9:30315005 ,
Chrl9:30314586-30315653 段点位置 30315366 303153661
Chrl4:96085100-96085700 Chrl4:96085140 Chrl4:96085140
表 8的结果为采用上机数据得到结果, 样本 L-170, L-800 , Genome均来自 同一肝癌样本, L- 170为插入片段 170bp, L-800为 800bp文库, genome为全基因 组测序。 从表 8中可以得出自制探针对于捕获基因片段的准确性, 以及片段长 度的影响。 通过本发明的方法完全可以得到稳定以及可靠的位点, 且所需数据 量仅为全基因组测序数据的 1%左右。 实施例 8 制备试剂盒
一种可用于检测病毒在待测样本中整合方式的试剂盒, 包括以下
(1)第一容器以及位于容器内核酸芯片;
(2)第二容器以及位于容器内的用于构建样本文库的接头;
(3)第三容器以及位于容器内的接头封闭分子;
(4)第四容器以及位于容器内的标签封闭分子;
(5)第五容器以及位于容器内的用于进行 PCR扩增所需的试剂;
(6)第六容器以及位于容器内的用于进行封闭反应所需的试剂;
(7)第七容器以及位于容器内的用于进行杂交反应所需的试剂;
(8)第八容器以及位于容器内的用于进行测序反应所需的试剂。
(9)检测说明书。
在本发明提及的所有文献都在本申请中引用作为参考, 就如同每一篇文献 被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后, 本领域技术人员可以对本发明作各种改动或修改, 这些等价形式同样落于本申 请所附权利要求书所限定的范围。

Claims

1. 一种核酸探针集, 包括多个探针, 其特征在于, 所述探针集具有以下特征:
(1)每个探针上具有 1个或多个生物素标记的 dNTP; 和 /或
(2)生物素标记的 dNTP在探针集中的丰度为 1 :6-1 :2; 和 /或
(3所述探针集的全部核酸序列覆盖对应病毒基因组序列的 70%-100%; 优选地, 所述探针集具有 1-20000个核酸探针; 较佳地, 所述探针集具有 1000-5000个核酸探针; 更佳地, 所述探针集具有 2500个核酸探针;
优选地, 所述生物素标记的 dNTP在所述探针集中的丰度为 1 :4;
优选地, 所述探针之间具有部分重叠;
优选地, 所述探针长度为 100-500bp; 较佳地, 所述探针长度为 200-300bp; 更 佳地, 所述探针长度为 250bp;
优选地, 所述探针是以病毒基因组作为模板, PCR法扩增获得, 较佳地, 所 述扩增模板为为乙型肝炎病毒 (HBV) 基因组、 丙型肝炎病毒 (HCV) 基因组、 艾滋病病毒 (HIV) 基因组、 乳头瘤病毒 (HPV) 基因组, 或其组合; 更佳地所述 样本为 B型 HB V基因组和 /或 C型 HB V基因组。
2. —种表面固定有权利要求 1所述探针集的核酸芯片。
3. 权利要求 1所述核酸探针集或权利要求 2所述核酸芯片的用途,其特征在于, 用于检测病毒在待样本中的整合方式; 较佳地, 所述的整合方式选自下组: 重排、 异位、 插入、 替换, 或其组合。
4. 一种制备权利要求 1所述核酸探针的方法, 其特征在于, 包括步骤:
(a) 获得探针来源样本;
(b) 对步骤 (a) 获得的样本进行 PCR扩增, PCR扩增体系的 dNTP为生物素 标记的 dNTP, 获得带有生物素标记的 PCR扩增产物;
(c)对步骤(b) 获得的生物素标记的 PCR扩增产物进行打断, 得到片段化的 生物素标记的 PCR扩增产物, 即为探针;
较佳地, 步骤 (a) 所述样本具有以下特征:
样本为含有核酸的病毒样本; 和 /或
所述样本为病毒粒子、 血清、 血液、 组织样本、 脱落细胞, 上皮细胞, 或其 组合; 和 /或
所述样本选自下组: 乙型肝炎病毒 (HBV) 、 丙型肝炎病毒 (HCV) 、 艾滋 病病毒 (HIV) 、 乳头瘤病毒 (HPV) , 或其组合; 和 /或
所述样本为 B型 HBV和 /或 C型 HBV;
较佳地, 步骤 (b) 具有以下特征:
步骤 (b) 所述的扩增为对样本中病毒 DNA全长进行扩增; 和 /或
步骤 (b) 所述标记的 dNTP为 biotin-dNTP, 且所述标记的 dNTP能够与链霉素 亲和磁珠结合; 和 /或
步骤(b)所述标记的 dNTP与非标记 dNTP的比例为 1 : 2-8; 优选比例为 1 : 3-6; 更优选比例为 1 : 4;
较佳地, 步骤 (c) 所述打断为超声法打断。
5. 如权利要求 1所述的方法, 其特征在于, 还包括步骤 (d) :
对步骤 (c) 获得的探针进行纯化和 /或定量。
6. 如权利要求 1所述的方法, 其特征在于, 所述探针的长度为 100-500bp; 较 佳地, 所述探针的长度为 200-300bp, 更佳地, 所述探针的长度为 250bp。
7.一种检测病毒在待测样本中基因整合方式的方法, 其特征在于, 包括步骤:
(i) 获得待测样本;
(ii) 对步骤 (i) 获得的样本进行文库构建;
(iii)将权利要求 1所述的探针集包括的探针与步骤(ii)获得的文库进行杂交, 捕获与病毒基因整合有关的核酸序列;
(iv) 对步骤 (iii) 捕获的核酸序列进行扩增, 获得与病毒整合有关的扩增产 物;
( V ) 对步骤 (iv ) 获得的扩增产物进行测序, 获得与病毒整合方式有关核酸
I Ή自、 .;
较佳地, 步骤 (i) 具有以下特征:
所述待测样本为组织、 血液、 脱落细胞, 上皮细胞; 和 /或
所述待测样本来源于人或非人哺乳动物, 较佳地来源于人; 和 /或
所述待测样本来源于 HBV感染者或肝癌患者;
优选地, 步骤 (iii) 具有以下特征: 所述探针为变性的单链 DNA; 和 /或
在杂交液中加入接头封闭分子和标签封闭分子; 和 /或
所述接头封闭分子的序列如 SEQ ID NO:7所示; 和 /或
所述标签封闭分子的序列如 SEQ ID NO:8和 SEQ ID NO:9所示。
8. 如权利要求 7所述的方法, 其特征在于, 在步骤 (V ) 中, 将所述的扩增产 物与固相载体上固定的测序探针进行杂交, 进行固相桥式 PCR扩增, 形成测序簇; 然后对所述测序簇用 "边合成-边测序"法进行测序, 从而得到与病毒整合方式有关 核酸信息。
9. 如权利要求 7所述的方法, 其特征在于, 在步骤 (ii) 中, 所述的文库构建 为: 对打断的基因组 DNA进行末端修复, 加入接头, 对具有接头的片段进行扩增, 获得的带有接头的扩增混合物即为样本文库;
较佳地, 所述的接头具有如 SEQ ID NO:l和 SEQ ID NO:2所示的序列; 和 /或 所构建的文库具有如 SEQ ID:3和 SEQ ID:4所示的标签序列。
10. 一种可用于权利要求 7-9任一所述方法的试剂盒, 其特征在于, 所述试剂 盒包括:
(1)第一容器以及位于容器内权利要求 2所述的核酸芯片, 或权利要求 1所述 的探针;
(2)第二容器以及位于容器内的用于构建样本文库的接头;
(3)第三容器以及位于容器内的接头封闭分子;
(4)第四容器以及位于容器内的标签封闭分子;
(5)检测说明书;
较佳地, 所述试剂盒还包括选自下组的试剂:
用于进行 PCR扩增所需的试剂、
用于进行封闭反应所需的试剂、
用于进行杂交反应所需的试剂、
用于进行测序反应所需的试剂、
或其组合。
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