WO2016082129A1 - 一种核酸的双接头单链环状文库的构建方法和试剂 - Google Patents

一种核酸的双接头单链环状文库的构建方法和试剂 Download PDF

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WO2016082129A1
WO2016082129A1 PCT/CN2014/092296 CN2014092296W WO2016082129A1 WO 2016082129 A1 WO2016082129 A1 WO 2016082129A1 CN 2014092296 W CN2014092296 W CN 2014092296W WO 2016082129 A1 WO2016082129 A1 WO 2016082129A1
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nucleic acid
sequence
acid molecule
linker sequence
linker
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PCT/CN2014/092296
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English (en)
French (fr)
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江媛
李巧玲
阿莱克谢耶夫•安德烈
贺罗维茨•埃文
赵霞
王童
董超
李栋
德马纳克•拉多杰
章文蔚
蒋慧
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深圳华大基因研究院
深圳华大基因科技有限公司
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Priority to PCT/CN2014/092296 priority Critical patent/WO2016082129A1/zh
Priority to US15/529,881 priority patent/US10479991B2/en
Priority to CN201480082967.7A priority patent/CN107002292B/zh
Publication of WO2016082129A1 publication Critical patent/WO2016082129A1/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
    • 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
    • C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C40B50/18—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the invention relates to the technical field of molecular biology, in particular to a method and a reagent for constructing a double-linker single-chain circular library of nucleic acids.
  • high-throughput sequencing is one of the important research methods in various fields such as molecular biology research and medical diagnosis. Since the birth of high-throughput second-generation sequencing technology, sequencing technology has grown by leaps and bounds, and the cost of sequencing for second-generation sequencing has been reduced by several orders of magnitude compared to previous expensive sequencing costs; shorten. The emergence of third-generation sequencers now makes the competition in the sequencing market more intense and more rapid. Therefore, each sequencing company and research team must reduce the cost of sequencing, shorten the process time and improve the accuracy of the results, and then have the opportunity to continue to develop in the competition.
  • CG Complete Genomics
  • Detection and sequencing of the individual genome provide accurate genetic information.
  • the library construction process of the CG platform takes too long, the cost of building a library is high, and the insertion of the library is short, which limits the output and analysis of subsequent data, which not only affects the development of scientific research projects, but also is not conducive to the large-scale CG platform.
  • the CG platform must optimize its database building process, shorten the time to build the library, increase the length of the library and reduce the cost to maintain the advantage in the current fierce competition.
  • the database construction process mainly includes: enzymatic cleavage of cyclized DNA, removal of phosphorylation, and end-repair, 3 12 steps of 'end-join connection, 5'-end linker connection, nick translation, polymer chain reaction, single-strand separation and cyclization, and each step of the enzymatic reaction is purified by magnetic beads.
  • the process has a long time and high cost, and the library insert generated by the enzyme digestion is only 26 bp, which does not utilize the large-scale application of the CG platform, nor does it meet the requirements of the CG next-generation sequencing platform.
  • the invention provides a method and a reagent for constructing a double-linker single-chain circular library of nucleic acid, which can increase the length of a library insert, simplify the process of building a library, shorten the time of building a library, and reduce the cost of building a library.
  • the present invention provides a method for constructing a double-linker single-stranded circular library of nucleic acid, comprising the steps of:
  • a first product having a first linker sequence at both ends is obtained by first PCR amplification, wherein the first PCR uses a primer sequence having a U base site with or without a nickase recognition sequence, and one of the primer sequences is Having a first affinity tag;
  • the first product is digested with the USER enzyme to produce sticky ends with or without gaps;
  • a second product having a second linker sequence at both ends is obtained by second PCR amplification
  • the second product is denatured to obtain a single-stranded nucleic acid molecule, and the single-stranded nucleic acid molecule is cyclized using a mediated sequence complementary to both ends of one of the single-stranded nucleic acid molecules to obtain a double-stranded single-stranded circular library.
  • the first affinity tag is a biotin tag
  • the second affinity tag is a streptavidin tag
  • the first linker sequence comprises a first 5' linker sequence and a first 3'L type linker sequence, respectively connecting the 3' end and the 5' end of each of the fragments;
  • the first 5' linker sequence comprises a long strand of phosphorylation at the 5' end and a complementary short strand, the 3' end of the short strand is dideoxy modified, and the short strand contains a U base site;
  • the first 3 'L linker sequence is adjacent to the ligated fragment a portion that is partially complementary to the first 5' linker sequence;
  • the first linker sequence is ligated to both ends of the nucleic acid fragment, and specifically includes:
  • the short base U base site of the first 5' linker sequence is digested with the USER enzyme
  • the first 3' L-type linker sequence is ligated to the 5' end of each strand of the phosphorylated nucleic acid fragment.
  • the first PCR uses a primer sequence having a U base site and a nickase recognition sequence; after cleavage of the U base site by USER, a sticky end is formed at both ends of the nucleic acid fragment.
  • the cohesive ends are cyclized to produce a circular nucleic acid molecule; the nicking enzyme nicking sequence is used to generate a nick.
  • one primer sequence used in the first PCR has two U base sites, and the other primer has a U base site; after the U base site is digested with USER enzyme A sticky end is formed at both ends of the nucleic acid fragment, and the sticky end is complementary to cyclization to produce a circular nucleic acid molecule.
  • the method further comprises: digesting the uncircularized nucleic acid molecule.
  • the length of the generated nick translation fragment is controlled by controlling at least one of a molar ratio of the dNTP to the nucleic acid molecule as a template, an enzyme reaction temperature, and time.
  • the portion of the circular nucleic acid molecule that does not undergo a restriction nick translation reaction is digested and removed, specifically comprising: first degrading using a double-stranded exonuclease until the gaps at both ends meet; then using a single-strand exonuclease Degrading a single strand; or directly excising a portion of a circular nucleic acid molecule that does not undergo a restriction nick translation reaction using an endonuclease.
  • the second linker sequence is a buccal linker sequence
  • the buccal linker sequence comprises a base sequence in which two end portions are complementary paired but the middle segment is not complementary paired, wherein the middle portion forms a bubbling shape, and the middle portion a U-base site; a 5' end of a chain of bubbling linker sequences has a prominent T base;
  • Connecting the second linker sequence at both ends of the linear nucleic acid molecule specifically includes:
  • the buccal linker sequence is ligated to both ends of the linear nucleic acid molecule by pairing the T base with the A base;
  • the U base site on the middle segment was digested with the USER enzyme.
  • the method further comprises: digesting the uncircularized single-stranded nucleic acid molecule.
  • the invention provides a construction reagent for a double linker single-stranded circular library of nucleic acids comprising the following components:
  • first linker sequence comprising a first 5' linker sequence and a first 3'L type linker sequence, respectively ligated to the 3' end and the 5' end of each of the strands;
  • the first 5' linker sequence comprises a 5 a 'terminal phosphorylated long chain and a complementary short chain, the short chain 3' terminal dideoxy modification, and the short chain contains a U base site;
  • the first 3 'L type linker sequence is adjacent to the joined fragment portion Partially complementary to the first 5' linker sequence;
  • a first PCR primer having a U base site with or without a nickase recognition sequence, and one of the primer sequences having a first affinity tag for obtaining a first linker sequence at both ends by first PCR amplification First product
  • a cyclase for cyclizing the digested first product to produce a circular nucleic acid molecule
  • Dephosphorylation enzyme for dephosphorylation of circular nucleic acid molecules with gaps on both strands; or nickase for nicking enzyme recognition sequences on one strand and gap or double on the other strand
  • a circular nucleic acid molecule having a nicking enzyme recognition sequence and having no nicks in the chain is subjected to enzymatic cleavage to generate a nick;
  • a component of the nick translation reaction for performing a restriction nick translation reaction from a nick and/or a gap using a circular nucleic acid molecule bound to a solid support as a template
  • a digestive enzyme for digesting and removing a portion of the circular nucleic acid molecule that does not undergo a restriction nick translation reaction, to obtain a linear nucleic acid molecule
  • the second linker sequence is a buccal linker sequence
  • the buccal linker sequence comprises a base sequence in which two end portions are complementary paired but the middle segment is not complementary paired, wherein the middle segment forms a bubbling shape, and the middle segment band There is a U base site; the 5' end of one strand of the buccal linker sequence has a prominent T base;
  • a second PCR primer for obtaining a second product having a second linker sequence at both ends by second PCR amplification
  • the mediated sequence is complementary to a single-stranded nucleic acid molecule in the single-stranded nucleic acid molecule after denaturation of the second product, and is used for cyclization of the single-stranded nucleic acid molecule to obtain a double-stranded single-stranded circular library.
  • the first affinity tag is a biotin tag; the second affinity tag is a chain Mold avidin label.
  • the method for constructing the double-linker single-stranded circular library of the nucleic acid of the present invention is combined with the enzymatic reaction carried out on the magnetic beads by a restriction nick translation reaction, and the restriction nick translation reaction replaces the III in the conventional method with a new nicking enzyme site.
  • FIG. 1 is a flow chart showing a process of binding from a magnetic bead to a single-stranded ring in a method for constructing a double-linker single-stranded circular library of nucleic acid according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram showing the basic principle of CNT slit creation according to an embodiment of the present invention
  • FIG. 3 is a basic schematic diagram of CNT notch generation according to an embodiment of the present invention.
  • Figure 5 is a comparison diagram of the conventional directional joint connection method and the L-type joint connection method of the present invention.
  • Figure 6 is a result of electrophoresis detection of the final product of four parallel experiments in one embodiment of the present invention, wherein M represents DNA Marker; 1, 2, 3, and 4 represent electrophoresis results of four parallel samples C22, D22, E22, and F22, respectively.
  • the first affinity tag and the second affinity tag may be a component of a biologically common biological binding reaction, such as an antigen or an antibody, a strand of a short-stranded DNA fragment, biotin or streptavidin Harmony, and so on.
  • the second affinity tag is selected An antibody that binds to the antigen, and vice versa; in the case where the first affinity tag selects one strand of a double-stranded DNA short segment, the second affinity tag selects another strand that is complementary to the strand, and vice versa
  • biotin is selected for the first affinity tag
  • the second affinity tag is selected from biotin-bound streptavidin and vice versa.
  • the first affinity tag is a biotin tag and the second affinity tag is a streptavidin tag, both of which have a strong binding capacity.
  • a method for constructing a double-linker single-stranded circular library of a nucleic acid includes the steps of: breaking genomic DNA to form a nucleic acid fragment for library construction; performing dephosphorylation and terminal repair reaction; 'Terminal A sequence; USER enzyme digestion and phosphorylation; ligation of 3'L type linker A sequence; PCR amplification of products with 5' linker A sequence and 3'L type linker A sequence at both ends, of which PCR
  • the primer sequence has a U base site and a nickase recognition sequence, and one primer has a biotin label; the USER enzyme is used to cleave the U base site to generate a sticky end, and the product of the USER enzyme is cyclized.
  • a circular nucleic acid molecule binding the circular nucleic acid molecule to streptavidin-labeled magnetic beads; using a nicking enzyme to cleave the incision at the nickase recognition sequence; and performing a restriction nick translation reaction from the incision (Controlled Nick Translation, CNT); use an endonuclease to cleave the nucleic acid strand at the nick (also first degraded with a double-stranded exonuclease until the gaps at both ends meet, then use a single-strand exonuclease Single-stranded), obtaining a linear nucleic acid molecule; performing terminal repair on the linear nucleic acid molecule and reacting with the 3' end plus A base; connecting the buccal linker sequence; using the USER enzyme to digest the U base site on the buccal linker sequence, Forming an L-shaped linker; PCR amplification results in a product having a different sequence at both ends; denaturation treatment results in a single-strande
  • the U-base site and the nickase recognition sequence are introduced on the primer sequence used in the first PCR, and the principle shown in FIG. 2 is employed.
  • An incision is made as a starting point for a restrictive nick translation reaction.
  • the existing method comprises introducing a class III endonuclease recognition sequence into a linker sequence, and after the linker sequence is ligated and cyclized, the double-stranded DNA is linearized by class III endonuclease digestion; and the method of the invention is The U-base site and the nickase recognition sequence were introduced into the primer sequence used in one PCR.
  • the U-base site was digested with USER to generate a sticky end, and double-stranded cyclization was performed, followed by nickase (for example, Nb.BsrDI, Nb.BsmI, Nt.BbvCI, Nb.Bbv.Nb.BtsI or Nt.BstNBI, etc.) cleaves a single strand of cyclized DNA, creating a nick on each single strand to provide an effective effect on CNTs. Starting site.
  • nickase For example, Nb.BsrDI, Nb.BsmI, Nt.BbvCI, Nb.Bbv.Nb.BtsI or Nt.BstNBI, etc.
  • a U base site is introduced into the primer of the first PCR, and The gap was generated by digestion with the USER enzyme as a starting point for the restriction nick translation reaction.
  • the basic principle of the generation of the gap is shown in Figure 3: (1) After the 5' linker A sequence and the 3'L type linker A sequence are ligated, the linker A is ligated with a primer with two U and one U, respectively; (2) The U base is digested with USER, and the phosphorylated 3' end and 5' end are formed at the incision; (3) the double-stranded cyclization is carried out by the sticky end produced by enzymatic cleavage, and the gap on one strand after cyclization (Gap 1, formed by USER digestion) is the 3' end and 5' end of the phosphorylation, and the gap on the other chain (Gap 2, which is formed by the absence of a matching base after cyclization) is dephosphorylation.
  • the reaction starting from the nick and/or the notch is called "restricted nick translation reaction” because factors such as the amount of dNTP in the reaction, the amount of the nucleic acid molecule as a template, the temperature and time of the enzyme reaction, and the like can be used.
  • Control the length of the target fragment generated by the reaction is controlled within a certain range, and the nucleic acid fragment of a certain length range is suitable for a specific sequencing platform.
  • the length of the target fragment in the present invention is controlled within a range of 50 to 250 bp. This length is several times longer than the length of the target segment obtained by the traditional CG sequencing platform.
  • the CNT technology of the present invention can control the library insert in a very concentrated range without performing the recovery of the gel, and effectively improves the operability of the nick translation reaction technique.
  • the existing method utilizes the cleavage property of class III endonuclease, and cleaves the genomic DNA at 25-27 bp on both sides of the linker A to form a DNA fragment of about 104 bp; the subsequent removal of more than 200 bp by the two-step magnetic bead purification method
  • the DNA fragment of the linker A at which time the digested product obtained by the selection of the magnetic bead fragment is further mixed with some non-target DNA fragments having a main band of 100-200 bp; after being ligated through the linker B, a primer is used with biotin-labeled
  • the primers of the bases are amplified and ligated to the DNA fragment of the linker B, wherein the single-stranded nucleic acid amplified by the primer with the biotin-labeled base is a non-target single-stranded nucleic acid; and the subsequent streptavidin magnetic
  • the method of the present invention on the one hand, binds a circular nucleic acid molecule to a streptavidin magnetic bead after cyclization with a biotin label carried on the first PCR primer, and the nucleic acid molecule of interest is always bound in the subsequent reaction.
  • the magnetic beads there is no need to add new magnetic beads after each step of reaction. Purification, only need to wash the reaction solution with washing reagent to carry out the next reaction, not only reduce the use of magnetic beads, but also save the experimental operation time; on the other hand, the incision enzyme is opened on the two links of the joint A An incision is then used to extend the nick from the junction A region to the junction A by using the nick translation function of the polymerase in the presence of dNTPs.
  • the incision is flexibly controlled by controlling the molar ratio of dNTP to template DNA, reaction temperature and reaction time.
  • the extension length, the size of the extension fragment can be controlled within the range of 50-250 bp, and then the non-target DNA fragment without the linker A is digested by one-step exonuclease digestion reaction, and the remaining DNA is the target DNA with the linker A.
  • Fragments, after PCR amplification of the linker B and without biotin-labeled primers, can be separated and cyclized by simple high-temperature denaturation, melting double-stranded DNA, and mediating the cyclization of the target single strand with a mediating sequence.
  • Target single-stranded DNA can be controlled by controlling the molar ratio of dNTP to template DNA, reaction temperature and reaction time.
  • the single-chain cyclization method of the present invention only needs thermal denaturation and mediated sequence hybridization to successfully separate and cyclize the single-stranded nucleic acid of interest, which is not only simple in steps, easy to operate, and does not require consumption of a large amount of expensive reagents, and the cost of building a library is obtained. reduce.
  • an L-joint connection is used in place of a conventional joint connection.
  • a comparison will be made between the conventional joint joining method and the joint joining method of the present invention.
  • the directional joint connection method adopted by the prior art method is to ensure the joint connection of the joints while minimizing the problem of interconnection between the DNA fragments, and the method of separately designing the 5' joint and the 3' joint by stepwise connection.
  • Each additional linker requires a linker sequence, a blocking sequence, and a primer sequence to perform.
  • the entire process requires dephosphorylation, terminal repair, addition of 5' linker, primer extension, addition of 3' linker, nick translation and ligation of the 6-step enzymatic reaction and 5 purification steps in order to add the sequence of linker A to the target DNA. Both ends.
  • the existing method steps are cumbersome, the cost of building the library (sequence cost, enzyme reagent cost, purification cost) is high, the cycle is long, and the sample loss is large, which is not in line with the requirements of efficient and simple library construction.
  • the L-joint connection method of the invention can improve the efficiency of building the library and reduce the cost of building the library under the premise of ensuring the directional connection of the joint.
  • the L-joint connection method also uses a step-by-step connection, the steps are simpler than the existing methods.
  • a 5' linker with a blocking sequence is added, wherein the blocking sequence is about 12 bp in length and is fully complementary to the 5' linker to form a partially complementary double-stranded structure such that the DNA fragment is ligated to the 5' linker. Since the 3' end of the blocking sequence has a dideoxy modification and the 5' end is a dephosphorylated base, both the 5' linker and the 3' end of the DNA fragment are directionally linked, and the closed sequence is not linked to the 5' end of the DNA fragment.
  • the U-base is located in the middle of the blocking sequence and is blocked by the USER enzyme.
  • the blocking sequence is "degraded" into two single-stranded DNA fragments of less than 8 bp and is uncoupled from the 5' linker. Then, an "L" type single-stranded 3' linker was added by post-hybridization ligation. It is also necessary to phosphorylate the 5' end of the DNA fragment to remove the cleavage before the L-type linker is added.
  • the experiment proves that The USER enzyme treatment can be carried out simultaneously with the phosphorylation reaction. After the reaction, the magnetic beads are purified, and the magnetic beads after washing are directly resuspended in the next step to the reaction buffer.
  • the design of the L-type adaptor is that the last 8 bases of the 3' end are complementary to the last 8 bases of the 5' end of the 5' linker, so that they can be directly hybridized to the 5' linker, and then the nickase is used to close the incision.
  • the L-type 3' linker was ligated to the 5' end of the DNA fragment. Since a part of the base of the L-type linker is complementary to a part of the base at the 5' end of the 5' linker, and the other bases are not complementary, they appear to be L-shaped, so they are called L-type linkers. After the reaction is completed, an appropriate amount of magnetic bead binding buffer is further added to the magnetic beads to purify and recover the ligation product of the added linker.
  • the whole process only needs to undergo dephosphorylation, terminal repair, 5' linker, USER digestion and phosphorylation one-step reaction, plus 5'L type link, 5 steps of enzyme reaction and 3 purification operations, which can make the joint faster.
  • the sequence orientation of A is added to both ends of the target DNA, the steps are simple, the cost of building the library is reduced, and the cycle is shortened.
  • the unique innovation of the invention lies in that the enzymatic reaction is carried out on the streptavidin magnetic beads, and the nucleic acid can be combined with the streptavidin magnetic beads without entrainment to carry out the enzymatic reaction; using a controllable gap
  • the translation reaction produces a nucleic acid duplex of a particular fragment length.
  • the circular nucleic acid molecule is bound by the biotin label and the streptavidin magnetic beads. After each step of the enzymatic reaction, the nucleic acid molecule is always bound to the magnetic beads, and only a simple washing step is needed in the middle to react the enzyme, Ions and the like are washed away, and the double-stranded nucleic acid-labeled nucleic acid is not eluted until the second PCR, the nucleic acid double strand is denatured into two single strands, and the single-strand collection without biotin labeling stand up.
  • the enzyme reaction is carried out in solution. After each step of the enzyme reaction, a new magnetic bead is added to the target fragment, the enzyme and buffer in the reaction are washed off, and the target fragment is washed. Take off and proceed to the next reaction.
  • the enzymatic reaction of the present invention is carried out on streptavidin magnetic beads, and only one magnetic bead needs to be added. After the double-stranded nucleic acid molecule is combined with the magnetic beads, it is not necessary to elute multiple times in the middle and repeatedly add new magnetic beads to recombine. As long as a simple washing step, the desired target fragment can be collected, and a lot of fragment purification is omitted.
  • magnetic beads are used as the solid phase carrier, but the solid phase carrier is not limited to the magnetic beads, and other solid phase carriers such as chips may be used as long as the streptavidin is fixed to the solid phase carrier.
  • the functions of the present invention can be implemented.
  • the circular DNA is carried out at a distance of 26 bp from the restriction site. After digestion, the circular DNA is transformed into two linear DNAs, and then the biotin label on the DNA is combined with the streptavidin magnetic beads to collect the target fragments.
  • the target fragment after digestion by this method is only 26 bp, which limits the fragment size of the library; and the enzyme reaction time is long, which takes 16 hours.
  • the gap generated by cyclization is used, or the recognition site of the class III enzyme is replaced with the recognition site of the nickase, and after the circular DNA and the streptavidin magnetic beads are combined, the enzyme is cleaved in two chains.
  • a gap or incision is formed on each of the cells, and then the 5'-3' polymerase activity and the 3'-5' exonuclease activity of the polymerase are used to achieve translation of the gap or the incision, so that the two strands of the target fragment are from the gap or Starting at the incision, the polymerization extension was carried out in the direction of 5'-3', the length of the library insert was increased, and the reaction conditions were controlled to control the length of the fragment.
  • the reaction conditions controlled include the amount of dNTP used, the amount of enzyme of the polymerase, temperature, time, and the like.
  • the DNA polymerase will continue to function as an exonuclease, continue cutting in the 3'-5' direction of the strand, creating a sufficiently large gap, and finally using the single-strand endonuclease to nick the gap.
  • the other single strand is cleaved to become a double-stranded nucleic acid at both ends.
  • the target fragment to be recovered has biotin labeling, which has already been combined with streptavidin magnetic beads, and the enzymatic reaction can be carried out on the magnetic beads, so only a simple washing step is required, and the enzyme and buffer in the reaction are needed. After removal, the target fragment can be obtained and the next step of reaction.
  • the enzyme reaction time in this process is approximately 2.5 hours. Compared with the traditional method, not only the time is shortened, but also the insert of the last library is improved, and the control of the length of the fragment is realized.
  • Genomic DNA interruption There are many ways to interrupt genomic DNA. Whether it is physical ultrasound or enzymatic reaction, there are very mature programs on the market. This embodiment employs a physical ultrasonic breaking method.
  • Breaking fragment selection magnetic bead purification or gel recovery method can be used. This embodiment uses magnetic bead purification.
  • reaction solution 7.2 ⁇ L of the reaction solution was added to the recovered product of the previous step, mixed, incubated at 37 ° C for 45 min, incubated at 65 ° C for 10 min, and the temperature was lowered to 4 ° C at a rate of 0.1 ° C per second.
  • Fragment end repair Prepare the system according to Table 3.
  • the system was mixed and added to the product of the previous step, mixed and incubated at 12 ° C for 20 min. Purification was carried out using 48 ⁇ L of Ampure XP magnetic beads, and 40 ⁇ L of TE buffer solution was dissolved to recover the product.
  • 5' linker A sequence ligation The 5' linker A sequence used in this example is as follows (the sequence in this example is from 5' to 3' end from left to right, "//" indicates a modifying group, “phos” shows phosphorylation, “dd” shows dideoxy, "bio” shows biotin, and the font shows the label sequence in bold.
  • a 5' linker A mixture (10 ⁇ M) was prepared according to the formulation of Table 4.
  • Reaction component Volume 1M tromethamine base 37.5 1M citric acid 9.6 1M magnesium chloride 35 1M trisodium citrate 20 100% glycerin 50 10% Tween-80 1 30% polyethylene glycol 8000 333 0.1M adenosine triphosphate 10 0.5M trichloroethyl phosphate (pH 7.0) 2 Enzyme-free pure water 1.9
  • reaction mixture was mixed with the mixture of the linker and the product, incubated at 25 ° C for 30 min, incubated at 65 ° C for 10 min, and cooled to 4 ° C.
  • 3' L-type linker A sequence ligation The 3' L-former A sequence used in this example is shown below: ACGTTCTCGACUCCTCAGCTT (SEQ ID NO: 3).
  • Ampure XP magnetic beads resuspended in a 48 ⁇ L 3' L-type linker reaction system were placed on an incubator at 300 rpm for 30 min at 25 °C. After the reaction, 43.2 ⁇ L of Ampure XP magnetic bead binding buffer was added, and after incubation at room temperature for 10 min, the supernatant was removed, washed twice with 70% ethanol, and air-dried for 5-10 min at room temperature, and then the product was dissolved in 30 ⁇ L of TE buffer solution.
  • the primer 1 sequence is as follows:
  • the primer 2 sequence is as follows:
  • ACGTTCTCGACUCCTCAGCTT (SEQ ID NO: 5).
  • the PCR system was prepared according to Table 9.
  • the above product was recovered in 50 ⁇ L (180 ng) of the above step, added to the above system, and after mixing, the reaction was carried out under the conditions shown in Table 10.
  • the above reaction solution was added to 60 ⁇ L (2 ⁇ g) of the reaction product in the step, mixed, and then incubated at 37 ° C for 1 h.
  • Double-chain cyclization The reaction system 1 shown in Table 12 below was prepared.
  • reaction product of the previous step was added to the reaction system 1, mixed and divided into 4 tubes, and placed in a water bath at 50 ° C for 15 minutes. After the reaction was completed, it was placed in a normal temperature water bath for 15 minutes.
  • reaction system 2 50 ⁇ L of the reaction system 2 was separately added to the aliquoted 4-tube reaction system 1, and incubated at room temperature for 1 h.
  • the product of the above step was added to the reaction system, and after mixing, it was incubated at 37 ° C for 1 h.
  • the starting site of the CNT reaction on the double-stranded circularized DNA formed in this embodiment is a nick type, that is, both are complete double-stranded circular DNA, and the linker A sequence has a recognition sequence of the nicking enzyme.
  • Binding of DNA to magnetic beads Take 500 ng of circular DNA, add streptavidin magnetic beads (Life Technologies), bind for 1 hour at room temperature, and bind DNA to Streptomyces using biotin labeling on circular DNA. Avidin wrapped on magnetic beads. Then placed on a magnetic stand, remove the supernatant, and wash with a high salt wash Wash once, wash once with low-salt lotion, and rinse once with 1 ⁇ NEB buffer 2.
  • the formulations of the high salt lotion and the low salt lotion are shown in Tables 16 and 17 below, respectively.
  • Tween 20 10% Tween 20 was added before use to bring the final concentration of Tween 20 to 0.05%.
  • Tween 20 10% Tween 20 was added before use to bring the final concentration of Tween 20 to 0.05%.
  • Nickel enzyme digestion reaction The system was prepared according to the formulation of Table 18 below.
  • reaction solution 80 ⁇ L was added to the magnetic beads of the previous step, and after mixing, the reaction was carried out at 37 ° C for 60 minutes.
  • the sample was placed on a magnetic stand, the supernatant was removed, washed once with a high salt wash, once with a low salt wash, and once with 1 x NEB buffer 2.
  • the amount of dNTPs and DNA polymerase I is variable and can be adjusted according to the length of the desired fragment obtained.
  • reaction solution 60 ⁇ L was added to the magnetic beads of the previous step, mixed and reacted at 25 ° C for 15 min, and EDTA (0.5 M, AMBION) 1.2 ⁇ L was added, and the reaction was carried out at 65 ° C for 15 min.
  • the sample was placed on a magnetic stand, the supernatant was removed, washed once with a high salt wash, once with a low salt wash, and once with 1 x NEB buffer 2.
  • reaction solution 90 ⁇ L of the reaction solution was added to the magnetic beads of the previous step, mixed and reacted at 25 ° C for 15 min, and 2 ⁇ L of EDTA (0.5 M, AMBION) was added. After the reaction, the sample was placed on a magnetic stand, the supernatant was removed, washed once with a high salt wash, washed twice with a low salt wash, and resuspended with 100 ⁇ L of a low salt wash.
  • EDTA 0.5 M, AMBION
  • Adhesive end fill and 3' end plus A The system was formulated according to the formulation of Table 21 below.
  • reaction solution 30 ⁇ L was added to the magnetic bead suspension of the previous step, mixed and reacted at 37 ° C for 60 min, and EDTA (0.5 M, AMBION) 2 ⁇ L was added. After the reaction, the sample was placed on a magnetic stand, the supernatant was removed, and the low-salt wash was washed three times, and the magnetic beads were resuspended with 70 ⁇ L of a low-salt wash.
  • connection connector B (bubble joint)
  • the linker B is formed by complementary pairing of the top chain L and the bottom chain S, and the sequence is as follows:
  • Top chain L /phos/AGTCGGAGGCCAAGCGTGCTTAGGACAT (SEQ ID NO: 6);
  • the bottom strand S GTCCTAAGCACUGTAGTGTACGATCCGACTT (SEQ ID NO: 7).
  • reaction solution 100 ⁇ L was added to the magnetic bead suspension of the previous step, mixed, and reacted at room temperature for 30 min, and then reacted at 65 ° C for 10 min.
  • the primers F and R sequences used in this example are as follows:
  • Primer F /bio/ATGTCCTAAGCACGCTTGGCC (SEQ ID NO: 8);
  • Primer R /phos/GTAGTGTACGATCCGACTT (SEQ ID NO: 9).
  • the system was formulated according to the formulation of Table 23 below.
  • the nucleic acid single-strand O can be ligated at both ends of the product of the previous step by using the corresponding complementary sequence.
  • the single-strand O sequence of the nucleic acid is as follows:
  • ATCGTACACTACATGTCCTAAGCA (SEQ ID NO: 10).
  • nucleic acid single-chain O 10 ⁇ M, manufactured
  • reaction solution 50 ⁇ L was added to a mixture of the PCR product and single-chain O, and the mixture was mixed and reacted at 37 ° C for 60 minutes.
  • reaction solution 8 ⁇ L was added to the ligation reaction solution of the previous step, mixed and reacted at 37 ° C for 30 min; and EDTA (0.5 M) 6 ⁇ L was added. Then, it was purified by using 170 ⁇ L of PEG32 magnetic beads, and 55 ⁇ L of TE buffer was reconstituted.
  • the concentration and total amount of the library are sufficient to meet the requirements of subsequent sequencing for the amount of library; simultaneous electrophoresis (Fig. 6) and the results of testing using LabChip GX instrument (automatic microfluidic electrophoresis instrument, Caliper) Figure 7-1010) shows that the DNA library after polymerase chain reaction is concentrated in a band size of 200 bp to 300 bp. The electrophoresis bands are concentrated and the main peaks are prominent, which is consistent with the requirements of subsequent sequencing for fragment range.

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Abstract

一种核酸的双接头单链环状文库的构建方法和试剂，该方法包括：将核酸打断成核酸片段；连接第一接头序列；扩增得到两端具有第一接头序列的第一产物，其中引物序列上具有U碱基位点且具有或不具有切口酶识别序列，并且其中一条引物序列上具有第一亲和标记；使用USER酶酶切第一产物；环化酶切后的第一产物；去磷酸化酶或切口酶处理环化产物；使用固相载体与环化分子结合；进行限制性缺口平移反应；消化除去未发生限制性缺口平移反应的部分；连接第二接头序列；扩增得到两端具有第二接头序列的第二产物；变性第二产物，并对单链核酸分子进行环化。该方法能够提高文库插入片段长度，简化建库流程，缩短建库时间，降低建库成本。

Description

一种核酸的双接头单链环状文库的构建方法和试剂 技术领域
本发明涉及分子生物学技术领域，尤其涉及一种核酸的双接头单链环状文库的构建方法和试剂。
背景技术
目前高通量测序是分子生物学研究、医学诊断等各领域的重要研究手段之一。自从高通量的第二代测序技术诞生之后，测序技术获得了突飞猛进的发展，与以往昂贵的测序费用相比，第二代测序的测序费用降低了好几个数量级；同时，测序的时间也大大缩短。现在第三代测序仪的出现使得测序市场的竞争更加激烈，发展更加迅速。所以各个测序公司、研究团队必须得减低测序成本，缩短流程时间和提高结果准确性，才有机会在竞争中继续发展。
在二代测序平台中，Complete Genomics(CG)公司开发出了一个精确度高、测序通量大的测序平台用于人全基因组测序，其数据准确度高达99.9998％，为癌症研究、低频突变的检测和个人基因组测序提供了准确的基因信息。但是CG平台的文库构建流程耗时太长，建库成本较高，而且文库的插入片段短，限制了后续数据的产出及分析，不仅影响了科研项目的发展，也不利于CG平台大规模大范围地使用和发展。CG平台必须优化其建库流程，缩短建库时间，增加文库长度和降低成本等才能在目前如此激烈的竞争中保持优势。在传统的CG平台的建库流程中，双链环化后至最后单链环化之间的步骤相当繁琐，建库流程主要包括：环化DNA的酶切，去除磷酸化，末端修复，3’端接头连接，5’端接头连接，缺口平移，聚合链式反应，单链分离和环化等12个步骤，每一步酶反应之后都要用磁珠纯化。该流程的时间长，成本高，酶切产生的文库插入片段只有26bp，既不利用CG平台的大规模应用，也不符合CG下一代测序平台对片段的要求。
发明内容
本发明提供一种核酸的双接头单链环状文库的构建方法和试剂，该方法能够提高文库插入片段长度，简化建库流程，缩短建库时间，降低建库成本。
根据本发明的第一方面，本发明提供一种核酸的双接头单链环状文库的构建方法，包括如下步骤：
将核酸打断成用于文库构建的核酸片段；
在核酸片段的两端连接第一接头序列；
通过第一PCR扩增得到两端具有第一接头序列的第一产物，其中第一PCR使用的引物序列上具有U碱基位点且具有或不具有切口酶识别序列，并且其中一条引物序列上具有第一亲和标记；
使用USER酶酶切第一产物，产生粘性末端且产生或不产生缺口；
对酶切后的第一产物进行环化，产生环状核酸分子；
使用去磷酸化酶处理双链上均具有缺口的环状核酸分子，或者使用切口酶处理一条链上具有切口酶识别序列且另一条链上具有缺口或双链均具有切口酶识别序列且均不具有缺口的环状核酸分子以产生切口；
使用带有第二亲和标记的固相载体与环状核酸分子结合；
以结合到固相载体上的环状核酸分子为模板，从切口和/或缺口处开始进行限制性缺口平移反应；
消化除去环状核酸分子上的未发生限制性缺口平移反应的部分，得到线性核酸分子；
在线性核酸分子的两端连接第二接头序列；
通过第二PCR扩增得到两端具有第二接头序列的第二产物；
对第二产物进行变性得到单链核酸分子，并使用与其中一条单链核酸分子两端均互补的介导序列对单链核酸分子进行环化，得到双接头单链环状文库。
作为本发明的优选方案，第一亲和标记为生物素标记；第二亲和标记为链霉亲和素标记。
作为本发明的优选方案，第一接头序列包括第一5’接头序列和第一3’L型接头序列，分别连接片段每条链的3’端和5’端；第一5’接头序列包括一条5’端磷酸化的长链和一条互补的短链，短链的3’末端双脱氧修饰，并且短链中包含U碱基位点；第一3’L型接头序列在邻近连接的片段的部分与第一5’接头序列有部分碱基互补；
在核酸片段的两端连接第一接头序列，具体包括：
对核酸片段进行去磷酸化；
对去磷酸化后的核酸片段进行末端修复；
在核酸片段的每条链的3’端连接第一5’接头序列；
使用USER酶酶切第一5’接头序列的短链的U碱基位点；
对USER酶酶切后的核酸片段进行磷酸化处理；
在磷酸化处理后的核酸片段每条链的5’端连接第一3’L型接头序列。
作为本发明的优选方案，第一PCR使用的引物序列上均具有一个U碱基位点和切口酶识别序列；使用USER酶酶切U碱基位点后，在核酸片段两端形成粘性末端，粘性末端互补发生环化，产生环状核酸分子；再使用切口酶酶切切口酶识别序列产生切口。
作为本发明的优选方案，第一PCR使用的引物序列中有一条引物序列具有两个U碱基位点，另一条引物具有一个U碱基位点；使用USER酶酶切U碱基位点后，在核酸片段两端形成粘性末端，粘性末端互补发生环化，产生环状核酸分子。
作为本发明的优选方案，对酶切后的第一产物进行环化之后，还包括：对未环化的核酸分子进行消化。
作为本发明的优选方案，在限制性缺口平移反应中，通过控制dNTP与作为模板的核酸分子的摩尔比、酶反应温度和时间中至少一个因素来控制生成的缺口平移片段的长度。
作为本发明的优选方案，消化除去环状核酸分子上的未发生限制性缺口平移反应的部分，具体包括：首先使用双链外切酶降解，直到两端的缺口相遇；然后使用单链外切酶降解单链；或使用核酸内切酶直接切掉环状核酸分子上的未发生限制性缺口平移反应的部分。
作为本发明的优选方案，第二接头序列为鼓泡接头序列，鼓泡接头序列包括两条两端部分互补配对但中间一段不互补配对的碱基序列，其中中间一段形成鼓泡状，并且中间一段带有U碱基位点；鼓泡接头序列的一条链的5’端有一个突出的T碱基；
在线性核酸分子的两端连接第二接头序列，具体包括：
对线性核酸分子进行末端修复和3’端加A碱基的反应；
通过T碱基与A碱基的配对，将鼓泡接头序列连接到线性核酸分子两端；
使用USER酶酶切中间一段上的U碱基位点。
作为本发明的优选方案，对单链核酸分子进行环化之后，还包括：对未环化的单链核酸分子进行消化。
根据本发明的第二方面，本发明提供一种核酸的双接头单链环状文库的构建试剂，包括如下组成部分：
第一接头序列，第一接头序列包括第一5’接头序列和第一3’L型接头序列，分别连接片段每条链的3’端和5’端；第一5’接头序列包括一条5’端磷酸化的长链和一条互补的短链，短链的3’末端双脱氧修饰，并且短链中包含U碱基位点；第一3’L型接头序列在邻近连接的片段的部分与所述第一5’接头序列有部分碱基互补；
第一PCR引物，具有U碱基位点且具有或不具有切口酶识别序列，并且其中一条引物序列上具有第一亲和标记，用于通过第一PCR扩增得到两端具有第一接头序列的第一产物；
USER酶，用于酶切第一产物，产生粘性末端且产生或不产生缺口；
环化酶，用于对所述酶切后的第一产物进行环化，产生环状核酸分子；
去磷酸化酶，用于对双链上均具有缺口的环状核酸分子进行去磷酸化酶处理；或者切口酶，用于对一条链上具有切口酶识别序列且另一条链上具有缺口或双链均具有切口酶识别序列且均不具有缺口的环状核酸分子进行酶切，以产生切口；
固相载体，带有第二亲和标记，用于与环状核酸分子结合；
缺口平移反应的组分，用于以结合到固相载体上的环状核酸分子为模板，从切口和/或缺口处开始进行限制性缺口平移反应；
消化酶，用于消化除去环状核酸分子上的未发生限制性缺口平移反应的部分，得到线性核酸分子；
第二接头序列，第二接头序列为鼓泡接头序列，鼓泡接头序列包括两条两端部分互补配对但中间一段不互补配对的碱基序列，其中中间一段形成鼓泡状，并且中间一段带有U碱基位点；鼓泡接头序列的一条链的5’端有一个突出的T碱基；
第二PCR引物，用于通过第二PCR扩增得到两端具有第二接头序列的第二产物；
介导序列，与第二产物变性后得到单链核酸分子中的一条单链核酸分子两端均互补，用于对单链核酸分子进行环化，得到双接头单链环状文库。
作为本发明的优选方案，第一亲和标记为生物素标记；第二亲和标记为链 霉亲和素标记。
本发明核酸的双接头单链环状文库的构建方法通过限制性缺口平移反应与在磁珠上进行的酶反应相结合，限制性缺口平移反应用新的切口酶位点代替传统方法中的III类内切酶位点，并从缺口或切口处开始进行可控制的核酸链延伸，实现文库插入片段长度的增加；另外将环状核酸分子与磁珠结合后，不需要洗脱核酸，而是直接加入酶反应液，让酶反应在磁珠上进行，一直到单链洗脱的步骤，中间不需要多次地结合、洗脱磁珠，缩短了建库时间，也节省了反复加入新磁珠的成本。
附图说明
图1为本发明一个实施例的核酸的双接头单链环状文库的构建方法中从磁珠结合到单链成环过程的流程图；
图2为本发明一个实施例的CNT切口产生基本原理图；
图3为本发明一个实施例的CNT缺口产生基本原理图；
图4为现有方法与本发明方法插入片段形成及单链分离环化原理对比图；
图5为现有定向接头连接法与本发明L型接头连接法的对比图；
图6为本发明一个实施例中四个平行实验的最终产物电泳检测结果，其中M表示DNA Marker；1、2、3、4分别表示四个平行样本C22、D22、E22和F22的电泳结果。
图7-10分别为本发明一个实施例中四个平行实验C22、D22、E22和F22的最终产物使用LabChip GX仪器(全自动微流体电泳仪，Caliper公司)测试的结果。
具体实施方式
下面通过具体实施例对本发明作进一步详细说明。除非特别说明，下面实施例中所使用的技术均为本领域内的技术人员已知的常规技术；所使用的仪器设备和试剂等，均为本领域内的技术人员可以通过公共途径如商购等获得的。
本发明中，任何情况下使用的“第一”和“第二”等概念都不应当理解为具有顺序和技术的含义，其作用仅在于将其与其它对象区别开来。
本发明中，第一亲和标记和第二亲和标记可以是生物学上常用的生物结合反应的一个组成部分，比如抗原或抗体，双链DNA短片段的一条链，生物素或链霉亲和素，等等。在第一亲和标记选用了抗原的情况下，第二亲和标记选用 与该抗原结合的抗体，反之亦然；在第一亲和标记选用了双链DNA短片段的一条链的情况下，第二亲和标记选用与该链互补配对的另一条链，反之亦然；在第一亲和标记选用了生物素的情况下，第二亲和标记选用与生物素结合的链霉亲和素，反之亦然。本发明的一个实施方案中，第一亲和标记是生物素标记，第二亲和标记是链霉亲和素标记，二者具有很强的结合能力。
请参考图1，本发明一个实施例的核酸的双接头单链环状文库的构建方法包括步骤：打断基因组DNA形成用于文库构建的核酸片段；进行去磷酸化和末端修复反应；连接5’接头A序列；USER酶酶切和磷酸化处理；连接3’L型接头A序列；PCR扩增得到两端具有5’接头A序列和3’L型接头A序列的产物，其中PCR使用的引物序列上具有U碱基位点和切口酶识别序列，并且一条引物上具有生物素标记；使用USER酶酶切U碱基位点产生粘性末端，并对USER酶酶切后的产物进行环化，产生环状核酸分子；将环状核酸分子与链霉亲和素标记的磁珠结合；使用切口酶在切口酶识别序列处酶切出切口；从切口处开始进行限制性缺口平移反应(Controlled Nick Translation，CNT)；使用核酸内切酶在切口处切断核酸链(也可以首先使用双链外切酶降解，直到两端的缺口相遇，然后使用单链外切酶降解单链)，得到线性核酸分子；对线性核酸分子进行末端修复和3’端加A碱基的反应；连接鼓泡接头序列；使用USER酶酶切鼓泡接头序列上的U碱基位点，形成L型接头；PCR扩增得到两端具有不同序列的产物；变性处理得到单链核酸分子，并使用与其中一条单链核酸分子两端均互补的介导序列对单链核酸分子进行环化，得到双接头单链环状文库。
本发明中，图1所示的双接头单链环状文库的构建方法中，在第一次PCR使用的引物序列上引入U碱基位点和切口酶识别序列，采用图2所示的原理产生切口，作为限制性缺口平移反应的起始点。现有方法是在接头序列中引入III类内切酶识别序列，接头序列连接并环化后，采用III类内切酶酶切双链产生线性化的双链DNA；而本发明的方法在第一次PCR使用的引物序列上引入U碱基位点和切口酶识别序列，PCR扩增后使用USER酶酶切U碱基位点产生粘性末端，并进行双链环化，然后使用切口酶(如Nb.BsrDI、Nb.BsmI、Nt.BbvCI、Nb.Bbv.Nb.BtsI或Nt.BstNBI等)酶切环化DNA的单链，在每条单链上产生切口，以为CNT提供有效的作用起始位点。
本发明中，作为替代方案，在第一次PCR的引物中引入U碱基位点，并使 用USER酶酶切产生缺口，作为限制性缺口平移反应的起始点。该种缺口产生的基本原理如图3所示：(1)连接5’接头A序列和3’L型接头A序列之后，用分别带两个U和一个U的引物扩增接头A连接产物；(2)用USER酶酶切U碱基，切口处形成磷酸化的3’末端和5’末端；(3)利用酶切产生的粘性末端进行双链环化，环化之后一条链上的缺口(缺口1，由USER酶切形成)为磷酸化的3’末端和5’末端，另一条链上的缺口(缺口2，因环化之后该处缺少一个匹配碱基而形成)为去磷酸化的3’末端和磷酸化的5’末端；(4)去磷酸化处理，使切口1的3’末端去磷酸化，以为CNT提供有效的作用起始位点。
本发明中，从切口和/或缺口处开始进行的反应，称为“限制性缺口平移反应”，因为可以通过对反应中dNTP用量、作为模板的核酸分子的用量、酶反应温度和时间等因素的控制，将反应生成的目的片段长度控制在一定的范围之内，一定长度范围的核酸片段适于特定的测序平台，一般本发明中目的片段的长度控制在50～250bp范围内是较好的，这样的长度比传统的CG测序平台建库方案得到的目的片段长度大几倍。而且本发明的CNT技术在不进行切胶回收的前提下，就可使文库插入片段控制在非常集中的范围，有效地提高了缺口平移反应技术的可操作性。
请参考图4，对现有方法和本发明方法进行比较。现有方法利用III类内切酶的切割特性，酶切接头A两侧25-27bp处的基因组DNA，形成约104bp的目的DNA片段；后续通过两步磁珠纯化法，去除200bp以上的不带接头A的DNA片段，此时经过磁珠片段选择得到的酶切产物中还混杂了一些主带在100-200bp的非目的DNA片段；经过接头B连接后，使用一条引物上带有生物素标记的碱基的引物扩增连接了接头B的DNA片段，其中带有生物素标记的碱基的引物扩增出的单链为非目的单链核酸；后续再通过一次链霉亲和素磁珠富集连接了接头B的DNA片段，通过一次特异序列杂交捕获进一步富集连接了接头A的DNA片段；最后通过碱变性使双链DNA解链，将目的单链核酸从链霉亲和素磁珠上洗脱下来，再利用介导序列环化目的单链核酸。现有方法的整个过程不但步骤繁琐，操作时间长，而且消耗的试剂(主要是每一步反应都需要用Ampure磁珠或者链霉亲和素磁珠)价格昂贵。本发明的方法，一方面，用第一PCR引物上带有的生物素标记，在环化之后将环状核酸分子结合到链霉亲和素磁珠上，后续反应中目的核酸分子一直结合在磁珠上，每一步反应后不需要加入新磁珠进 行纯化，只需要用洗涤试剂将反应液洗掉即可进行下一步反应，不仅减少了磁珠的使用，也节省了实验操作时间；另一方面，切口酶在接头A两条链上分别打开一个切口，然后利用聚合酶在dNTP存在时的切口平移功能，将切口从接头A区域延伸至接头A两侧，通过控制dNTP与模板DNA的摩尔比例、反应温度和反应时间等条件，灵活控制切口延伸长度，延伸片段主带大小可控制在50～250bp范围内，后续再通过一步外切酶消化反应，将不带接头A的非目的DNA片段消化掉，剩余的即为带接头A的目的DNA片段，经过接头B连接及不带生物素标记引物PCR扩增之后，通过简单的高温变性，解链双链DNA，再用介导序列介导目的单链的环化，即可分离和环化目的单链DNA。可见本发明的单链环化方法只需要热变性和介导序列杂交即可成功地分离和环化目的单链核酸，不但步骤简单，易于操作，且不需要消耗大量昂贵试剂，建库成本得到降低。
在本发明的一个优选实施例中，采用L型接头连接替代传统的接头连接。请参考图5，比较说明现有的接头连接法和本发明的接头连接法。现有方法采用的定向接头连接法，此方法为保证接头定向连接的同时，最大程度地降低DNA片段间相互连接问题，采用将5’接头和3’接头分开设计，分步连接的方法。每加一端接头，都需要接头序列、封闭序列、引物序列共同作用来完成。整个过程需要经过去磷酸化、末端修复、加5’接头、引物延伸、加3’接头、切口平移及连接这6步酶反应及5次纯化操作，才能将接头A的序列定向加入到目的DNA两端。现有方法步骤繁琐，建库成本(序列成本、酶反应试剂成本、纯化成本)高，周期长，样品损耗大，不符合文库构建高效简便的要求。而本发明的L型接头连接法，能够在保证接头定向连接的前提下，提高建库效率，降低建库成本。L型接头连接法虽也是采用分步连接，但步骤相对现有方法简单。首先，加入带有封闭序列的5’接头，其中封闭序列长度为12bp左右，与5’接头完全互补，形成部分互补的双链结构，以便DNA片段与5’接头连接。由于封闭序列3’端有双脱氧修饰，5’端为去磷酸化碱基，既可保证5’接头与DNA片段3’末端定向连接，又保证封闭序列不能与DNA片段5’末端连接。封闭序列中间位置带有一个U碱基，通过USER酶处理，封闭序列被“降解”成两段小于8bp的单链DNA片段，并解链脱离5’接头。然后，通过杂交后连接法加入“L”型单链3’接头。在加入L型接头之前，还需要将DNA片段的5’末端磷酸化，以解除封闭。实验证明， USER酶处理可以与磷酸化反应同时进行，反应后磁珠纯化，直接重悬洗涤之后的磁珠于下一步连接反应缓冲液中。L型接头的设计巧妙之处为3’末端最后8个碱基与5’接头5’末端最后8个碱基互补，这样可以直接杂交到5’接头上，再用连接酶封闭切口，即可将L型3’接头连接到DNA片段的5’末端。由于L型接头的一部分碱基与5’接头5’末端的一部分碱基互补，而其它碱基不互补，所以看上去呈L型，故称为L型接头。反应结束之后，在磁珠中再加入适量磁珠结合缓冲液，即可纯化回收加好接头的连接产物。整个过程只需要经过去磷酸化、末端修复、加5’接头、USER酶切与磷酸化一步反应、加5’L型接头这5步酶反应及3次纯化操作，即可较快速地将接头A的序列定向加入到目的DNA两端，步骤简单，建库成本降低，周期缩短。
本发明的独特性创新点主要在于：酶反应在链霉亲和素磁珠上进行，核酸与链霉亲和素磁珠结合后不需要洗脱，即可进行酶反应；采用可控制的缺口平移反应，产生特定片段长度的核酸双链。
环状核酸分子通过生物素标记和链霉亲和素磁珠相结合，之后每一步的酶反应中，核酸分子一直绑定在磁珠上，中间只需要简单的洗涤步骤将反应中的酶、离子等洗掉，而带有生物素标记的核酸双链不会被洗脱下来，直到第二次PCR以后，核酸双链才被变性成两条单链，将没有生物素标记的单链收集起来。
在传统的Complete Genomics公司的实验操作中，酶反应在溶液中进行，每一步酶反应后都需要加入新的磁珠结合目的片段，洗涤掉反应中的酶和缓冲液等，最后将目的片段洗脱下来再进行下一步的反应。而本发明的酶反应在链霉亲和素磁珠上进行，只需要加入一次磁珠即可。双链核酸分子与磁珠结合后，中间不用多次的洗脱和反复多次加入新的磁珠重新结合，只要简单的洗涤步骤，即可收集到需要的目的片段，省去了很多片段纯化的步骤，不仅节约了实验操作的时间，也减少了磁珠的用量，从而节约了成本。同时由于避免了样品与磁珠间反复的结合、洗脱，减少了实验中样品的损失，提高了最后目的片段的得率。本发明一个实施例中，使用磁珠作为固相载体，但是固相载体并不局限于磁珠，也可以使用芯片等其它固相载体，只要将链霉亲和素固定到固相载体上即可实现本发明的功能。
在传统的Complete Genomics公司的文库构建中，环状DNA上有III类酶酶切位点，III类酶识别酶切位点后，会在距离酶切位点26bp处对环状DNA进行 酶切，将环状DNA变成两段线性DNA，然后再通过DNA上的生物素标记与链霉亲和素磁珠结合，达到对目的片段的收集。该方法酶切后的目的片段只有26bp，限制了文库的片段大小；并且酶反应时间长，需要16个小时。而本发明用环化时产生的缺口，或者将III类酶的识别位点替换为切口酶的识别位点，在环状DNA和链霉亲和素磁珠结合后，酶切在两条链上分别形成一个缺口或者切口，再通过聚合酶的5’-3’聚合酶活性和3’-5’外切酶活性，来实现缺口或者切口的平移，使目的片段的两条链从缺口或者切口处开始，以5’-3’为方向进行聚合延伸，提高文库插入片段长度，并控制反应条件来控制片段的长度。控制的反应条件包括dNTP的使用量、聚合酶的酶量、温度、时间等。当dNTP用尽后，DNA聚合酶会继续发挥外切酶的作用，沿着这条链的3’-5’方向继续切割，产生足够大的缺口，最后再用单链内切酶将缺口处的另一条单链切断成为两端核酸双链。其中需要回收的目的片段上有生物素标记，早已经和链霉亲和素磁珠结合，并且酶反应可以在磁珠上进行，所以只需要简单的洗涤步骤，将反应中的酶、缓冲液等去除，就能得到目的片段，并进入下一步反应。这个流程中酶反应时间大约为2.5个小时。与传统方法相比，不仅缩短了时间，还提高了最后文库的插入片段，并实现了片段长度的可控制化。
下面通过实施例详细说明本发明。
1、基因组DNA打断：基因组DNA打断有多种方式，无论是物理超声法还是酶反应法，市场上有非常成熟的方案。本实施例采用的是物理超声打断法。
取96孔PCR板一块，加入一根聚四氟乙烯线，加入基因组DNA 1μg，加入TE缓冲溶液或无酶纯水补齐100μL。将板封膜后置于E220超声打断仪上超声打断。打断条件设置如表1。
表1
参数 数值
填充系数 21％
压力(PIP) 500
脉冲系数 500
打断时间 20s，2次
2、打断片段选择：可以采用磁珠纯化法或凝胶回收法，本实施例采用磁珠纯化法。
取打断后的DNA，加入45μL Ampure XP磁珠，混匀后放置7-15min；置入磁力架后收集上清，在上清中加入18μL Ampure XP磁珠，混匀后放置7-15min；置入磁力架吸去上清，用75％乙醇洗磁珠两次；晾干后加入30μL TE缓冲溶液，混匀后放置7-15min溶解回收产物。
3、片段去磷酸化反应：取上步骤回收产物，按表2配制体系。
表2
反应成分 体积(μL)
10×NEB缓冲液2 3.6
虾碱性磷酸酶(1U/μL) 3.6
总共 7.2
将7.2μL反应液加入前一步的回收产物中，混匀，置于37℃孵育45min，65℃孵育10min，按照每秒降低0.1℃的速率，梯度降温到4℃。
4、片段末端修复：按表3配制体系。
表3
反应成分 体积(μL)
无酶水 7.32
10×NEB缓冲液2 1.08
0.1M三磷酸腺苷 0.48
dNTPs(25mM，Enzymatic) 0.48
牛血清白蛋白(10mg/ml) 0.24
T4脱氧核糖核酸聚合酶(3U/μL) 1.2
总共 10.8
将体系混匀后加入上一步骤产物中，混匀后置于12℃孵育20min。使用48μL Ampure XP磁珠进行纯化，40μL TE缓冲溶液溶解回收产物。
5、5’接头A序列连接：本实施例中使用的5’接头A序列如下(本实施例中的序列从左到右为5’端至3’端，“//”示修饰基团，“phos”示磷酸化，“dd”示双脱氧，“bio”示生物素，字体加粗示标签序列)。
5’接头A序列：
/5phos/AAGCTGAGGGTACTGTGTCATAAATAGCACGAGACGTTCTCGACT(SEQ ID NO：1)；
5’封闭序列：TACCCUCAGCT/3ddT/(SEQ ID NO：2)。
5’接头A混合液(10μM)按表4配方配制。
表4
反应成分 体积(μL)
5’接头A序列(100μM) 12
5’封闭序列(100μM) 10
TE缓冲液 78
总共 100
将4.5μL配制好的接头A混合液(10μM)加入上一步骤产物中，充分混匀。连接反应体系按以下表5配方配制。
表5
反应成分 体积(μL)
无酶纯水 13.1
2×连接缓冲液1 60
T4 DNA连接酶(快速)(600U/μL) 2.4
总共 75.5
其中，本实施例使用的2×连接缓冲液1配方如表6所示。
表6
反应成分 体积(μL)
1M氨基丁三醇碱 37.5
1M柠檬酸 9.6
1M氯化镁 35
1M柠檬酸三钠盐 20
100％甘油 50
10％吐温-80 1
30％聚乙二醇8000 333
0.1M三磷酸腺苷 10
0.5M磷酸三氯乙酯(pH7.0) 2
无酶纯水 1.9
总共 500
将连接反应体系与接头和产物的混合液混匀，置于25℃孵育30min，65℃孵育10min，降温至4℃。
6、USER酶切与磷酸化一步反应：在上一步反应液中加入1.2μL USER酶(1U/μL)，1.2μL T4多聚核苷酸激酶(10U/μL)，混匀后置于37℃孵育20min。用108μL Ampure XP磁珠(Agencourt)进行纯化，用70％乙醇清洗两次后，吸干清洗液，室温晾干2min，将Ampure XP磁珠重悬于48μL 3’L型接头反应体系中。
7、3’L型接头A序列连接：本实施例采用的3’L型接头A序列如下所示：ACGTTCTCGACUCCTCAGCTT(SEQ ID NO：3)。
按表7配制3’L型接头反应体系：
表7
反应成分 体积(μL)
无酶纯水 28.98
3×连接缓冲液2 16.02
L型接头序列(100μM) 1.8
T4 DNA连接酶(快速)(600U/μL) 1.2
总共 48
本实施例中使用的3×连接缓冲液2配方如表8所示。
表8
反应成分 体积(μL)
聚乙二醇-8000(50％) 60
Tris-Cl，pH7.8(2M) 7.5
三磷酸腺苷(100mM) 3
牛血清蛋白(10mg/mL) 1.5
氯化镁(1M) 3
双对氯苯基三氯乙烷(DDT)(1M) 0.15
无酶纯水 24.9
将重悬于48μL 3’L型接头反应体系的Ampure XP磁珠置于孵育仪上以300rpm的转速，25℃孵育30min。反应完之后，加入43.2μL Ampure XP磁珠结合缓冲液，室温孵育10min后，去上清，用70％乙醇洗涤两次，室温晾干5-10min之后，用30μL TE缓冲溶液溶解回收产物。
8、聚合酶链式反应：
引物1序列如下：
AGTCGAGAACGUCTCG/iBiodT/GCT(SEQ ID NO：4)；
引物2序列如下：
ACGTTCTCGACUCCTCAGCTT(SEQ ID NO：5)。
按表9配制PCR体系。
表9
反应成分 体积(μL)
无酶纯水 186.5
2×PfuTurbo Cx缓冲液 275
PfuTurbo Cx热启动核酸聚合酶(2.5U/μL) 11
20μM引物1 13.75
20μM引物2 13.75
总体积 500
将上步骤50μL(180ng)回收产物，加入到以上体系中，混匀后按表10的条件进行反应。
表10
Figure PCTCN2014092296-appb-000001
反应完成后，使用550μL Ampure XP磁珠进行纯化，80μL TE缓冲液溶解回收产物。取1μL回收产物，用Qubit dsDNA HS分析试剂盒(Invitrogen公司)定量产物浓度。取2μg产物进行下一步反应。
9、去尿嘧啶：配制以下表11所示的反应液。
表11
反应成分 体积(μL)
无酶纯水 25.8
10×Taq缓冲液 11
USER酶(1U/μL) 13.2
总体积 50
将以上反应液加入60μL(2μg)上步骤反应产物中，混匀后置于37℃孵育1h。
10、双链环化：配制以下表12所示的反应体系1。
表12
反应成分 体积(μL)
无酶纯水 1520
10×TA缓冲液 180
总体积 1700
将上一步骤反应产物加入反应体系1中，混匀后平分成4管，置于50℃水浴反应15min。反应完成后置于常温水浴反应15min。
配制以下表13所示的反应体系2。
表13
反应成分 体积(μL)
无酶纯水 98
20×Circ缓冲液 100
T4 DNA连接酶(快速)(600U/μL) 2
总体积 200
本实施例使用的20×Circ缓冲液配方如表14所示。
表14
反应成分 浓度
Tris-Cl，pH 7.5 66mM
醋酸钾 132mM
醋酸镁 20mM
双对氯苯基三氯乙烷(DDT) 1mM
三磷酸腺苷 20mM
将50μL反应体系2分别加入平分的4管反应体系1中，置于室温孵育1h。
每管反应产物(500μL)，加入330μL Ampure XP磁珠，混匀后放置7-15min；置入磁力架后收集上清，在上清中加入170μL Ampure XP磁珠，混匀后放置7-15min；置入磁力架吸去上清，用75％乙醇洗磁珠两次；晾干后加入65μL TE缓冲液溶解4管纯化产物。
11、线性消化：配制以下表15所示的反应体系。
表15
Figure PCTCN2014092296-appb-000002
将上步骤产物加入反应体系中，混匀后置于37℃孵育1h。
使用80μL Ampure XP磁珠纯化，使用82μL TE缓冲液溶解回收产物。取1μL回收产物，用Qubit dsDNA HS分析试剂盒(Invitrogen公司)定量产物浓度。取700ng产物进行下一步反应。本实施例形成的双链环化DNA上CNT反应的起始位点为切口型，即两条均为完整的双链环状DNA，接头A序列上有切口酶的识别序列。
12、环状DNA与磁珠结合：取500ng环状DNA，加入链霉亲和素磁珠(Life Technologies)，室温结合1小时，利用环状DNA上的生物素标记，将DNA结合到链霉亲和素包裹的磁珠上。然后置于磁力架上，去掉上清，用高盐洗液洗 涤一次，低盐洗液洗涤一次，1×NEB缓冲液2润洗一次。高盐洗液和低盐洗液成分配方分别如下表16和表17所示。
表16
反应成分 体积(μL)
Tris-Cl，pH 7.5(1M，SIGMA) 5000
氯化钠(5M，SIGMA公司) 10000
无酶纯水 35000
总共 50000
使用前加入10％的吐温20，使吐温20的终浓度为0.05％。
表17
反应成分 体积(μL)
Tris-Cl，pH 7.5(1M，SIGMA) 5000
氯化钠(5M，SIGMA公司) 3000
无酶纯水 42000
总共 50000
使用前加入10％的吐温20，使吐温20的终浓度为0.05％。
13、切口酶酶切反应：按如下表18的配方配制体系。
表18
反应成分 体积(μL)
无酶水 66.3
10×NEB缓冲液2 8
Nt.BvbCI 1.7
总共 80
将80μL反应液加入到上一步骤的磁珠中，混匀后37℃反应60min。
反应后置于磁力架上，去掉上清，用高盐洗液洗涤一次，低盐洗液洗涤一次，1×NEB缓冲液2润洗一次。
14、限制性缺口平移反应：按如下表19的配方配制体系。
表19
反应成分 体积(μL)
无酶纯水 48
10×NEB缓冲液2 6
dNTPs(0.014mM，Enzymatic) 4.3
DNA聚合酶I(大肠杆菌)(10U/μL，NEB) 1.8
总共 60
其中，dNTPs和DNA聚合酶I的用量是可变的，可根据所需要获得的目的片段长度进行调整。
将60μL反应液加入到上一步骤的磁珠中，混匀后25℃反应15min，加入EDTA(0.5M，AMBION)1.2μL，65℃反应15min。
反应后置于磁力架上，去掉上清，用高盐洗液洗涤一次，低盐洗液洗涤一次，1×NEB缓冲液2润洗一次。
15、核酸内切酶在缺口酶切：按如下表20的配方配制体系。
表20
反应成分 体积(μL)
无酶纯水 78
10×NEB缓冲液2 9
T7核酸内切酶I(10U/μL，NEB) 3
总共 90
将90μL反应液加入到上一步骤的磁珠中，混匀后25℃反应15min，加入EDTA(0.5M，AMBION公司)2μL。反应后置于磁力架上，去掉上清，用高盐洗液洗涤一次，低盐洗液洗涤两次，用100μL的低盐洗液重悬磁珠。
16、粘性末端补平及3’端加A：按如下表21的配方配制体系。
表21
反应成分 体积(μL)
无酶纯水 0.8
5×Klex NTA mix 26
Klenow片段(3′→5′exo-)(5U/μL，NEB) 3.2
总共 30
将30μL反应液加入到上一步骤的磁珠重悬液中，混匀后37℃反应60min，加入EDTA(0.5M，AMBION公司)2μL。反应后置于磁力架上，去掉上清，低盐洗液洗涤三次，用70μL的低盐洗液重悬磁珠。
17、连接接头B(鼓泡接头)：
接头B由顶链L和底链S互补配对而成，其序列如下：
顶链L：/phos/AGTCGGAGGCCAAGCGTGCTTAGGACAT(SEQ ID NO：6)；
底链S：GTCCTAAGCACUGTAGTGTACGATCCGACTT(SEQ ID NO：7)。
按如下表22的配方配制体系。
表22
反应成分 体积(μL)
无酶纯水 21
3×连接缓冲液2 56.8
接头B(10μM) 20
T4连接酶(600U/μL，Enzymatics) 3.2
总共 100
将100μL反应液加入到上一步骤的磁珠重悬液中，混匀后室温反应30min，然后65℃反应10min。
18、USER酶切：加入1μL的USER酶(1U/μL，NEB)，混匀后37℃反应60min；然后加入EDTA(0.5M，AMBION)4.5μL。反应后置于磁力架上，去掉上清，低盐洗液洗涤三次，使用40μL的0.1M氢氧化钠将没有生物素标记的单链分离下来，加入酸性缓冲液中和获得的分离产物，中和后产物总体积60μL；有生物素标记的另一条链依旧结合在磁珠上。
19、聚合酶链式反应：
本实施例使用的引物F、R序列如下：
引物F：/bio/ATGTCCTAAGCACGCTTGGCC(SEQ ID NO：8)；
引物R：/phos/GTAGTGTACGATCCGACTT(SEQ ID NO：9)。
按如下表23的配方配制体系。
表23
反应成分 体积(μL)
上一步骤单链DNA 30
无酶纯水 160
2×PfuCx缓冲液 219.8
PfuCx热启动核酸聚合酶(2.5U/μL，Agilen) 8.2
引物F(20μM，生工) 11
引物R(20μM，生工) 11
总体积 440
混匀后按如下表24条件进行反应：
表24
Figure PCTCN2014092296-appb-000003
反应完成后使用400μL Ampure XP磁珠(Agencourt)进行纯化，80μL TE缓冲溶液溶解回收产物。
20、单链环化：核酸单链O利用相应互补序列，可将上一步骤产物两端连接起来。核酸单链O序列如下：
ATCGTACACTACATGTCCTAAGCA(SEQ ID NO：10)。
取100ng上一步骤的PCR产物，加入10μL核酸单链O(10μM，生工)，混匀后放置于95℃，3min；然后迅速放于冰上冷却。按如下表25的配方配制体系。
表25
反应成分 体积(μL)
无酶纯水 36.4
10×TA缓冲液(epicentre公司) 12
100mM三磷酸腺苷(epicentre公司) 1.2
T4连接酶(600U/μL，enzymatics) 0.4
总共 50
将50μL反应液加入到PCR产物和单链O的混合液中，混匀后37℃反应60min。
21、线性DNA消化：按如下表26的配方配制体系。
表26
反应成分 体积(μL)
无酶纯水 2
10×TA缓冲液(epicentre公司) 0.8
核酸外切酶1(20U/μL，NEB公司) 3.9
核酸外切酶3(100U/μL，NEB公司) 1.3
总共 8
将8μL反应液加入到上一步骤的连接反应液中，混匀后37℃反应30min；加入EDTA(0.5M)6μL。然后，使用170μL的PEG32磁珠纯化回收，55μL的TE缓冲液回溶。
本实施例四个平行实验的最终产物浓度和总量情况如下表27所示。电泳结果见图6。
表27
样品名称 浓度(ng/μL) 总量(ng)
C22 0.33 18.33
D22 0.32 17.87
E22 0.32 17.87
F22 0.31 17.15
从上表可以看出，文库的浓度、总量，足以满足后续测序对于文库量的要求；同时电泳(图6)和使用LabChip GX仪器(全自动微流体电泳仪，Caliper公司)测试的结果(图7-10)显示：聚合酶链式反应后的DNA文库的条带集中，片段大小为200bp-300bp之间，电泳条带集中，主峰突出，符合后续测序对于片段范围的要求。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换。

Claims (12)

  1. 一种核酸的双接头单链环状文库的构建方法，包括如下步骤：
    将核酸打断成用于文库构建的核酸片段；
    在所述核酸片段的两端连接第一接头序列；
    通过第一PCR扩增得到两端具有所述第一接头序列的第一产物，其中所述第一PCR使用的引物序列上具有U碱基位点且具有或不具有切口酶识别序列，并且其中一条引物序列上具有第一亲和标记；
    使用USER酶酶切所述第一产物，产生粘性末端且产生或不产生缺口；
    对所述酶切后的第一产物进行环化，产生环状核酸分子；
    使用去磷酸化酶处理双链上均具有缺口的环状核酸分子，或者使用切口酶处理一条链上具有切口酶识别序列且另一条链上具有缺口或双链均具有切口酶识别序列且均不具有缺口的环状核酸分子以产生切口；
    使用带有第二亲和标记的固相载体与所述环状核酸分子结合；
    以结合到所述固相载体上的环状核酸分子为模板，从所述切口和/或缺口处开始进行限制性缺口平移反应；
    消化除去所述环状核酸分子上的未发生限制性缺口平移反应的部分，得到线性核酸分子；
    在所述线性核酸分子的两端连接第二接头序列；
    通过第二PCR扩增得到两端具有所述第二接头序列的第二产物；
    对所述第二产物进行变性得到单链核酸分子，并使用与其中一条单链核酸分子两端均互补的介导序列对所述单链核酸分子进行环化，得到双接头单链环状文库。
  2. 根据权利要求1所述的方法，其特征在于，所述第一亲和标记为生物素标记；所述第二亲和标记为链霉亲和素标记。
  3. 根据权利要求1所述的方法，其特征在于，所述第一接头序列包括第一5’接头序列和第一3’L型接头序列，分别连接所述片段每条链的3’端和5’端；所述第一5’接头序列包括一条5’端磷酸化的长链和一条互补的短链，所述短链的3’末端双脱氧修饰，并且所述短链中包含U碱基位点；所述第一3’L型接头序列在邻近连接的片段的部分与所述第一5’接头序列有部分碱基互补；
    在所述核酸片段的两端连接第一接头序列，具体包括：
    对所述核酸片段进行去磷酸化；
    对去磷酸化后的核酸片段进行末端修复；
    在所述核酸片段的每条链的3’端连接所述第一5’接头序列；
    使用USER酶酶切所述第一5’接头序列的短链的U碱基位点；
    对USER酶酶切后的核酸片段进行磷酸化处理；
    在所述磷酸化处理后的核酸片段每条链的5’端连接所述第一3’L型接头序列。
  4. 根据权利要求1所述的方法，其特征在于，所述第一PCR使用的引物序列上均具有一个U碱基位点和切口酶识别序列；使用USER酶酶切U碱基位点后，在核酸片段两端形成粘性末端，所述粘性末端互补发生环化，产生环状核酸分子；再使用切口酶酶切所述切口酶识别序列产生切口。
  5. 根据权利要求1所述的方法，其特征在于，所述第一PCR使用的引物序列中有一条引物序列具有两个U碱基位点，另一条引物具有一个U碱基位点；使用USER酶酶切U碱基位点后，在核酸片段两端形成粘性末端，所述粘性末端互补发生环化，产生环状核酸分子。
  6. 根据权利要求1所述的方法，其特征在于，对所述酶切后的第一产物进行环化之后，还包括：对未环化的核酸分子进行消化。
  7. 根据权利要求1所述的方法，其特征在于，在所述限制性缺口平移反应中，通过控制dNTP与作为模板的核酸分子的摩尔比、酶反应温度和时间中至少一个因素来控制生成的缺口平移片段的长度。
  8. 根据权利要求1所述的方法，其特征在于，所述消化除去所述环状核酸分子上的未发生限制性缺口平移反应的部分，具体包括：首先使用双链外切酶降解，直到两端的缺口相遇；然后使用单链外切酶降解单链；或使用核酸内切酶直接切掉所述环状核酸分子上的未发生限制性缺口平移反应的部分。
  9. 根据权利要求1所述的方法，其特征在于，所述第二接头序列为鼓泡接头序列，所述鼓泡接头序列包括两条两端部分互补配对但中间一段不互补配对的碱基序列，其中所述中间一段形成鼓泡状，并且所述中间一段带有U碱基位点；所述鼓泡接头序列的一条链的5’端有一个突出的T碱基；
    在所述线性核酸分子的两端连接第二接头序列，具体包括：
    对所述线性核酸分子进行末端修复和3’端加A碱基的反应；
    通过所述T碱基与所述A碱基的配对，将所述鼓泡接头序列连接到所述线 性核酸分子两端；
    使用USER酶酶切所述中间一段上的U碱基位点。
  10. 根据权利要求1所述的方法，其特征在于，对所述单链核酸分子进行环化之后，还包括：对未环化的单链核酸分子进行消化。
  11. 一种核酸的双接头单链环状文库的构建试剂，包括如下组成部分：
    第一接头序列，所述第一接头序列包括第一5’接头序列和第一3’L型接头序列，分别连接片段每条链的3’端和5’端；所述第一5’接头序列包括一条5’端磷酸化的长链和一条互补的短链，所述短链的3’末端双脱氧修饰，并且所述短链中包含U碱基位点；所述第一3’L型接头序列在邻近连接的片段的部分与所述第一5’接头序列有部分碱基互补；
    第一PCR引物，具有U碱基位点且具有或不具有切口酶识别序列，并且其中一条引物序列上具有第一亲和标记，用于通过第一PCR扩增得到两端具有所述第一接头序列的第一产物；
    USER酶，用于酶切所述第一产物，产生粘性末端且产生或不产生缺口；
    环化酶，用于对所述酶切后的第一产物进行环化，产生环状核酸分子；
    去磷酸化酶，用于对双链上均具有缺口的环状核酸分子进行去磷酸化酶处理；或者切口酶，用于对一条链上具有切口酶识别序列且另一条链上具有缺口或双链均具有切口酶识别序列且均不具有缺口的环状核酸分子进行酶切，以产生切口；
    固相载体，带有第二亲和标记，用于与所述环状核酸分子结合；
    缺口平移反应的组分，用于以结合到所述固相载体上的环状核酸分子为模板，从所述切口和/或缺口处开始进行限制性缺口平移反应；
    消化酶，用于消化除去所述环状核酸分子上的未发生限制性缺口平移反应的部分，得到线性核酸分子；
    第二接头序列，所述第二接头序列为鼓泡接头序列，所述鼓泡接头序列包括两条两端部分互补配对但中间一段不互补配对的碱基序列，其中所述中间一段形成鼓泡状，并且所述中间一段带有U碱基位点；所述鼓泡接头序列的一条链的5’端有一个突出的T碱基；
    第二PCR引物，用于通过第二PCR扩增得到两端具有所述第二接头序列的第二产物；
    介导序列，与所述第二产物变性后得到单链核酸分子中的一条单链核酸分子两端均互补，用于对所述单链核酸分子进行环化，得到双接头单链环状文库。
  12. 根据权利要求11所述的试剂，其特征在于，所述第一亲和标记为生物素标记；所述第二亲和标记为链霉亲和素标记。
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018121634A1 (zh) * 2016-12-30 2018-07-05 安诺优达基因科技(北京)有限公司 用于dna片段的非特异性复制的方法及试剂盒
CN111455469A (zh) * 2020-04-07 2020-07-28 深圳易倍科华生物科技有限公司 一种单链快速建库方法及建库仪器
CN112226821A (zh) * 2020-10-16 2021-01-15 鲲羽生物科技(江门)有限公司 一种基于双链环化的mgi测序平台测序文库的构建方法
WO2021023123A1 (zh) * 2019-08-02 2021-02-11 北京贝瑞和康生物技术有限公司 一种非特异性扩增天然短片段核酸的方法和试剂盒
CN113736850A (zh) * 2021-08-13 2021-12-03 纳昂达(南京)生物科技有限公司 基于双链环化的文库构建方法及其在测序中的应用
WO2023109887A1 (zh) * 2021-12-15 2023-06-22 南京金斯瑞生物科技有限公司 一种整合位点的检测方法
WO2024138517A1 (zh) * 2022-12-29 2024-07-04 深圳华大生命科学研究院 提升测序通量的文库接头设计

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107124888B (zh) 2014-11-21 2021-08-06 深圳华大智造科技股份有限公司 鼓泡状接头元件和使用其构建测序文库的方法
CN107002292B (zh) * 2014-11-26 2019-03-26 深圳华大智造科技有限公司 一种核酸的双接头单链环状文库的构建方法和试剂
CN106554957B (zh) * 2015-09-30 2020-04-21 中国农业科学院深圳农业基因组研究所 测序文库及其制备和应用
CN111315895A (zh) * 2017-09-14 2020-06-19 豪夫迈·罗氏有限公司 用于产生环状单链dna文库的新型方法
CN109957492A (zh) * 2017-12-26 2019-07-02 安诺优达基因科技(北京)有限公司 一种用于二代测序dna文库构建的自动化液体处理工作站
CN110734967B (zh) * 2018-07-19 2023-02-17 深圳华大智造科技股份有限公司 一种接头组合物及其应用
CN110791813B (zh) * 2018-08-01 2023-06-16 广州华大基因医学检验所有限公司 对单链dna进行处理的方法及应用
WO2020061903A1 (zh) * 2018-09-27 2020-04-02 深圳华大生命科学研究院 测序文库的构建方法和得到的测序文库及测序方法
CN111074353B (zh) * 2018-10-18 2023-10-13 深圳华大智造科技股份有限公司 全基因组甲基化文库单链建库方法和得到的全基因组甲基化文库
CN112795620B (zh) * 2019-11-13 2024-08-13 深圳华大基因股份有限公司 双链核酸环化方法、甲基化测序文库构建方法和试剂盒
CN114807123B (zh) * 2021-01-29 2023-12-01 深圳华大基因科技服务有限公司 一种dna分子的扩增引物设计和连接方法
CN113832549A (zh) * 2021-11-03 2021-12-24 纳昂达(南京)生物科技有限公司 低频引入突变的酶切打断建库方法和试剂盒
CN114736951A (zh) * 2022-04-20 2022-07-12 深圳大学 一种小分子rna的高通量测序文库构建方法
WO2024138572A1 (zh) * 2022-12-29 2024-07-04 深圳华大生命科学研究院 测序接头、测序接头复合物、靶核酸序列多次纳米孔测序的方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001020039A2 (en) * 1999-09-16 2001-03-22 Curagen Corporation Method of sequencing a nucleic acid
CN102016068A (zh) * 2008-01-09 2011-04-13 生命科技公司 制备用于核酸测序的配对标签文库的方法
CN102534811A (zh) * 2010-12-16 2012-07-04 深圳华大基因科技有限公司 一种dna文库及其制备方法、一种dna测序方法和装置
CN102628079A (zh) * 2012-03-31 2012-08-08 盛司潼 一种通过环化方式构建测序文库的方法
CN103103624A (zh) * 2011-11-15 2013-05-15 深圳华大基因科技有限公司 高通量测序文库的构建方法及其应用
CN103119162A (zh) * 2010-09-02 2013-05-22 学校法人久留米大学 用于产生由单分子dna形成的环状dna的方法
CN103290106A (zh) * 2007-12-05 2013-09-11 考利达基因组股份有限公司 测序反应中碱基的有效确定
CN103806111A (zh) * 2012-11-15 2014-05-21 深圳华大基因科技有限公司 高通量测序文库的构建方法及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2750879C (en) * 2009-01-30 2018-05-22 Oxford Nanopore Technologies Limited Adaptors for nucleic acid constructs in transmembrane sequencing
JP6017458B2 (ja) * 2011-02-02 2016-11-02 ユニヴァーシティ・オブ・ワシントン・スルー・イッツ・センター・フォー・コマーシャリゼーション 大量並列連続性マッピング
CN107002292B (zh) * 2014-11-26 2019-03-26 深圳华大智造科技有限公司 一种核酸的双接头单链环状文库的构建方法和试剂

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001020039A2 (en) * 1999-09-16 2001-03-22 Curagen Corporation Method of sequencing a nucleic acid
CN103290106A (zh) * 2007-12-05 2013-09-11 考利达基因组股份有限公司 测序反应中碱基的有效确定
CN102016068A (zh) * 2008-01-09 2011-04-13 生命科技公司 制备用于核酸测序的配对标签文库的方法
CN103119162A (zh) * 2010-09-02 2013-05-22 学校法人久留米大学 用于产生由单分子dna形成的环状dna的方法
CN102534811A (zh) * 2010-12-16 2012-07-04 深圳华大基因科技有限公司 一种dna文库及其制备方法、一种dna测序方法和装置
CN103103624A (zh) * 2011-11-15 2013-05-15 深圳华大基因科技有限公司 高通量测序文库的构建方法及其应用
CN102628079A (zh) * 2012-03-31 2012-08-08 盛司潼 一种通过环化方式构建测序文库的方法
CN103806111A (zh) * 2012-11-15 2014-05-21 深圳华大基因科技有限公司 高通量测序文库的构建方法及其应用

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018121634A1 (zh) * 2016-12-30 2018-07-05 安诺优达基因科技(北京)有限公司 用于dna片段的非特异性复制的方法及试剂盒
WO2021023123A1 (zh) * 2019-08-02 2021-02-11 北京贝瑞和康生物技术有限公司 一种非特异性扩增天然短片段核酸的方法和试剂盒
CN111455469A (zh) * 2020-04-07 2020-07-28 深圳易倍科华生物科技有限公司 一种单链快速建库方法及建库仪器
CN111455469B (zh) * 2020-04-07 2023-08-18 深圳易倍科华生物科技有限公司 一种单链快速建库方法及建库仪器
CN112226821A (zh) * 2020-10-16 2021-01-15 鲲羽生物科技(江门)有限公司 一种基于双链环化的mgi测序平台测序文库的构建方法
CN112226821B (zh) * 2020-10-16 2024-02-06 鲲羽生物科技(江门)有限公司 一种基于双链环化的mgi测序平台测序文库的构建方法
CN113736850A (zh) * 2021-08-13 2021-12-03 纳昂达(南京)生物科技有限公司 基于双链环化的文库构建方法及其在测序中的应用
WO2023109887A1 (zh) * 2021-12-15 2023-06-22 南京金斯瑞生物科技有限公司 一种整合位点的检测方法
WO2024138517A1 (zh) * 2022-12-29 2024-07-04 深圳华大生命科学研究院 提升测序通量的文库接头设计

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