WO2016058127A1 - 一种转座酶打断核酸并加接头的方法和试剂 - Google Patents

一种转座酶打断核酸并加接头的方法和试剂 Download PDF

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WO2016058127A1
WO2016058127A1 PCT/CN2014/088542 CN2014088542W WO2016058127A1 WO 2016058127 A1 WO2016058127 A1 WO 2016058127A1 CN 2014088542 W CN2014088542 W CN 2014088542W WO 2016058127 A1 WO2016058127 A1 WO 2016058127A1
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linker
transposase
reagent
sequence
modification
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PCT/CN2014/088542
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English (en)
French (fr)
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耿春雨
陈若莹
郭荣荣
阿莱克谢耶夫安德烈
张迎新
蒋慧
章文蔚
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深圳华大基因科技有限公司
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Priority to ES14903871T priority Critical patent/ES2821434T3/es
Priority to PCT/CN2014/088542 priority patent/WO2016058127A1/zh
Priority to US15/519,147 priority patent/US20170292153A1/en
Priority to CN201480082154.8A priority patent/CN106715691B/zh
Priority to EP14903871.3A priority patent/EP3208335B1/en
Priority to JP2017539480A priority patent/JP2017534297A/ja
Priority to DK14903871.3T priority patent/DK3208335T3/da
Priority to PCT/CN2015/091859 priority patent/WO2016058517A1/en
Priority to EP15850902.6A priority patent/EP3207169B1/en
Priority to US15/519,149 priority patent/US20180044668A1/en
Publication of WO2016058127A1 publication Critical patent/WO2016058127A1/zh

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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
    • C12Q1/6844—Nucleic acid amplification reactions
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    • C12N15/09—Recombinant DNA-technology
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    • 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
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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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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the invention relates to the technical field of molecular biology, in particular to a method and a reagent for transposing a nucleic acid to interrupt a nucleic acid and adding a linker.
  • Sample fragmentation is mainly achieved by physical methods (such as ultrasonic shearing) or enzymatic methods (ie, non-specific endonuclease treatment).
  • the physical method is based on Covaris, a patent-based Adaptive Focused Acoustic (AFA) technology. Under isothermal conditions, the acoustic energy of a wavelength of 1 mm is focused on the sample by a geometrically focused acoustic energy using a spherical solid-state ultrasonic sensor of >400 kHz. This method ensures that the integrity of the nucleic acid sample is retained and high recovery is achieved.
  • Covaris' instruments include the economical M-Series, the single-tube full-power S-series, and the higher-throughput E and L-series.
  • NEB Next dsDNA Fragmentase from NEB.
  • the reagent firstly generates a random nicking site in the double-stranded DNA, and then recognizes the nicking site by another enzyme to cleave the complementary DNA strand, thereby achieving the purpose of interruption.
  • This reagent can be used for genomic DNA, whole genome amplification products and PCR products, etc., and the randomness is also good, but some artificial short fragment insertions and deletions are generated, and it is inevitable that subsequent separate end treatment and joints are required. And PCR and the corresponding purification operations.
  • the transposase interrupting kit led by Epigentra's Nextera kit (acquired by Illumina), uses DNA transposase to simultaneously complete DNA fragmentation and linker addition, thereby reducing sample processing time.
  • transposase realizes transposition Rely on a specific 19 bp Me sequence.
  • transposases can be embedded by two completely different linker sequences
  • a different linker sequence is added to the 5' end and the 3' end of the target sequence, but the linker needs to contain a specific sequence of Me, thereby causing an effect that both ends of the fragment produced by the break are symmetrical.
  • a Me sequence and due to the special action of the transposase, there is a gap of 9 nt base deletion between the sequence of interest (or disrupted fragment) and the Me sequence.
  • the Me sequence which is completely consistent with the two ends of the target sequence, will affect some downstream applications.
  • the Me sequences on both sides of the same chain are complementary sequences, which easily cause single chain. Annealing occurs inside the molecule and is not conducive to anchoring the primer.
  • the invention provides a method and a reagent for interrupting a nucleic acid and adding a linker by a transposase, which introduces a nucleic acid product after transposition of the transposase into another sequence different from the transposase recognition sequence, and realizes both ends of the interrupted nucleic acid.
  • Different linker sequences are ligated such that the application of the disrupted product is not limited by the consensus of the transposase recognition sequences at both ends.
  • a method of transposing a nucleic acid to interrupt a nucleic acid and adding a linker comprising the steps of:
  • the nucleic acid is randomly disrupted using a transposase-embedded complex comprising a transposase and a first linker comprising a transposase recognition sequence, the disrupted nucleic acid being ligated to the first linker at both ends and Forming a gap;
  • the PCR reaction was carried out using primers respectively targeting the first linker and the second linker to obtain a product in which different linker sequences were ligated to both ends.
  • the first joint is provided with protection Modifications that are self-connected or interconnected with a second connector.
  • the modification on the first linker comprises a combination of any one or more of the following:
  • the first linker consists of a complete sequence that internally complements to form a 3'-5' phosphodiester bond cross-linked double-stranded sequence.
  • the modification on the first linker is a 3' terminal base dideoxy modification of the first linker.
  • the second joint in order to prevent self-linking of the joint, is provided with a modification preventing self-joining.
  • the modification on the second linker is a 3'-end base dideoxy modification.
  • one of the primers used in the PCR reaction is a terminal biotin-labeled primer for passing biotin.
  • - Streptavidin affinity reaction to obtain a single chain molecule. Specifically, after the PCR reaction, a single-stranded molecule carrying biotin at the end is separated by binding to streptavidin on the surface of the magnetic bead.
  • the purification is magnetic bead purification or column purification.
  • Magnetic bead purification or column purification can completely remove the transposase in the system.
  • Ampure XP beads were used for magnetic bead purification and subjected to column purification using a QIAGEN PCR purification column. Needless to say, any similar magnetic bead purification or column purification product can be used in the present invention.
  • the chemical agent is treated to dissociate from the target sequence by denaturation or digestion of the transposase. Since the transposase is chemically a protein, it can be dissociated from the target sequence using the corresponding denaturation or digestion methods. Although the transposase thus treated may still remain in the system, it has been lost. Its biological activity will not adversely affect subsequent reactions.
  • the chemical reagent includes a first reagent and a second reagent; wherein the first reagent includes one or more of a protease solution, a sodium dodecyl sulfate (SDS) solution, and an NT buffer, The adsorption of the target sequence for breaking the transposase and the nucleic acid; the second reagent comprising a Triton-X100 solution for attenuating the effect of the first reagent on the subsequent enzymatic reaction.
  • the first reagent includes one or more of a protease solution, a sodium dodecyl sulfate (SDS) solution, and an NT buffer
  • the first reagent is used for treatment and then the second reagent is used.
  • the first reagent is used to treat the reaction product after the transposase is interrupted, so as to break the adsorption of the transposase and the target sequence of the nucleic acid, instead of Traditional complex and costly magnetic bead purification or column purification steps are then treated with a second reagent to attenuate the effect of the first reagent on subsequent enzymatic reactions, ensuring smooth downstream PCR amplification.
  • the first reagent may be one or more of the above solutions, and the plurality of may be two or three, such as a protease solution and an SDS solution, an SDS solution and an NT buffer, a protease solution and an NT buffer. Liquid, protease solution, SDS solution and NT buffer. Among them, the NT buffer can be used in the NT buffer of the Tuprep kit S5 series.
  • the first reagent includes a protease solution
  • the first reagent is added for treatment, and further treated with an ethylenediaminetetraacetic acid (EDTA) solution.
  • EDTA inhibits the activity of proteases and prevents proteases from degrading enzymes in subsequent PCR reactions.
  • the second reagent comprises a Triton-X100 solution.
  • Triton-X100 also known as Triton X-100, chemically known as octyl phenyl ethoxylate, acts as a nonionic surfactant in the present invention to attenuate the effect of the first reagent on subsequent enzymatic reactions. .
  • the second reagent further comprises a Tween-20 solution.
  • Tween-20 can further attenuate the effect of SDS on subsequent enzymatic reactions and enhance the PCR effect.
  • Tween-20 can be used as a second reagent in a form mixed with Triton-X100; it can also be provided separately in the form separated from Triton-X100, in which case the second reagent refers to a Triton-X100 solution. And Tween-20 solution.
  • first reagent and the second reagent in the present invention are not limited to a single object, and may also refer to a combination of a plurality of objects.
  • concepts such as “first” and “second” are used in any case. Neither should be construed as having a meaning of order and technology, but only to distinguish it from other objects.
  • the working concentrations of the first reagent and the second reagent can be determined empirically by those skilled in the art.
  • the working concentration of the protease is preferably from 50 to 5000 mAU/mL, preferably from 75 to 3750 mAU/mL, most preferably 1500 mAU/mL; and the working concentration of EDTA is preferably from 1 to 50 mmol/L, preferably 14 mmol. /L;
  • the working concentration of the SDS is preferably 0.01% to 1.5% by volume, preferably 1% by volume; and the final concentration of the NT buffer may be 1 ⁇ .
  • the working concentration of Triton-X100 is preferably 0.1% to 2% (volume ratio), preferably 1% (volume ratio); and the working concentration of Tween-20 is 0.1% to 2% (volume ratio). Preferably, it is preferably 0.5% by volume.
  • sequence of the second linker is not limited and may be any sequence as long as it differs from the sequence of the first linker.
  • the nucleic acid to be interrupted may be genomic DNA, whole genome amplification product or PCR product, may be DNA or cDNA, and has no limitation on the source of the nucleic acid, and may be a nucleic acid sample of animal, plant or microbial origin.
  • a reagent for transposing a nucleic acid to cleave a nucleic acid and to a linker comprising the following components:
  • transposase and a first linker comprising a transposase recognition sequence for forming a transposase-embedded complex to randomly interrupt the nucleic acid, connecting the first linker at both ends of the interrupted nucleic acid and forming a gap;
  • the PCR primers are respectively targeted to bind the first linker and the second linker, and are used for performing a PCR reaction to obtain a product in which different linker sequences are respectively connected at both ends.
  • the first joint is provided with modifications to prevent self-connection or interconnection with the second joint.
  • the modification on the first linker comprises a combination of any one or more of the following:
  • the first linker consists of a complete sequence that internally complements to form a 3'-5' phosphodiester bond. Linked double-stranded sequence.
  • the second linker is provided with a modification to prevent self-ligation; preferably, the modification on the second linker is a 3'-end base dideoxy modification.
  • one of the primers in the PCR primer is a terminal biotin-labeled primer for obtaining a single-stranded molecule by biotin-streptavidin affinity reaction.
  • the method of the present invention modifies the sequence on both sides of the product after the transposase is interrupted by means of a second linker, and realizes that the structure of the final disrupted product or the PCR product has different specific sequences on both sides, thereby
  • the subsequent application process is not limited to the traditional two-terminal shared transposase recognition sequence (19bp Me), and is more flexible in application, such as molecular cyclization, enzymatic cleavage or ligation.
  • FIG. 1 is a schematic flow chart of a technical scheme for transposing a nucleic acid and a gap junction (ie, a second linker) in the present invention
  • Example 2 is a result of gel electrophoresis detection of a PCR product after the gap junction (ie, the second joint) is connected in Example 1 of the present invention, wherein 1 is an annealing product at 60 ° C after the gap joint is broken after the single joint-2 is broken; 2 is a single joint -2 After annealing, the voided joint is annealed at 55 °C; 3 is an annealing product at 60 °C after the single joint-3 is broken; and 4 is an annealing product at 55 °C after the single joint-3 is broken.
  • 5 is an annealing product at 60 °C after the single joint-1 is broken, and 6 is an annealing product at 55 °C after the single joint-1 is broken, and 7 is a direct joint PCR 60 °C annealing product after the double joint is broken; 8 is a direct PCR at 55 ° C after the double joint is broken;
  • M1 is DL2000 DNA Marker;
  • M2 is 50 bp DNA Marker;
  • N is a negative control;
  • FIG. 3 is a diagram showing the base quality of the ligation method of the first embodiment of the present invention.
  • Example 4 is a result of electrophoresis detection of a PCR product after the single-linker transposase complex of Example 1 of the present invention interrupts nucleic acid and introduces a second linker, wherein D2000 is a DNA Ladder lane; Lane 1 is 2 ⁇ L of protease +1%.
  • Triton-X100 treatment results lane 2 was treated with NT buffer + 1% Triton-X100; lane 3 was 1% SDS + 1% Triton-X100 + 0.5% Tween-20 treatment; lane 4 was 2 ⁇ L protease + 14 mM EDTA + 1% Triton-X100 treatment results; Lane 5 was 1 x PBI, 1.3 x Ampure XP beads treatment results; Lane 6 was a negative control (no template) results.
  • the terms used in the present invention are as follows: the first joint is referred to as the first joint in the specific embodiment; the second joint is referred to as the second joint or the void joint in the specific embodiment; the first reagent is referred to in the specific embodiment As the No. 1 reagent; the second reagent is referred to as the No. 2 reagent in the specific embodiment.
  • the method of the present invention mainly comprises: (1) using a transposase to embed a specific modified sequence of a single linker for randomizing nucleic acid sequences such as genome, whole genome amplification products and PCR product sequences.
  • interrupted DNA is ligated to the first linker at both ends and forms a gap of 9 nt base deletion; (2) removal of the effect of the transposase in the system on the subsequent reaction by purification or chemical treatment; (3) In the 9nt gap, the second linker is introduced to realize the introduction of the second linker, and the sequence of the linker adjacent to the fragmented target sequence is changed, so that the sequences on both sides of the target sequence are completely different, and one of the segments retains the recognition of the transposase.
  • the sequence of the first linker sequence, and the other is completely arbitrarily designed for the second linker sequence; (4) direct special primers (primers are targeted to bind to the first linker and the second linker) mediated PCR reaction to obtain the target A PCR product with completely different sequences attached to both ends of the sequence can be used in subsequent molecular biology experiments.
  • a transposase kit of a domestically produced (S50 series of the Truprep kit of Nanjing Nuoweizan) was used for the experiment, and the kit contains two kinds of genomic DNA and 50 ng of genomic DNA.
  • the invention independently designs a plurality of embedding linker sequences (linker No. 1), and uses the transposase and the embedding linker sequence to prepare the transposase complex.
  • 5 ng or 50 ng of high-quality genomic DNA is first disrupted by using an embedded transposase complex; the free unembedded first linker is removed after magnetic bead purification or column purification; A second adaptor (gap junction) was ligated and purified to remove the free ligated linker to construct a linear genomic sequence with different linker sequences ligated at both ends; PCR primers designed for the first and second linkers were used. Amplification, thereby enriching PCR products with different linker sequences linked at both ends.
  • One application of the PCR product of this example is to label a PCR primer by introducing a biotin label, and then obtain a single-stranded molecule of a specific sequence, by single-stranded cyclization or a short nucleic acid sequence as a bridge-mediated cyclization mode.
  • the preparation of a single-stranded circular molecule is carried out.
  • the formed single-stranded circular molecule can be used for the preparation of solid dense DNA nanospheres.
  • sequence A and sequence B design multiple pairs of primer sequences (sequence A and sequence B) with a 19 bp transposase recognition sequence for the preparation of a single connector for embedding (link No. 1), three different tests were tested in this example.
  • Single linker sequences ie, single linker 1 sequence, single linker 2 sequence, and single linker 3 sequence
  • a standard double linker sequence sequence A + sequence B; sequence A + sequence C).
  • dUTP is introduced into one strand of the single linker 1 sequence (A chain) for subsequent USER digestion of the excess linker;
  • the single linker 2 sequence of the 19 bp transposase recognition sequence introduces a base pair, wherein the 3' terminal base The base is a dideoxy-modified base;
  • the entire double-stranded sequence of the single linker 3 sequence consists of one complete sequence, which internally complements to form a double-stranded sequence cross-linked by a 3'-5' phosphodiester bond.
  • the modification schemes of the above three No. 1 linkers each have a 3'-end dideoxy modification in at least one chain, which is advantageous for preventing the self-joining of the No. 1 linker and its interconnection with the No. 2 linker.
  • the sequence of each linker is as follows:
  • Single linker 1 sequence B GCTTCGACTGGAGACAGATGTGTATAAGAGACAG (SEQ ID NO: 2);
  • Single linker 2 sequence B GCTTCGACTGGAGACAGATGTGTATAAGAGACAG ddC (SEQ ID NO: 4);
  • Double linker sequence A CTGTCTCTTATACACATCT (SEQ ID NO: 6);
  • Double linker sequence B TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG (SEQ ID NO: 7);
  • Double linker sequence C GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO: 8).
  • Method 1 Add 1 volume of PBI (Qiagen PCR Purification Kit) and mix with 1.3 times Ampure XP beads (automated operation); Method 2: Purify with QIAGEN PCR Column purification. After purification, it is remelted with pure water.
  • the purified product was ligated to the gap junction (ie, the second linker) according to the following system (Table 5), and ligated at 25 ° C for 60 minutes to complete the joint connection.
  • Component content water 8 ⁇ L 3 ⁇ connection buffer 20 ⁇ L Connector 2 (5 ⁇ M) 10 ⁇ L Ligase 2 ⁇ L DNA 20 ⁇ L total 30 ⁇ L
  • the second linker sequence A p AAGTCGGAGGCCAAGCGGTCGT ddC (SEQ ID NO: 9); the second linker sequence B: TTGGCCTCCGACT ddT (SEQ ID NO: 10); wherein p represents a 5'-terminal phosphorylation modification, and dd represents a 3'-end double Deoxygenation modification.
  • Method 1 Add 1 volume of PBI and mix with 1.3 times of Ampure XP beads (automated operation);
  • Method 2 Purify with QIAGEN PCR column . After purification, it is remelted with pure water.
  • PCR amplification was carried out according to the following PCR reaction system (Table 6) and reaction conditions (Table 7).
  • PCR primer sequences are as follows:
  • Double linker primer 1 AATGACATCGGCGACCACCGA (SEQ ID NO: 13);
  • Double linker primer 2 CAAGCAGAAGACGGCATACGA (SEQ ID NO: 14).
  • the single-chain cyclization of the target strand is carried out after single-strand separation of the PCR product, thereby obtaining a single-stranded circular DNA molecule, which is used for rolling-loop replication on the whole genome sequencing platform to prepare DNA nanometers.
  • the ball and the junction were sequenced.
  • the method of single chain separation and cyclization is as follows:
  • Double linker-mediated sequence CGCCGTATCATTCAAGCAGAAGAC (SEQ ID NO: 16).
  • connection buffer 3.7 ⁇ L 20U/ ⁇ L exonuclease I 11.1 ⁇ L 100U/ ⁇ L exonuclease III 5.2 ⁇ L total 20 ⁇ L
  • Sequencing can be sequenced from the 5' and 3' ends, and the target fragment with different sequences linked at both ends is only present at one end due to the 19 bp transposase recognition sequence, thereby avoiding specific annealing of the 19 bp transposase recognition sequence at both ends.
  • competition with the sequencing linker has greatly improved the quality of the sequencing, and the results are shown in FIG.
  • the data shown in Fig. 3 is mostly between 80 and 90, and is generally considered to be acceptable at 75 or more.
  • the conventional method of sequencing the 19 bp transposase recognition sequence at both ends generally does not achieve such high data. Even between 30 and 40, this indicates that the 19 bp sequence complementary sequencing probe of the present invention can be well matched to the sequencing template, that is, the effect of two 19 bp reverse complements of the transposase on sequencing is solved.
  • PCR primer was used for amplification, and a certain concentration of Triton X-100 was added to the PCR reaction system.
  • Linker single linker sequence A TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG (SEQ ID NO: 17);
  • Linker single linker sequence B CTGTCTCTTATACACATC ddT (SEQ ID NO: 18, dd indicates dideoxy modification).
  • the first linker and the transposase were embedded into a transposase complex, gently pipetted 20 times, and incubated at 30 ° C for 1 hour to complete the complex embedding.
  • the complex was stored at -20 °C.
  • Method 1 Add 0.1-5 ⁇ L protease (750 mAU/mL) for treatment. This method is preferably 2 ⁇ L protease, and 0.1 ⁇ L and 5 ⁇ L protease are tested separately.
  • Method 2 Add a final concentration of commercial 1 ⁇ NT buffer (matching reagent in the Trouprep kit S5 series);
  • Method 3 Add 0.01%-1.5% (by volume) of SDS, preferably 1% by volume in this example.
  • Method 4 Add 0.1-5 ⁇ L of protease for treatment, then add EDTA at a final concentration of 1-50 mM, this example is preferred 2 ⁇ L of protease and a final concentration of 14 mM EDTA were tested simultaneously with 0.1 ⁇ L of protease plus 1 mM EDTA and 5 ⁇ L of protease plus 50 mM EDTA; Method 5: Add 1 volume of PBI (a commercial reagent in Qiagen PCR Purification Kit) After homogenization, it was purified by 1.3 times of Ampure XP beads, and pure water was used for remelting.
  • PBI a commercial reagent in Qiagen PCR Purification Kit
  • Triton-X100 0.1% to 2% by volume of Triton-X100 is added, preferably 1% by volume in this embodiment, and 0.1% by volume and 2% by volume. The test was carried out using a Triton-X100.
  • Component content water 8 ⁇ L 3 ⁇ connection buffer 20 ⁇ L Connector (5 ⁇ M) 10 ⁇ L Ligase 2 ⁇ L DNA 20 ⁇ L total 30 ⁇ L
  • Linker sequence A 5'-pAAGTCGGAGGCCAAGCGGTCGT ddC-3' (SEQ ID NO: 9);
  • Linker sequence B 5'-TTGGCCTCCGACT ddT-3' (SEQ ID NO: 10) (p indicates phosphorylation Modification; dd means dideoxy modification).
  • PCR amplification was carried out according to the following PCR reaction system (Table 15) and reaction conditions (Table 16).
  • a specific concentration of Tween-20 is also needed in the PCR system to partially improve the efficiency of PCR, and the working concentration of Tween-20 can be adjusted to be different, such as 0.1%-2% (volume). This example is preferably 0.5% while testing Tween-20 at a working concentration of 0.1% by volume and 2% by volume.

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Abstract

提供一种转座酶打断核酸并加接头的方法和试剂。该方法包括如下步骤：使用转座酶包埋复合体对核酸进行随机打断，其中转座酶包埋复合体包含转座酶和含转座酶识别序列的第一接头，打断的核酸两端连接第一接头并且形成缺口；通过纯化或化学试剂处理的方式，去除体系中的转座酶对后续反应的影响；使用连接酶在缺口处连接上第二接头，第二接头的序列不同于第一接头；使用分别靶向结合第一接头和第二接头的引物进行PCR反应，得到两端分别连接有不同接头序列的产物。

Description

一种转座酶打断核酸并加接头的方法和试剂 技术领域
本发明涉及分子生物学技术领域，尤其涉及一种转座酶打断核酸并加接头的方法和试剂。
背景技术
自从罗氏发明了焦磷酸测序方法开辟了二代测序以来，直至现在，二代测序经历了一段高速发展期。但随着高通量测序的发展，高通量和低成本的样本制备环节逐渐成了测序领域的一个重点考虑的因素。各种原理的样本处理方法及自动化装置不断被研发出来，主要包括：样本片段化、核酸分子的末端处理及接头连接并最终的出库。
其中样本片段化主要分为物理方法(如超声剪切)或酶学方法(即非特异的核酸内切酶处理)来实现。其中物理方法以基于专利的自适应聚焦超声(Adaptive Focused Acoustic，AFA)技术的Covaris为主。在等温的条件下，利用几何聚焦声波能量，通过＞400kHz的球面固态超声传感器，将波长为1mm的声波能量聚焦在样品上。该方法确保了核酸样品的完整性得以保留，并能实现高回收率。Covaris的仪器包括经济的M系列、单管全功率的S系列以及更高通量的E和L系列。基于物理方法打断的片段随机性良好，但是通量上也要依赖大量的Covaris打断仪，同时需要后续单独进行末端处理、加接头和PCR以及各种纯化操作。其中酶学方法有一种NEB公司推出的NEB Next dsDNA Fragmentase。该试剂首先在双链DNA产生随机的切刻位点，然后通过另一种酶识别切刻位点来切割互补的DNA链，从而实现打断的目的。这种试剂可以用于基因组DNA、全基因组扩增产物和PCR产物等，随机性也较好，但是会产生一些人工短片段插入和缺失，同时也不可避免的需要后续单独进行末端处理、加接头和PCR以及相应的纯化操作。另外以Epicentra公司(已被Illumina收购)的Nextera试剂盒领衔的转座酶打断试剂盒，利用转座酶同时完成DNA片段化和接头的添加，从而减少样品处理的时间。
从各种操作的简便性来看，转座酶打断的方式无疑在通量及操作简便性上远远胜过其它方法，但是这种打断方式也有自身的缺点：转座酶实现转座依赖特定的19bp Me序列。因此，虽然转座酶可以通过包埋两种完全不同的接头序 列而在靶序列的5’端和3’端加上不同的接头序列，但是接头均需要含有Me特定序列，从而带来的一个影响即打断所产生的片段的两端会对称的各有一个Me序列，并且由于转座酶的特殊作用使得目的序列(或打断片段)与Me序列之间存在一个9nt碱基缺失的缺口。靶序列邻近的两端完全一致的Me序列会对下游的一些技术应用带来影响，比如基于连接法的二代测序技术，同一条链两侧的Me序列为互补的序列，从而容易引起单链分子内部出现退火而不利于锚定引物的结合。
曾经有相关专利申请(申请公布号：CN 102703426 A，申请公布日为2012年10月3日)提出了一种解决办法，即将打断后的序列进行特定内切酶酶切，从而去除9nt序列和Me序列，但是这种方法只是利用了转座酶打断的优势将核酸序列进行随机打断，但是引入了后续接头需要单独进行加入的缺点，步骤繁琐而不易于更高通量的应用。
迄今为止，未有专利及其它文献报道一种能够极其高效快速的采用转座酶技术打断靶序列并将打断后的序列修正为两段完全不同序列的分子生物学实验方法。
发明内容
本发明提供一种转座酶打断核酸并加接头的方法和试剂，该方法使转座酶打断后的核酸产物引入不同于转座酶识别序列的其它序列，实现打断的核酸两端连有不同的接头序列，从而使得打断产物的应用不受限于两端共有转座酶识别序列的影响。
根据本发明的第一方面，本发明提供一种转座酶打断核酸并加接头的方法，包括如下步骤：
使用转座酶包埋复合体对核酸进行随机打断，其中转座酶包埋复合体包含转座酶和含转座酶识别序列的第一接头，打断的核酸两端连接第一接头并且形成缺口；
通过纯化或化学试剂处理的方式，去除体系中的转座酶对后续反应的影响；
使用连接酶在缺口处连接上第二接头，第二接头的序列不同于第一接头；
使用分别靶向结合第一接头和第二接头的引物进行PCR反应，得到两端分别连接有不同接头序列的产物。
作为本发明的优选方案，为了防止接头的自连或互连，第一接头上带有防 止自连或与第二接头互连的修饰。
作为本发明的优选方案，第一接头上的修饰方式包括如下任一种或多种的组合：
(a)第一接头的3’端碱基双脱氧修饰；
(b)第一接头的一条链中引入dUTP，用于之后的USER酶切过量接头；
(c)第一接头的转座酶识别序列外侧引入一个碱基对，其中3’端碱基双脱氧修饰；和
(d)第一接头由一条完整序列组成，内部互补形成一段3’-5’磷酸二酯键交联的双链序列。
需要说明的是：可以单独使用(a)～(d)任一种修饰方式，也可组合使用两种或两种以上的修饰方式，尤其是可以将(a)修饰方式分别与(b)、(c)或(d)修饰方式组合使用，以取得更好的防止接头的自连或互连的效果。
作为本发明的优选方案，第一接头上的修饰方式为第一接头的3’端碱基双脱氧修饰。
作为本发明的优选方案，为了防止接头的自连，第二接头上带有防止自连的修饰。
作为本发明的优选方案，第二接头上的修饰为3’端碱基双脱氧修饰。
本发明中，所谓“自连”是指同一种接头的不同分子之间的连接，比如第一接头的不同分子之间的连接或第二接头的不同分子之间的连接；所谓“互连”是指不同种接头的分子之间的连接，比如第一接头的分子与第二接头的分子之间的连接。
作为本发明的优选方案，为了便于PCR反应后单链分子的获取以用于后续的单链分子操作实验，PCR反应使用的引物中的一条引物是末端生物素标记的引物，用于通过生物素-链霉亲和素亲和反应而获取单链分子。具体地，PCR反应后，末端带有生物素的单链分子通过与磁珠表面的链霉亲和素结合而分离出来。
作为本发明的优选方案，纯化为磁珠纯化或过柱纯化。磁珠纯化或过柱纯化能够彻底去除体系中的转座酶。本发明的一个实施例中使用了Ampure XP beads进行磁珠纯化，使用了QIAGEN PCR纯化柱进行过柱纯化。毫无疑问，任何类似的磁珠纯化或过柱纯化产品均可用于本发明。
作为本发明的优选方案，化学试剂处理为使转座酶变性或消化而从靶序列上解离下来的处理。由于转座酶在化学性质上属于蛋白质，因此可以使用相应的变性或消化手段将其从靶序列上解离下来，虽然这样处理后的转座酶仍然可能在体系中有残留，但是已经失去了其生物活性，对后续反应也不会有不利的影响。
作为本发明的优选方案，化学试剂包括第一试剂和第二试剂；其中，第一试剂，包括蛋白酶溶液、十二烷基硫酸钠(SDS)溶液和NT缓冲液中的一种或多种，用于打破转座酶与核酸的靶序列的吸附作用；第二试剂，包含Triton-X100溶液，用于减弱第一试剂对后续酶促反应的影响。
一般而言，先使用第一试剂处理再使用第二试剂处理，首先采用第一试剂处理转座酶打断核酸后的反应产物，以打破转座酶与核酸的靶序列的吸附作用，替代了传统的复杂且成本较高的磁珠纯化或过柱纯化步骤，然后采用第二试剂处理，以减弱第一试剂对后续酶促反应的影响，保证下游的PCR扩增顺利进行。
需要说明的是：第一试剂可以是上述溶液中的一种或多种，其中多种可以是两种或三种，比如蛋白酶溶液和SDS溶液，SDS溶液和NT缓冲液，蛋白酶溶液和NT缓冲液，蛋白酶溶液、SDS溶液和NT缓冲液。其中，NT缓冲液可以采用Truprep试剂盒S5系列中配套的NT缓冲液。
作为本发明的优选方案，如果第一试剂包括蛋白酶溶液，则加入第一试剂进行处理后，进一步加入乙二胺四乙酸(EDTA)溶液处理。EDTA抑制蛋白酶的活性，防止蛋白酶对后续PCR反应中的酶产生降解作用。
作为本发明的优选方案，第二试剂包含Triton-X100溶液。Triton-X100又称曲拉通X-100，化学名为辛基苯基聚氧乙烯醚，作为非离子型表面活性剂，在本发明中的作用是减弱第一试剂对后续酶促反应的影响。
作为本发明的优选方案，如果第一试剂包括SDS溶液，则第二试剂还包含吐温-20溶液。吐温-20的加入能够进一步减弱SDS对后续酶促反应的影响，提升PCR效果。需要说明的是：吐温-20可以以与Triton-X100混合的形式作为第二试剂使用；也可以以与Triton-X100分离的形式单独提供，在这种情况下第二试剂指Triton-X100溶液和吐温-20溶液。
应当理解，本发明中第一试剂和第二试剂并不限定指单一的对象，也可以指多个对象的组合。并且，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，第一试剂和第二试剂的工作浓度，可由本领域技术人员根据经验确定。一般地，在第一试剂中，蛋白酶的工作浓度以50～5000mAU/mL为佳，优选75～3750mAU/mL，最优选1500mAU/mL；EDTA的工作浓度以1～50mmol/L为佳，优选14mmol/L；SDS的工作浓度以0.01％～1.5％(体积比)为佳，优选1％(体积比)；NT缓冲液的终浓度按照1×使用即可。在第二试剂中，Triton-X100的工作浓度以0.1％～2％(体积比)为佳，优选1％(体积比)；吐温-20的工作浓度以0.1％～2％(体积比)为佳，优选0.5％(体积比)。
本发明中，第二接头的序列不受限制，可以是任何序列，只要与第一接头的序列不同即可。
本发明中，待打断的核酸可以是基因组DNA、全基因组扩增产物或PCR产物，可以是DNA或cDNA，并且对于核酸的来源没有限制，可以是动物、植物或微生物来源的核酸样品。
根据本发明的第二方面，本发明提供一种转座酶打断核酸并加接头的试剂，包括如下组成部分：
转座酶和含转座酶识别序列的第一接头，用于形成转座酶包埋复合体以对核酸进行随机打断，在打断的核酸两端连接第一接头并且形成缺口；
第二接头和连接酶组分，用于在缺口处连接上第二接头；和
PCR引物，分别靶向结合第一接头和第二接头，用于进行PCR反应，得到两端分别连接有不同接头序列的产物。
作为本发明的优选方案，第一接头上带有防止自连或与第二接头互连的修饰。
作为本发明的优选方案，第一接头上的修饰方式包括如下任一种或多种的组合：
(a)第一接头的3’端碱基双脱氧修饰；
(b)第一接头的一条链中引入dUTP，用于之后的USER酶切过量接头；
(c)第一接头的转座酶识别序列外侧引入一个碱基对，其中3’端碱基双脱氧修饰；和
(d)第一接头由一条完整序列组成，内部互补形成一段3’-5’磷酸二酯键交 联的双链序列。
作为本发明的优选方案，第二接头上带有防止自连的修饰；优选地，第二接头上的修饰为3’端碱基双脱氧修饰。
作为本发明的优选方案，PCR引物中的一条引物是末端生物素标记的引物，用于通过生物素-链霉亲和素亲和反应而获取单链分子。
本发明的方法将转座酶打断后的产物两侧的序列通过连接第二接头的方式进行了修改，实现了最终打断产物或PCR产物的结构中两侧具有不同的特异序列，从而在后续应用过程中不受限于传统的两端共有转座酶识别序列(19bp Me)的影响，在应用上更加具有灵活性，比如分子的环化、酶切或连接等。
附图说明
图1为本发明中转座酶打断核酸和连接空隙接头(即二号接头)的技术方案流程示意图；
图2为本发明实施例1中连接空隙接头(即二号接头)后PCR产物凝胶电泳检测结果，其中1为单接头-2打断后空隙接头连接后60℃退火产物；2为单接头-2打断后空隙接头连接后55℃退火产物；3为单接头-3打断后空隙接头连接后60℃退火产物；4为单接头-3打断后空隙接头连接后55℃退火产物；5为单接头-1打断后空隙接头连接后60℃退火产物；6为单接头-1打断后空隙接头连接后55℃退火产物；7为双接头打断后直接PCR 60℃退火产物；8为双接头打断后直接PCR 55℃退火产物；M1为DL2000 DNA Marker；M2为50bp DNA Marker；N为阴性对照；
图3为本发明实施例1的连接法测序碱基质量图；
图4为本发明实施例2的一号接头单接头转座酶复合体打断核酸并引入二号接头后的PCR产物电泳检测结果，其中D2000为DNA Ladder泳道；泳道1为2μL蛋白酶+1％Triton-X100处理结果；泳道2为NT缓冲液+1％Triton-X100处理结果；泳道3为1％SDS+1％Triton-X100+0.5％吐温-20处理结果；泳道4为2μL蛋白酶+14mM EDTA+1％Triton-X100处理结果；泳道5为1×PBI，1.3×Ampure XP beads处理结果；泳道6为阴性对照(未加模板)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。
本发明中用到的术语说明如下：第一接头在具体实施方式中称作一号接头；第二接头在具体实施方式中称作二号接头或空隙接头；第一试剂在具体实施方式中称作一号试剂；第二试剂在具体实施方式中称作二号试剂。
请参考图1，本发明的方法操作流程主要包括：(1)利用转座酶包埋特定修饰序列的一号接头用于对基因组、全基因组扩增产物和PCR产物序列等核酸序列进行随机打断，打断的DNA两端连接第一接头并且形成9nt碱基缺失的缺口；(2)通过纯化或化学试剂处理的方式，去除体系中的转座酶对后续反应的影响；(3)通过在9nt缺口进行二号接头连接的方式，实现二号接头的引入，改变片段化的靶序列毗邻的接头碱基序列，使得靶序列两侧的序列完全不同，其中一段保留有含转座酶识别序列的一号接头序列，而另一段完全是任意设计的二号接头序列；(4)直接进行特殊引物(引物分别靶向结合一号接头和二号接头)介导的PCR反应，从而得到靶序列两端连接有完全不同的序列的PCR产物，这种产物可以用于后续的分子生物学实验。
本发明中采用了一家国产(南京诺唯赞公司的Truprep试剂盒的S50系列)的转座酶试剂盒进行实验，试剂盒包含5ng基因组DNA和50ng基因组DNA用量两种。
本发明自主设计了多种包埋接头序列(一号接头)，用转座酶和该种包埋接头序列进行转座酶复合体的制备。
实施例1
本实施例首先用包埋的转座酶复合体将5ng或者50ng高质量的基因组DNA进行打断操作；经过磁珠纯化或过柱纯化后去除游离的未包埋的一号接头；然后创新性地进行二号接头(间隙接头)连接，并经过纯化去除游离的二号接头，从而构建出两端连接有不同接头序列的线性基因组序列；通过针对一号接头和二号接头设计的PCR引物进行扩增，从而富集两端连接有不同接头序列的PCR产物。
本实施例的PCR产物的一个应用是，通过对PCR引物引入生物素标记的方式进行标记，然后获取特定序列的单链分子，通过单链环化或者短核酸序列作为桥介导的环化方式进行单链环状分子的制备。形成的单链环状分子可以用于实心致密DNA纳米球的制备。
1、设计订购带有19bp转座酶识别序列的多对引物序列(序列A和序列B)，用于制备包埋用的单接头(一号接头)，在本实施例中测试了三种不同的单接头序列(即单接头1序列、单接头2序列和单接头3序列)和一种标准的双接头序列(序列A+序列B；序列A+序列C)。
其中，单接头1序列的一条链(A链)中引入dUTP，用于之后的USER酶切过量接头；单接头2序列的19bp转座酶识别序列外侧引入一个碱基对，其中3’端碱基为双脱氧修饰的碱基；单接头3序列的整条双链序列由一条完整序列组成，其内部互补形成一段为3’-5’磷酸二酯键交联的双链序列。此外，上述三种一号接头的修饰方案均在至少一条链含有3’端双脱氧修饰，利于防止一号接头自连以及它与二号接头的互连。各一号接头序列表示如下：
单接头1序列A：CTGTCUCTTAUACACATC ddT(SEQ ID NO：1)；
单接头1序列B：GCTTCGACTGGAGACAGATGTGTATAAGAGACAG(SEQ ID NO：2)；
单接头2序列A：GCTGTCTCTTATACACATC ddT(SEQ ID NO：3)；
单接头2序列B：GCTTCGACTGGAGACAGATGTGTATAAGAGACAG ddC(SEQ ID NO：4)；
单接头3序列：
GCTTCGACTGGAGACAGATGTGTATAAGAGACAGCTGTCTCTTATAC ACATC ddT(SEQ ID NO：5)；
双接头序列A：CTGTCTCTTATACACATCT(SEQ ID NO：6)；
双接头序列B：TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG(SEQ ID NO：7)；
双接头序列C：GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG(SEQ ID NO：8)。
2、将每对单接头序列均稀释到100μM，充分混合后离心，于PCR仪中按如下程序(表1)退火得到一号接头(储存于-20℃)，用于包埋复合体的制备。将双接头序列A、B和C分别稀释到100μM，序列A+序列B组合，序列A+序列C组合，充分混合后离心，于PCR仪中按如下程序(表1)退火得到一号接头(储存于-20℃)，用于包埋复合体的制备。
表1
Figure PCTCN2014088542-appb-000001
3、按照如下体系(表2)将一号接头与转座酶包埋成转座酶复合体，轻轻吹打20次混合，30℃孵育1小时后完成复合体包埋，该复合体储存于-20℃。
表2
组分 含量
转座酶 85μL
一号接头 30μL
偶联缓冲液 85μL
合计 200μL
4、按照如下体系(表3)将50ng的高质量基因组和转座酶复合体进行混合，轻轻吹打20次混合，55℃孵育10分钟后降温至4℃，完成基因组的打断。
表3
组分 含量
水 5μL
5×打断缓冲液 2μL
gDNA(50ng/μL) 1μL
打断酶复合体 2μL
合计 10μL
5、按照如下两种方法进行纯化，方法1：加入1倍体积的PBI(Qiagen PCR纯化试剂盒)混匀后再用1.3倍数的Ampure XP beads纯化(自动化操作)；方法2：用QIAGEN PCR纯化柱纯化。纯化后，采用纯水回融。
6、对于单接头1的打断后加入USER酶进行酶切消化，然后进行同上一步骤的纯化方法，消化体系如下(表4)：
表4
组分 含量
DNA 10μL
10×Buffer 2μL
USER酶 1μL
水 7μL
合计 20μL
7、纯化后的产物按照如下体系(表5)进行空隙接头(即二号接头)的连接，25℃孵育60分钟完成接头连接。
表5
组分 含量
水 8μL
3×连接buffer 20μL
二号接头(5μM) 10μL
连接酶 2μL
DNA 20μL
合计 30μL
注：二号接头序列如下：
二号接头序列A：p AAGTCGGAGGCCAAGCGGTCGT ddC(SEQ ID NO：9)；二号接头序列B：TTGGCCTCCGACT ddT(SEQ ID NO：10)；其中，p表示5’端磷酸化修饰，dd表示3’端双脱氧修饰。
8、连接反应后的产物按照如下两种方法进行纯化，方法1：加入1倍体积的PBI混匀后再用1.3倍数的Ampure XP beads纯化(自动化操作)；方法2：用QIAGEN PCR纯化柱纯化。纯化后，采用纯水回融。
9、按照如下PCR反应体系(表6)及反应条件(表7)进行PCR扩增。
表6
组分 含量
纯化后的产物DNA 30μL
5×PCR缓冲液 10μL
10mM dNTP 1μL
引物1 2μL
引物2 2μL
PCR酶 1μL
纯水 4μL
合计 50μL
注：PCR引物序列如下：
单接头引物1：
AGACAAGCTCGAGCTCGAGCGATCGGGCTTCGACTGGAGAC(SEQ ID NO：11)；
单接头引物2：TCCTAAGACCGCTTGGCCTCCGACT(SEQ ID NO：12)；
双接头引物1：AATGATACGGCGACCACCGA(SEQ ID NO：13)；
双接头引物2：CAAGCAGAAGACGGCATACGA(SEQ ID NO：14)。
表7
Figure PCTCN2014088542-appb-000002
10、连接空隙接头(即二号接头)后的PCR产物检测结果如图2所示，PCR产物浓度测定结果如下(表8)：
表8
Figure PCTCN2014088542-appb-000003
Figure PCTCN2014088542-appb-000004
PCR结果说明：本发明的方法成功实现了空隙接头的引入。
11、按照目前测序的通用手段，对PCR产物经过单链分离后将目的条带进行单链环化，从而得到单链环状的DNA分子，用于全基因组测序平台上滚环复制制备DNA纳米球和连接测序。单链分离及环化操作方法如下：
(1)将PCR产物进行95℃热变性，然后立即冰浴5min；
(2)将3pmol的PCR产物变性后的单链分子按照如下反应体系(表9)进行单链环化操作；
表9
组分 含量
介导序列(20μM) 20μL
纯水 158.3μL
10×连接缓冲液 35μL
100mM ATP 3.5μL
连接酶 1.2μL
变性后PCR产物 112μL
合计 350μL
注：介导序列如下：
单接头介导序列：TCGAGCTTGTCTTCCTAAGACCGC(SEQ ID NO：15)；
双接头介导序列：CGCCGTATCATTCAAGCAGAAGAC(SEQ ID NO：16)。
(3)消化未环化单链，按如下体系(表10)配置反应体系，混匀，短暂离心后，取20μL加入上一步反应体系中，继续37℃孵育30分钟。之后用1.8倍数的Ampure XP磁珠纯化即制备完成用于连接测序的单链环状分子。
表10
组分 含量
10×连接缓冲液 3.7μL
20U/μL外切酶I 11.1μL
100U/μL外切酶III 5.2μL
合计 20μL
12、测序可以从5’端和3’端进行测序，两端连接有不同序列的目的片段由于19bp转座酶识别序列只存在于一端，从而避免了两端19bp转座酶识别序列的特异退火而与测序接头产生竞争，进而大幅度地提高了测序质量，结果如图3所示。图3所示的数据大都在80～90之间，一般在75以上认为是可以接受的，而传统的两端有19bp转座酶识别序列的方法的测序结果该数据一般达不到这样高，甚至只有30～40之间，这说明本发明的19bp序列互补的测序探针可以很好的匹配到测序模板，也就是解决了转座酶两条19bp反向互补序列对测序的影响。
实施例2
本实施例首先用包埋的转座酶复合体将50ng高质量的基因组DNA进行打断操作，经过蛋白酶、SDS、NT或蛋白酶和EDTA组合物来处理，去除结合在DNA上的转座酶蛋白；通过空隙接头连接后，直接用PCR引物进行扩增，PCR反应体系中加入一定浓度的TritonX-100。
1、设计订购带有19bp转座酶识别序列的一对引物序列，序列A和序列B，用于制备包埋用的一号接头单接头：
一号接头单接头序列A：TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG(SEQ ID NO：17)；
一号接头单接头序列B：CTGTCTCTTATACACATC ddT(SEQ ID NO：18，dd表示双脱氧修饰)。
2、将序列A和序列B稀释到100μM，充分混合后离心，于PCR仪中按如下程序(表11)退火得到一号接头(储存于-20℃)，用于包埋复合体的制备。
表11
Figure PCTCN2014088542-appb-000005
3、按照如下体系(表12)将一号接头与转座酶包埋成转座酶复合体，轻轻吹打20次混合，30℃孵育1小时后完成复合体包埋。该复合体储存于-20℃。
表12
组分 含量
转座酶 85μL
一号接头 30μL
偶联缓冲液 85μL
合计 200μL
4、按照如下体系(表13)将50ng的高质量基因组和转座酶复合体进行混合，轻轻吹打20次混合，55℃孵育10分钟后降温至4℃，完成基因组的打断。
表13
组分 含量
水 5μL
5×打断缓冲液 2μL
gDNA(50ng/μL) 1μL
打断酶复合体 2μL
合计 10μL
5、打断后的样本处理方法有以下几种选择，方法1：加入0.1-5μL蛋白酶(750 mAU/mL)进行处理，本实施优选2μL蛋白酶，同时对0.1μL和5μL蛋白酶用量分别进行了测试；方法2：加入终浓度的商业1×NT缓冲液(Truprep试剂盒S5系列中配套试剂)；方法3：加入0.01％-1.5％(体积比)的SDS，本实施例优选1％(体积比)的SDS，同时对0.01％(体积比)和1.5％(体积比)浓度分别进行了测试；方法4：加入0.1-5μL蛋白酶进行处理，然后加入终浓度1-50mM的EDTA，本实施例优选2μL蛋白酶和终浓度14mM的EDTA，同时对0.1μL蛋白酶加1mM EDTA和5μL蛋白酶加50mM EDTA组合进行测试；方法5：加入1倍体积的PBI(Qiagen PCR纯化试剂盒中的一种商业试剂)混匀后再用1.3倍数的Ampure XP beads纯化，回融采用纯水。
6、在上述处理以后的产物中，加入0.1％-2％(体积比)的Triton-X100，本实施例中优选1％(体积比)，同时对0.1％(体积比)和2％(体积比)用量的Triton-X100进行测试。
7、上述Triton-X100处理后的产物按照如下体系(表14)进行空隙接头(二号接头)的连接，25℃孵育60分钟完成接头连接。
表14
组分 含量
水 8μL
3×连接buffer 20μL
接头(5μM) 10μL
连接酶 2μL
DNA 20μL
合计 30μL
注：二号接头序列A：5’-pAAGTCGGAGGCCAAGCGGTCGT ddC-3’(SEQ ID NO：9)；二号接头序列B：5’-TTGGCCTCCGACT ddT-3’(SEQ ID NO：10)(p表示磷酸化修饰；dd表示双脱氧修饰)。
8、按照如下PCR反应体系(表15)及反应条件(表16)进行PCR扩增。对于加入了SDS的实验组，在该PCR体系中还需要加入特定浓度的吐温-20来部分提高PCR的效率，吐温-20的工作浓度可以调整到不同，如0.1％-2％(体积比)，本实施例优选0.5％，同时对0.1％(体积比)和2％(体积比)工作浓度的吐温-20进行了测试。
表15
组分 含量
处理后的DNA样品 30μL
5×PCR缓冲液 10μL
10mM dNTP 1μL
引物1 2μL
引物2 2μL
PCR酶(DNA聚合酶) 1μL
纯水 4μL
合计 50μL
注：一号接头单接头引物1：
AGACAAGCTCGAGCTCGAGCGATCGGGATCTACACGACTCACTGATCGTCGGCAGCGTC(SEQ ID NO：19)；一号接头单接头引物2： TCCTAAGACCGCTTGGCCTCCGACT(SEQ ID NO：20)。
表16
Figure PCTCN2014088542-appb-000006
9、单接头包埋复合体打断及空隙接头连接后PCR产物检测结果如图4所示，PCR产物浓度测定结果如表17。
表17
Figure PCTCN2014088542-appb-000007
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 (15)

  1. 一种转座酶打断核酸并加接头的方法，包括如下步骤：
    使用转座酶包埋复合体对核酸进行随机打断，其中所述转座酶包埋复合体包含转座酶和含转座酶识别序列的第一接头，打断的核酸两端连接所述第一接头并且形成缺口；
    通过纯化或化学试剂处理的方式，去除体系中的转座酶对后续反应的影响；
    使用连接酶在所述缺口处连接上第二接头，所述第二接头的序列不同于所述第一接头；
    使用分别靶向结合所述第一接头和所述第二接头的引物进行PCR反应，得到两端分别连接有不同接头序列的产物。
  2. 根据权利要求1所述的方法，其特征在于，所述第一接头上带有防止自连或与所述第二接头互连的修饰。
  3. 根据权利要求2所述的方法，其特征在于，所述第一接头上的修饰方式包括如下任一种或多种的组合：
    (a)所述第一接头的3’端碱基双脱氧修饰；
    (b)所述第一接头的一条链中引入dUTP，用于之后的USER酶切过量接头；
    (c)所述第一接头的转座酶识别序列外侧引入一个碱基对，其中3’端碱基双脱氧修饰；和
    (d)所述第一接头由一条完整序列组成，内部互补形成一段3’-5’磷酸二酯键交联的双链序列。
  4. 根据权利要求3所述的方法，其特征在于，所述第一接头上的修饰方式为所述第一接头的3’端碱基双脱氧修饰。
  5. 根据权利要求1所述的方法，其特征在于，所述第二接头上带有防止自连的修饰；
    优选地，所述第二接头上的修饰为3’端碱基双脱氧修饰。
  6. 根据权利要求1所述的方法，其特征在于，所述PCR反应使用的引物中的一条引物是末端生物素标记的引物，用于通过生物素-链霉亲和素亲和反应而获取单链分子。
  7. 根据权利要求1-6任一项所述的方法，其特征在于，所述纯化为磁珠纯化或过柱纯化。
  8. 根据权利要求1-6任一项所述的方法，其特征在于，所述化学试剂处理为使转座酶变性或消化而从靶序列上解离下来的处理。
  9. 根据权利要求8所述的方法，其特征在于，所述化学试剂包括第一试剂和第二试剂；其中，所述第一试剂，包括蛋白酶溶液、SDS溶液和NT缓冲液中的一种或多种，用于打破所述转座酶与所述核酸的靶序列的吸附作用；所述第二试剂，包含Triton-X100溶液，用于减弱所述第一试剂对后续酶促反应的影响。
  10. 根据权利要求9所述的方法，其特征在于，所述第一试剂还包括含有EDTA的附加试剂；
    优选地，所述第二试剂还包含吐温-20溶液。
  11. 一种转座酶打断核酸并加接头的试剂，包括如下组成部分：
    转座酶和含转座酶识别序列的第一接头，用于形成转座酶包埋复合体以对核酸进行随机打断，在打断的核酸两端连接所述第一接头并且形成缺口；
    第二接头和连接酶组分，用于在所述缺口处连接上第二接头；和
    PCR引物，分别靶向结合所述第一接头和所述第二接头，用于进行PCR反应，得到两端分别连接有不同接头序列的产物。
  12. 根据权利要求11所述的试剂，其特征在于，所述第一接头上带有防止自连或与所述第二接头互连的修饰。
  13. 根据权利要求12所述的试剂，其特征在于，所述第一接头上的修饰方式包括如下任一种或多种的组合：
    (a)所述第一接头的3’端碱基双脱氧修饰；
    (b)所述第一接头的一条链中引入dUTP，用于之后的USER酶切过量接头；
    (c)所述第一接头的转座酶识别序列外侧引入一个碱基对，其中3’端碱基双脱氧修饰；和
    (d)所述第一接头由一条完整序列组成，内部互补形成一段3’-5’磷酸二酯键交联的双链序列。
  14. 根据权利要求11所述的试剂，其特征在于，所述第二接头上带有防止自连的修饰；
    优选地，所述第二接头上的修饰为3’端碱基双脱氧修饰。
  15. 根据权利要求11所述的试剂，其特征在于，所述PCR引物中的一条引 物是末端生物素标记的引物，用于通过生物素-链霉亲和素亲和反应而获取单链分子。
PCT/CN2014/088542 2014-10-14 2014-10-14 一种转座酶打断核酸并加接头的方法和试剂 WO2016058127A1 (zh)

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