WO2016082130A1 - 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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WO2016082130A1
WO2016082130A1 PCT/CN2014/092297 CN2014092297W WO2016082130A1 WO 2016082130 A1 WO2016082130 A1 WO 2016082130A1 CN 2014092297 W CN2014092297 W CN 2014092297W WO 2016082130 A1 WO2016082130 A1 WO 2016082130A1
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nucleic acid
sequence
linker sequence
linker
acid molecule
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PCT/CN2014/092297
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English (en)
French (fr)
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江媛
赵霞
阿莱克谢耶夫•安德烈
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 EP14907081.5A priority Critical patent/EP3225721B1/en
Priority to CN201480082966.2A priority patent/CN107002291B/zh
Priority to US15/529,867 priority patent/US20170349893A1/en
Priority to PCT/CN2014/092297 priority patent/WO2016082130A1/zh
Publication of WO2016082130A1 publication Critical patent/WO2016082130A1/zh

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C40B40/04—Libraries containing only organic compounds
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    • 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

Definitions

  • the library insert of the CG sequencing platform is too small (2 ⁇ 19-35 bp), which makes subsequent genome assembly work difficult, which limits the application of the platform in the direction of genome de novo sequencing.
  • the library construction cycle is too long, which also seriously hinders the progress of scientific research and projects, coupled with the challenges of the rapid development of various next-generation sequencing technologies, improving library insert size and simplifying the database construction process through technological improvement and innovation. It is an urgent and important research and development task to shorten the application time and expand the application scope and energy efficiency of the CG sequencing platform.
  • the insertion method of the existing double-linker single-stranded circular library construction process of the CG platform greatly limits the length of the library insert, and the NT (Nick Translation) technique used by the SOLiD (Sequencing by Oligonucleotide Ligation and Detection) platform.
  • the length of the library insert is increased, but there is a disadvantage that the insert span is large, and the fragment selection is required by the method of gel extraction, which increases the cumbersome operation, and the recovery of the gelation affects the recovery efficiency of the insert.
  • sequencing linker of the existing double-linker single-stranded circular library construction method of CG platform needs to be subjected to multi-step enzyme reaction and purification to be connected to the genomic DNA fragment to be tested, which is time-consuming, reagent-consuming and has large sample loss, which limits the sample.
  • the speed of processing, and the initial amount of sample (3 ⁇ g) is not advantageous over other sequencing platforms.
  • the invention provides a method and a reagent for constructing a double-linker single-stranded circular library of nucleic acid, which method can
  • the length of the library insert can be increased, and no gel recovery is required; the single-stranded nucleic acid molecule can be directly cyclized after thermal denaturation.
  • a first product having the 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;
  • the first product is digested with the USER enzyme to form a cohesive end and then cyclized to generate a gap; or the primer sequence used in the first PCR further has a nickase recognition sequence, and the first product is digested with the USER enzyme. Cyclization is carried out after forming a sticky end, and then the nicking product is cleaved using a nicking enzyme to produce an incision;
  • a second product having the 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 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;
  • a 5' linker sequence comprises a 5'-terminally phosphorylated long chain and a complementary short strand, the middle portion of the long chain having a tag sequence, the 3' end of the short strand being dideoxy modified, and the short a U base site is included in the strand;
  • the first 3 'L linker sequence is partially complementary to the first 5' linker sequence in a portion adjacent to the ligated fragment;
  • 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 nickase recognition sequence and a U base site; after cleavage of the U base site by the USER enzyme, the nucleic acid fragment is formed at both ends of the nucleic acid fragment. A cohesive end that is cyclized to complement a circular nucleic acid molecule.
  • one primer sequence used in the first PCR has two U base sites, and the other primer has one U base site; the U base position is digested with USER enzyme. After the point, sticky ends are formed at both ends of the nucleic acid fragment, and the sticky ends are complementary to cyclization to produce a circular nucleic acid molecule.
  • the method further comprises: digesting the uncircularized nucleic acid molecule.
  • one of the primer sequences used in the first PCR has a biotin label; before the restriction nick/gap translation reaction, the streptavidin-labeled magnetic beads and the first The products of one PCR are combined such that subsequent reactions are carried out on the magnetic beads.
  • 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 a time. .
  • the digestion removes a portion of the circular nucleic acid molecule that does not undergo a restriction nick/gap translation reaction, and specifically includes: firstly using an enzyme having 5'-3' exonuclease activity to degrade, Until the gaps at both ends meet; then the single strand is degraded using an enzyme with 3'-5' exonuclease activity or a single-stranded exonuclease.
  • the second linker sequence comprises a second 5' linker sequence and a second 3'L type linker sequence, respectively connecting the 3' end and the 5' end of each strand of the linear nucleic acid molecule;
  • the second 5' linker sequence comprises a 5'-terminally phosphorylated long chain 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 second 3'L-type linker sequence is partially complementary to the second 5' linker sequence at a portion of the adjacent ligated fragment;
  • Connecting the second linker sequence at both ends of the linear nucleic acid molecule specifically includes:
  • the short base U base site of the second 5' linker sequence is digested with the USER enzyme
  • the second 3'-L linker sequence is ligated at the 5' end of each strand of the linearized nucleic acid molecule after the phosphorylation treatment.
  • the method further comprises: digesting the uncircularized single-stranded nucleic acid molecule.
  • a first adaptor sequence comprising a first 5' linker sequence and a first 3'L type linker sequence, respectively joining the 3' end and the 5' end of each strand of the fragment;
  • the first 5' linker The sequence comprises a 5'-terminally phosphorylated long chain and a complementary short chain, the middle portion of the long chain having a tag sequence, the 3' end of the short chain being dideoxy modified, and the short chain comprising U a base site;
  • the first 3'L-type linker sequence is partially complementary to the first 5' linker sequence in a portion adjacent to the ligated fragment;
  • a first PCR primer having a U base site or having a nickase recognition sequence and a U base site for obtaining a first product having the first linker sequence at both ends by first PCR amplification;
  • a component of the nick translation reaction for performing a restriction nick/gap translation reaction from the nick or gap using the circular nucleic acid molecule 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/nick translation reaction to obtain a linear nucleic acid molecule
  • a second linker sequence comprising a second 5' linker sequence and a second 3'L type linker sequence, respectively ligated to the 3' end and the 5' end of each strand of the linear nucleic acid molecule;
  • the linker sequence comprises a 5'-phosphorylated long chain 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 second 3 The 'L-type linker sequence is partially complementary to the second 5' linker sequence at a portion of the adjacent ligated fragment;
  • a second PCR primer for obtaining a second product having the second linker sequence at both ends by second PCR amplification
  • a method for constructing a double-linker single-stranded circular library of the nucleic acid of the present invention by introducing a nicking enzyme recognition sequence and/or a U-base site on a primer sequence of the first PCR, and then incision or nicking by restriction enzyme digestion
  • the nick/gap translation reaction generates a fragment of a certain length, and the length of the fragment of the library is increased while controlling the length of the fragment to a certain extent, so that the selected fragment is not required to be recovered by gelation; the finally formed single-stranded nucleic acid molecule can be directly circularized after thermal denaturation. No filtering is required, thus simplifying the database building process.
  • the connection is performed using a special L-type connector sequence, and the steps are simple and the cycle is shortened.
  • FIG. 1 is a flow chart showing 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 basic schematic diagram of CNT notch generation according to an embodiment of the present invention.
  • FIG. 3 is a basic schematic diagram of the CNT cut 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.
  • FIG. 6 is a schematic diagram showing a strategy for constructing a CNT on beads on a magnetic bead according to an embodiment of the present invention
  • Figure 7 is a graph showing the results of detection of PCR products using Agilent 2100 after addition of linker B in one embodiment of the present invention.
  • 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; the USER enzyme is used to cleave the U base site to generate a gap, and the product after digestion with the USER enzyme is cyclized to generate a circular nucleic acid molecule; the restriction gap is started from the gap Controlled Nick Translation (CNT) uses a 5'-3' circumcision The enzymatic activity of the enzyme is degraded until the gaps at both ends meet, and then the single strand is degraded using an enzyme having a 3'-5'
  • CNT Controlled Nick Translation
  • a U-base site is introduced into the primer of the first PCR, and a gap is generated by USER enzyme digestion, as a restriction gap translation. The starting point of the reaction.
  • the basic principle of the generation of the gap is shown in Figure 2: (1) After the 5' linker A sequence and the 3'L type linker A sequence are ligated, the linker A ligated product is amplified with primers carrying 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 3' end and the phosphorylated 5' end; (4) dephosphorylation, dephosphorylation of the 3' end of gap 1 to provide an effective starting site for CNTs.
  • a slit can also be produced using the principle shown in Fig. 3 as a starting point for the translational reaction of the restriction nick.
  • a class III endonuclease recognition sequence is introduced into the sequence of the linker A.
  • the linker A sequence is ligated and cyclized, the double-stranded DNA is linearized by class III endonuclease digestion; and the method of the present invention
  • a nickase recognition sequence is introduced into the sequence of the linker A.
  • a nicking enzyme such as Nb.BsrDI, Nb.BsmI, Nt.BbvCI, Nb.Bbv.Nb.BtsI or Nt.BstNBI, etc.
  • the single strand of the circularized DNA is cleaved to create a nick on each single strand to provide an effective starting site for the CNT.
  • the reaction starting from the slit or the notch is called "restriction nick/gap 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 improve the nick translation reaction. Technical operability.
  • 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 uses a nicking enzyme to open a nick on each of the two strands of the linker A, and then uses the nicking function of the polymerase in the presence of dNTPs to extend the nick from the region of the linker A to the side of the linker A, by controlling dNTPs and template DNA.
  • Molar ratio, reaction temperature and reaction time, etc., flexible control of the extension length of the incision, the size of the extension fragment can be controlled within the range of 50-250 bp, followed by two-step exonuclease digestion reaction, the non-joint A
  • the DNA fragment of interest is digested, and the remaining DNA fragment containing the linker A is ligated by a linker B and amplified without a biotin-labeled primer, and then double-stranded DNA is cleaved by simple high temperature denaturation.
  • the sequence mediates the cyclization of the single strand of interest, ie, the single stranded DNA of interest can be isolated and cyclized.
  • 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, and the cost of building the library (sequence cost, enzyme reaction reagent cost, purification cost) High, long cycle, and large sample loss do not meet the requirements for 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 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 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.
  • one of the primer sequences used in the first PCR amplification has a biotin label; the streptavidin-labeled magnetic beads are used prior to the restriction nick/gap translation reaction. Binding to the product of the first PCR amplification allows subsequent reactions to be performed on the magnetic beads.
  • FIG. 6 for explaining the on beads reaction of the present invention: the product of the first PCR amplification has a biotin label on one strand, and the product after cyclization can be combined with streptavidin magnetic beads. Combine. From step 8 to step 14, each step of the enzymatic reaction is carried out on the streptavidin magnetic beads.
  • the one-step enzymatic reaction replaces the purification reaction carried out in each step of the conventional enzyme-building method, which saves the purification reagent, and the magnetic bead washing method has a shorter operation time than the conventional purification method.
  • 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.
  • the ligation reaction system was formulated as shown in Table 5 below.
  • 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 Total 500
  • 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: CGTTCTCGACUCAGCAGT (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.
  • This step completes the ligation of the nucleic acid fragment of interest to linker A.
  • the total amount and yield of the products before and after the connection are shown in Table 8.
  • the primer 1 sequence is as follows:
  • the primer 2 sequence is as follows:
  • 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 13 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.
  • Binding streptavidin magnetic beads Magnetic bead binding and washing buffer were prepared according to the systems shown in Tables 17-19.
  • reaction system 60 ⁇ L was quickly added to the pre-cooled magnetic beads, quickly mixed well, and then incubated at 8 ° C for 15 min.
  • 35 ⁇ L of the reaction system was quickly added to the previous step magnetic beads, quickly mixed well, and then incubated at 37 ° C for 30 min. Add 0.8 ⁇ L of 0.5 M EDTA and mix well.
  • the magnetic beads were washed once with 350 ⁇ L of HSWB and 350 ⁇ L of LSWB, respectively, and the magnetic beads were equilibrated with 200 ⁇ L of 1 ⁇ Exo VII reaction buffer (containing 0.025% Tween-20), and the supernatant was completely discarded.
  • linker B sequence ligation on beads reaction The sequence of linker B used in this example is as follows:
  • 5' linker B sequence /5Phos/AAGTCGGAGGCCAAGCGTGCTTAGGA (SEQ ID NO: 6);
  • the 5' linker B mixture (10 ⁇ M) was prepared according to the system shown in Table 25 below.
  • the ligation reaction system was prepared according to the system shown in Table 26 below.
  • reaction system 78 ⁇ L of the reaction system was added to the previous step magnetic beads, mixed well, and 2 ⁇ L of T4 DNA ligase (fast) (600 U/ ⁇ L) was quickly added and mixed well, then incubated at 25 ° C for 60 min, incubated at 65 ° C for 10 min, and cooled to 4 ° C. 1.5 ⁇ L of T4 polynucleotide kinase (10 U/ ⁇ L) was added, mixed well and incubated at 37 ° C for 20 min. Add 2 ⁇ L of 0.5M EDTA and mix well.
  • the magnetic beads were washed once with 350 ⁇ L of HSWB and 350 ⁇ L of LSWB, respectively, and the magnetic beads were equilibrated with 200 ⁇ L of 1 ⁇ NEBuffer 2 reaction buffer (containing 0.025% Tween-20), and the supernatant was completely discarded.
  • 3' L-type linker B sequence ligation on beads reaction 3' L-type linker B sequence used in this example: /5Phos/CATGTAGTGTACGATCCGACTT (SEQ ID NO: 8).
  • the 3' L type linker reaction system was prepared in accordance with the system shown in Table 27.
  • reaction system 80 ⁇ L of the reaction system was added to the upper magnetic beads, thoroughly mixed, incubated at 25 ° C for 60 min, and cooled to 14 ° C at a rate of 0.1 ° C per second. Add 2 ⁇ L of 0.5M EDTA and mix well. The magnetic beads were washed twice with 350 ⁇ L of HSWB and 350 ⁇ L of LSWB, respectively, and the supernatant was completely discarded.
  • Primer 1 sequence /5Phos/CATGTAGTGTACGATCCGACTT (SEQ ID NO: 9);
  • Primer 2 sequence TCCTAAGCACGCTTGGCCT (SEQ ID NO: 10).
  • the PCR system was prepared according to the system shown in Table 28.
  • the product was recovered in 30 ⁇ L of the above step, added to the above system, and after mixing, the reaction was carried out under the conditions shown in Table 29.
  • the mediated fragment has a corresponding complementary sequence for ligation of both ends of the single strand, the sequence of which is as follows: GTACACTACATGTCCTAAGCACGC (SEQ ID NO: 11).
  • reaction mixture 50 ⁇ L was added to the mixture of the PCR product and the mediated fragment, mixed, and incubated at 37 ° C for 60 min.
  • the concentration and total amount of the final product in this example are shown in Table 32.
  • Fig. 7 The results of detection of the ligated linker B-linked product after PCR using an Agilent 2100 capillary electrophoresis apparatus are shown in Fig. 7.
  • the abscissa indicates the size of the base pair and the ordinate indicates the intensity of the detected fluorescent signal. When the ordinate value is 0, in order to detect the baseline signal, the higher the value of the ordinate, the stronger the fluorescence signal of the detected sample.
  • 35 and 1030 correspond to DNA standards of 35 bp and 1030 bp, which are reference materials for detecting the size of a sample fragment.
  • 218 and 219 in the figure are the sizes of the main bands of product 1 and product 2, namely 218 bp and 219 bp, respectively.
  • the rectangular strip on the right side shows the distribution of the strips of the segment to be tested simulated according to the detection signal. From the results, the resulting target strips (indicated by the arrows) are very concentrated, indicating that the method of CNT can effectively control the inserts to a very narrow 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；用USER酶酶切第一产物，环化产生缺口；或引物序列上还具有切口酶识别序列，用USER酶酶切第一产物，环化并使用切口酶酶切产生切口；从切口或缺口处进行限制性切口/缺口平移反应；消化除去未发生限制性切口/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，并使用介导序列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，并且无需切胶回收；单链核酸分子热变性后可以直接进行环化。

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
自从上世纪九十年代AB公司推出毛细管电泳测序仪，人类基因组计划启动之后，DNA(Deoxyribonucleic acid，脱氧核糖核酸)测序技术便开始以难以想象的速度高速发展。第二、第三代测序仪也相继推出，面向市场。在第二代测序平台中，Complete Genomics公司(以下简称CG)的Blackbird测序平台凭借测序准确性相对其它平台更高(99.9998％)、测序通量相对其它平台更大的优势，在分子诊断等临床研究领域的应用中占据巨大的市场。然而，由于样品处理方法的限制，CG测序平台的文库插入片段太小(2×19～35bp)，导致后续的基因组组装工作困难，限制了该平台在基因组de novo测序方向的应用。另外，文库构建周期太长，也严重阻滞了科研、项目的开展进度，加上各种新一代测序技术迅速兴起所面临的挑战，通过技术改良和创新提高文库插入片段大小、简化建库流程、缩短建库时间，以拓展CG测序平台的应用范围和能效是迫在眉睫的重要研发任务。
CG平台现有的双接头单链环状文库构建流程的插入片段引入方法极大地限制了文库插入片段长度，SOLiD(Sequencing by Oligonucleotide Ligation and Detection)平台使用的NT(Nick Translation，切口平移)技术，文库插入片段长度有所提高，但是存在插入片段跨度很大的缺点，需要通过切胶回收的方法来进行片段选择，增加了操作的繁琐性，并且切胶回收会影响插入片段的回收效率。
此外，CG平台现有双接头单链环状文库构建方法的测序接头需要经过多步骤的酶反应及纯化才能连接到待测基因组DNA片段上，耗时、耗试剂且样品损耗大，限制了样品处理的速度，并且样品起始量(3μg)相对其它测序平台不具优势。
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，并且无需切胶回收；单链核酸分子热变性后可以直接进行环化。
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；
用USER酶酶切所述第一产物，形成粘性末端后进行环化产生缺口；或所述第一PCR使用的引物序列上还具有切口酶识别序列，用USER酶酶切所述第一产物，形成粘性末端后进行环化，然后使用切口酶酶切环化产物产生切口；
以所述环状核酸分子为模板，从所述切口或缺口处开始进行限制性切口/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切口/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。
作为本发明的优选方案，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，所述长链的中间部分有一段标签序列，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。
作为本发明的优选方案，所述第一PCR使用的引物序列上均具有一个切口酶识别序列和一个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。
作为本发明的优选方案，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。
作为本发明的优选方案，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。
作为本发明的优选方案，所述第一PCR使用的引物序列中有一条引物序列具有生物素标记；在所述限制性切口/缺口平移反应之前，使用链霉亲和素标记的磁珠与第一PCR的产物结合，使得后续反应在所述磁珠上进行。
作为本发明的优选方案，在所述限制性切口/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，所述消化除去所述环状核酸分子上的未发生限制性切口/缺口平移反应的部分，具体包括：首先使用具5’-3’外切酶活性的酶降解，直到两端的缺口相遇；然后使用具3’-5’外切酶活性的酶或单链外切酶降解单链。
作为本发明的优选方案，所述第二接头序列包括第二5’接头序列和第二3’L型接头序列，分别连接所述线性核酸分子每条链的3’端和5’端；所述第二5’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；所述第二3’L型接头序列在邻近连接的片段的部分与所述第二5’接头序列有部分碱基互补；
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去磷酸化；
对去磷酸化后的线性核酸分子进行末端修复；
在所述线性核酸分子的每条链的3’端连接所述第二5’接头序列；
使用USER酶酶切所述第二5’接头序列的短链的U碱基位点；
对USER酶酶切后的片段进行磷酸化处理；
在所述磷酸化处理后的线性核酸分子每条链的5’端连接所述第二3’L型接头序列。
作为本发明的优选方案，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。
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，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，所述长链的中间部分有一段标签序列，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碱基位点或者具有切口酶识别序列和U碱基位点，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；
切口酶，用于酶切所述第一产物，产生切口；
USER酶，用于酶切所述第一产物，产生用于环化的粘性末端和缺口；
缺口平移反应的组分，用于以所述环状核酸分子为模板，从所述切口或缺口处开始进行限制性切口/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切口/缺口平移反应的部分，得到线性核酸分子；
第二接头序列，所述第二接头序列包括第二5’接头序列和第二3’L型接头序列，分别连接线性核酸分子每条链的3’端和5’端；所述第二5’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；所述第二3’L型接头序列在邻近连接的片段的部分与所述第二5’接头序列有部分碱基互补；
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。
本发明核酸的双接头单链环状文库的构建方法，通过在第一PCR的引物序列上引入切口酶识别序列和/或U碱基位点，经酶切后产生切口或缺口，通过限制性切口/缺口平移反应生成一定长度的片段，在提高文库插入片段长度的同时将片段长度控制在一定范围，因此无需切胶回收选择片段；最终形成的单链核酸分子热变性后可以直接进行环化，无需进行筛选操作，因此简化了建库过程。此外，使用特别的L型接头序列进行连接，步骤简单，周期缩短。
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流程图；
图2为本发明一个实施例的CNT缺口产生基本原理图；
图3为本发明一个实施例的CNT切口产生基本原理图；
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的磁珠上限制性切口/缺口平移反应(CNT on beads)建库策略示意图；
图7为本发明一个实施例中加完接头B后的PCR产物使用Agilent 2100检测结果图。
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。
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；使用USER酶酶切U碱基位点产生缺口，并对USER酶酶切后的产物进行环化，产生环状核酸分子；从缺口处开始进行限制性缺口平移反应(Controlled Nick Translation，CNT)使用具5’-3’外切 酶活性的酶降解，直到两端的缺口相遇，然后使用具3’-5’外切酶活性的酶或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去磷酸化和末端修复；连接5’接头B序列，磷酸化后再连接3’L型接头B序列；PCR扩增得到两端具有5’接头B序列和3’L型接头B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2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，使缺口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，还可以采用图3所示的原理产生切口，作为限制性切口平移反应的起始点。现有方法是在接头A序列中引入III类内切酶识别序列，接头A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；而本发明的方法在接头A序列中引入切口酶识别序列，接头A序列连接并环化后，采用切口酶(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，从切口或缺口处开始进行的反应，称为“限制性切口/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(主要是链霉亲和素磁珠)价格昂贵。本发明方法用切口酶在接头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，后续再通过两步外切酶消化反应，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。
在本发明的一个优选实施例中，第一次PCR扩增使用的引物序列中有一条引物序列具有生物素标记；在限制性切口/缺口平移反应之前，使用链霉亲和素标记的磁珠与第一次PCR扩增的产物结合，使得后续反应在所述磁珠上进行。请参考图6，对本发明的磁珠上(on beads)反应进行说明：第一次PCR扩增的产物一条链上带有生物素标记，环化之后的产物可与链霉亲和素磁珠结合。从步骤8到步骤14，每步酶反应都是在链霉亲和素磁珠上进行，反应之后只需要用磁力架吸附磁珠，去除反应液后再洗涤磁珠上的杂质即可进行下一步酶反应，替代了常规建库方法中每一步酶反应都要进行的纯化反应，节省了纯化试剂，且磁珠洗涤法相比常规纯化法的操作时间短。
以上分析可知，在本发明一个优选实施例中，通过独特的CNT技术、L型 接头连接技术以及简单的热变性后直接单链环化技术，成功实现对CG测序平台双接头建库法的文库构建流程的进行改进和优化，使文库插入片段大小提高至原来的2-10倍，整个建库周期和成本降低了约40％，建库起始量从3μg降低至500ng。
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
25mM脱氧核糖核苷三磷酸 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/ACTGCTGACGTACTGTGTCATAAATAGCACGAGACGTTCTCGACT/3ddC/(SEQ ID NO：1)；
5’封闭序列：TACGUCAGCAG/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
T4DNA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：CGTTCTCGACUCAGCAGT(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
T4DNA连接酶(快速)(600U/μL) 1.2
总共 48
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。
此步骤完成了目的核酸片段与接头A的连接。连接前后产物总量与得率如表8所示。
表8
  浓度(ng/μL) 总量(ng) 得率
产物1 5.95 178.5 59.5％
产物2 6.11 183.3 61.1％
产物3 5.92 177.6 59.2％
8、聚合酶链式反应：
引物1序列如下：
GTCGAGAACGUCTCG/iBiodT/GCTATTTATGACACAGTACGUCAGCAG(SEQ ID NO：4)；
引物2序列如下：
ACGTTCTCGACUCAGCAG(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 PCTCN2014092297-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 dsDNAHS分析试剂盒(Invitrogen公司)定量产物浓度(表11)。取2μg产物进行下一步反应。
表11
  浓度(ng/μL) 总量(ng) PCR扩增效率
产物1 57.4 4592 1.59
产物2 52 4160 1.57
9、去尿嘧啶：配制以下表12所示的反应液。
表12
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13所示的反应体系1。
表13
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14所示的反应体系2。
表14
反应成分 体积(μL)
无酶纯水 98
20×Circ缓冲液 100
T4DNA连接酶(快速)(600U/μL) 2
总体积 200
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 PCTCN2014092297-appb-000002
Figure PCTCN2014092297-appb-000003
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反应的起始位点为缺口型。
12、去磷酸化处理：配制以下表16所示的反应体系。
表16
反应成分 体积(μL)
无酶纯水 39
10×NEBuffer 3 14
Endo IV(10U/μL) 7
总体积 60
将80μL(700ng)上步骤产物加入反应体系中，混匀后置于37℃孵育1h。
用210μL Ampure XP磁珠纯化，使用55μL TE缓冲液溶解回收产物。用Qubit dsDNA HS分析试剂盒(Invitrogen公司)定量产物浓度。取500ng产物进行下一步反应。
13、结合链霉亲和素磁珠：按照表17～19所示的体系配制磁珠结合及清洗缓冲液。
表17 2×LSBB(Low Salt Bead Bindingbuffer，低盐磁珠结合缓冲液)
组分 含量
三羟甲基氨基甲烷(pH7.5) 100mM
氯化钠 300mM
吐温-20 0.005％
表18 LSWB(Low Salt Bead Wash buffer，低盐磁珠洗涤缓冲液)
组分 含量
三羟甲基氨基甲烷(pH7.5) 50mM
氯化钠 150mM
吐温-20 0.05％
表19 HSWB(High Salt Bead Wash buffer，高盐磁珠洗涤缓冲液)
组分 含量
三羟甲基氨基甲烷(pH7.5) 50mM
氯化钠 500mM
吐温-20 0.05％
取75μL MyOne Streptavidin C1磁珠，吸弃上清，用350μL 1×LSBB清洗磁珠后用100μL 2×LSBB重悬磁珠，加入100μL用无酶纯水稀释的去磷酸化环化DNA(570ng)。在每分30r的转速下，室温孵育磁珠和DNA的混合液1h。
分别用350μL HSWB，350μL LSWB清洗磁珠一次，再用200μL 1×NEBuffer2(含0.025％吐温-20)平衡磁珠，每次清洗之后都需要吸弃上清，最后一次平衡之后，需要将上清彻底吸干净。将磁珠放在冰上预冷至少10min。
14、CNT on beads反应：配制以下表20所示的反应体系。
表20
反应成分 体积(μL)
无酶纯水 51.2
10×NEBuffer2 6
2.5mM dNTP(1∶175) 2
PolI DNA聚合酶(10U/μL) 0.8
总体积 60
将60μL反应体系迅速加入预冷的磁珠中，快速充分混匀后置于8℃孵育15min。
加入1.5μL 0.5M EDTA，充分混匀。分别用350μL HSWB，350μL LSWB清洗磁珠一次，再用200μL 1×NEBuffer4(含0.025％吐温-20)平衡磁珠，彻底吸弃上清。
15、3’-5’外切酶消化on beads反应：配制以下表21所示的反应体系。
表21
反应成分 体积(μL)
无酶纯水 65.5
10×NEBuffer4 8
T7Exo(10U/μL) 6.5
总体积 80
将80μL反应体系迅速加入上步磁珠中，快速充分混匀后置于25℃孵育1h。加入2μL 0.5M EDTA，充分混匀。分别用350μL HSWB，350μL LSWB清洗磁珠一次，再用200μL 1×Exo VII反应缓冲液(含0.025％吐温-20)平衡磁珠，彻底吸弃上清。
16、单链消化on beads反应：配制以下表22所示的反应体系。
表22
反应成分 体积(μL)
无酶纯水 27.8
5×ExoVII反应缓冲液 7
Exo VII(10U/μL) 0.2
总体积 35
将35μL反应体系迅速加入上步磁珠中，快速充分混匀后置于37℃孵育30min。加入0.8μL 0.5M EDTA，充分混匀。分别用350μL HSWB，350μL LSWB清洗磁珠一次，再用200μL 1×Exo VII反应缓冲液(含0.025％吐温-20)平衡磁珠，彻底吸弃上清。
17、末端修复on beads反应：配制以下表23所示的反应体系。
表23
反应成分 体积(μL)
无酶纯水 69.2
10×NEBuffer2 8
25mM dNTP D.8
T4脱氧核糖核酸聚合酶(3U/μL) 2
总体积 80
将80μL反应体系迅速加入上步磁珠中，快速充分混匀后置于12℃孵育20min。加入2μL 0.5M EDTA，充分混匀。分别用350μL HSWB，350μL LSWB清洗磁珠一次，再用200μL1×NEBuffer2反应缓冲液(含0.025％吐温-20)平衡磁珠，彻底吸弃上清。
18、去磷酸化on beads反应：配制以下表24所示的反应体系。
表24
反应成分 体积(μL)
无酶纯水 48
10×NEBuffer2 6
FastAp(1U/μL) 6
总体积 60
将60μL反应体系迅速加入上步磁珠中，快速充分混匀后置于37℃孵育45min。加入1.5μL 0.5M EDTA，充分混匀。分别用350μL HSWB，350μL LSWB清洗磁珠一次，再用200μL 1×NEBuffer2反应缓冲液(含0.025％吐温-20)平衡磁珠，彻底吸弃上清。
19、5’接头B序列连接on beads反应：本实施例中使用的接头B序列如下：
5’接头B序列：/5Phos/AAGTCGGAGGCCAAGCGTGCTTAGGA(SEQ ID NO：6)；
5’封闭序列：GCCUCCGACT/ddT/(SEQ ID NO：7)。
5’接头B混合液(10μM)按以下表25所示的体系配制。
表25
反应成分 体积(μL)
5’接头B序列(100μM) 12
5’封闭序列(100μM) 10
TE缓冲液 78
总共 100
连接反应体系按以下表26所示的体系配制。
表26
反应成分 体积(μL)
无酶纯水 43
3×连接缓冲液2 27
10μM 5’接头B混合液 8
总共 78
将78μL反应体系加入上步磁珠中，充分混匀，快速加入2μL T4DNA连接酶(快速)(600U/μL)充分混匀后置于25℃孵育60min，65℃孵育10min，冷却至4℃。加入1.5μL T4多聚核苷酸激酶(10U/μL)，充分混匀后置于37℃孵育20min。加入2μL 0.5M EDTA，充分混匀。分别用350μL HSWB，350μL LSWB清洗磁珠一次，再用200μL 1×NEBuffer2反应缓冲液(含0.025％吐温-20)平衡磁珠，彻底吸弃上清。
20、3’L型接头B序列连接on beads反应：本实施例使用的3’L型接头B序列：/5Phos/CATGTAGTGTACGATCCGACTT(SEQ ID NO：8)。
按表27所示的体系配制3’L型接头反应体系。
表27
反应成分 体积(μL)
无酶纯水 44.5
3×连接缓冲液2 27
L型接头序列(100μM) 6.5
T4DNA连接酶(快速)(600U/μL) 2
总共 80
将80μL反应体系加入上步磁珠中，充分混匀后置于25℃孵育60min，按照每秒降低0.1℃的速度降温至14℃。加入2μL 0.5M EDTA，充分混匀。分别用350μL HSWB，350μL LSWB清洗磁珠两次，彻底吸弃上清。
加入40μL 0.1M氢氧化钠，混匀之后室温孵育10min。吸取上清，加入20μL 0.3M酸性缓冲液中和获得的单链分离产物，中和后产物总体积60μL。此步完成了目的核酸片段与接头B的连接，并碱变性分离得到目的单链DNA。
21、聚合酶链式反应：
引物1序列：/5Phos/CATGTAGTGTACGATCCGACTT(SEQ ID NO：9)；
引物2序列：TCCTAAGCACGCTTGGCCT(SEQ ID NO：10)。
按表28所示的体系配制PCR体系。
表28
反应成分 体积(μL)
无酶纯水 160
2×PfuTurbo Cx缓冲液 219.8
PfuTurbo Cx热启动核酸聚合酶(2.5U/μL) 8.2
20μM引物1 11
20μM引物2 11
总体积 410
将上步骤30μL回收产物，加入到以上体系中，混匀后按表29所示的条件进行反应。
表29
Figure PCTCN2014092297-appb-000004
反应完成后使用440μL 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100ng产物进行下一步反应。PCR产物片段大小用Agilent 2100HS试剂盒进行电泳检测，结果如图7所示。
22、单链环化：
介导片段具有相应互补序列用于连接单链两端，其序列如下：GTACACTACATGTCCTAAGCACGC(SEQ ID NO：11)。
取100ng上一步骤的PCR产物，加入10μL介导片段(10μM)，混匀后放置于95℃孵育3min，然后迅速放于冰上冷却。配制以下表30所示的反应体系。
表30
反应成分 体积(μL)
无酶水 36.4
10×TA缓冲液(epicentre公司) 12
100mM三磷酸腺苷 1.2
T4Ligase(600U/μL，enzymatics) 0.4
总共 50
将50μL反应液加入到PCR产物和介导片段的混合液中，混匀后置于37℃孵育60min。
23、线性DNA消化：配制表31所示的体系。
表31
反应成分 体积(μL)
无酶水 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，混匀后置于37℃孵育60min。加入6μL 0.5M EDTA，混匀。使用170μL PEG32磁珠纯化回收，55μL TE缓冲液回溶。
本实施例最终产物浓度和总量情况如表32所示。
表32
  浓度(ng/μL) 总量(ng) 分子量(pmol)
产物1 0.36 19.8 0.27
产物2 0.34 18.7 0.26
从结果来看，各产物浓度与总量满足了后续测序要求(分子量≥0.12pmol)。环化前接头B连接产物PCR后使用Agilent公司2100毛细管电泳仪检测的结果如图7所示，横坐标表示碱基对的大小，纵坐标表示检测到的荧光信号强度。纵坐标值为0时，为检测基线信号，纵坐标的值越高说明检测的样品的荧光信号越强。图中35和1030相当于35bp和1030bp的DNA标准品，是检测样品片段大小的参照物。图中218和219是产物1和产物2主带的大小，即分别为218bp和219bp。右侧的长方形条状框显示根据检测信号模拟的待测片段的条带分布情况。从结果来看，所得到的目的条带(箭头指示)很集中，说明通过CNT的方法可以有效地将插入片段控制在非常窄的范围内。另外，根据PCR产物主带大小及接头A的大小(54bp)、接头B的大小(48bp)，可以计 算出插入片段的主带大小分别为：218-54-48＝116bp＝58bp*2和219-54-48＝117bp≈59bp*2，证明本实施例成功得到了双接头单链环状文库。
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 (11)

  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；
    用USER酶酶切所述第一产物，形成粘性末端后进行环化产生缺口；或所述第一PCR使用的引物序列上还具有切口酶识别序列，用USER酶酶切所述第一产物，形成粘性末端后进行环化，然后使用切口酶酶切环化产物产生切口；
    以所述环状核酸分子为模板，从所述切口或缺口处开始进行限制性切口/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切口/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，所述长链的中间部分有一段标签序列，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。
  3. 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切口酶识别序列和一个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。
  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。
  5. 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。
  6. 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生物素标记；在所述限制性切口/缺口平移反应之前，使用链霉亲和素标记的磁珠与第一PCR的产物结合，使得后续反应在所述磁珠上进行。
  7. 根据权利要求1所述的方法，其特征在于，在所述限制性切口/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切口/缺口平移反应的部分，具体包括：首先使用具5’-3’外切酶活性的酶降解，直到两端的缺口相遇；然后使用具3’-5’外切酶活性的酶或单链外切酶降解单链。
  9. 根据权利要求1所述的方法，其特征在于，所述第二接头序列包括第二5’接头序列和第二3’L型接头序列，分别连接所述线性核酸分子每条链的3’端和5’端；所述第二5’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；所述第二3’L型接头序列在邻近连接的片段的部分与所述第二5’接头序列有部分碱基互补；
    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去磷酸化；
    对去磷酸化后的线性核酸分子进行末端修复；
    在所述线性核酸分子的每条链的3’端连接所述第二5’接头序列；
    使用USER酶酶切所述第二5’接头序列的短链的U碱基位点；
    对USER酶酶切后的片段进行磷酸化处理；
    在所述磷酸化处理后的线性核酸分子每条链的5’端连接所述第二3’L型接头序列。
  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，所述长链的中间部分有一段标签序列，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碱基位点或者具有切口酶识别序列和U碱基位点，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；
    切口酶，用于酶切所述第一产物，产生切口；
    USER酶，用于酶切所述第一产物，产生用于环化的粘性末端和缺口；
    缺口平移反应的组分，用于以所述环状核酸分子为模板，从所述切口或缺口处开始进行限制性切口/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切口/缺口平移反应的部分，得到线性核酸分子；
    第二接头序列，所述第二接头序列包括第二5’接头序列和第二3’L型接头序列，分别连接线性核酸分子每条链的3’端和5’端；所述第二5’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；所述第二3’L型接头序列在邻近连接的片段的部分与所述第二5’接头序列有部分碱基互补；
    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。
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