WO2022001149A1 - 一种用于构建测序文库的酶反应液及其应用 - Google Patents

一种用于构建测序文库的酶反应液及其应用 Download PDF

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WO2022001149A1
WO2022001149A1 PCT/CN2021/078865 CN2021078865W WO2022001149A1 WO 2022001149 A1 WO2022001149 A1 WO 2022001149A1 CN 2021078865 W CN2021078865 W CN 2021078865W WO 2022001149 A1 WO2022001149 A1 WO 2022001149A1
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reaction solution
enzyme reaction
enzyme
dna polymerase
optionally
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PCT/CN2021/078865
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French (fr)
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聂俊伟
瞿志鹏
张力军
韩锦雄
江明扬
邱翠
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江苏康科斯医疗科技有限公司
南京诺唯赞生物科技股份有限公司
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Priority to US17/999,296 priority Critical patent/US20230193252A1/en
Priority to EP21832203.0A priority patent/EP4130256A4/en
Publication of WO2022001149A1 publication Critical patent/WO2022001149A1/zh

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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
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01078—Polynucleotide 5'-hydroxyl-kinase (2.7.1.78)
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
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    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present application belongs to the field of biotechnology, and relates to an enzyme reaction solution for constructing a sequencing library and its application.
  • the high-throughput sequencing market is growing rapidly as the cost of high-throughput sequencing has plummeted.
  • DNA sequencing accounts for about half of the entire high-throughput sequencing market, so there is an increasingly urgent need for a simple, fast, and high-throughput DNA library construction technology.
  • the first and most critical step in the DNA library construction process is to unbiased fragmentation of large DNA fragments into small DNA fragments of a specific length.
  • the current mainstream DNA fragmentation method is ultrasonic mechanical interruption.
  • ultrasonic mechanical interruption instruments are often expensive, and many industrial, scientific research, and medical laboratories cannot be equipped with this instrument; the operation of ultrasonic mechanical fragmentation of DNA is cumbersome and time-consuming, and real-time sampling is required to detect the fragmentation effect of samples; and ultrasonic mechanical interruption
  • the number of samples produced is small, and the input amount of a single processing sample is small, which cannot meet the needs of high-throughput processing samples in the industrial and medical fields. Fragmentase can solve the above problems well.
  • the enzymatic-based DNA sample processing method can be completed in a thermal cycler, and the operation process is simple and fast.
  • Fragmentase can perform non-specific random cutting of DNA molecules, so it is expected to replace The traditional ultrasonic mechanical method unbiased fragmentation of large fragments of DNA molecules into small fragments of DNA molecules of a specific length, which can be applied to DNA library construction in high-throughput sequencing.
  • the existing enzymatic-based DNA fragmentation technologies include: 1) randomly generating a nick on a double-stranded DNA molecule using Vvn, and at the same time using T7 endonuclease to cut another DNA molecule at the nick site; 2) using DNaseI breaks double-stranded DNA in the presence of Mn 2+ or Mg 2+ ; 3) Multiple restriction endonucleases (such as MspI, AluI, CviQI, MseI, MlucI, HaeIII, etc. ) mixed, and the cleavage of DNA molecules was carried out.
  • the initial input amount of samples for constructing DNA libraries ranges from 100pg to 1 ⁇ g, and there are 5 orders of magnitude difference, and the commercial kits on the market are not enough to meet the market demand in terms of input amount compatibility.
  • the lower compatibility limit of commercial kits is above 100 pg, and on the other hand, different starting amounts require different reaction times.
  • the reaction time needs to be adjusted for different starting amounts of samples, which not only increases the complexity of the experiment and the demand for instruments, but also limits the application of library construction methods in large-throughput samples.
  • CN108998434A discloses a fragmentation enzyme interruption buffer and a method for improving the fragmentation efficiency of the fragmentation enzyme.
  • the fragmentation enzyme interruption buffer includes buffer components suitable for the fragmentation enzyme to function and the final concentration is below 10% PEG8000, and Mg 2+ with a final concentration of 10-25 mM, by adding different proportions of PEG and Mg 2+ in the reaction buffer system to adjust the efficiency and fragment size of DNA fragmentation, to obtain PE100 that can meet the BGISEQ-500 platform
  • the interruption buffer required by the PE150 paired-end sequencing library is still incompatible with samples with a starting amount of 100pg to 1 ⁇ g.
  • the present application provides an enzyme reaction solution for constructing a sequencing library and an application thereof.
  • the enzyme reaction solution is balanced by optimizing the type and amount of the enzyme composition and cooperating with the metal ions and the buffer medium in the reaction buffer.
  • the enzyme cleavage reaction rate and the end repair reaction rate in the process of library construction are realized, and the technical effect of obtaining a sequencing library of the same length under the condition of different sample starting amount and the same processing time is realized, which has a wide range of applicability and convenience. operability.
  • the application provides an enzyme reaction solution for constructing a sequencing library, the enzyme reaction solution comprising an enzyme composition and a reaction buffer;
  • the enzyme composition includes endonucleases, DNA polymerases and polynucleotide kinases;
  • the reaction buffer includes metal salts, substrates and aqueous buffer media.
  • Enzyme compositions of the present application include endonucleases for cleavage, DNA polymerases for end repair and 3' A-tailing, and polynucleotide kinases for 5' phosphorylation, all of which
  • the enzyme composition is concentrated in one reaction system, and the cleavage, end repair and A-tail treatment of nucleic acid samples are realized through one-step reaction. It also contains the substrate molecules dNTPs, dATP and ATP required for various reactions. Various components cooperate with each other within a certain concentration range to balance the enzyme cleavage reaction rate and the end repair reaction rate.
  • the technical effect of obtaining a sequencing library of the same length when the sample input amount is 100pg-1 ⁇ g and the processing time is the same.
  • the present application uses an endonuclease to cut a nucleic acid sample
  • the endonuclease can be, for example, endonuclease dsDNase, T7 endonuclease, salt-active endonuclease SAN, endonuclease Vvn or endonuclease Any one or a combination of at least two of the Dicer DNaseI.
  • the endonuclease may be, for example, a shrimp-derived endonuclease dsDNase, a T7 phage-derived endonuclease T7 Endonuclease, a salt-active endonuclease SAN, a V. vulnificus-derived endonuclease Any one or a combination of at least two of the endonuclease Vvn or the endonuclease DNaseI derived from bovine pancreas.
  • the final concentration of the endonuclease in the enzyme reaction solution is 0.003-0.05U/ ⁇ L, for example, it can be 0.003U/ ⁇ L, 0.004U/ ⁇ L, 0.005U/ ⁇ L, 0.006U / ⁇ L, 0.007U/ ⁇ L, 0.008U/ ⁇ L, 0.009U/ ⁇ L, 0.01U/ ⁇ L, 0.02U/ ⁇ L, 0.03U/ ⁇ L, 0.04U/ ⁇ L or 0.05U/ ⁇ L, preferably 0.004 ⁇ 0.03U/ ⁇ L, more preferably 0.005 to 0.01 U/ ⁇ L.
  • the final concentration of the endonuclease Vvn in the enzyme reaction solution is 0.1-0.5ng/ ⁇ L, for example, it can be 0.1ng/ ⁇ L, 0.2ng/ ⁇ L, 0.3ng/ ⁇ L, 0.4ng/ ⁇ L or 0.5ng/ ⁇ L.
  • the present application is aimed at different kinds of endonucleases, by optimizing the concentration of the endonuclease in the reaction system and controlling the amount of the endonuclease, so as to adjust the cutting rate of the nucleic acid by the endonuclease.
  • thermostable DNA polymerase can be Taq DNA polymerase, for example, to carry out A tailing reaction at the 3' end of the cut nucleic acid, so that the subsequent reaction is based on The TA ligation principle performs the linker ligation reaction.
  • the term “heat-stable DNA polymerase” may be Taq DNA polymerase, the residual activity of which is greater than 90% of the original after the reaction at 70°C for 2 hours, and the residual activity of which is 60% of the original after the reaction at 93°C for 2 hours, Its residual activity is 40% of the original after 2 hours of reaction at 95°C.
  • the term “low temperature DNA polymerase” refers to a DNA polymerase without thermostable activity relative to "heat-resistant DNA polymerase".
  • the final concentration of the low-temperature DNA polymerase in the enzyme reaction solution is 0.01-0.05U/ ⁇ L, for example, 0.01U/ ⁇ L, 0.02U/ ⁇ L, 0.03U/ ⁇ L, 0.04U / ⁇ L or 0.05U/ ⁇ L, preferably 0.02-0.04U/ ⁇ L.
  • the final concentration of the thermostable DNA polymerase in the enzyme reaction solution is 0.03-1.2 U/ ⁇ L, for example, 0.03 U/ ⁇ L, 0.04 U/ ⁇ L, 0.05 U/ ⁇ L, 0.06 U/ ⁇ L U/ ⁇ L, 0.07U/ ⁇ L, 0.08U/ ⁇ L, 0.09U/ ⁇ L, 0.1U/ ⁇ L, 0.2U/ ⁇ L, 0.3U/ ⁇ L, 0.4U/ ⁇ L, 0.5U/ ⁇ L, 0.6U/ ⁇ L, 0.7 U/ ⁇ L, 0.8U/ ⁇ L, 0.9U/ ⁇ L, 1.0U/ ⁇ L, 1.1U/ ⁇ L or 1.2U/ ⁇ L, preferably 0.04-1.1U/ ⁇ L, more preferably 0.05-1.0U/ ⁇ L, still more preferably It is 0.06 ⁇ 0.9U/ ⁇ L.
  • the present application is aimed at different kinds of DNA polymerases, by optimizing the concentration of the DNA polymerase in the reaction system and controlling the amount of the DNA polymerase, so as to adjust the end repair rate of the DNA polymerase for the cut nucleic acid.
  • the enzyme reaction solution of the present application also includes a polynucleotide kinase, such as T4 polynucleotide kinase (T4 PNK), which performs 5' phosphorylation on the cleaved nucleic acid, and the polynucleotide kinase
  • T4 PNK T4 polynucleotide kinase
  • the final concentration in the enzyme reaction solution is 0.05-0.2U/ ⁇ L, such as 0.05U/ ⁇ L, 0.06U/ ⁇ L, 0.07U/ ⁇ L, 0.08U/ ⁇ L, 0.09U/ ⁇ L, 0.1U/ ⁇ L or 0.2U/ ⁇ L.
  • the present application adopts an accessory protein to regulate the cleavage rate of endonuclease and the end repair rate of DNA polymerase
  • the accessory protein includes bovine serum albumin (BSA) and/or single-stranded binding protein, preferably, the single-stranded Binding proteins include the single-chain binding protein E. coli SSB derived from Escherichia coli and/or the single-chain binding protein T4 GP32 derived from T4 phage.
  • the final concentration of the auxiliary protein in the enzyme reaction solution is 0.05-1 ⁇ g/ ⁇ L, such as 0.05 ⁇ g/ ⁇ L, 0.06 ⁇ g/ ⁇ L, 0.07 ⁇ g/ ⁇ L, 0.08 ⁇ g/ ⁇ L, or 1 ⁇ g/ ⁇ L, preferably 0.05 to 0.5 ⁇ g/ ⁇ L, more preferably 0.1 to 0.4 ⁇ g/ ⁇ L.
  • the present application is aimed at different kinds of accessory proteins, by optimizing the concentration of accessory proteins in the reaction system and controlling the amount of accessory proteins, so as to adjust the cleavage rate of endonuclease and the end repair rate of DNA polymerase.
  • the metal cations and the buffer medium jointly adjust the reaction system to be in a suitable acid-base environment, and control the enzymatic activities of endonuclease and DNA polymerase, so as to balance the enzyme cleavage reaction rate and end-repair reaction rate.
  • the final concentration of Mg 2+ in the enzyme reaction solution is 0-20 mM, such as 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM , 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM or 20 mM, preferably 2 to 18 mM, more preferably 3 to 17 mM, still more preferably 4 to 16 mM or 5 to 15 mM.
  • the final concentration of Mn 2+ in the enzyme reaction solution is 0.05-1 mM, such as 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM or 1 mM, preferably 0.07 to 0.8 mM, more preferably 0.09 to 0.6 mM, still more preferably 0.1 to 0.5 mM.
  • the final concentration of Na+ in the enzyme reaction solution is 0-50mM, for example, it can be 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM or 50mM, preferably 0-30mM, more preferably 5-30mM, still more preferably 5-20mM or 5 ⁇ 15mM.
  • the final concentration of the Ca 2+ in the enzyme reaction solution is 0-10 mM, such as 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM, preferably 0 to 8 mM, 0 to 5 mM, 0 to 3 mM, 1 to 10 mM, 1 to 5 mM, 1 to 3 mM, 2 to 10 mM, or 2 to 5 mM.
  • the substrate comprises any one or a combination of at least two of dNTPs, dATP or ATP.
  • the final concentration of the dNTPs in the enzyme reaction solution is 0.05-0.5mM, such as 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.1mM, 0.2mM, 0.3mM mM, 0.4mM or 0.5mM, preferably 0.05 ⁇ 0.4mM, 0.05 ⁇ 0.3mM, 0.05 ⁇ 0.2mM, 0.05 ⁇ 0.1mM, 0.07 ⁇ 0.5mM, 0.07 ⁇ 0.4mM, 0.07 ⁇ 0.3mM, 0.07 ⁇ 0.2mM, 0.07-0.1 mM, 0.09-0.5 mM, 0.09-0.4 mM, 0.09-0.3 mM, 0.09-0.2 mM or 0.09-0.1 mM.
  • the concentration of dNTPs is limited within a certain range, which is beneficial to regulate the end-repair rate of DNA polymerase.
  • the final concentration of the dATP in the enzyme reaction solution is 0.1-2 mM, such as 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM , 0.9mM, 1mM, 1.1mM, 1.2mM, 1.3mM, 1.4mM, 1.5mM, 1.6mM, 1.7mM, 1.8mM, 1.9mM or 2mM, preferably 0.1 ⁇ 1.5mM, 0.1 ⁇ 1mM, 0.3 ⁇ 2mM, 0.3-1.5 mM, 0.3-1 mM, 0.5-2 mM, 0.5-1.5 mM, 0.5-1 mM or 0.5-0.8 mM.
  • the final concentration of the ATP in the enzyme reaction solution is 1-10 mM, such as 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM, preferably 1 mM ⁇ 9mM, 1 ⁇ 8mM, 1 ⁇ 7mM, 1 ⁇ 6mM, 1 ⁇ 5mM, 3 ⁇ 10mM, 3 ⁇ 9mM, 3 ⁇ 8mM, 3 ⁇ 7mM or 3 ⁇ 6mM.
  • the buffer medium includes 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and/or tris(hydroxymethylaminomethane) (TRIS).
  • HPES 4-hydroxyethylpiperazineethanesulfonic acid
  • TMS tris(hydroxymethylaminomethane)
  • the final concentration of the buffer medium in the enzyme reaction solution is 10-50 mM, such as 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM, preferably 10-50 mM. 40 mM, 10-30 mM, 15-50 mM, 15-45 mM, 15-40 mM or 15-30 mM.
  • the pH of the enzyme reaction solution is 7.0-8.5, for example, it can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5, preferably 7.5 to 8.1.
  • the enzyme composition comprises T4 PNK, BSA, T4 GP32, Vvn, T7 endonuclease, Taq DNA polymerase, and Klenow.
  • the enzyme composition comprises T4 PNK, BSA, E. coli SSB, dsDNase, Taq DNA polymerase, and Klenow.
  • the enzyme composition comprises T4 PNK, BSA, T4 GP32, SAN, Taq DNA polymerase, and E. coli DNA polymerase I.
  • the enzyme composition comprises T4 PNK, BSA, T4 GP32, DNaseI, Taq DNA polymerase, and T4 DNA polymerase.
  • the reaction buffer comprises 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), MgCl 2, CaCl 2, NaCl, MnCl 2, dNTPs, dATP and ATP.
  • HEPES 4-hydroxyethylpiperazine ethanesulfonic acid
  • the reaction buffer contains Tris or (TRIS), MgCl 2, CaCl 2, NaCl, MnCl 2, dNTPs, dATP and ATP.
  • the enzymatic reaction solution further comprises glycerol.
  • the enzyme reaction solution comprises tris(TRIS), MgCl 2 , CaCl 2 , NaCl, MnCl 2 , dNTPs, dATP, ATP, T4 PNK, BSA, T4 GP32, DNaseI, Taq DNA polymerase, T4 DNA polymerase and glycerol.
  • the enzyme reaction solution comprises 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), MgCl 2 , CaCl 2 , NaCl, MnCl 2 , dNTPs, dATP, ATP, T4 PNK, BSA, T4 GP32 , DNaseI, Taq DNA polymerase, T4 DNA polymerase and glycerol.
  • HEPES 4-hydroxyethylpiperazineethanesulfonic acid
  • the enzyme reaction solution comprises 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), MgCl 2 , CaCl 2 , NaCl, MnCl 2 , dNTPs, dATP, ATP, T4 PNK, BSA, T4 GP32 , DNaseI, Taq DNA polymerase, T4 DNA polymerase and glycerol.
  • HEPES 4-hydroxyethylpiperazineethanesulfonic acid
  • the enzyme reaction solution comprises tris (TRIS), MgCl 2 , CaCl 2 , NaCl, MnCl 2 , dNTPs, dATP, ATP, T4 PNK, BSA, T4 GP32, SAN, Taq DNA polymerase, E. coli DNA polymerase I and glycerol.
  • the enzyme reaction solution comprises Tris (TRIS) 30 mM, MgCl 2 15 mM, CaCl 2 5 mM, NaCl 10 mM, MnCl 2 0.1 mM, dNTPs 0.1 mM, dATP 0.6 mM, ATP 1 mM, T4 PNK 0.1 U/ ⁇ L, BSA 0.3 ⁇ g/ ⁇ L, T4 GP32 0.1 ⁇ g/ ⁇ L, DNaseI 0.06 U/ ⁇ L, Taq DNA polymerase 0.3 U/ ⁇ L, T4 DNA polymerase 0.9 U/ ⁇ L and glycerol.
  • Tris Tris
  • the enzymatic reaction solution includes 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) 20 mM, MgCl 2 15 mM, CaCl 2 5 mM, NaCl 10 mM, MnCl 2 0.5 mM, dNTPs 0.1 mM, dATP 0.6 mM , ATP 1mM, T4 PNK 0.1U/ ⁇ L, BSA 0.3 ⁇ g/ ⁇ L, T4 GP32 0.1 ⁇ g/ ⁇ L, DNaseI 0.06U/ ⁇ L, Taq DNA polymerase 0.3U/ ⁇ L, T4 DNA polymerase 0.9U/ ⁇ L and glycerol.
  • HEPES 4-hydroxyethylpiperazine ethanesulfonic acid
  • the enzymatic reaction solution includes 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) 20 mM, MgCl 2 5 mM, CaCl 2 5 mM, NaCl 10 mM, MnCl 2 0.1 mM, dNTPs 0.1 mM, dATP 0.6 mM , ATP 1mM, T4 PNK 0.1U/ ⁇ L, BSA 0.3 ⁇ g/ ⁇ L, T4 GP32 0.1 ⁇ g/ ⁇ L, DNaseI 0.06U/ ⁇ L, Taq DNA polymerase 0.3U/ ⁇ L, T4 DNA polymerase 0.9U/ ⁇ L and glycerol.
  • HEPES 4-hydroxyethylpiperazine ethanesulfonic acid
  • the enzyme reaction solution comprises Tris (TRIS) 30 mM, MgCl 2 15 mM, CaCl 2 5 mM, NaCl 10 mM, MnCl 2 0.5 mM, dNTPs 0.1 mM, dATP 0.6 mM, ATP 1 mM, T4 PNK 0.1 U/ ⁇ L, BSA 0.3 ⁇ g/ ⁇ L, T4 GP32 0.1 ⁇ g/ ⁇ L, SAN 0.06 U/ ⁇ L, Taq DNA polymerase 0.3 U/ ⁇ L, E. coli DNA polymerase I 0.06 U/ ⁇ L and glycerol.
  • Tris Tris
  • the type and concentration of metal ions in the reaction system and the type and concentration of the buffer medium cooperate with each other within a certain range to maintain the pH value of the enzyme reaction solution in a relatively stable and suitable state, and affect the endonuclease and DNA polymerization.
  • the activity of the enzyme ensures that the enzyme cleavage reaction rate and the end repair reaction rate are consistent, so that the length of the constructed sequencing library is suitable for the next-generation sequencing platform.
  • the application provides a method for constructing a sequencing library, the method comprising:
  • the incubation product is ligated with the sequencing adapter
  • the ligation product was subjected to PCR to obtain a sequencing library.
  • the target nucleic acid is added in an amount of 100 pg to 1 ⁇ g, for example, 100 pg, 1 ng, 10 ng, 100 ng or 1 ⁇ g.
  • the incubation conditions are 35-40°C for 10-20 minutes, 60-70°C for 20-40 minutes, preferably 37°C for 15 minutes, and 65°C for 30 minutes.
  • the present application provides a kit for constructing a sequencing library, the kit comprising the enzyme reaction solution described in the first aspect.
  • the kit further includes any one or a combination of at least two of sequencing adapters, ligation reaction reagents or PCR reagents.
  • reaction buffer and the enzyme composition in the enzyme reaction solution of the present application may be separately packaged, and the final concentrations of the reaction buffer and the enzyme composition after mixing are consistent with or substantially consistent with the present application , also within the scope of protection of this application.
  • the application provides a sequencing library, the sequencing library is constructed using the method described in the second aspect or the kit described in the third aspect;
  • the length of the sequencing file is 300-500 bp.
  • the present application integrates nucleic acid sample fragmentation, end repair, 3' addition of dA tail and 5' phosphorylation modification into a one-step reaction
  • the enzyme composition includes an endonuclease for cleavage, end repair and
  • the DNA polymerase with 3' A tail and the polynucleotide kinase used for 5' phosphorylation cooperate with the reaction buffer within a certain concentration range to control the rate of the enzyme cleavage reaction and the rate of the end repair reaction.
  • the enzyme reaction solution of the present application further simplifies the enzymatic-based DNA fragmentation and library construction methods, and has wider applicability and more convenient operability.
  • Fig. 1 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 1;
  • Fig. 2 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 2;
  • Fig. 3 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 3;
  • Fig. 4 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 4;
  • Fig. 5 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 5;
  • Fig. 6 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 6;
  • Figure 8 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 8;
  • Fig. 9 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 9;
  • Figure 10 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 10;
  • Figure 11 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 11;
  • Figure 12 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 12;
  • Figure 13 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 13;
  • Figure 15 is the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 15;
  • Figure 16 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 16.
  • the enzyme reaction solution is first prepared, including two parts: the reaction buffer shown in Table 1 and the enzyme composition shown in Table 2, and the reaction buffer shown in Table 1 and the enzyme composition shown in Table 2 are crossed Combination, the enzyme reaction solutions of different formulas as shown in Table 3 are obtained, the manufacturer and the article number of the protease are shown in Table 4, and the amino acid sequence of Vibrio vulnificus nuclease Vvn is shown in SEQ ID NO: 1.
  • the salmon sperm gDNA was used as the template, and the initial input amounts were 100 pg, 1 ng, 10 ng, 100 ng and 1 ⁇ g, respectively.
  • the different enzyme reaction solutions of Example 1 were used to cut the gDNA, end repair, add A tail to the 3' and 5 ' phosphorylated and used Vayzme #ND607 in subsequent examples Universal DNA Library Prep Kit for Rapid DNA Ligation Buffer and Rapid DNA Ligase in V3 for adapter ligation, using Vazyme #N401 DNA Clean Beads purify adapter ligation products and PCR amplification products using Vayzme #ND607 Universal DNA Library Prep Kit for The VAHTS HiFi Amplification Mix and PCR Primer Mix 3 for Illumina in V3 are used to amplify and enrich the purified ligation products, where the adaptor used for the ligation reaction is DNA Adapters for (Vazyme #N801).
  • the reaction system of fragmentation, end repair, 5' phosphorylation and 3' addition of dA tail of target DNA is shown in Table 5, wherein the formula of the enzyme composition adopts one of the combinations 1 to 16 of Example 1, and the target
  • the input amount of DNA X ⁇ L corresponds to 100pg, 1ng, 10ng, 100ng and 1 ⁇ g, respectively.
  • the reaction conditions are incubation at 37°C for 15min and 65°C for 30min.
  • the obtained fragmented products are used for sequencing library construction.
  • the fragmented product prepared in Example 2 was subjected to adapter ligation reaction, PCR amplification reaction and purification to construct a sequencing library.
  • the adapter ligation reaction system was shown in Table 6. Adapter ligation; use 60 ⁇ L of VAHTS DNA Clean Beads to purify the ligation product with an elution volume of 20/22.5 ⁇ L;
  • the purified product was subjected to PCR amplification, the system was shown in Table 7, the conditions were shown in Table 8, and 45 ⁇ L of VAHTS DNA Clean Beads were used to purify the amplified product, and the elution volume was 20/22.5 ⁇ L.
  • Linker ligation purified product 20 ⁇ L PCR amplification enzymes and their buffers (VAHTS HiFi Amplification Mix) 25 ⁇ L Primers (PCR Primer Mix 3 for Illumina) 5 ⁇ L
  • the Agilent 2100 DNA1000 chip was used to detect the length distribution of the DNA library. The results are shown in Figure 1 to Figure 16. It can be seen that each group of enzyme reaction solutions can achieve effective cutting and end repair of DNA with different input amounts within the same processing time. , 5' phosphorylation modification and 3' dA tail.
  • the effect of the enzyme reaction solution of combination 16 is the best, it can be fully compatible with the initial DNA input amount of 100pg ⁇ 1 ⁇ g, and the distribution range of the library fragment length is consistent, mainly in the range of 400 ⁇ 500bp
  • the enzyme composition is 0.3U DNaseI, 45U T4 DNA polymerase, 1.5U Taq DNA polymerase, 5U T4 PNK, 15 ⁇ g BSA, and 5 ⁇ g T4 GP32, with reaction buffer (300 mM Tris, 150 mM MgCl 2 , 30 mM CaCl 2 ) , 100 mM NaCl, 1 mM MnCl 2 , 1 mM dNTPs, 6 mM dATP and 50 mM ATP), which can perfectly balance the enzyme digestion reaction rate and the end repair reaction rate, and prepare a sequencing library with the same fragment length;
  • the enzyme reaction solutions of combination 8, combination 11 and combination 4 also have good effects, and have good compatibility with DNA with an initial input amount of 100 pg to 1 ⁇ g, and the prepared fragment length It is generally consistent, indicating that when the enzyme composition is 0.004-0.008U/ ⁇ L endonuclease, 0.05-0.9U/ ⁇ L DNA polymerase, 0.01-0.05U/ ⁇ L Taq DNA polymerase, 0.05-0.3U/ ⁇ L T4 PNK and 0.2 ⁇ 0.6 ⁇ g/ ⁇ L accessory protein, with reaction buffer 100 ⁇ 400mM buffer medium, 100 ⁇ 300mM Mg 2+ , 1 ⁇ 6mM Mn 2+ , 50 ⁇ 250mM Na + , 5 ⁇ 50mM Ca 2+ , 0.5 ⁇ 5mM dNTPs, 1-10 mM dATP and 20-40 mM ATP cooperate with each other to regulate the reaction rate of enzyme cleavage and end-repair reaction, so that the fragment lengths of the
  • the suitable initial input amount of the enzyme reaction solution of combination 1 is 100pg to 10ng, and further increase of the initial input amount will affect the length of the fragment; it can be seen from Figure 2 that the enzyme reaction solution of combination 2 is suitable for The initial input amount is 100ng to 1 ⁇ g; it can be seen from Figure 3 that the appropriate initial input amount of the enzyme reaction solution of combination 3 is 1ng to 10ng; it can be seen from Figure 5 that the appropriate initial input amount of the enzyme reaction solution of combination 5 is The input amount is 100pg to 100ng; it can be seen from Figure 6 that the appropriate initial input amount of the enzyme reaction solution of combination 6 is 10ng to 100ng; it can be seen from Figure 7 that the appropriate initial input amount of the enzyme reaction solution of combination 7 It can be seen from Figure 9 that the appropriate initial input amount of the enzyme reaction solution of combination 9 is 100pg to 10ng; it can be seen from Figure 10 that the appropriate initial input amount of the enzyme reaction solution of combination 10 is 100pg It can be seen from Figure 12 that the appropriate initial input amount of the
  • the present application optimizes the enzyme cleavage reaction rate and the end repair reaction rate in the process of library construction by optimizing the formula of the enzyme reaction solution, the enzyme composition and the reaction buffer cooperate with each other, and realizes the difference in the sample starting amount. , Under the same processing time, the technical effect of obtaining a sequencing library of the same length has a wide range of applicability and convenient operation.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

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Abstract

本申请提供了一种用于构建测序文库的酶反应液及其应用。所述酶反应液包括酶组合物和反应缓冲液；其中酶组合物包括核酸内切酶、DNA聚合酶和多聚核苷酸激酶；其中反应缓冲液包括金属盐、底物和缓冲介质水溶液。本申请通过优化酶反应液的配方，通过一步反应实现对核酸样本的切割、末端修复和加A尾处理，在适宜的缓冲体系中，酶切反应速率和末端修复反应速率达到平衡，在样本起始量为100pg-1μg、处理时间相同的情况下，获得了长度分布一致的测序文库。

Description

一种用于构建测序文库的酶反应液及其应用 技术领域
本申请属于生物技术领域，涉及一种用于构建测序文库的酶反应液及其应用。
背景技术
随着高通量测序成本的大幅下降，高通量测序市场迅速增长。其中，DNA测序约占据整个高通量测序市场的一半，因此人们对于流程简便、快速、高通量的DNA建库技术的需求越来越迫切。DNA建库流程中第一步也是最关键的一步在于将大片段DNA分子无偏向性地片段化为特定长度的小片段DNA，目前主流的DNA片段化方式为超声机械打断。然而超声机械打断仪往往价格昂贵，许多工业、科研、医疗实验室无法配备此仪器；超声机械片段化DNA的操作繁琐、耗时长，需要实时取样检测样品的片段化效果；并且超声机械打断的样品数量少、单次处理样本的投入量少，无法满足工业、医疗领域对大通量处理样品的需求。片段化酶可以很好地解决上述问题，基于酶法的DNA样品处理方法可在热循环仪中完成，操作流程简便、快速；片段化酶能够对DNA分子进行非特异性的随机切割，因此有望替代传统的超声机械法将大片段DNA分子无偏向性地片段化为特定长度的小片段DNA分子，从而应用于高通量测序中的DNA文库构建。
目前已有的基于酶法的DNA片段化技术包括：1)使用Vvn在双链DNA分子上随机产生缺刻、同时利用T7核酸内切酶在缺刻位点将另一条DNA分子切开；2)使用DNaseI在Mn 2+或Mg 2+存在的情况下将双链DNA断裂；3)将多种识别特定碱基序列的限制性核酸内切酶(比如MspI、AluI、CviQI、MseI、MlucI、HaeIII等)混合，进行对DNA分子的切割。
当前，构建DNA文库的样本起始投入量从100pg～1μg不等，存在5个数量级的差异，而市面上的商业化试剂盒在起始量兼容性方面均不足以满足市场需求。一方面商业化的试剂盒的兼容下限均在100pg以上，另一方面不同的起始量需要使用不同的反应时间。在实际的实验过程中，从不同的样本中获得的核酸量是不同的，不可避免地会使用不同起始量的样本进行文库构建。因此， 针对不同的样本起始量，需要调整反应时间，不仅增加了实验的繁琐程度和对仪器的需求量，而且也限制了文库构建方法在大通量样本方面的应用。
CN108998434A公开了片段化酶打断缓冲液及提高片段化酶打断效率的方法，所述片段化酶打断缓冲液包括适于片段化酶发挥作用的缓冲液组分和终浓度为10％以下的PEG8000，以及终浓度为10-25mM的Mg 2+，通过在反应缓冲液体系中添加不同比例的PEG及Mg 2+来调节DNA片段化的效率及片段大小，得到可满足BGISEQ-500平台PE100和PE150双端测序文库要求的打断缓冲液，但是仍然不能兼容起始量为100pg～1μg的样本。
因此，有必要对现有的文库构建试剂盒进行改进。
发明内容
本申请提供了一种用于构建测序文库的酶反应液及其应用，所述酶反应液通过优化酶组合物的种类和用量，并与反应缓冲液中的金属离子和缓冲介质相互配合，平衡了文库构建过程中的酶切反应速率和末端修复反应速率，实现了在样本起始量不同、处理时间相同的情况下，获得长度一致的测序文库的技术效果，具有广泛的适用性和便捷的操作性。
第一方面，本申请提供了一种用于构建测序文库的酶反应液，所述酶反应液包括酶组合物和反应缓冲液；
所述酶组合物包括核酸内切酶、DNA聚合酶和多聚核苷酸激酶；
所述反应缓冲液包括金属盐、底物和缓冲介质水溶液。
本申请的酶组合物包括用于进行切割的核酸内切酶、用于进行末端修复和3’加A尾的DNA聚合酶、以及用于进行5’磷酸化的多聚核苷酸激酶，所述酶组合物集中在一个反应体系中，通过一步反应实现对核酸样本的切割、末端修复和加A尾处理，反应缓冲液中既包含切割反应所需的离子缓冲液，又包含末端修复所需的离子缓冲液，而且还含有各类反应所需的底物分子dNTPs、dATP和ATP，各种组分在一定的浓度范围内相互配合，平衡酶切反应速率和末端修复反应速率，实现了在样本起始量为100pg-1μg、处理时间相同的情况下，获得长度一致的测序文库的技术效果。
本申请采用核酸内切酶对核酸样本进行切割，所述核酸内切酶例如可以是核酸内切酶dsDNase、T7核酸内切酶、盐活性核酸内切酶SAN、核酸内切酶 Vvn或核酸内切酶DNaseI中的任意一种或至少两种的组合。
在一些实施方案中，所述核酸内切酶例如可以是来源于虾的核酸内切酶dsDNase、来源于T7噬菌体的核酸内切酶T7 Endonuclease、盐活性核酸内切酶SAN、来源于创伤弧菌的核酸内切酶Vvn或来源于牛胰的核酸内切酶DNaseI中的任意一种或至少两种的组合。
在一些实施方案中，所述核酸内切酶在所述酶反应液中的终浓度为0.003～0.05U/μL，例如可以是0.003U/μL、0.004U/μL、0.005U/μL、0.006U/μL、0.007U/μL、0.008U/μL、0.009U/μL、0.01U/μL、0.02U/μL、0.03U/μL、0.04U/μL或0.05U/μL，优选为0.004～0.03U/μL，进一步优选为0.005～0.01U/μL。
在一些实施方案中，当所述核酸内切酶为Vvn时，所述核酸内切酶Vvn在所述酶反应液中的终浓度为0.1～0.5ng/μL，例如可以是0.1ng/μL、0.2ng/μL、0.3ng/μL、0.4ng/μL或0.5ng/μL。
本申请针对不同种类的核酸内切酶，通过优化核酸内切酶在反应体系中的浓度、控制核酸内切酶的用量，从而调节核酸内切酶对核酸的切割速率。
本申请采用DNA聚合酶对已切割的核酸进行末端修复或在已切割核酸的3’端进行加A尾反应，所述DNA聚合酶包括低温DNA聚合酶和/或耐热DNA聚合酶，所述低温DNA聚合酶例如可以是来源于T4噬菌体的DNA聚合酶、来源于T7噬菌体的DNA聚合酶、来源于大肠杆菌的DNA聚合酶I或来源于大肠杆菌的DNA聚合酶I大片段Klenow中的任意一种或至少两种的组合，对已切割的核酸进行末端修复，所述耐热DNA聚合酶例如可以是Taq DNA聚合酶，在已切割核酸的3’端进行加A尾反应，以便后续基于TA连接原理进行接头连接反应。
本申请中，术语“耐热DNA聚合酶”可以是Taq DNA聚合酶，在70℃反应2h后其残留活性大于原来的90％，在93℃下反应2h后其残留活性是原来的60％，在95℃下反应2h后其残留活性是原来的40％，术语“低温DNA聚合酶”是相对于“耐热DNA聚合酶”而言，不具有耐热活性的DNA聚合酶。
在一些实施方案中，所述低温DNA聚合酶在所述酶反应液中的终浓度为0.01～0.05U/μL，例如可以是0.01U/μL、0.02U/μL、0.03U/μL、0.04U/μL或0.05U/μL，优选为0.02～0.04U/μL。
在一些实施方案中，所述耐热DNA聚合酶在所述酶反应液中的终浓度为 0.03～1.2U/μL，例如可以是0.03U/μL、0.04U/μL、0.05U/μL、0.06U/μL、0.07U/μL、0.08U/μL、0.09U/μL、0.1U/μL、0.2U/μL、0.3U/μL、0.4U/μL、0.5U/μL、0.6U/μL、0.7U/μL、0.8U/μL、0.9U/μL、1.0U/μL、1.1U/μL或1.2U/μL，优选为0.04～1.1U/μL，进一步优选为0.05～1.0U/μL，再优选为0.06～0.9U/μL。
本申请针对不同种类的DNA聚合酶，通过优化DNA聚合酶在反应体系中的浓度、控制DNA聚合酶的用量，从而调节DNA聚合酶对已切割的核酸的末端修复速率。
本申请的酶反应液中还包括多聚核苷酸激酶，例如可以是T4多聚核苷酸激酶(T4 PNK)，对已切割的核酸进行5’磷酸化，所述多聚核苷酸激酶在所述酶反应液中的终浓度为0.05～0.2U/μL，例如可以是0.05U/μL、0.06U/μL、0.07U/μL、0.08U/μL、0.09U/μL、0.1U/μL或0.2U/μL。
在一些实施方案中，所述酶组合物还包括辅助蛋白。
本申请采用辅助蛋白来调控核酸内切酶的切割速率和DNA聚合酶的末端修复速率，所述辅助蛋白包括牛血清白蛋白(BSA)和/或单链结合蛋白，优选地，所述单链结合蛋白包括来源于大肠杆菌的单链结合蛋白E.coli SSB和/或来源于T4噬菌体的单链结合蛋白T4 GP32。
在一些实施方案中，所述辅助蛋白在所述酶反应液中的终浓度为0.05～1μg/μL，例如可以是0.05μg/μL、0.06μg/μL、0.07μg/μL、0.08μg/μL、0.09μg/μL、0.1μg/μL、0.2μg/μL、0.3μg/μL、0.4μg/μL、0.5μg/μL、0.6μg/μL、0.7μg/μL、0.8μg/μL、0.9μg/μL或1μg/μL，优选为0.05～0.5μg/μL，进一步优选为0.1～0.4μg/μL。
本申请针对不同种类的辅助蛋白，通过优化辅助蛋白在反应体系中的浓度、控制辅助蛋白的用量，从而调节核酸内切酶的切割速率和DNA聚合酶的末端修复速率。
在一些实施方案中，所述金属阳离子包括Mg 2+、Mn 2+、Na +或Ca 2+中的任意一种或至少两种的组合，所述含有Mg 2+的盐可以是MgCl 2和/或Mg 2SO 4，所述含有Mn 2+的盐可以是MnCl 2，所述含有Na +的盐可以是NaCl，所述含有Ca 2+的盐可以是CaCl 2。
本申请通过调节酶反应液中的金属阳离子种类、浓度，金属阳离子与缓冲介质共同调节反应体系处于适宜的酸碱环境中，控制核酸内切酶和DNA聚合酶 的酶活性，从而平衡酶切反应速率和末端修复反应速率。
在一些实施方案中，所述Mg 2+在所述酶反应液中的终浓度为0～20mM，例如可以是1mM、2mM、3mM、4mM、5mM、6mM、7mM、8mM、9mM、10mM、11mM、12mM、13mM、14mM、15mM、16mM、17mM、18mM、19mM或20mM，优选为2～18mM，进一步优选为3～17mM，再优选为4～16mM或5～15mM。
在一些实施方案中，所述Mn 2+在所述酶反应液中的终浓度为0.05～1mM，例如可以是0.05mM、0.06mM、0.07mM、0.08mM、0.09mM、0.1mM、0.2mM、0.3mM、0.4mM、0.5mM、0.6mM、0.7mM、0.8mM、0.9mM或1mM，优选为0.07～0.8mM，进一步优选为0.09～0.6mM，再优选为0.1～0.5mM。
在一些实施方案中，所述Na +在所述酶反应液中的终浓度为0～50mM，例如可以是1mM、2mM、3mM、4mM、5mM、6mM、7mM、8mM、9mM、10mM、11mM、12mM、13mM、14mM、15mM、16mM、17mM、18mM、19mM、20mM、25mM、30mM、35mM、40mM、45mM或50mM，优选为0～30mM，进一步优选为5～30mM，再优选为5～20mM或5～15mM。
在一些实施方案中，所述Ca 2+在所述酶反应液中的终浓度为0～10mM，例如可以是1mM、2mM、3mM、4mM、5mM、6mM、7mM、8mM、9mM或10mM，优选为0～8mM、0～5mM、0～3mM、1～10mM、1～5mM、1～3mM、2～10mM或2～5mM。
在一些实施方案中，所述底物包括dNTPs、dATP或ATP中的任意一种或至少两种的组合。
在一些实施方案中，所述dNTPs在所述酶反应液中的终浓度为0.05～0.5mM，例如可以是0.05mM、0.06mM、0.07mM、0.08mM、0.09mM、0.1mM、0.2mM、0.3mM、0.4mM或0.5mM，优选为0.05～0.4mM、0.05～0.3mM、0.05～0.2mM、0.05～0.1mM、0.07～0.5mM、0.07～0.4mM、0.07～0.3mM、0.07～0.2mM、0.07～0.1mM、0.09～0.5mM、0.09～0.4mM、0.09～0.3mM、0.09～0.2mM或0.09～0.1mM。
本申请中，将dNTPs的浓度限定在一定的范围内，有利于调节DNA聚合酶的末端修复速率。
在一些实施方案中，所述dATP在所述酶反应液中的终浓度为0.1～2mM， 例如可以是0.1mM、0.2mM、0.3mM、0.4mM、0.5mM、0.6mM、0.7mM、0.8mM、0.9mM、1mM、1.1mM、1.2mM、1.3mM、1.4mM、1.5mM、1.6mM、1.7mM、1.8mM、1.9mM或2mM，优选为0.1～1.5mM、0.1～1mM、0.3～2mM、0.3～1.5mM、0.3～1mM、0.5～2mM、0.5～1.5mM、0.5～1mM或0.5～0.8mM。
在一些实施方案中，所述ATP在所述酶反应液中的终浓度为1～10mM，例如可以是1mM、2mM、3mM、4mM、5mM、6mM、7mM、8mM、9mM或10mM，优选为1～9mM、1～8mM、1～7mM、1～6mM、1～5mM、3～10mM、3～9mM、3～8mM、3～7mM或3～6mM。
在一些实施方案中，所述缓冲介质包括4-羟乙基哌嗪乙磺酸(HEPES)和/或三羟甲基氨基甲烷(TRIS)。
在一些实施方案中，所述缓冲介质在所述酶反应液中的终浓度为10～50mM，例如可以是10mM、15mM、20mM、25mM、30mM、35mM、40mM、45mM或50mM，优选为10～40mM、10～30mM、15～50mM、15～45mM、15～40mM或15～30mM。
在一些实施方案中，所述酶反应液的pH为7.0～8.5，例如可以是7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4或8.5，优选为7.5～8.1。
在一些实施方案中，所述酶组合物包含T4 PNK、BSA、T4 GP32、Vvn、T7核酸内切酶、Taq DNA聚合酶和Klenow。
在一些实施方案中，所述酶组合物包含T4 PNK、BSA、E.coli SSB、dsDNase、Taq DNA聚合酶和Klenow。
在一些实施方案中，所述酶组合物包含T4 PNK、BSA、T4 GP32、SAN、Taq DNA聚合酶和大肠杆菌DNA聚合酶I。
在一些实施方案中，所述酶组合物包含T4 PNK、BSA、T4 GP32、DNaseI、Taq DNA聚合酶和T4 DNA聚合酶。
在一些实施方案中，所述反应缓冲液包含4-羟乙基哌嗪乙磺酸(HEPES)、MgCl 2、CaCl 2、NaCl、MnCl 2、dNTPs、dATP和ATP。
在一些实施方案中，所述反应缓冲液包含或三羟甲基氨基甲烷(TRIS)、MgCl 2、CaCl 2、NaCl、MnCl 2、dNTPs、dATP和ATP。
在一些实施方案中，所述酶反应液还包含甘油。
在一些实施方案中，所述酶反应液包含三羟甲基氨基甲烷(TRIS)、MgCl 2、CaCl 2、NaCl、MnCl 2、dNTPs、dATP、ATP、T4 PNK、BSA、T4 GP32、DNaseI、Taq DNA聚合酶、T4 DNA聚合酶和甘油。
在一些实施方案中，所述酶反应液包含4-羟乙基哌嗪乙磺酸(HEPES)、MgCl 2、CaCl 2、NaCl、MnCl 2、dNTPs、dATP、ATP、T4 PNK、BSA、T4 GP32、DNaseI、Taq DNA聚合酶、T4 DNA聚合酶和甘油。
在一些实施方案中，所述酶反应液包含4-羟乙基哌嗪乙磺酸(HEPES)、MgCl 2、CaCl 2、NaCl、MnCl 2、dNTPs、dATP、ATP、T4 PNK、BSA、T4 GP32、DNaseI、Taq DNA聚合酶、T4 DNA聚合酶和甘油。
在一些实施方案中，所述酶反应液包含三羟甲基氨基甲烷(TRIS)、MgCl 2、CaCl 2、NaCl、MnCl 2、dNTPs、dATP、ATP、T4 PNK、BSA、T4 GP32、SAN、Taq DNA聚合酶、大肠杆菌DNA聚合酶I和甘油。
在一些实施方案中，所述酶反应液包含三羟甲基氨基甲烷(TRIS)30mM、MgCl 2 15mM、CaCl 2 5mM、NaCl 10mM、MnCl 2 0.1mM、dNTPs 0.1mM、dATP 0.6mM、ATP 1mM、T4 PNK 0.1U/μL、BSA 0.3μg/μL、T4 GP32 0.1μg/μL、DNaseI 0.06U/μL、Taq DNA聚合酶0.3U/μL、T4 DNA聚合酶0.9U/μL和甘油。
在一些实施方案中，所述酶反应液包4-羟乙基哌嗪乙磺酸(HEPES)20mM、MgCl 2 15mM、CaCl 2 5mM、NaCl 10mM、MnCl 2 0.5mM、dNTPs 0.1mM、dATP 0.6mM、ATP 1mM、T4 PNK 0.1U/μL、BSA 0.3μg/μL、T4 GP32 0.1μg/μL、DNaseI 0.06U/μL、Taq DNA聚合酶0.3U/μL、T4 DNA聚合酶0.9U/μL和甘油。
在一些实施方案中，所述酶反应液包4-羟乙基哌嗪乙磺酸(HEPES)20mM、MgCl 2 5mM、CaCl 2 5mM、NaCl 10mM、MnCl 2 0.1mM、dNTPs 0.1mM、dATP 0.6mM、ATP 1mM、T4 PNK 0.1U/μL、BSA 0.3μg/μL、T4 GP32 0.1μg/μL、DNaseI 0.06U/μL、Taq DNA聚合酶0.3U/μL、T4 DNA聚合酶0.9U/μL和甘油。
在一些实施方案中，所述酶反应液包含三羟甲基氨基甲烷(TRIS)30mM、MgCl 2 15mM、CaCl 2 5mM、NaCl 10mM、MnCl 2 0.5mM、dNTPs 0.1mM、dATP 0.6mM、ATP 1mM、T4 PNK 0.1U/μL、BSA 0.3μg/μL、T4 GP32 0.1μg/μL、SAN 0.06U/μL、Taq DNA聚合酶0.3U/μL、大肠杆菌DNA聚合酶I 0.06U/μL和甘油。
本申请中，反应体系中的金属离子种类、浓度和缓冲介质的种类、浓度在 一定范围内相互配合，维持酶反应液的pH值处于较稳定和适宜的状态，影响核酸内切酶和DNA聚合酶的活性，保证酶切反应速率和末端修复反应速率相一致，使构建的测序文库的长度适用于二代测序平台。
第二方面，本申请提供了一种测序文库的构建方法，所述方法包括：
将靶核酸加入第一方面所述的酶反应液中，孵育，进行靶核酸的片段化、末端修复、5’磷酸化和3’加A尾；
将孵育产物与测序接头进行连接反应；
将连接产物进行PCR，得到测序文库。
优选地，所述靶核酸的添加量为100pg～1μg，例如可以是100pg、1ng、10ng、100ng或1μg。
优选地，所述孵育的条件为35～40℃保持10～20min，60～70℃保持20～40min，优选为37℃保持15min，65℃保持30min。
第三方面，本申请提供了一种用于构建测序文库的试剂盒，所述试剂盒包括第一方面所述的酶反应液。
优选地，所述试剂盒还包括测序接头、连接反应试剂或PCR试剂中的任意一种或至少两种的组合。
本领域技术人员可以理解的是，本申请的酶反应液中的反应缓冲液和酶组合物可各自以独立的包装存在，反应缓冲液和酶组合物混合后终浓度与本申请一致或基本一致，亦在本申请的保护范围内。
第四方面，本申请提供了一种测序文库，所述测序文库采用第二方面所述的方法或第三面所述的试剂盒构建；
所述测序文件的长度为300～500bp。
与现有技术相比，本申请具有如下有益效果：
(1)本申请将核酸样本片段化、末端修复、3’加dA尾和5’磷酸化修饰整合为一步反应，酶组合物包括用于进行切割的核酸内切酶、用于进行末端修复和3’加A尾的DNA聚合酶、以及用于进行5’磷酸化的多聚核苷酸激酶，与反应缓冲液在一定的浓度范围内相互配合，控制酶切反应速率和末端修复反应速率，实现了在样本起始量为100pg-1μg、处理时间相同的情况下，获得长度一致的测序文库的技术效果，提高了文库构建的成功率；
(2)本申请的酶反应液使基于酶法的DNA片段化与文库构建方法得到了 进一步简化，具有更加广泛的适用性和更加便捷的操作性。
附图说明
图1为使用组合1的酶反应液构建的测序文库的长度分布；
图2为使用组合2的酶反应液构建的测序文库的长度分布；
图3为使用组合3的酶反应液构建的测序文库的长度分布；
图4为使用组合4的酶反应液构建的测序文库的长度分布；
图5为使用组合5的酶反应液构建的测序文库的长度分布；
图6为使用组合6的酶反应液构建的测序文库的长度分布；
图7为使用组合7的酶反应液构建的测序文库的长度分布；
图8为使用组合8的酶反应液构建的测序文库的长度分布；
图9为使用组合9的酶反应液构建的测序文库的长度分布；
图10为使用组合10的酶反应液构建的测序文库的长度分布；
图11为使用组合11的酶反应液构建的测序文库的长度分布；
图12为使用组合12的酶反应液构建的测序文库的长度分布；
图13为使用组合13的酶反应液构建的测序文库的长度分布；
图14为使用组合14的酶反应液构建的测序文库的长度分布；
图15为使用组合15的酶反应液构建的测序文库的长度分布；
图16为使用组合16的酶反应液构建的测序文库的长度分布。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1酶反应液的配制
本实施例首先配制酶反应液，包括两个部分：如表1所示的反应缓冲液和如表2所示的酶组合物，将表1的反应缓冲液和表2的酶组合物进行交叉组合，得到如表3所示的不同配方的酶反应液，蛋白酶的厂家及货号见表4，创伤弧菌 核酸酶Vvn的氨基酸序列如SEQ ID NO:1所示。
表1反应缓冲液
Figure PCTCN2021078865-appb-000001
表2酶组合物
Figure PCTCN2021078865-appb-000002
表3酶反应液
组合编号 反应缓冲液+酶组合物
组合1 反应缓冲液A+酶组合物1
组合2 反应缓冲液A+酶组合物2
组合3 反应缓冲液A+酶组合物3
组合4 反应缓冲液A+酶组合物4
组合5 反应缓冲液B+酶组合物1
组合6 反应缓冲液B+酶组合物2
组合7 反应缓冲液B+酶组合物3
组合8 反应缓冲液B+酶组合物4
组合9 反应缓冲液C+酶组合物1
组合10 反应缓冲液C+酶组合物2
组合11 反应缓冲液C+酶组合物3
组合12 反应缓冲液C+酶组合物4
组合13 反应缓冲液D+酶组合物1
组合14 反应缓冲液D+酶组合物2
组合15 反应缓冲液D+酶组合物3
组合16 反应缓冲液D+酶组合物4
表4蛋白/酶厂家及货号
蛋白/酶名称 产家 货号
T4 PNK Vazyme N102-01
BSA Sigma V900933
T4 GP32 NEB M0300L
Vvn 自表达 序列见下方
T7 Endonuclease Vazyme EN303
Taq DNA聚合酶 Vazyme P101
Klenow Vazyme N104
dsDNase thermo EN0771
SAN arcticzymes 70910
DNaseI Vazyme EN401
E.ColI SSB abcam ab123224
创伤弧菌核酸酶Vvn的氨基酸序列(SEQ ID NO:1)：
Figure PCTCN2021078865-appb-000003
实施例2 DNA模板的片段化和末端修复
本实施例以鲑鱼精gDNA为模板，起始投入量分别为100pg、1ng、10ng、100ng和1μg，采用实施例1的不同酶反应液对gDNA进行切割、末端修复、3’加A尾和5’磷酸化，并在随后的实施例中使用Vayzme#ND607
Figure PCTCN2021078865-appb-000004
Universal DNA Library Prep Kit for
Figure PCTCN2021078865-appb-000005
V3中的Rapid DNA Ligation Buffer和Rapid DNA Ligase进行接头连接反应，使用Vazyme#N401
Figure PCTCN2021078865-appb-000006
DNA Clean Beads对接头连接产物和PCR扩增产物进行纯化，使用Vayzme#ND607
Figure PCTCN2021078865-appb-000007
Universal DNA Library Prep Kit for
Figure PCTCN2021078865-appb-000008
V3中的VAHTS HiFi Amplification Mix和PCR Primer Mix 3 for Illumina对纯化的连接产物进行扩增富集，其中用于连接反应的接头(Adaptor)为
Figure PCTCN2021078865-appb-000009
DNA Adapters for
Figure PCTCN2021078865-appb-000010
(Vazyme#N801)。
靶DNA的片段化、末端修复、5’磷酸化和3’加dA尾反应体系如表5所示，其中，酶组合物的配方采用实施例1的组合1～16中的其中一种，靶DNA的投入量XμL分别对应100pg、1ng、10ng、100ng和1μg，反应条件为37℃孵育15min、65℃孵育30min，得到的片段化产物进行测序文库构建。
表5片段化反应体系
组分 用量
反应缓冲液 5μL
酶组合物 10μL
靶DNA XμL
灭菌ddH 2O 补齐至50μL
实施例3构建测序文库
本实施例对实施例2制备的片段化产物进行接头连接反应、PCR扩增反应和纯化，构建测序文库，接头连接反应体系如表6所示，配制好的体系在20℃下孵育15min，进行接头连接；使用60μL VAHTS DNA Clean Beads对连接产物进行纯化，洗脱体积为20/22.5μL；
将纯化产物进行PCR扩增，体系如表7所示，条件如表8所示，使用45μL VAHTS DNA Clean Beads对扩增产物进行纯化，洗脱体积为20/22.5μL。
表6接头连接反应体系
组分 体积
片段化纯化产物 50μL
连接反应缓冲液(Rapid DNA Ligation Buffer) 25μL
DNA连接酶(Rapid DNA Ligase) 5μL
接头(DNA Adaptor) 5μL
灭菌ddH 2O 15μL
表7 PCR体系
组分 体积
接头连接纯化产物 20μL
PCR扩增用酶及其缓冲液(VAHTS HiFi Amplification Mix) 25μL
引物(PCR Primer Mix 3 for Illumina) 5μL
表8 PCR反应程序
温度 时间
95℃ 3min
98℃ 20sec
60℃ 15sec
72℃ 30sec
72℃ 5min
4℃ 保存
不同起始DNA投入量对应的接头稀释倍数和扩增循环数如表9所示。
表9不同起始DNA投入量对应的接头稀释倍数和扩增循环数
gDNA投入量 接头稀释倍数 扩增循环数
100pg 200倍 14
1ng 100倍 12
10ng 10倍 8
100ng 原液 3
1μg 原液 2
采用Agilent 2100 DNA1000芯片检测DNA文库的长度分布，结果如图1～图16，可以看出，各组酶反应液均可以在相同的处理时间内实现对不同投入量的DNA的有效切割、末端修复、5’磷酸化修饰和3’加dA尾。
其中，如图16所示，组合16的酶反应液的效果最优，能够完全兼容100pg～1μg的起始DNA投入量，文库片段长度的分布范围一致性好，主要在400～500bp范围内，说明当酶组合物为0.3U DNaseI、45U T4 DNA聚合酶、1.5U Taq DNA聚合酶、5U T4 PNK、15μg BSA和5μg T4 GP32时，与反应缓冲液(300mM Tris、150mM MgCl 2、30mM CaCl 2、100mM NaCl、1mM MnCl 2、1mM dNTPs、6mM dATP和50mM ATP)相互配合，可以完美平衡酶切反应速率和末端修复反应速率，制备得到片段长度一致的测序文库；
如图8、图11和图4所示，组合8、组合11和组合4的酶反应液的效果也较好，对起始投入量为100pg～1μg的DNA兼容性较好，制备得到片段长度大体一致，说明当酶组合物为0.004～0.008U/μL核酸内切酶、0.05～0.9U/μL DNA聚合酶、0.01～0.05U/μL Taq DNA聚合酶、0.05～0.3U/μL T4 PNK和0.2～0.6μg/μL辅助蛋白时，与反应缓冲液100～400mM缓冲介质、100～300mM Mg 2+、1～6mM Mn 2+、50～250mM Na +、5～50mM Ca 2+、0.5～5mM dNTPs、1～10mM dATP和20～40mM ATP相互配合，调控酶切反应速率和末端修复反应速率，使制备得到的测序文库的片段长度较一致。
从图1可以看出，组合1的酶反应液适宜的起始投入量为100pg～10ng，起始投入量进一步增加会影响片段长度；从图2可以看出，组合2的酶反应液适宜的起始投入量为100ng～1μg；从图3可以看出，组合3的酶反应液适宜的起始投入量为1ng～10ng；从图5可以看出，组合5的酶反应液适宜的起始投入量 为100pg～100ng；从图6可以看出，组合6的酶反应液适宜的起始投入量为10ng～100ng；从图7可以看出，组合7的酶反应液适宜的起始投入量为100pg～10ng；从图9可以看出，组合9的酶反应液适宜的起始投入量为100pg～10ng；从图10可以看出，组合10的酶反应液适宜的起始投入量为100pg～10ng；从图12可以看出，组合12的酶反应液适宜的起始投入量为1ng～100ng；从图13可以看出，组合13的酶反应液适宜的起始投入量为1ng～100ng；从图15可以看出，组合15的酶反应液适宜的起始投入量为10ng～100ng。
综上所述，本申请通过优化酶反应液的配方，酶组合物和反应缓冲液相互配合，优化了文库构建过程中的酶切反应速率和末端修复反应速率，实现了在样本起始量不同、处理时间相同的情况下，获得长度一致的测序文库的技术效果，具有广泛的适用性和便捷的操作性。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (15)

  1. 一种用于构建测序文库的酶反应液，其包括酶组合物和反应缓冲液；其中，
    所述酶组合物包括核酸内切酶、DNA聚合酶和多聚核苷酸激酶；
    所述反应缓冲液包括金属盐、底物和缓冲介质水溶液。
  2. 根据权利要求1所述的酶反应液，其中，所述核酸内切酶包括核酸内切酶dsDNase、T7核酸内切酶、盐活性核酸内切酶SAN、核酸内切酶Vvn或核酸内切酶DNaseI中的任意一种或至少两种的组合。
  3. 根据权利要求1或2所述的酶反应液，其中，所述DNA聚合酶包括低温DNA聚合酶和/或耐热DNA聚合酶。
  4. 根据权利要求1-3任一项所述的酶反应液，其中，所述低温DNA聚合酶包括T4 DNA聚合酶、T7 DNA聚合酶、DNA聚合酶I或DNA聚合酶I大片段Klenow中的任意一种或至少两种的组合。
  5. 根据权利要求1-4任一项所述的酶反应液，其中，所述耐热DNA聚合酶包括Taq DNA聚合酶。
  6. 根据权利要求1-5任一项所述的酶反应液，其中，所述多聚核苷酸激酶包括T4多聚核苷酸激酶。
  7. 根据权利要求1-6任一项所述的酶反应液，其中，所述酶组合物还包括辅助蛋白；
    任选地，所述辅助蛋白包括牛血清白蛋白和/或单链结合蛋白；
    任选地，所述单链结合蛋白包括大肠杆菌单链结合蛋白和/或T4噬菌体单链结合蛋白。
  8. 根据权利要求1-7任一项所述的酶反应液，其中，所述金属盐包括金属阳离子，所述金属阳离子包括Mg 2+、Mn 2+、Na +或Ca 2+中的任意一种或至少两种的组合。
  9. 根据权利要求1-8任一项所述的酶反应液，其中，所述底物包括dNTPs、dATP或ATP中的任意一种或至少两种的组合。
  10. 根据权利要求1-9任一项所述的酶反应液，其中，所述缓冲介质包括4-羟乙基哌嗪乙磺酸和/或三羟甲基氨基甲烷。
  11. 根据权利要求1-10任一项所述的酶反应液，其中，所述核酸内切酶在所述酶反应液中的终浓度为0.003～0.05U/μL或0.1～0.5ng/μL；
    任选地，所述低温DNA聚合酶在所述酶反应液中的终浓度为0.01～0.05U/μL；
    任选地，所述耐热DNA聚合酶在所述酶反应液中的终浓度为0.03～1.2U/μL；
    任选地，所述多聚核苷酸激酶在所述酶反应液中的终浓度为0.05～0.2U/μL；
    任选地，所述辅助蛋白在所述酶反应液中的终浓度为0.05～1μg/μL；
    任选地，所述Mg 2+在所述酶反应液中的终浓度为0～20mM；
    任选地，所述Mn 2+在所述酶反应液中的终浓度为0.05～1mM；
    任选地，所述Na +在所述酶反应液中的终浓度为0～50mM；
    任选地，所述Ca 2+在所述酶反应液中的终浓度为0～10mM；
    任选地，所述dNTPs在所述酶反应液中的终浓度为0.05～0.5mM；
    任选地，所述dATP在所述酶反应液中的终浓度为0.1～2mM；
    任选地，所述ATP在所述酶反应液中的终浓度为1～10mM；
    任选地，所述缓冲介质在所述酶反应液中的终浓度为10～50mM。
  12. 一种测序文库的构建方法，其包括：
    (1)将靶核酸加入权利要求1-11任一项所述的酶反应液中，孵育，进行靶核酸的片段化、末端修复、5’磷酸化和3’加A尾；
    (2)将孵育产物与测序接头进行连接反应；和
    (3)将连接产物进行PCR，得到测序文库。
  13. 根据权利要求12所述的构建方法，其中，所述靶核酸的添加量为100pg～1μg。
  14. 根据权利要求12或13所述的构建方法，其中，所述孵育的条件为35～40℃保持10～20min，60～70℃保持20～40min。
  15. 一种用于构建测序文库的试剂盒，其包括权利要求1-11任一项所述的酶反应液；
    任选地，所述试剂盒还包括测序接头、连接反应试剂或PCR试剂中的任意一种或至少两种的组合。
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