WO2012079490A1 - Method for constructing dna sequencing library and use thereof - Google Patents

Method for constructing dna sequencing library and use thereof Download PDF

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Publication number
WO2012079490A1
WO2012079490A1 PCT/CN2011/083786 CN2011083786W WO2012079490A1 WO 2012079490 A1 WO2012079490 A1 WO 2012079490A1 CN 2011083786 W CN2011083786 W CN 2011083786W WO 2012079490 A1 WO2012079490 A1 WO 2012079490A1
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reagent
dna
reagent according
fragment
sample
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PCT/CN2011/083786
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French (fr)
Chinese (zh)
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栾合密
张俊青
程玲
孔淑娟
张秀清
杨焕明
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深圳华大基因科技有限公司
深圳华大基因研究院
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Publication of WO2012079490A1 publication Critical patent/WO2012079490A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to the field of biotechnology, and in particular to the field of DNA sequencing technology, in particular to a method for constructing a DNA sequencing library and its use. More specifically, the present invention provides two reagents, a method of constructing a DNA sequencing library, a DNA sequencing library, a method of determining sequence information of a DNA sample, and a kit for constructing a DNA sequencing library. Background technique
  • DNA deoxyribonucleic acid
  • DNA is a molecular compound of a double-helical structure composed of two nucleotide chains in a complementary pairing principle, which is a carrier of life genetic information. Sequencing and analysis of DNA nucleotide sequences not only provides important data for basic biological research such as gene expression and regulation, but also plays an important role in applied research such as disease diagnosis and gene therapy (see Gilbert W. DNA sequencing and gene structure). In: Forsen S. Nobel Lectures in Chemistry 1971-1980. 1980.; Singapore: World Scientific Publishing Co, 1993, 408-426.; Sanger F, Coulson A R. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase [J]. J Mol Biol, 1975, 94: 441-448., which is incorporated herein by reference in its entirety.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the invention provides methods of constructing DN A sequencing libraries and uses thereof.
  • the invention provides a reagent comprising a buffer salt, dithiothreitol, MgCl 2 .
  • the inventors have found that by using the reagent as a buffer system, it is possible to efficiently configure a reaction system required for constructing a sequencing library to efficiently add a base A to the 3' end of the DNA fragment, and to efficiently bind the linker with 3' A reaction system in which a DNA fragment of base A is linked is added. Thereby, the step of recycling can be saved. Thereby, the efficiency of constructing the sequencing library can be improved.
  • the present invention provides an agent.
  • the reagent comprises: 10 mM to 1000 mM of a buffer salt; 10 mM to 100 mM of a soluble salt; 10 mM to 300 mM of MgCl 2 ; 1 mM to 50 mM of bis-threitol; 1 mM to 10 mM of dATP; And a Klenow fragment (3' ⁇ 5' exo) of 1 U/ml to 40 U/ml, wherein the pH of the reagent is 7.6 to 7.9.
  • the reagent is sometimes referred to as a first reagent, which is used in the method of constructing a DNA sequencing library of the present invention to process a DNA fragment subjected to end repair to obtain 3'.
  • a DNA fragment of base A is added at the end.
  • the invention also provides an agent.
  • the reagent comprises: 10 mM to 200 mM buffer salt; 1 mM to 100 mM dithiothreitol; 10 mM to 400 mM MgCl 2 ; 1 mM to 40 mM ATP; and 1 U/ml to 200 U/ml
  • the DNA ligase wherein the reagent has a pH of 7.6 to 7.9.
  • the inventors have surprisingly found that with this reagent, the linker can be efficiently ligated to the DNA fragment to which the base A is added at the 3' end, and the obtained product has good quality and high purity, and can be effectively applied to subsequent treatment.
  • the reagent is sometimes referred to as a second reagent, which is used in the method for constructing a DNA sequencing library of the present invention to add a DNA fragment of the base A to the 3' end.
  • the joints are joined to obtain the ligation product.
  • the present invention provides a method of constructing a DNA sequencing library.
  • the method comprises the steps of: fragmenting DNA to obtain a DNA fragment; performing end repair of the DNA fragment to obtain a DNA fragment subjected to end repair; using a first reagent according to an embodiment of the present invention, The end-repaired DNA fragment is treated to obtain a DNA fragment having a base A added at the 3' end; and a DNA fragment having a base A added to the 3' end is ligated to the linker using a second reagent according to an embodiment of the present invention, To obtain a ligation product; the ligation product is subjected to fragment selection to obtain a fragment of interest; the target fragment is subjected to PCR amplification to obtain an amplification product; and the amplification product is isolated and purified, and the amplification product constitutes a DNA sequencing library.
  • the method for constructing a DNA sequencing library according to an embodiment of the present invention is simple, easy to operate, less time-consuming, and reproducible, and the method can reduce library loss, reduce library construction cost, and improve library construction efficiency.
  • the quality of the library obtained was very good.
  • the DNA sequencing library of the sample can be conveniently and efficiently constructed, and the DNA sequencing library can be effectively applied to a high-throughput sequencing platform such as an Illumina sequencing platform, and the obtained sequencing result is accurate and reproducible. .
  • the present invention provides a DNA sequencing library constructed by a method of constructing a DNA sequencing library according to an embodiment of the present invention.
  • the DNA sequencing library can have Effectively applied to high-throughput sequencing platforms, and the sequencing results are accurate and repeatable.
  • the present invention provides a method of determining sequence information of a DNA sample.
  • the method comprises the steps of: constructing a DNA sequencing library of a DNA sample according to a method of constructing a DNA sequencing library according to an embodiment of the present invention; and sequencing the DNA sequencing library to determine sequence information of the DNA sample .
  • the method can conveniently and effectively determine the sequence information of the DNA sample, and has high efficiency, accurate result and good repeatability.
  • the present invention also provides a kit for constructing a DNA sequencing library.
  • the kit comprises: a first reagent; and a second reagent.
  • the first reagent and the second reagent are both the first reagent and the second reagent according to the embodiments of the present invention as described above.
  • the kit can be used to construct a DNA sequencing library conveniently and efficiently, and the reproducibility is good, and the obtained library is of good quality, so that it can be effectively applied to a high-throughput sequencing platform such as the Illumina sequencing platform.
  • Figure 1 shows a schematic flow diagram showing a library construction method for small fragments of DNA based on the Illumina sequencing platform.
  • Figure 2 shows a DNA sequencing library constructed according to one embodiment of the present invention via Agilent Bioanalyzer
  • Figure 3 A flow diagram showing a method of constructing a DNA sequencing library in accordance with one embodiment of the present invention. Detailed description of the invention
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first” and “second” may include one or more of the features, either explicitly or implicitly. Further, in the description of the present invention, “multiple” means two or more unless otherwise stated.
  • the invention provides a reagent comprising a buffer salt, dithiothreitol, MgCl 2 .
  • the inventors have found that by using the reagent as a buffer system, it is possible to efficiently configure a reaction system required for constructing a sequencing library to efficiently add a base A to the 3' end of the DNA fragment, and to efficiently bind the linker with A reaction system in which a DNA fragment of base A is linked to the 3' end is added. Thereby, the recovery step before the linker is added to the DNA fragment of the base A at the 3' end after the base A is added to the 3' end of the DNA fragment can be omitted. Thereby, the efficiency of constructing the sequencing library can be improved.
  • the invention provides an agent.
  • the reagent may comprise: 10 mM to 1000 mM buffer salt; 10 mM to 100 mM soluble salt; 10 mM to 300 mM MgCl 2 ; 1 mM to 50 mM dithiothreitol; 1 mM to 10 mM dATP; And a Klenow fragment (3' ⁇ 5' exo) of 1 U/ml to 40 U/ml, wherein the reagent has a H of 7.6 to 7.9.
  • the base A can be efficiently added to the 3' end of the DNA fragment, and the obtained product is excellent in quality and high in purity, and can be effectively used for subsequent treatment such as a joint.
  • the type of the buffer salt contained in the reagent is not particularly limited.
  • the buffer salt may be at least one selected from the group consisting of Tris-HCl and phosphate, preferably For Tris-HCl.
  • the concentration of the buffer salt in the reagent may be from 10 mM to 1000 mM, and preferably from 50 mM to 500 mM, more preferably 100 mM, according to a specific example of the present invention.
  • the type of the soluble salt contained in the reagent is not particularly limited.
  • the soluble salt may be at least one selected from the group consisting of sodium chloride and potassium chloride, preferably Sodium chloride.
  • the concentration of the soluble salt contained in the reagent may be 10 mM to 100 mM, and is preferably 25 mM to 75 mM, more preferably 50 mM, according to a specific example of the present invention.
  • the reagent may comprise 50 mM to 200 mM of MgCl 2 , and according to a specific example, preferably contains 100 mM of MgCl 2 .
  • the reagent may comprise from 2.5 mM to 7.5 mM of dATP, and according to a specific example, preferably comprises 5 mM of dATP.
  • the reagent may comprise a Klenow fragment (3' ⁇ 5' exo) of 10 U/ml to 30 U/ml, and according to a specific example, preferably comprises a KUow fragment of 20 U/ml (3' ⁇ 5' Exo ).
  • the pH of the reagent is 7.9.
  • the first reagent of the present invention may have a pH of 7.6 to 7.9, preferably 7.9, the solvent is water, and the solute may be a mixture of the following final concentrations: 10 mM to 1 OO mM, Preferably, 25 mM to 75 mM, more preferably 50 mM soluble salt; 10 to 300 mM, preferably 50 to 200 mM, more preferably 100 mM MgCl 2 ; 10 mM to 1000 mM, preferably 50 mM to 500 mM, more preferably 100 mM buffer salt; 1 mM to 50 mM Excellent 5 mM to 15 mM, more preferably 10 mM bisthreitol; 1 mM to 10 mM, preferably 2.5 mM to 7.5 mM, more preferably 5 mM dATP, 1 U/ml to 40 U/ml, preferably 10 U/ml ⁇ 30 U/ml, more
  • the first reagent of the present invention can be efficiently added to the 3' end of the DNA fragment, and the obtained product is of good quality, high purity, and can be effectively used for subsequent processing such as ligation without purification. Connector.
  • the first reagent can be used in the method of constructing a DNA sequencing library of the present invention to process the DNA fragment subjected to end repair to obtain a DNA fragment having a base A added at the 3' end, which may also be included in the present invention.
  • the kit of the invention is used to construct a DNA sequencing library in order to perform the same function.
  • the present invention also provides an agent.
  • the reagent may comprise: 10 mM to 200 mM of a buffer salt; 1 mM to 100 mM of dithiothreitol; 10 mM to 400 mM of MgCl 2 ; 1 mM to 40 mM of ATP; and 1 U/ml to 200 U/ Ml DNA ligase, wherein the pH of the reagent is 7.6 ⁇ 7.9.
  • the linker can be efficiently linked to the DNA fragment to which the base A is added at the 3' end, and the obtained product has good quality and high purity, and can be effectively used for subsequent treatment.
  • the type of the buffer salt contained in the reagent is not particularly limited.
  • the buffer salt may be at least one selected from the group consisting of Tris-HCl and phosphate, preferably For Tris-HCl.
  • the concentration of the buffer salt in the reagent may be from 10 mM to 200 mM, and preferably from 50 mM to 150 mM, more preferably 100 mM, according to a specific example of the present invention.
  • the reagent may contain 10 mM to 90 mM of dithiothreitol, and according to a specific example, preferably contains 50 mM of dithiothreitol.
  • the reagent may comprise 50 mM to 200 mM of MgCl 2 , and according to a specific example, preferably contains 100 mM of MgCl 2 .
  • the reagent may comprise 5 mM to 20 mM ATP, and according to a specific example, preferably comprises 10 mM ATP.
  • the reagent may comprise from 50 U/ml to 150 U/ml of DNA ligase, and according to a specific example, preferably comprises 72 U/ml of DNA ligase.
  • the reagent has a pH of 7.6.
  • the second reagent of the present invention may have a pH of 7.6 to 7.9, preferably 7.6, the solvent is water, and the solute may be a mixture of the following final concentrations: 10 to 200 mM, preferably 50 to 150 mM.
  • 100 mM buffer salt 1 to 100 mM, preferably 10 to 90 mM, more preferably 50 mM dithiothreitol; 1 to 40 mM, preferably 5 to 20 mM, more preferably 10 mM ATP; 10 to 400 mM, preferably 50 ⁇ 200 mM, more preferably 100 mM MgCl 2 ; 1 U/ml to 200 U/ml, preferably 50 U/ml to 150 U/ml, more preferably 72 U/ml DNA ligase.
  • the buffer salt may be Tris-HCl or phosphate, preferably Tris-HCl.
  • the linker can be efficiently linked to the DNA fragment to which the base A is added at the 3' end, and the obtained product is excellent in quality and high in purity, and can be effectively used for subsequent treatment.
  • the second reagent can be used in the method of constructing a DN A sequencing library of the present invention to link a DNA fragment of the base A with a base A to a linker to obtain a ligation product, which can also be included in the present invention.
  • the kit in order to play the same role, to construct a DNA sequencing library.
  • the present invention provides a method of constructing a DNA sequencing library.
  • the method may include the following steps:
  • the DNA is fragmented to obtain a DNA fragment.
  • the method of fragmenting DNA is not particularly limited. According to a specific example of the present invention, it may be carried out by at least one selected from the group consisting of ultrasonication and enzymatic treatment, wherein the ultrasonication method can be carried out using a Covaris ultrasonic interrupter.
  • the DNA fragment may have a length of 200 to 800 bp.
  • the DNA fragment is end-repaired to obtain a DNA fragment that has been repaired at the end.
  • the method of performing end repair of the DNA fragment is not particularly limited.
  • terminal repair can be performed using Klenow fragment, T4 DNA polymerase, and T4 polynucleotide kinase, wherein the Klenow fragment has 5' ⁇ 3' polymerase activity and 3' ⁇ 5' polymerase activity. However, it lacks 5' ⁇ 3' exonuclease activity.
  • the end-repaired DNA fragment is treated using the first reagent according to an embodiment of the present invention to obtain a DNA fragment in which the base A is added at the 3' end.
  • the end-repaired DNA fragment can be treated at 12 ° C to 40 ° C using the first reagent according to an embodiment of the present invention.
  • the treatment is preferably carried out at 37 °C.
  • the joint comprises a first strand and a second strand
  • the DNA fragment in which the 3' end is added to the base A can be ligated to the linker at 4 ° C to 22 ° C using a second reagent according to an embodiment of the present invention.
  • the treatment is preferably carried out at 16 °C.
  • the inventors have surprisingly found that by using the first reagent and the second reagent, it is possible to directly carry out the ligation reaction after the addition of the base A at the 3' end, without purification and recovery. Thereby, the efficiency of preparing the sequencing library can be significantly improved, thereby improving the efficiency of subsequent sequencing and analysis, and avoiding the loss of nucleic acid samples. Then, the ligation product is subjected to fragment selection to obtain a fragment of interest.
  • the method of performing fragment selection of the ligation product is not particularly limited.
  • the ligation product can be subjected to fragment selection using agarose gel electrophoresis.
  • the target segment may be 100-1000 bp in length.
  • it is preferred that the length of the target fragment is 200-800 bp.
  • the target fragment is subjected to PCR amplification to obtain an amplification product.
  • the target fragment is subjected to PCR amplification using the first PCR primer and the second PCR primer, wherein the sequence of the first PCR primer is:
  • CTCTTCCGATCT the sequence of the second PCR primer is: 5' AATGATACGGCGACCACCGAGATCTA CACTCTTTCCCTACACGACGCTCTTCCGATCT.
  • the amplified product is isolated and purified, and the amplified product constitutes the DNA sequencing library.
  • the method of isolating and purifying the amplification product is not particularly limited. According to a specific example of the present invention, it was carried out by 2% agarose gel electrophoresis.
  • a DNA sequencing library of a sample can be conveniently and efficiently constructed, and the obtained library is of good quality, can be effectively applied to a high-throughput sequencing platform such as an Illumina sequencing platform, and the sequencing result is accurate. , repeatability is good.
  • the method of constructing a DNA sequencing library of the present invention may comprise the following steps:
  • the target fragment is subjected to PCR amplification, and the amplified product is isolated and purified, and the amplified product constitutes a DNA sequencing library.
  • the first reagent in the step (3) may have a pH of 7.6 to 7.9, preferably 7.9, the solvent is water, and the solute may be a final concentration of the substance: 10 mM to 100 mM, preferably 25 mM to 75 mM, more preferably 50 mM of soluble salt.
  • the second reagent in the step (4) may have a pH of 7.6 to 7.9, preferably 7.6, the solvent is water, and the solute may be
  • the final concentration material is: 10 to 200 mM, preferably 50 to 150 mM, more preferably 100 mM buffer salt; 1 to 100 mM, preferably 10 to 90 mM, more preferably 50 mM dithiothreitol; 1 to 40 mM, preferably 5 to 20 mM, more preferably 10 mM ATP; 10 to 400 mM, preferably 50 to 200 mM, more preferably 100 mM MgCl 2 ; 1 U/ml to 200 U/ml, preferably 50 U/ml to 150 U/ml, more preferably 72 U/ Ml DNA ligase.
  • the method may further comprise the step (7): detecting the library concentration and fragment size of the DNA sequencing library obtained in the step (6), preferably, using Agilent Bioanalyzer 2100 and Q-PCR DNA for DNA The sequencing library was tested.
  • the method may further comprise the step (8): sequencing the DNA sequencing library, preferably using an Illumina sequencing platform.
  • DNA in the step (1), may be fragmented by ultrasonic or enzymatic cleavage.
  • the length of the DNA fragment in step (1) may be from 200 bp to 800 bp.
  • the step of purifying the DNA fragment may be further included before performing step (2).
  • the end repair in the step (2), can be carried out by mixing the DNA fragment obtained in the step (1) with the terminal repair reagent to form a blunt-ended DNA fragment.
  • the step of purifying the blunt-ended DNA fragment may be further included prior to performing step (3).
  • the buffer salt in the first reagent may be Tris-HCl or a phosphate salt, preferably Tris-HCl.
  • the soluble salt in the first reagent may be sodium chloride or potassium chloride, preferably sodium chloride.
  • the incubation may be carried out at a temperature of from 12 ° C to 40 ° C, preferably at 37 ° C.
  • the linker in the step (4), may be any linker having a T base end, and preferably, the linker comprises the first chain and the second chain as follows:
  • the first chain is:
  • the second chain is:
  • the buffer salt in the second reagent may be Tris-HCl or phosphate, preferably Tris-HCL according to the present invention.
  • the incubation may be carried out at a temperature of 4 ° C to 22 ° C, preferably at a temperature of 16 °c.
  • the DNA fragment to which the adaptor is attached in the step (5), can be subjected to fragment selection by subjecting the DNA fragment to which the adaptor is attached to agarose gel electrophoresis, and then cutting the gel to recover the target fragment.
  • the segment of interest may be 100 _ 1000 bp in length, for example 200, 400 bp and 800 bp.
  • the primers used for PCR amplification are:
  • the present invention provides a DNA sequencing library constructed by the above method of constructing a DNA sequencing library according to an embodiment of the present invention.
  • the inventors found that the DNA sequencing library is high in purity, high in quality, and can be effectively applied to a high-throughput sequencing platform, and the sequencing results are accurate and reproducible.
  • the DNA sequencing library constructed for small fragment DNA can also be effectively applied to subsequent sequencing and the like.
  • the present invention also provides a kit for constructing a DNA sequencing library.
  • the kit may comprise: a first reagent; and a second reagent. Wherein the first reagent and the second reagent are both the first reagent and the second reagent according to the embodiments of the present invention as described above.
  • the first reagent may comprise: 10 mM to 1000 mM buffer salt; 10 mM to 100 mM soluble salt; 10 mM to 300 mM MgCl 2 ; 1 mM to 50 mM dithiothreitol; 1 mM to 10 mM dATP; Klenow fragment (3' ⁇ 5' exo ) of /ml ⁇ 40U/ml, wherein the pH of the reagent is 7.6 to 7.9.
  • the second reagent may comprise: 10 mM to 200 mM buffer salt; 1 mM to 100 mM dithiothreitol; 10 mM to 400 mM MgCl 2 ; 1 mM to 40 mM ATP; and 1 U/ml to 200 U/ml DNA ligase, Among them, the pH of the reagent is 7.6 ⁇ 7.9. Other features and advantages regarding the first reagent and the second reagent have been described in detail above and will not be described again.
  • the first reagent and the second reagent may be respectively disposed in different containers.
  • the kit can be used to construct a DNA sequencing library conveniently and efficiently, and the reproducibility is good, and the obtained library is of good quality, so that it can be effectively applied to a high-throughput sequencing platform such as an Illumina sequencing platform.
  • a high-throughput sequencing platform such as an Illumina sequencing platform.
  • those skilled in the art can understand that other components for constructing a DNA sequencing library can also be included in the kit, and details are not described herein.
  • the present invention provides a method of determining sequence information of a DNA sample.
  • the method may comprise the steps of: constructing a DNA sequencing library of a DNA sample according to the method of constructing a DNA sequencing library according to an embodiment of the present invention; and sequencing the DNA sequencing library to determine the sequence of the DNA sample information.
  • the DNA sequencing library prior to sequencing the DNA sequencing library, may be further detected using at least one of Agilent Bioanalyzer 2100 and Q-PCR.
  • sequencing can be performed using an Illumina sequencing platform. The method can conveniently and effectively determine the sequence information of the DNA sample, and has the advantages of simple operation, less time, high efficiency, accurate result and good repeatability.
  • the first reagent pH is 7.9, the solvent is water, and the solute is the following final concentration: 50 mM sodium chloride (National Pharmaceutical Group Chemical Co., Ltd.), lOO mM Tris-HCl (National Pharmaceutical Group Chemical Reagent Co., Ltd.), 100 mM MgCl 2 (National Pharmaceutical Group Chemical Reagent Co., Ltd.;), 10 mM dithiothreitol (Sigma), 5 mM dATP (Enzymatics), 20 U/ml Klenow fragment (3 '-5 'exo).
  • the second reagent pH is 7.6, the solvent is water, and the solute is the following final concentration: 72 U/ml T4 DNA polymerase, 10 mM ATP, 500 mM Tris-HCl, 100 mM MgCl 2 , 50 mM dicequititol .
  • the DNA fragments of sample X and sample Y were purified and recovered by Agencourt's Ampure magnetic beads, respectively, to obtain purified products, and then the purified products of the two samples were separately dissolved in 45 ⁇ l of ultrapure water. Each of the 42 ⁇ l was placed in two PCR tubes and used.
  • the prepared end-recovery reaction system of sample X and sample ⁇ was placed on a PCR machine and placed at 20 ° C for 30 min to obtain a terminal repair product, which was then purified and dissolved in 20 ⁇ l and 32 ⁇ l respectively using Ampure magnetic beads. Ultra pure water, spare.
  • the total volume of 50 ⁇ 1 is to add the base ⁇ reaction system of the prepared sample X and the 3′ end of the sample ,, and react at 15 ° C to 40 ° C for 30 min respectively to obtain the product of adding the base A at the 3 ' end, and then The product of the base A was added to the 3' end of the sample X without purification, and the product of the base A of the sample Y was purified by using Ampure magnetic beads and dissolved in 37 ⁇ l of ultrapure water for use.
  • the linker used in the reaction system of sample X and sample oxime is a double-stranded oligonucleotide comprising the first strand and the second strand as follows:
  • the first chain is:
  • the prepared sample reaction system of sample X and sample was separately placed at 16 ° C for 14-18 hours to obtain a ligation product, which was then purified by Ampure magnetic beads and washed with ultrapure water. Take off, spare.
  • the first PCR primer the first PCR primer:
  • a PCR amplification reaction was carried out in a PCR machine to obtain an amplification product, and then the amplification products of the two samples were separately separated by 2% agarose gel electrophoresis.
  • the 400 bp and 800 bp amplification products were separately fractionated and dissolved in 30 ⁇ l of ultrapure water.
  • the 400 bp and 800 bp amplification products of sample X constituted the DNA sequencing library
  • the 400 bp and 800 bp amplification products of sample Y constituted DNA sequencing library.
  • the conditions of the PCR amplification reaction are:
  • a DNA sequencing library of sample X was prepared by the method for constructing a DNA sequencing library according to an embodiment of the present invention, and a DNA sequencing library of sample Y was constructed by using a method for constructing a DNA sequencing library provided by the Illumina sequencing platform.
  • the second reagent pH is 7.6, the solvent is water, and the solute is the following final concentration: T4 DNA ligase (Enzymatics), ATP (NEB), 100 mM buffer salt, 100 mM MgCl 2 (National Pharmaceutical Group Chemical Reagent Co., Ltd.; ), 50 mM dicequititol (Sigma).
  • the prepared sample X and the 3' end of the sample were added to the base ⁇ reaction system and reacted at 37 ° C for 30 min to obtain the product of the base A added at the 3 ' end, and then, the 3 ' end of the sample X
  • the product of the base A was added without purification, and the product of the base A of the sample Y was purified by the QlAquick PCR Purification Kit (QIAGEN) and dissolved in a 16.4 ⁇ l hydrazine eluate for use.
  • the sequence of the linker is the same as in the first embodiment.
  • a DNA sequencing library of sample X and sample is obtained, that is, a DNA sequencing library of sample X is prepared by the method of constructing a DNA sequencing library according to an embodiment of the present invention, and a DNA sequencing library provided by the Illumina sequencing platform is constructed. Method, a DNA sequencing library of sample Y was constructed.
  • the second reagent pH is 7.6, the solvent is water, and the solute is the following final concentration: T4 DNA ligase ( Takara ), 10 mM ATP ( NEB ), 500 mM Tris-HCl, lOOmM MgCl 2 (National Pharmaceutical Group Chemical Reagent Co., Ltd.) ), 50 mM dicequititol (Sigma).
  • sample X and sample Y Two 50 ⁇ g/ ⁇ Fosmid samples (supplied by Shenzhen Huada Gene Research Institute) were taken in two Covaris sample tubes, named sample X and sample Y, respectively, and then tested according to the following steps.
  • sample X and sample Y were end-repaired according to the instructions in the Agilent SureSelect platform manual G3360-90020 to obtain the end repair product.
  • the total volume of 35 ⁇ 1 is to add the base ⁇ reaction system of the prepared sample X and the 3′ end of the sample ,, and respectively react at 37 ° C for 30 min to obtain the product of adding the base A at the 3′ end, and then, the sample X
  • the product of the base A added at the 3' end was not purified, and the product of the base A added to the 3' end of the sample Y was purified by column and dissolved in 37 ⁇ l of the eluate for use.
  • the sequence of the linker is the same as in the first embodiment.
  • the ligation products of sample X and sample Y were respectively formulated into a PCR amplification reaction system, and then the PCR amplification reaction systems of sample X and sample Y were respectively subjected to a PCR instrument.
  • the PCR amplification reaction was carried out to obtain an amplification product, and then the amplification products of the two samples were separately separated by 2% agarose gel electrophoresis, and the 200 bp amplification product was recovered by gelation and dissolved in 30 ⁇ l of ultrapure water. Thereby, a DNA sequencing library of the sample X and a DNA sequencing library of the sample were obtained.
  • the two reagents of the present invention can be effectively applied to the construction of the DNA sequencing library of the sample DNA.
  • sequencing and the obtained library is of good quality and the sequencing results are accurate.

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Abstract

Provided are two reagents, a method for constructing a DNA sequencing library, a DNA sequencing library, a method for determining the sequence information in a DNA sample and a kit for constructing a DNA sequencing library, wherein one reagent comprises 10 mM to 1000 mM of a buffer salt, 10 mM to 100 mM of a soluble salt, 10 mM to 300 mM of MgCl2, 1 mM to 50 mM of dithiothreitol, 1 mM to 10 mM of dATP and 1 U/ml to 40 U/ml of klenow fragment, with the pH of the reagent being 7.6 to 7.9, and the other reagent comprises 10 mM to 200 mM of a buffer salt, 1 mM to 100 mM of dithiothreitol, 10 mM to 400 mM of MgCl2, 1 mM to 40 mM of ATP and 1 U/ml to 200 U/ml of DNA ligase, with the pH of the reagent being 7.6 to 7.9.

Description

构建 DNA测序文库的方法及其用途  Method for constructing DNA sequencing library and use thereof
优先权信息 Priority information
本申请请求 2010 年 12 月 15 日向中国国家知识产权局提交的、 专利申请号为 201010588936.2的专利申请的优先权和权益， 并且通过参照将其全文并入此处。 技术领域  The present application claims priority to and the benefit of the patent application No. 201010588936.2, filed on Dec. 15, 2010, to the Chinese National Intellectual Property Office, the entire disclosure of which is hereby incorporated by reference. Technical field
本发明涉及生物技术领域， 具体地， 涉及 DNA测序技术领域， 特别是构建 DNA 测序文库的方法及其用途。 更具体地， 本发明提供了两种试剂、 一种构建 DNA测序文 库的方法、 一种 DNA测序文库、 一种确定 DNA样品的序列信息的方法、 一种用于构 建 DNA测序文库的试剂盒。 背景技术  The present invention relates to the field of biotechnology, and in particular to the field of DNA sequencing technology, in particular to a method for constructing a DNA sequencing library and its use. More specifically, the present invention provides two reagents, a method of constructing a DNA sequencing library, a DNA sequencing library, a method of determining sequence information of a DNA sample, and a kit for constructing a DNA sequencing library. Background technique
DNA (脱氧核糖核酸） ， 是两条核苷酸链以互补配对原则所构成的双螺旋结构的 分子化合物， 其是生命遗传信息的载体。 对 DNA核苷酸序列进行测序分析， 不仅能够 为基因表达和调控等生物学基础研究提供重要数据，而且对疾病诊断学和基因治疗等应 用研究意义重大 (可参见 Gilbert W. DNA sequencing and gene structure. In: Forsen S. Nobel Lectures in Chemistry 1971-1980. 1980.； Singapore: World Scientific Publishing Co, 1993, 408-426.； Sanger F, Coulson A R. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase[J]. J Mol Biol, 1975, 94:441—448. , 通过参照将 其全文并入本文） 。  DNA (deoxyribonucleic acid) is a molecular compound of a double-helical structure composed of two nucleotide chains in a complementary pairing principle, which is a carrier of life genetic information. Sequencing and analysis of DNA nucleotide sequences not only provides important data for basic biological research such as gene expression and regulation, but also plays an important role in applied research such as disease diagnosis and gene therapy (see Gilbert W. DNA sequencing and gene structure). In: Forsen S. Nobel Lectures in Chemistry 1971-1980. 1980.; Singapore: World Scientific Publishing Co, 1993, 408-426.; Sanger F, Coulson A R. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase [J]. J Mol Biol, 1975, 94: 441-448., which is incorporated herein by reference in its entirety.
然而， 目前常用的 DNA测序平台， 例如 Illumina第二代高通量测序平台， 各个步 骤均独立进行， 且每步之后都需要进行纯化， 因而过程繁瑣、 产物损失多、 成本高， 仍 需改进。 发明内容  However, currently used DNA sequencing platforms, such as Illumina's second-generation high-throughput sequencing platform, are performed independently and require purification after each step, resulting in cumbersome processes, high product losses, high costs, and improvements. Summary of the invention
本发明旨在至少解决现有技术中存在的技术问题之一。 为此， 本发明提供了构建 DN A测序文库的方法及其用途。  The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the invention provides methods of constructing DN A sequencing libraries and uses thereof.
根据本发明的一个方面， 本发明提出了一种试剂， 该试剂包含緩冲盐、二硫苏糖醇、 MgCl2。 发明人发现， 通过釆用该试剂作为緩冲体系， 可以有效地配置构建测序文库中所需 要的能够有效地将 DNA片段的 3'端添加碱基 A的反应体系， 以及能够有效地将接头与 3' 端添加碱基 A的 DNA片段相连的反应体系。 由此， 可以节省回收的步骤。 从而能够提高 构建测序文库的效率。 According to one aspect of the invention, the invention provides a reagent comprising a buffer salt, dithiothreitol, MgCl 2 . The inventors have found that by using the reagent as a buffer system, it is possible to efficiently configure a reaction system required for constructing a sequencing library to efficiently add a base A to the 3' end of the DNA fragment, and to efficiently bind the linker with 3' A reaction system in which a DNA fragment of base A is linked is added. Thereby, the step of recycling can be saved. Thereby, the efficiency of constructing the sequencing library can be improved.
在上述试剂作为緩冲体系的基础上， 根据本发明的又一个方面， 本发明提供了一种 试剂。 根据本发明的实施例， 该试剂包含： 10mM〜1000mM的緩冲盐； 10mM〜100mM的可 溶性盐； 10mM〜300mM的 MgCl2; lmM〜50mM的二^ ¾苏糖醇； lmM〜10mM的 dATP; 以 及 lU/ml〜 40U/ml的 Klenow片段（ 3'→5' exo ), 其中， 该试剂的 pH为 7.6〜7.9。 发明人惊 奇地发现， 利用该试剂， 能够有效地将 DNA片段的 3'端添加碱基 A, 所得产物质量好、 纯 度高， 无需经过纯化即可用于后续处理例如与接头相连。 需要说明的是， 在本文中， 有时 也将该试剂称为第一试剂， 其在本发明的构建 DNA测序文库的方法中， 被用于对经过末端 修复的 DNA片段进行处理， 以便获得 3'端添加碱基 A的 DNA片段。 根据本发明的另一 方面， 本发明还提供了一种试剂。 根据本发明的实施例， 该试剂包含： 10mM〜200mM 的 緩冲盐； lmM〜100mM的二硫苏糖醇； 10mM〜400mM的 MgCl2; lmM〜40mM的 ATP; 以 及 lU/ml〜 200U/ml的 DNA连接酶， 其中， 该试剂的 pH为 7.6 ~ 7.9。 发明人惊奇地发现， 利用该试剂， 能够有效地将接头与 3'端添加碱基 A的 DNA片段相连， 所得产物质量好、 纯度高， 能够有效地应用于后续处理。 需要说明的是， 在本文中， 有时也将该试剂称为第 二试剂，其在本发明的构建 DNA测序文库的方法中，被用于将所述 3'端添加碱基 A的 DNA 片段与接头进行连接， 以便获得连接产物。 Based on the above reagent as a buffer system, in accordance with still another aspect of the present invention, the present invention provides an agent. According to an embodiment of the present invention, the reagent comprises: 10 mM to 1000 mM of a buffer salt; 10 mM to 100 mM of a soluble salt; 10 mM to 300 mM of MgCl 2 ; 1 mM to 50 mM of bis-threitol; 1 mM to 10 mM of dATP; And a Klenow fragment (3'→5' exo) of 1 U/ml to 40 U/ml, wherein the pH of the reagent is 7.6 to 7.9. The inventors have surprisingly found that with this reagent, the base A can be efficiently added to the 3' end of the DNA fragment, and the resulting product is of good quality and high purity, and can be used for subsequent treatment such as attachment to a linker without purification. It should be noted that, in this context, the reagent is sometimes referred to as a first reagent, which is used in the method of constructing a DNA sequencing library of the present invention to process a DNA fragment subjected to end repair to obtain 3'. A DNA fragment of base A is added at the end. According to another aspect of the invention, the invention also provides an agent. According to an embodiment of the present invention, the reagent comprises: 10 mM to 200 mM buffer salt; 1 mM to 100 mM dithiothreitol; 10 mM to 400 mM MgCl 2 ; 1 mM to 40 mM ATP; and 1 U/ml to 200 U/ml The DNA ligase, wherein the reagent has a pH of 7.6 to 7.9. The inventors have surprisingly found that with this reagent, the linker can be efficiently ligated to the DNA fragment to which the base A is added at the 3' end, and the obtained product has good quality and high purity, and can be effectively applied to subsequent treatment. It should be noted that, in this context, the reagent is sometimes referred to as a second reagent, which is used in the method for constructing a DNA sequencing library of the present invention to add a DNA fragment of the base A to the 3' end. The joints are joined to obtain the ligation product.
根据本发明的再一方面， 本发明提供了一种构建 DNA测序文库的方法。 根据本发明的 实施例， 该方法包含以下步骤： 将 DNA片段化， 以便获得 DNA片段； 将 DNA片段进行 末端修复， 以便获得经过末端修复的 DNA片段； 使用根据本发明实施例的第一试剂， 对经 过末端修复的 DNA片段进行处理， 以便获得 3'端添加碱基 A的 DNA片段； 使用根据本发 明实施例的第二试剂， 将 3'端添加碱基 A的 DNA片段与接头进行连接， 以便获得连接产 物； 将连接产物进行片段选择， 以便获得目的片段； 将目的片段进行 PCR扩增， 以便获得 扩增产物； 以及分离纯化扩增产物， 该扩增产物构成 DNA测序文库。  According to still another aspect of the present invention, the present invention provides a method of constructing a DNA sequencing library. According to an embodiment of the present invention, the method comprises the steps of: fragmenting DNA to obtain a DNA fragment; performing end repair of the DNA fragment to obtain a DNA fragment subjected to end repair; using a first reagent according to an embodiment of the present invention, The end-repaired DNA fragment is treated to obtain a DNA fragment having a base A added at the 3' end; and a DNA fragment having a base A added to the 3' end is ligated to the linker using a second reagent according to an embodiment of the present invention, To obtain a ligation product; the ligation product is subjected to fragment selection to obtain a fragment of interest; the target fragment is subjected to PCR amplification to obtain an amplification product; and the amplification product is isolated and purified, and the amplification product constitutes a DNA sequencing library.
发明人发现， 根据本发明实施例的构建 DNA测序文库的方法， 步骤简便、 易于操作、 需时少、 可重复性好， 且该方法能够减少文库损失、 降低建库成本、 提高文库构建效率， 获得的文库质量非常好。根据本发明的实施例，利用该方法能够方便高效地构建样品的 DNA 测序文库，并且该 DNA测序文库能够有效地应用于高通量测序平台例如 Illumina测序平台， 所得测序结果准确、 可重复性好。  The inventors have found that the method for constructing a DNA sequencing library according to an embodiment of the present invention is simple, easy to operate, less time-consuming, and reproducible, and the method can reduce library loss, reduce library construction cost, and improve library construction efficiency. The quality of the library obtained was very good. According to an embodiment of the present invention, the DNA sequencing library of the sample can be conveniently and efficiently constructed, and the DNA sequencing library can be effectively applied to a high-throughput sequencing platform such as an Illumina sequencing platform, and the obtained sequencing result is accurate and reproducible. .
根据本发明的另一方面， 本发明提供了一种 DNA测序文库， 其是通过根据本发明实 施例的构建 DNA测序文库的方法构建的。 根据本发明的实施例， 该 DNA测序文库能够有 效地应用于高通量测序平台， 并且测序结果准确、 可重复性好。 According to another aspect of the present invention, the present invention provides a DNA sequencing library constructed by a method of constructing a DNA sequencing library according to an embodiment of the present invention. According to an embodiment of the present invention, the DNA sequencing library can have Effectively applied to high-throughput sequencing platforms, and the sequencing results are accurate and repeatable.
根据本发明的又一方面， 本发明提供了一种确定 DNA样品的序列信息的方法。 根据本 发明的实施例， 该方法包括下列步骤： 根据本发明实施例的构建 DNA测序文库的方法， 构 建 DNA样品的 DNA测序文库； 以及对该 DNA测序文库进行测序， 以便确定 DNA样品的 序列信息。利用该方法能够方便有效地确定 DNA样品的序列信息，并且效率高、结果准确、 可重复性好。  According to still another aspect of the present invention, the present invention provides a method of determining sequence information of a DNA sample. According to an embodiment of the present invention, the method comprises the steps of: constructing a DNA sequencing library of a DNA sample according to a method of constructing a DNA sequencing library according to an embodiment of the present invention; and sequencing the DNA sequencing library to determine sequence information of the DNA sample . The method can conveniently and effectively determine the sequence information of the DNA sample, and has high efficiency, accurate result and good repeatability.
根据本发明的再一方面， 本发明还提供了一种用于构建 DNA测序文库的试剂盒。 根据 本发明的实施例， 该试剂盒包含： 第一试剂； 以及第二试剂。 其中第一试剂和第二试剂均 为前面所述的根据本发明实施例的第一试剂和第二试剂。 利用该试剂盒能够方便有效地构 建 DNA测序文库， 并且可重复性好， 获得的文库质量好， 从而能够有效地应用于高通量测 序平台例如 Illumina测序平台。  According to still another aspect of the present invention, the present invention also provides a kit for constructing a DNA sequencing library. According to an embodiment of the invention, the kit comprises: a first reagent; and a second reagent. Wherein the first reagent and the second reagent are both the first reagent and the second reagent according to the embodiments of the present invention as described above. The kit can be used to construct a DNA sequencing library conveniently and efficiently, and the reproducibility is good, and the obtained library is of good quality, so that it can be effectively applied to a high-throughput sequencing platform such as the Illumina sequencing platform.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得 明显， 或通过本发明的实践了解到。 附图说明  The additional aspects and advantages of the invention will be set forth in part in the description which follows. DRAWINGS
本发明的上述和 /或附加的方面和优点从结合下面附图对实施例的描述中将变得明 显和容易理解， 其中：  The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from
图 1 : 显示了基于 Illumina测序平台的小片段 DNA的文库构建方法的流程示意图。 图 2: 显示了根据本发明一个实施例的构建的 DNA测序文库经 Agilent Bioanalyzer Figure 1 shows a schematic flow diagram showing a library construction method for small fragments of DNA based on the Illumina sequencing platform. Figure 2: shows a DNA sequencing library constructed according to one embodiment of the present invention via Agilent Bioanalyzer
2100检测结果。 2100 test results.
图 3: 显示了根据本发明一个实施例的构建 DNA测序文库的方法的流程示意图。 发明详细描述  Figure 3: A flow diagram showing a method of constructing a DNA sequencing library in accordance with one embodiment of the present invention. Detailed description of the invention
下面详细描述本发明的实施例， 所述实施例的示例在附图中示出， 其中自始至终相 图描述的实施例是示例性的， 仅用于解释本发明， 而不能理解为对本发明的限制。  The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the accompanying drawings, wherein the embodiments described in the drawings are intended to be illustrative only and not to limit the invention. .
需要说明的是， 术语 "第一" 、 "第二" 仅用于描述目的， 而不能理解为指示或暗 示相对重要性或者隐含指明所指示的技术特征的数量。 由此， 限定有 "第一"、 "第二" 的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中， 除非另有说明， "多个" 的含义是两个或两个以上。 试剂 It should be noted that the terms "first" and "second" are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining "first" and "second" may include one or more of the features, either explicitly or implicitly. Further, in the description of the present invention, "multiple" means two or more unless otherwise stated. Reagent
根据本发明的一个方面， 本发明提出了一种试剂， 该试剂包含緩冲盐、二硫苏糖醇、 MgCl2。 发明人发现， 通过釆用该试剂作为緩冲体系， 可以有效地配置构建测序文库中所需 要的能够有效地将 DNA片段的 3'端添加碱基 A的反应体系， 以及能够有效地将接头与 3' 端添加碱基 A的 DNA片段相连的反应体系。 由此，可以省去在将 DNA片段的 3 '端添加碱 基 A之后， 在将接头与 3'端添加碱基 A的 DNA片段相连之前的回收步骤。 从而能够提高 构建测序文库的效率。 According to one aspect of the invention, the invention provides a reagent comprising a buffer salt, dithiothreitol, MgCl 2 . The inventors have found that by using the reagent as a buffer system, it is possible to efficiently configure a reaction system required for constructing a sequencing library to efficiently add a base A to the 3' end of the DNA fragment, and to efficiently bind the linker with A reaction system in which a DNA fragment of base A is linked to the 3' end is added. Thereby, the recovery step before the linker is added to the DNA fragment of the base A at the 3' end after the base A is added to the 3' end of the DNA fragment can be omitted. Thereby, the efficiency of constructing the sequencing library can be improved.
在该緩冲体系的基础上， 根据本发明的另一个方面， 本发明提供了一种试剂。 根据 本发明的实施例，该试剂可以包含： 10mM〜1000mM的緩冲盐； 10mM〜100mM的可溶性盐； 10mM〜300mM的 MgCl2; lmM〜50mM的二硫苏糖醇； lmM〜10mM的 dATP; 以及 lU/ml〜 40U/ml的 Klenow片段（ 3'→5' exo ), 其中， 该试剂的 H为 7.6〜7.9。 利用该试剂 , 能够有 效地将 DNA片段的 3'端添加碱基 A, 所得产物质量好、 纯度高， 能够有效地用于后续处理 例如连接接头。 Based on this buffer system, in accordance with another aspect of the invention, the invention provides an agent. According to an embodiment of the present invention, the reagent may comprise: 10 mM to 1000 mM buffer salt; 10 mM to 100 mM soluble salt; 10 mM to 300 mM MgCl 2 ; 1 mM to 50 mM dithiothreitol; 1 mM to 10 mM dATP; And a Klenow fragment (3'→5' exo) of 1 U/ml to 40 U/ml, wherein the reagent has a H of 7.6 to 7.9. By using this reagent, the base A can be efficiently added to the 3' end of the DNA fragment, and the obtained product is excellent in quality and high in purity, and can be effectively used for subsequent treatment such as a joint.
根据本发明的实施例， 该试剂中所包含的緩冲盐的类型不受特别限制， 根据本发明的 一些具体示例， 緩冲盐可以为选自 Tris-HCl和磷酸盐的至少一种， 优选为 Tris-HCl。根据本 发明的一些实施例， 该试剂中緩冲盐的浓度可以为 10mM〜1000mM, 根据本发明的具体示 例， 优选为 50mM〜500mM, 更优选为 100mM。  According to an embodiment of the present invention, the type of the buffer salt contained in the reagent is not particularly limited. According to some specific examples of the present invention, the buffer salt may be at least one selected from the group consisting of Tris-HCl and phosphate, preferably For Tris-HCl. According to some embodiments of the present invention, the concentration of the buffer salt in the reagent may be from 10 mM to 1000 mM, and preferably from 50 mM to 500 mM, more preferably 100 mM, according to a specific example of the present invention.
根据本发明的实施例， 该试剂中所包含的可溶性盐的类型不受特别限制， 根据本发明 的一些具体示例， 可溶性盐可以为选自氯化钠和氯化钾的至少一种， 优选为氯化钠。 根据 本发明的一些实施例， 该试剂中包含的可溶性盐的浓度可以为 10mM〜100mM , 根据本发 明的具体示例， 优选为 25mM〜75mM, 更优选为 50mM。  According to an embodiment of the present invention, the type of the soluble salt contained in the reagent is not particularly limited. According to some specific examples of the present invention, the soluble salt may be at least one selected from the group consisting of sodium chloride and potassium chloride, preferably Sodium chloride. According to some embodiments of the present invention, the concentration of the soluble salt contained in the reagent may be 10 mM to 100 mM, and is preferably 25 mM to 75 mM, more preferably 50 mM, according to a specific example of the present invention.
根据本发明的一些实施例，该试剂中可以包含 50mM〜200mM的 MgCl2,根据具体示例， 优选包含 lOOmM的 MgCl2。 根据本发明的一些实施例， 该试剂中可以包含 2.5mM〜7.5mM 的 dATP , 根据具体示例， 优选包含 5mM的 dATP。 根据本发明的一些实施例， 该试剂中 可以包含 10U/ml〜 30U/ml的 Klenow片段（ 3'→5' exo ), 根据具体示例， 优选包含 20U/ml 的 Klenow片段（ 3'→5' exo )。 According to some embodiments of the present invention, the reagent may comprise 50 mM to 200 mM of MgCl 2 , and according to a specific example, preferably contains 100 mM of MgCl 2 . According to some embodiments of the invention, the reagent may comprise from 2.5 mM to 7.5 mM of dATP, and according to a specific example, preferably comprises 5 mM of dATP. According to some embodiments of the present invention, the reagent may comprise a Klenow fragment (3'→5' exo) of 10 U/ml to 30 U/ml, and according to a specific example, preferably comprises a KUow fragment of 20 U/ml (3'→5' Exo ).
才艮据本发明的具体示例， 优选地， 该试剂的 pH为 7.9。  According to a specific example of the present invention, preferably, the pH of the reagent is 7.9.
具体地， 才艮据本发明的实施例， 本发明的第一试剂， 其 pH可以为 7.6〜7.9 , 优选 7.9, 溶剂是水，溶质可以为如下终浓度物质的混合物： 10 mM〜1 OOmM ,优选 25mM〜75mM , 更优选 50mM的可溶性盐； 10〜300mM, 优选 50〜200mM, 更优选 lOOmM的 MgCl2; 10mM〜1000mM, 优选 50 mM 〜500mM, 更优选 1 OOmM的緩冲盐； ImM 〜50mM, 优 选 5mM〜15mM, 更优选 lOmM的二^ ¾苏糖醇； ImM〜10mM, 优选 2.5mM〜7.5mM, 更优选 5mM的 dATP , 1 U/ml 〜40 U/ml, 优选 10 U/ml 〜30 U/ml, 更优选 20 U/ml的 Klenow(3' -5' exo)酶。 其中所述緩冲盐可以为 Tris-HCl或磷酸盐， 优选是 Tris-HCl; 其中所述可溶性盐可以为氯化钠或氯化钾， 优选是氯化钠。 Specifically, according to an embodiment of the present invention, the first reagent of the present invention may have a pH of 7.6 to 7.9, preferably 7.9, the solvent is water, and the solute may be a mixture of the following final concentrations: 10 mM to 1 OO mM, Preferably, 25 mM to 75 mM, more preferably 50 mM soluble salt; 10 to 300 mM, preferably 50 to 200 mM, more preferably 100 mM MgCl 2 ; 10 mM to 1000 mM, preferably 50 mM to 500 mM, more preferably 100 mM buffer salt; 1 mM to 50 mM Excellent 5 mM to 15 mM, more preferably 10 mM bisthreitol; 1 mM to 10 mM, preferably 2.5 mM to 7.5 mM, more preferably 5 mM dATP, 1 U/ml to 40 U/ml, preferably 10 U/ml 〜30 U/ml, more preferably 20 U/ml of Klenow (3'-5' exo) enzyme. Wherein the buffer salt may be Tris-HCl or phosphate, preferably Tris-HCl; wherein the soluble salt may be sodium chloride or potassium chloride, preferably sodium chloride.
发明人惊奇地发现， 利用本发明的第一试剂， 能够有效地将 DNA片段的 3'端添加碱 基 A, 且所得产物质量好、 纯度高， 无需纯化即可有效地用于后续处理例如连接接头。 从 而， 该第一试剂能够用于本发明的构建 DNA测序文库的方法中， 以对经过末端修复的 DNA片段进行处理， 以便获得 3'端添加碱基 A的 DNA片段， 其还可以包含在本发明的试 剂盒中， 以备发挥同样的作用， 用于构建 DNA测序文库。  The inventors have surprisingly found that with the first reagent of the present invention, the base A can be efficiently added to the 3' end of the DNA fragment, and the obtained product is of good quality, high purity, and can be effectively used for subsequent processing such as ligation without purification. Connector. Thus, the first reagent can be used in the method of constructing a DNA sequencing library of the present invention to process the DNA fragment subjected to end repair to obtain a DNA fragment having a base A added at the 3' end, which may also be included in the present invention. The kit of the invention is used to construct a DNA sequencing library in order to perform the same function.
根据本发明的又一方面， 本发明还提供了一种试剂。 根据本发明的实施例， 该试剂 可以包含： 10mM〜200mM的緩冲盐； ImM-lOOmM的二硫苏糖醇； 10mM〜400mM的 MgCl2; lmM〜40mM的 ATP;以及 lU/ml〜 200U/ml的 DNA连接酶，其中，该试剂的 pH为 7.6 ~ 7.9。 利用该试剂， 能够有效地将接头与 3'端添加碱基 A的 DNA片段相连， 所得产物质量好、 纯度高， 能够有效地用于后续处理。 According to still another aspect of the present invention, the present invention also provides an agent. According to an embodiment of the present invention, the reagent may comprise: 10 mM to 200 mM of a buffer salt; 1 mM to 100 mM of dithiothreitol; 10 mM to 400 mM of MgCl 2 ; 1 mM to 40 mM of ATP; and 1 U/ml to 200 U/ Ml DNA ligase, wherein the pH of the reagent is 7.6 ~ 7.9. By using this reagent, the linker can be efficiently linked to the DNA fragment to which the base A is added at the 3' end, and the obtained product has good quality and high purity, and can be effectively used for subsequent treatment.
根据本发明的实施例， 该试剂中所包含的緩冲盐的类型不受特别限制， 根据本发明的 一些具体示例， 緩冲盐可以为选自 Tris-HCl和磷酸盐的至少一种， 优选为 Tris-HCl。根据本 发明的一些实施例，该试剂中緩冲盐的浓度可以为 10mM〜200mM,根据本发明的具体示例， 优选为 50mM〜150mM, 更优选为 100mM。  According to an embodiment of the present invention, the type of the buffer salt contained in the reagent is not particularly limited. According to some specific examples of the present invention, the buffer salt may be at least one selected from the group consisting of Tris-HCl and phosphate, preferably For Tris-HCl. According to some embodiments of the present invention, the concentration of the buffer salt in the reagent may be from 10 mM to 200 mM, and preferably from 50 mM to 150 mM, more preferably 100 mM, according to a specific example of the present invention.
根据本发明的一些实施例， 该试剂中可以包含 10mM〜90mM 的二硫苏糖醇， 根据具体 示例， 优选包含 50mM的二硫苏糖醇。  According to some embodiments of the present invention, the reagent may contain 10 mM to 90 mM of dithiothreitol, and according to a specific example, preferably contains 50 mM of dithiothreitol.
根据本发明的一些实施例，该试剂中可以包含 50mM〜200mM的 MgCl2,根据具体示例， 优选包含 lOOmM的 MgCl2。 According to some embodiments of the present invention, the reagent may comprise 50 mM to 200 mM of MgCl 2 , and according to a specific example, preferably contains 100 mM of MgCl 2 .
根据本发明的一些实施例， 该试剂中可以包含 5mM〜20mM的 ATP,根据具体示例， 优 选包含 10mM的 ATP。  According to some embodiments of the invention, the reagent may comprise 5 mM to 20 mM ATP, and according to a specific example, preferably comprises 10 mM ATP.
根据本发明的一些实施例， 该试剂中可以包含 50U/ml〜 150U/ml的 DNA连接酶， 根据 具体示例， 优选包含 72U/ml的 DNA连接酶。  According to some embodiments of the present invention, the reagent may comprise from 50 U/ml to 150 U/ml of DNA ligase, and according to a specific example, preferably comprises 72 U/ml of DNA ligase.
根据本发明的一个实施例， 该试剂的 pH为 7.6。  According to one embodiment of the invention, the reagent has a pH of 7.6.
具体地， 根据本发明的实施例， 本发明的第二试剂， 其 pH可以为 7.6〜7.9 , 优选 7.6, 溶剂是水， 溶质可以为如下终浓度物质的混合物： 10〜200mM, 优选 50〜150mM, 更优 选 100 mM的緩冲盐； l〜100mM,优选 10〜90mM,更优选 50mM的二硫苏糖醇； l〜40mM, 优选 5〜20mM, 更优选 10mM的 ATP; 10〜400mM, 优选 50〜200mM, 更优选 lOOmM 的 MgCl2; 1 U/ml〜200 U/ml,优选 50 U/ml〜150 U/ml, 更优选 72 U/ml的 DNA连接酶。 其中緩冲盐可以为 Tris-HCl或磷酸盐， 优选是 Tris-HCl。 Specifically, according to an embodiment of the present invention, the second reagent of the present invention may have a pH of 7.6 to 7.9, preferably 7.6, the solvent is water, and the solute may be a mixture of the following final concentrations: 10 to 200 mM, preferably 50 to 150 mM. More preferably, 100 mM buffer salt; 1 to 100 mM, preferably 10 to 90 mM, more preferably 50 mM dithiothreitol; 1 to 40 mM, preferably 5 to 20 mM, more preferably 10 mM ATP; 10 to 400 mM, preferably 50 ~200 mM, more preferably 100 mM MgCl 2 ; 1 U/ml to 200 U/ml, preferably 50 U/ml to 150 U/ml, more preferably 72 U/ml DNA ligase. The buffer salt may be Tris-HCl or phosphate, preferably Tris-HCl.
发明人惊奇地发现， 利用本发明的第二试剂， 能够有效地将接头与 3'端添加碱基 A的 DNA片段相连， 且所得产物质量好、 纯度高， 能够有效地用于后续处理。 从而， 该第二试 剂能够用于本发明的构建 DN A测序文库的方法中，以将 3 '端添加碱基 A的 DNA片段与 接头进行连接， 以便获得连接产物， 其还可以包含在本发明的试剂盒中， 以备发挥同样的 作用， 用于构建 DNA测序文库。  The inventors have surprisingly found that by using the second reagent of the present invention, the linker can be efficiently linked to the DNA fragment to which the base A is added at the 3' end, and the obtained product is excellent in quality and high in purity, and can be effectively used for subsequent treatment. Thus, the second reagent can be used in the method of constructing a DN A sequencing library of the present invention to link a DNA fragment of the base A with a base A to a linker to obtain a ligation product, which can also be included in the present invention. In the kit, in order to play the same role, to construct a DNA sequencing library.
DNA测序文库及其构建方法、 试剂盒 DNA sequencing library and its construction method and kit
根据本发明的再一方面， 本发明提供了一种构建 DNA测序文库的方法。 根据本发明 的实施例， 参照图 3 , 该方法可以包含以下步骤：  According to still another aspect of the present invention, the present invention provides a method of constructing a DNA sequencing library. According to an embodiment of the present invention, referring to FIG. 3, the method may include the following steps:
首先， 将 DNA片段化， 以便获得 DNA片段。 根据本发明的实施例， 将 DNA片段化 的方法不受特别限制。 根据本发明的具体示例， 可以通过选自超声打断法和酶切处理的至 少一种进行，其中，超声打断法可以利用 Covaris超声打断仪进行。根据本发明的具体示例， DNA片段的长度可以为 200-800bp。  First, the DNA is fragmented to obtain a DNA fragment. According to an embodiment of the present invention, the method of fragmenting DNA is not particularly limited. According to a specific example of the present invention, it may be carried out by at least one selected from the group consisting of ultrasonication and enzymatic treatment, wherein the ultrasonication method can be carried out using a Covaris ultrasonic interrupter. According to a specific example of the present invention, the DNA fragment may have a length of 200 to 800 bp.
其次， 将 DNA片段进行末端修复， 以便获得经过末端修复的 DNA片段。 根据本发明 的实施例， 将 DNA片段进行末端修复的方法不受特别限制。 根据本发明的具体示例， 可以 利用 Klenow片段、 T4 DNA聚合酶和 T4多聚核苷酸激酶进行末端修复， 其中， Klenow片 段具有 5'→3'聚合酶活性和 3'→5'聚合酶活性， 但缺少 5'→3'外切酶活性。  Next, the DNA fragment is end-repaired to obtain a DNA fragment that has been repaired at the end. According to an embodiment of the present invention, the method of performing end repair of the DNA fragment is not particularly limited. According to a specific example of the present invention, terminal repair can be performed using Klenow fragment, T4 DNA polymerase, and T4 polynucleotide kinase, wherein the Klenow fragment has 5'→3' polymerase activity and 3'→5' polymerase activity. However, it lacks 5'→3' exonuclease activity.
接着， 使用根据本发明实施例的第一试剂， 对经过末端修复的 DNA片段进行处理， 以 便获得 3'端添加碱基 A的 DNA片段。 根据本发明的具体示例， 使用根据本发明实施例的 第一试剂， 对经过末端修复的 DNA片段进行处理可以在 12°C〜40°C下进行。 根据本发明的 一些实施例， 优选在 37 °C下进行该处理。  Next, the end-repaired DNA fragment is treated using the first reagent according to an embodiment of the present invention to obtain a DNA fragment in which the base A is added at the 3' end. According to a specific example of the present invention, the end-repaired DNA fragment can be treated at 12 ° C to 40 ° C using the first reagent according to an embodiment of the present invention. According to some embodiments of the invention, the treatment is preferably carried out at 37 °C.
接下来， 使用根据本发明实施例的第二试剂， 将 3'端添加碱基 A的 DNA片段与接头 进行连接， 以便获得连接产物。 根据本发明的一些具体示例， 该接头包含第一链和第二链，  Next, using the second reagent according to an embodiment of the present invention, a DNA fragment to which the base A is added at the 3' end is ligated to a linker to obtain a ligation product. According to some specific examples of the invention, the joint comprises a first strand and a second strand,
明的具体示例， 使用根据本发明实施例的第二试剂， 将所述 3'端添加碱基 A的 DNA片段 与接头进行连接可以在 4°C〜22°C下进行。 根据本发明的一些实施例， 优选在 16°C下进行该 处理。 发明人惊奇地发现， 通过利用第一试剂和第二试剂， 可以在完成 3'端添加碱基 A后， 不需要进行纯化回收， 就能够直接进行连接反应。 由此， 可以显著提高制备测序文库的效 率， 进而提高后续测序以及分析的效率， 避免核酸样本的损失。 然后， 将连接产物进行片段选择， 以便获得目的片段。 根据本发明的实施例， 将连接 产物进行片段选择的方法不受特别限制。 根据本发明的具体示例， 可以利用琼脂糖凝胶电 泳对连接产物进行片段选择。 根据本发明的实施例， 目的片段的长度可以为 100-1000bp。 根据本发明的具体示例， 优选目的片段的长度为 200-800bp。 To be specific, the DNA fragment in which the 3' end is added to the base A can be ligated to the linker at 4 ° C to 22 ° C using a second reagent according to an embodiment of the present invention. According to some embodiments of the invention, the treatment is preferably carried out at 16 °C. The inventors have surprisingly found that by using the first reagent and the second reagent, it is possible to directly carry out the ligation reaction after the addition of the base A at the 3' end, without purification and recovery. Thereby, the efficiency of preparing the sequencing library can be significantly improved, thereby improving the efficiency of subsequent sequencing and analysis, and avoiding the loss of nucleic acid samples. Then, the ligation product is subjected to fragment selection to obtain a fragment of interest. According to an embodiment of the present invention, the method of performing fragment selection of the ligation product is not particularly limited. According to a specific example of the present invention, the ligation product can be subjected to fragment selection using agarose gel electrophoresis. According to an embodiment of the invention, the target segment may be 100-1000 bp in length. According to a specific example of the present invention, it is preferred that the length of the target fragment is 200-800 bp.
接着， 将目的片段进行 PCR扩增， 以便获得扩增产物。 根据本发明的实施例， 使用第 一 PCR引物和第二 PCR引物，将目的片段进行 PCR扩增， 其中， 第一 PCR引物的序列为：  Next, the target fragment is subjected to PCR amplification to obtain an amplification product. According to an embodiment of the present invention, the target fragment is subjected to PCR amplification using the first PCR primer and the second PCR primer, wherein the sequence of the first PCR primer is:
CTCTTCCGATCT, 第二 PCR引物的序列为： 5'AATGATACGGCGACCACCGAGATCTA CACTCTTTCCCTACACGACGCTCTTCCGATCT。 CTCTTCCGATCT, the sequence of the second PCR primer is: 5' AATGATACGGCGACCACCGAGATCTA CACTCTTTCCCTACACGACGCTCTTCCGATCT.
最后，分离纯化扩增产物，该扩增产物构成所述 DNA测序文库。根据本发明的实施例， 分离纯化扩增产物的方法不受特别限制。 才艮据本发明的具体示例， 利用 2%琼脂糖凝胶电泳 进行。  Finally, the amplified product is isolated and purified, and the amplified product constitutes the DNA sequencing library. According to an embodiment of the present invention, the method of isolating and purifying the amplification product is not particularly limited. According to a specific example of the present invention, it was carried out by 2% agarose gel electrophoresis.
利用根据本发明的实施例的构建 DNA测序文库的方法， 能够方便高效地构建样品的 DNA测序文库， 所得文库质量好， 能够有效地应用于高通量测序平台例如 Illumina测序平 台， 且测序结果准确、 可重复性好。  By using the method for constructing a DNA sequencing library according to an embodiment of the present invention, a DNA sequencing library of a sample can be conveniently and efficiently constructed, and the obtained library is of good quality, can be effectively applied to a high-throughput sequencing platform such as an Illumina sequencing platform, and the sequencing result is accurate. , repeatability is good.
具体地， 本发明的构建 DNA测序文库的方法可以包括以下步骤：  Specifically, the method of constructing a DNA sequencing library of the present invention may comprise the following steps:
(1) 将 DNA片段化， 以便获得 DNA片段；  (1) Fragmenting DNA to obtain a DNA fragment;
(2) 将 DNA片段进行末端修复， 得到平末端 DNA片段；  (2) end-repairing the DNA fragment to obtain a blunt-ended DNA fragment;
(3) 将平末端 DNA片段与第一试剂混合， 得到 3 '端添加碱基 A的 DNA片段； (3) mixing the blunt-ended DNA fragment with the first reagent to obtain a DNA fragment having a base A added at the 3' end;
(4) 将 3'端添加碱基 A的 DNA片段与第二试剂以及接头混合， 进行反应， 然后纯 化， 得到加接头的 DNA片段； (4) mixing the DNA fragment of the base A with the second reagent and the linker at the 3' end, reacting, and then purifying to obtain a DNA fragment to which the linker is added;
(5) 将加接头的 DNA片段进行片段选择， 以便获得目的片段；  (5) selecting a DNA fragment of the adaptor to obtain a fragment of interest;
(6) 将目的片段进行 PCR扩增， 并分离纯化扩增产物， 该扩增产物构成 DNA测序 文库。  (6) The target fragment is subjected to PCR amplification, and the amplified product is isolated and purified, and the amplified product constitutes a DNA sequencing library.
其中步骤 (3)中的第一试剂， 其 pH可以为 7.6〜7.9, 优选 7.9 , 溶剂是水， 溶质可以 为如下终浓度物质： 10 mM〜100mM, 优选 25mM〜75mM, 更优选 50mM的可溶性盐； 10〜300mM, 优选 50〜200mM, 更优选 lOOmM的 MgCl2; 10mM〜1000mM, 优选 50 mM 〜500mM, 更优选 lOOmM的緩冲盐； lmM〜50mM, 优选 5mM〜15mM, 更优选 10mM 的二 Α 苏糖醇； lmM〜10mM, 优选 2.5mM〜7.5mM, 更优选 5mM的 dATP , 1 U/ml〜40 U/ml, 优选 10 U/ml〜30 U/ml, 更优选 20 U/ml的 Klenow(3 '-5 ' exo)酶。 The first reagent in the step (3) may have a pH of 7.6 to 7.9, preferably 7.9, the solvent is water, and the solute may be a final concentration of the substance: 10 mM to 100 mM, preferably 25 mM to 75 mM, more preferably 50 mM of soluble salt. 10 to 300 mM, preferably 50 to 200 mM, more preferably 100 mM MgCl 2 ; 10 mM to 1000 mM, preferably 50 mM to 500 mM, more preferably 100 mM buffer salt; 1 mM to 50 mM, preferably 5 mM to 15 mM, more preferably 10 mM Threitol; lmM~10mM, preferably 2.5mM~7.5mM, more preferably 5mM dATP, 1 U/ml~40 U/ml, preferably 10 U/ml~30 U/ml, more preferably 20 U/ml Klenow (3 '-5 ' exo) enzyme.
其中步骤 (4)中的第二试剂， 其 pH可以为 7.6〜7.9, 优选 7.6 , 溶剂是水， 溶质可以 为如下终浓度物质： 10〜200mM,优选 50〜150mM,更优选 100 mM的緩冲盐； l〜100mM, 优选 10〜90mM, 更优选 50mM的二硫苏糖醇； l〜40mM, 优选 5〜20mM, 更优选 10mM 的 ATP; 10〜400mM, 优选 50〜200mM, 更优选 lOOmM的 MgCl2; 1 U/ml〜200 U/ml, 优选 50 U/ml〜150 U/ml, 更优选 72 U/ml的 DNA连接酶。 Wherein the second reagent in the step (4) may have a pH of 7.6 to 7.9, preferably 7.6, the solvent is water, and the solute may be The final concentration material is: 10 to 200 mM, preferably 50 to 150 mM, more preferably 100 mM buffer salt; 1 to 100 mM, preferably 10 to 90 mM, more preferably 50 mM dithiothreitol; 1 to 40 mM, preferably 5 to 20 mM, more preferably 10 mM ATP; 10 to 400 mM, preferably 50 to 200 mM, more preferably 100 mM MgCl 2 ; 1 U/ml to 200 U/ml, preferably 50 U/ml to 150 U/ml, more preferably 72 U/ Ml DNA ligase.
根据本发明的实施例， 该方法可以进一步包括步骤 (7): 对步骤 (6)所得到的 DNA测 序文库的文库浓度及片段大小进行检测， 优选地， 利用 Agilent Bioanalyzer 2100 和 Q-PCRDNA对 DNA测序文库进行检测。  According to an embodiment of the present invention, the method may further comprise the step (7): detecting the library concentration and fragment size of the DNA sequencing library obtained in the step (6), preferably, using Agilent Bioanalyzer 2100 and Q-PCR DNA for DNA The sequencing library was tested.
根据本发明的实施例， 该方法还可以进一步包括步骤 (8): 对 DNA测序文库进行测 序， 优选地， 利用 Illumina测序平台进行测序。  According to an embodiment of the invention, the method may further comprise the step (8): sequencing the DNA sequencing library, preferably using an Illumina sequencing platform.
根据本发明的实施例， 在步骤（1)中， 可以利用超声波或者酶切的方式将 DNA片段 化。 根据本发明的一些实施例， 步骤（1)中的 DNA片段的长度可以为 200 bp〜800 bp。 根据本发明的具体示例， 任选地， 在进行步骤 (2)之前可以进一步包括对 DNA片段进行 纯化的步骤。  According to an embodiment of the present invention, in the step (1), DNA may be fragmented by ultrasonic or enzymatic cleavage. According to some embodiments of the present invention, the length of the DNA fragment in step (1) may be from 200 bp to 800 bp. According to a specific example of the present invention, optionally, the step of purifying the DNA fragment may be further included before performing step (2).
根据本发明的实施例， 在步骤 (2)中， 可以通过如下方法进行末端修复： 将步骤（1) 所得到的 DNA片段与末端修复试剂混合进行反应， 以形成平末端的 DNA片段。 根据 本发明的一些实施例， 任选地， 在进行步骤 (3)之前可以进一步包括将平末端 DNA片段 进行纯化的步骤。  According to an embodiment of the present invention, in the step (2), the end repair can be carried out by mixing the DNA fragment obtained in the step (1) with the terminal repair reagent to form a blunt-ended DNA fragment. According to some embodiments of the invention, optionally, the step of purifying the blunt-ended DNA fragment may be further included prior to performing step (3).
根据本发明的实施例， 在步骤 (3)中， 第一试剂中的緩冲盐可以为 Tris-HCl或磷酸 盐， 优选是 Tris-HCl。 根据本发明的一些实施例， 在步骤 (3)中， 第一试剂中的可溶性盐 可以为氯化钠或氯化钾， 优选是氯化钠。 根据本发明的具体示例， 在步骤 (3)中， 将平 末端 DNA片段与第一试剂混合后， 可以在 12°C〜40°C的温度下进行孵育， 优选的温度 为 37 °C。  According to an embodiment of the present invention, in the step (3), the buffer salt in the first reagent may be Tris-HCl or a phosphate salt, preferably Tris-HCl. According to some embodiments of the invention, in step (3), the soluble salt in the first reagent may be sodium chloride or potassium chloride, preferably sodium chloride. According to a specific example of the present invention, in the step (3), after the blunt-end DNA fragment is mixed with the first reagent, the incubation may be carried out at a temperature of from 12 ° C to 40 ° C, preferably at 37 ° C.
根据本发明的实施例， 在步骤 (4)中， 接头可以为带有 T碱基末端的任意接头， 优 选地， 接头包含如下的第一链和第二链：  According to an embodiment of the present invention, in the step (4), the linker may be any linker having a T base end, and preferably, the linker comprises the first chain and the second chain as follows:
第一链为： 第二链为： 根据本发明的实施例， 在步骤 (4)中， 第二试剂中的緩冲盐可以为 Tris-HCl或磷酸 盐， 优选是 Tris-HCL 根据本发明的一些实施例， 在步骤 (4)中， 将 3 '端添加碱基 A的 DNA 片段与第二试剂混合后， 可以在 4 °C〜22°C的温度下进行孵育， 优选的温度为 16 °c。 The first chain is: The second chain is: According to an embodiment of the present invention, in step (4), the buffer salt in the second reagent may be Tris-HCl or phosphate, preferably Tris-HCL according to the present invention. In some embodiments, in step (4), after mixing the DNA fragment of the base A with the base A and the second reagent, the incubation may be carried out at a temperature of 4 ° C to 22 ° C, preferably at a temperature of 16 °c.
根据本发明的实施例， 在步骤 (5)中， 可以通过如下方法将加接头的 DNA片段进行 片段选择： 将加接头的 DNA片段进行琼脂糖凝胶电泳， 然后切胶回收目的片段。 根据 本发明的一些实施例， 目的片段的长度可以为 100 _ 1000bp , 例如可以为 200、 400bp 和 800bp。  According to an embodiment of the present invention, in the step (5), the DNA fragment to which the adaptor is attached can be subjected to fragment selection by subjecting the DNA fragment to which the adaptor is attached to agarose gel electrophoresis, and then cutting the gel to recover the target fragment. According to some embodiments of the invention, the segment of interest may be 100 _ 1000 bp in length, for example 200, 400 bp and 800 bp.
根据本发明的实施例， 在步骤 (6)中， PCR扩增所使用的引物为：  According to an embodiment of the present invention, in step (6), the primers used for PCR amplification are:
第一 PCR引物：  First PCR primer:
GTGCTCTTCCGATCT; GTGCTCTTCCGATCT;
第二 PCR引物：  Second PCR primer:
GATCT。 GATCT.
根据本发明的另一方面， 本发明提供了一种 DNA测序文库， 其是通过上述根据本发 明实施例的构建 DNA测序文库的方法构建的。 发明人发现， 该 DNA测序文库纯度高、 质 量好， 并且能够有效地应用于高通量测序平台， 且测序结果准确、 可重复性好。 而针对小 片段 DNA构建的 DNA测序文库， 同样能够有效地应用于后续的测序等处理。  According to another aspect of the present invention, the present invention provides a DNA sequencing library constructed by the above method of constructing a DNA sequencing library according to an embodiment of the present invention. The inventors found that the DNA sequencing library is high in purity, high in quality, and can be effectively applied to a high-throughput sequencing platform, and the sequencing results are accurate and reproducible. The DNA sequencing library constructed for small fragment DNA can also be effectively applied to subsequent sequencing and the like.
根据本发明的再一方面， 本发明还提供了一种用于构建 DNA测序文库的试剂盒。 根据 本发明的实施例， 该试剂盒可以包含： 第一试剂； 以及第二试剂。 其中， 第一试剂和第二 试剂均为前面所述的根据本发明实施例的第一试剂和第二试剂。 简言之， 第一试剂可以包 含： 10mM〜1000mM 的緩冲盐； 10mM〜100mM 的可溶性盐； 10mM〜300mM 的 MgCl2; lmM〜50mM的二硫苏糖醇； lmM〜10mM的 dATP; 以及 lU/ml〜 40U/ml的 Klenow片段（ 3' →5' exo ), 其中， 该试剂的 pH为 7.6〜7.9。 第二试剂可以包含： 10mM〜200mM的緩冲盐； lmM〜100mM的二硫苏糖醇； 10mM〜400mM的 MgCl2; lmM〜40mM的 ATP; 以及 lU/ml〜 200U/ml的 DNA连接酶， 其中， 该试剂的 pH为 7.6 ~ 7.9。 关于该第一试剂和第二试剂的其 他特征和优点在前面已经进行了详细描述， 不再赘述。 According to still another aspect of the present invention, the present invention also provides a kit for constructing a DNA sequencing library. According to an embodiment of the invention, the kit may comprise: a first reagent; and a second reagent. Wherein the first reagent and the second reagent are both the first reagent and the second reagent according to the embodiments of the present invention as described above. Briefly, the first reagent may comprise: 10 mM to 1000 mM buffer salt; 10 mM to 100 mM soluble salt; 10 mM to 300 mM MgCl 2 ; 1 mM to 50 mM dithiothreitol; 1 mM to 10 mM dATP; Klenow fragment (3' → 5' exo ) of /ml~40U/ml, wherein the pH of the reagent is 7.6 to 7.9. The second reagent may comprise: 10 mM to 200 mM buffer salt; 1 mM to 100 mM dithiothreitol; 10 mM to 400 mM MgCl 2 ; 1 mM to 40 mM ATP; and 1 U/ml to 200 U/ml DNA ligase, Among them, the pH of the reagent is 7.6 ~ 7.9. Other features and advantages regarding the first reagent and the second reagent have been described in detail above and will not be described again.
根据本发明的实施例， 第一试剂和第二试剂可以分别设置在不同的容器中。 由此， 利 用该试剂盒， 能够方便有效地构建 DNA测序文库， 并且可重复性好， 获得的文库质量好， 从而能够有效地应用于高通量测序平台例如 Illumina测序平台。 当然，本领域技术人员能够 理解， 试剂盒中还可以包含其他用于构建 DNA测序文库的常规组件， 在此不再赘述。  According to an embodiment of the invention, the first reagent and the second reagent may be respectively disposed in different containers. Thus, the kit can be used to construct a DNA sequencing library conveniently and efficiently, and the reproducibility is good, and the obtained library is of good quality, so that it can be effectively applied to a high-throughput sequencing platform such as an Illumina sequencing platform. Of course, those skilled in the art can understand that other components for constructing a DNA sequencing library can also be included in the kit, and details are not described herein.
确定 DNA样品的序列信息的方法 Method for determining sequence information of a DNA sample
根据本发明的又一方面， 本发明提供了一种确定 DNA样品的序列信息的方法。 根据本 发明的实施例，该方法可以包括下列步骤：才艮据本发明实施例的构建 DNA测序文库的方法， 构建 DNA样品的 DNA测序文库； 以及对该 DNA测序文库进行测序， 以便确定 DNA样品 的序列信息。 根据本发明的一些实施例， 对 DNA测序文库进行测序前， 可以进一步包括利 用 Agilent Bioanalyzer 2100和 Q-PCR的至少一种对 DNA测序文库进行检测。根据本发明的 具体示例， 可以利用 Illumina测序平台进行测序。 利用该方法能够方便有效地确定 DNA样 品的序列信息， 并且操作简便、 需时少、 效率高、 结果准确、 可重复性好。 According to still another aspect of the present invention, the present invention provides a method of determining sequence information of a DNA sample. According to this In an embodiment of the invention, the method may comprise the steps of: constructing a DNA sequencing library of a DNA sample according to the method of constructing a DNA sequencing library according to an embodiment of the present invention; and sequencing the DNA sequencing library to determine the sequence of the DNA sample information. According to some embodiments of the present invention, prior to sequencing the DNA sequencing library, the DNA sequencing library may be further detected using at least one of Agilent Bioanalyzer 2100 and Q-PCR. According to a specific example of the invention, sequencing can be performed using an Illumina sequencing platform. The method can conveniently and effectively determine the sequence information of the DNA sample, and has the advantages of simple operation, less time, high efficiency, accurate result and good repeatability.
需要说明的是，根据本发明实施例的确定 DNA样品的序列信息的方法是本申请的发明 人经过艰苦的创造性劳动和优化工作才完成的。 下面将结合实施例对本发明的方案进行解释。 本领域技术人员将会理解， 下面的实施 例仅用于说明本发明， 而不应视为限定本发明的范围。 实施例中未注明具体技术或条件的， 按照本领域内的文献所描述的技术或条件（例如参考 J.萨姆布鲁克等著， 黄培堂等译的《分 子克隆实验指南》， 第三版， 科学出版社）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， 均为可以通过市购获得的常规产品， 例如可以釆购自 Illumina公司。  It should be noted that the method of determining the sequence information of the DNA sample according to the embodiment of the present invention is completed by the inventor of the present application through arduous creative labor and optimization work. The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not specified by the manufacturer, and are conventional products that are commercially available, for example, from Illumina.
实施例 1:  Example 1:
1.1试剂  1.1 reagent
10xT4 多聚核苷酸激酶緩冲液 ( Enzymatics ) , dNTP ( Enzymatics ), T4 DNA聚合酶 ( Enzymatics ), T4多聚核苷酸激醉 ( Enzymatics ), Klenow片段 ( 3 '-5 'exo ) ( Enzymatics ), Klenow 片段 （Enzymatics ) , 10xblue 緩冲液 （Enzymatics ) ， lOxDNA 连接酶緩冲液 (Enzymatics) , Phusion DNA聚合酶（NEB )。  10xT4 Polynucleotide Kinase Buffer (Enzymatics), dNTP (Enzymatics), T4 DNA Polymerase (Enzymatics), T4 Polynucleotide Enzymatics, Klenow Fragment (3 '-5 'exo ) ( Enzymatics), Klenow fragments (Enzymatics), 10xblue buffer (Enzymatics), lOxDNA ligase buffer (Enzymatics), Phusion DNA polymerase (NEB).
第一试剂： pH为 7.9 , 溶剂是水， 溶质为如下终浓度物质： 50mM氯化钠（国药集团化 学试剂有限公司）， lOO mM Tris-HCl (国药集团化学试剂有限公司）， 100 mM MgCl2 (国药 集团化学试剂有限公司；)， 10mM二硫苏糖醇 (Sigma) , 5mM dATP ( Enzymatics ), 20 U/ml Klenow片段 ( 3 '-5 'exo )。 The first reagent: pH is 7.9, the solvent is water, and the solute is the following final concentration: 50 mM sodium chloride (National Pharmaceutical Group Chemical Co., Ltd.), lOO mM Tris-HCl (National Pharmaceutical Group Chemical Reagent Co., Ltd.), 100 mM MgCl 2 (National Pharmaceutical Group Chemical Reagent Co., Ltd.;), 10 mM dithiothreitol (Sigma), 5 mM dATP (Enzymatics), 20 U/ml Klenow fragment (3 '-5 'exo).
第二试剂： pH为 7.6 , 溶剂是水， 溶质为如下终浓度物质： 72 U/ml T4 DNA聚合酶， lO mM ATP , 500 mM Tris-HCl, lOOmM MgCl2 , 50 mM 二石克苏糖醇。 The second reagent: pH is 7.6, the solvent is water, and the solute is the following final concentration: 72 U/ml T4 DNA polymerase, 10 mM ATP, 500 mM Tris-HCl, 100 mM MgCl 2 , 50 mM dicequititol .
1.2 实验步骤  1.2 Experimental steps
将两份 50 ng/μΐ 的 Fosmid样品（深圳华大基因研究院）各吸取 40 μΐ分别置于 Covaris 样品管 （Covaris公司） 中， 分别命名为样品 X和样品 Y, 然后按下列步骤进行实验。  Two 50 ng/μΐ Fosmid samples (Shenzhen Huada Genetic Research Institute) were each taken 40 μM into Covaris sample tubes (Covaris), named as Sample X and Sample Y, respectively, and then tested as follows.
1.2.1 片段化  1.2.1 Fragmentation
利用 Covaris公司生产 E210型号打断仪， 将样品 X和样品 Y的 DNA进行片段化， 以 便获得 DNA片段， 其中打断仪的参数设置如下：
Figure imgf000012_0001
Fragmentation of DNA from sample X and sample Y using Covaris' E210 model interrupter The DNA fragment is obtained, and the parameters of the interrupter are set as follows:
Figure imgf000012_0001
1.2.2 纯化 DNA片段  1.2.2 Purification DNA fragment
根据生产商的说明书， 利用 Agencourt公司的 Ampure磁珠分别对样品 X和样品 Y的 DNA片段进行纯化、 回收， 以便获得纯化产物， 然后将 2种样品的纯化产物分别溶于 45μ1 超纯水中， 各取 42μ1分别置于 2个 PCR管中， 备用。  According to the manufacturer's instructions, the DNA fragments of sample X and sample Y were purified and recovered by Agencourt's Ampure magnetic beads, respectively, to obtain purified products, and then the purified products of the two samples were separately dissolved in 45 μl of ultrapure water. Each of the 42 μl was placed in two PCR tubes and used.
1.2.3 末端修复  1.2.3 End Repair
按照下表中的配比分别准备样品 X和样品 Υ的末端修复反应体系：  Prepare the end-repair reaction system for sample X and sample 按照 according to the ratios in the table below:
Figure imgf000012_0002
Figure imgf000012_0002
将准备好的样品 X和样品 Υ的末端修复反应体系分别置于 PCR仪上， 在 20°C下放置 30min, 以便获得末端修复产物， 然后利用 Ampure磁珠将其纯化并分别溶于 20μ1和 32μ1 的超纯水中， 备用。  The prepared end-recovery reaction system of sample X and sample 分别 was placed on a PCR machine and placed at 20 ° C for 30 min to obtain a terminal repair product, which was then purified and dissolved in 20 μl and 32 μl respectively using Ampure magnetic beads. Ultra pure water, spare.
1.2.4 3'端添加碱基 A  1.2.4 Adding bases at the 3' end A
按照下表中的配比分别准备样品 X和样品 Y的 3'端添加碱基 A反应体系：  Prepare Samples X and Y at the 3' end of the sample Y according to the ratios in the table below.
样品 X的反应体系  Sample X reaction system
Figure imgf000012_0003
ImM dATP ΙΟμΙ
Figure imgf000012_0003
ImM dATP ΙΟμΙ
总体积 50μ1 将准备好的样品 X和样品 Υ的 3 '端添加碱基 Α反应体系， 分别置于 15 °C〜40 °C下反应 30min, 以便获得 3 '端添加碱基 A的产物， 然后， 样品 X的 3 '端添加碱基 A的产物不经纯 化， 而利用 Ampure磁珠将样品 Y的 3 '端添加碱基 A的产物进行纯化并溶于 37μ1的超纯水 中， 备用。  The total volume of 50μ1 is to add the base Α reaction system of the prepared sample X and the 3′ end of the sample ,, and react at 15 ° C to 40 ° C for 30 min respectively to obtain the product of adding the base A at the 3 ' end, and then The product of the base A was added to the 3' end of the sample X without purification, and the product of the base A of the sample Y was purified by using Ampure magnetic beads and dissolved in 37 μl of ultrapure water for use.
1.2.5 添加接头  1.2.5 Adding a connector
按照下表中的配比分别准备样品 X和样品 Υ的添加接头反应体系：  Prepare the joint reaction system for sample X and sample 按照 according to the ratios in the table below:
样品 X的反应体系  Sample X reaction system
Figure imgf000013_0002
Figure imgf000013_0002
Figure imgf000013_0003
Figure imgf000013_0003
其中， 样品 X和样品 Υ的反应体系中所釆用的接头均为包含如下第一链和第二链的双 链寡核苷酸：  Wherein, the linker used in the reaction system of sample X and sample oxime is a double-stranded oligonucleotide comprising the first strand and the second strand as follows:
第一链为： The first chain is:
Figure imgf000013_0001
Figure imgf000013_0001
将准备好的样品 X和样品 Υ 的添加接头反应体系， 分别置于 16 °C下过夜进行反应 14-18h, 以便获得连接产物， 然后利用 Ampure磁珠将其纯化， 并利用超纯水溶解洗脱， 备 用。  The prepared sample reaction system of sample X and sample , was separately placed at 16 ° C for 14-18 hours to obtain a ligation product, which was then purified by Ampure magnetic beads and washed with ultrapure water. Take off, spare.
1.2.6 PCR扩增 按照下表中的配比分别准备样品 X和样品 Y的 PCR扩增反应体系 1.2.6 PCR amplification Prepare PCR amplification reaction system for sample X and sample Y according to the ratios in the table below.
Figure imgf000014_0002
Figure imgf000014_0002
其中， 第一 PCR引物:  Among them, the first PCR primer:
5'CAAGCAGA  5'CAAGCAGA
GCTCTTCCGATCT; GCTCTTCCGATCT;
第二 PCR引物：  Second PCR primer:
TCT。 TCT.
利用准备好的 2种样品的 PCR扩增反应体系， 于 PCR仪中进行 PCR扩增反应， 以便 获得扩增产物， 然后利用 2 %琼脂糖凝胶电泳分别将 2种样品的扩增产物进行分离， 分别切 胶回收 400bp和 800bp的扩增产物，并分别溶于 30μ1的超纯水中，样品 X的 400bp和 800bp 扩增产物构成其 DNA测序文库，样品 Y的 400bp和 800bp扩增产物构成其 DNA测序文库。  Using the PCR amplification reaction system of the prepared two samples, a PCR amplification reaction was carried out in a PCR machine to obtain an amplification product, and then the amplification products of the two samples were separately separated by 2% agarose gel electrophoresis. The 400 bp and 800 bp amplification products were separately fractionated and dissolved in 30 μl of ultrapure water. The 400 bp and 800 bp amplification products of sample X constituted the DNA sequencing library, and the 400 bp and 800 bp amplification products of sample Y constituted DNA sequencing library.
其中， PCR扩增反应的条件为：  Among them, the conditions of the PCR amplification reaction are:
98 °C 30s 10个循环 98 °C 30s 10 cycles
Figure imgf000014_0001
Figure imgf000014_0001
72 °C 5min  72 °C 5min
4°C oo  4°C oo
由此， 本实施例利用根据本发明实施例的构建 DNA测序文库的方法制备了样品 X的 DNA测序文库， 利用 Illumina测序平台提供的构建 DNA测序文库的方法， 构建了样品 Y 的 DNA测序文库。  Thus, in this example, a DNA sequencing library of sample X was prepared by the method for constructing a DNA sequencing library according to an embodiment of the present invention, and a DNA sequencing library of sample Y was constructed by using a method for constructing a DNA sequencing library provided by the Illumina sequencing platform.
1.2.7 Q-PCR检测  1.2.7 Q-PCR detection
分别将获得的样品 X和样品 Y的 DNA测序文库中 400bp和 800bp的扩增产物进行 Q-PCR检测， 结果如下表： 样品 样品 X 400bp 样品 X 800bp 样品 Y 400bp 样品 Y 800bpThe 400 bp and 800 bp amplification products in the DNA sequencing libraries of Sample X and Sample Y obtained were subjected to Q-PCR detection, and the results are shown in the following table: Sample sample X 400bp sample X 800bp sample Y 400bp sample Y 800bp
Q-PCR浓度 ( ng/μΐ ) 2.3 1.45 2.38 1.5 由上表可知， 相比之下， 样品 X和样品 Y的 DNA测序文库中相同长度的扩增产物的 浓度没有显著差别， 表明利用根据本发明实施例的构建 DNA测序文库的方法和 Illumina测 序平台提供的构建 DNA测序文库的方法， 构建的样品 DNA的测序文库没有显著的质量差 异。 实施例 2: Q-PCR concentration (ng/μΐ) 2.3 1.45 2.38 1.5 As can be seen from the above table, there is no significant difference in the concentration of amplification products of the same length in the DNA sequencing library of sample X and sample Y, indicating utilization according to the present invention. The method of constructing a DNA sequencing library of the embodiment and the method of constructing a DNA sequencing library provided by the Illumina sequencing platform did not have a significant quality difference in the sequenced library of the constructed sample DNA. Example 2:
2.1 试剂  2.1 Reagents
lOxblue緩冲液（ Enzymatics ), pUC18DNA ( Takara )。  lOxblue buffer (Enzymatics), pUC18DNA ( Takara).
第一试剂： 同实施例 1的 "第一试剂 "。  First reagent: Same as "first reagent" of Example 1.
第二试剂： pH为 7.6 ,溶剂是水，溶质为如下终浓度物质： T4 DNA连接酶（ Enzymatics ), ATP ( NEB ), 100 mM緩冲盐， 100 mM MgCl2 (国药集团化学试剂有限公司；)， 50 mM二 石克苏糖醇（ Sigma )。 The second reagent: pH is 7.6, the solvent is water, and the solute is the following final concentration: T4 DNA ligase (Enzymatics), ATP (NEB), 100 mM buffer salt, 100 mM MgCl 2 (National Pharmaceutical Group Chemical Reagent Co., Ltd.; ), 50 mM dicequititol (Sigma).
2.2 实验步骤  2.2 Experimental steps
分别吸取两份以 pUC18DNA (深圳华大基因研究院提供） 为模板扩增的 PCR产物 (200bp)各 19.7μ1, 置于 2个 ΕΡ管中， 分别命名为样品 X和样品 Υ, 然后按下列步骤进行实 验。  Two PCR products (200 bp) amplified by pUC18DNA (supplied by Shenzhen Huada Gene Research Institute) were each taken in 19.1 μl, placed in two fistulas, named sample X and sample 分别, respectively, and then followed the steps below. conduct experiment.
2.2.1 3 '端添加碱基 A  2.2.1 Adding bases at the 3 'end A
按照下表中的配比分别准备样品 X和样品 Y的 3 '端添加碱基 A反应体系：  Prepare the base A reaction system at the 3' end of sample X and sample Y according to the ratios in the table below:
Figure imgf000015_0001
Figure imgf000015_0001
将准备好的样品 X和样品 Υ的 3 '端添加碱基 Α反应体系，分别置于 37 °C下反应 30min, 以便获得 3 '端添加碱基 A的产物， 然后， 样品 X的 3 '端添加碱基 A的产物不经纯化， 而利 用 QlAquick PCR纯化试剂盒（QIAGEN公司）将样品 Y的 3 '端添加碱基 A的产物进行纯 化并溶于 16.4μ1 的 ΕΒ洗脱液中， 备用。  The prepared sample X and the 3' end of the sample were added to the base Α reaction system and reacted at 37 ° C for 30 min to obtain the product of the base A added at the 3 ' end, and then, the 3 ' end of the sample X The product of the base A was added without purification, and the product of the base A of the sample Y was purified by the QlAquick PCR Purification Kit (QIAGEN) and dissolved in a 16.4 μl hydrazine eluate for use.
2.2.2 添加接头  2.2.2 Adding a connector
按照下表中的配比分别准备样品 X和样品 Υ的添加接头反应体系： 样品 X的反应体系 Prepare the joint reaction system for sample X and sample 分别 according to the ratios in the table below: Sample X reaction system
Figure imgf000016_0001
Figure imgf000016_0001
其中， 接头的序列同实施例 1。  The sequence of the linker is the same as in the first embodiment.
将准备好的样品 X和样品 Υ的添加接头反应体系，分别置于 16°C下过夜进行反应 18h, 以便获得连接产物， 然后利用 Ampure磁珠将其纯化， 并利用 30μ1 EB液溶解洗脱， 由此， 获得样品 X和样品 Υ的 DNA测序文库，即本实施例利用根据本发明实施例的构建 DNA测 序文库的方法制备了样品 X的 DNA测序文库， 利用 Illumina测序平台提供的构建 DNA测 序文库的方法， 构建了样品 Y的 DNA测序文库。  The prepared sample reaction system of sample X and sample ruthenium was separately placed at 16 ° C for 18 h to obtain a ligation product, which was then purified by Ampure magnetic beads and eluted by using 30 μl of EB solution. Thus, a DNA sequencing library of sample X and sample , is obtained, that is, a DNA sequencing library of sample X is prepared by the method of constructing a DNA sequencing library according to an embodiment of the present invention, and a DNA sequencing library provided by the Illumina sequencing platform is constructed. Method, a DNA sequencing library of sample Y was constructed.
2.2.3 Q-PCR检测  2.2.3 Q-PCR detection
分别将获得的样品 X和样品 Y的 DNA测序文库进行 Q-PCR检测， 结果如下表:
Figure imgf000016_0002
The obtained DNA sequencing libraries of Sample X and Sample Y were subjected to Q-PCR detection, and the results are as follows:
Figure imgf000016_0002
由上表可知，样品 X和样品 Y的 DNA测序文库的 CT值没有明显差异，表明利用根据 本发明实施例的构建 DNA测序文库的方法和 Illumina测序平台提供的构建 DNA测序文库 的方法， 构建的样品 DNA的测序文库的浓度相当。 实施例 3:  As can be seen from the above table, there is no significant difference in the CT values of the DNA sequencing libraries of Sample X and Sample Y, indicating that the method for constructing a DNA sequencing library according to an embodiment of the present invention and the method for constructing a DNA sequencing library provided by the Illumina sequencing platform are constructed. The concentration of the sequencing library of sample DNA is comparable. Example 3:
3.1 试剂  3.1 Reagents
T4 DNA连接酶 (Rapid, L603-HC-L) ( Enzymatics )„ 第一试剂： 同实施例 1的 "第一试剂 "。 T4 DNA ligase (Rapid, L603-HC-L) ( Enzymatics ) „ First reagent: Same as "first reagent" of Example 1.
第二试剂： pH为 7.6, 溶剂是水， 溶质为如下终浓度物质： T4 DNA连接酶（ Takara ), 10 mM ATP ( NEB ), 500 mM Tris-HCl, lOOmM MgCl2 (国药集团化学试剂有限公司）， 50 mM 二石克苏糖醇（ Sigma )。 The second reagent: pH is 7.6, the solvent is water, and the solute is the following final concentration: T4 DNA ligase ( Takara ), 10 mM ATP ( NEB ), 500 mM Tris-HCl, lOOmM MgCl 2 (National Pharmaceutical Group Chemical Reagent Co., Ltd.) ), 50 mM dicequititol (Sigma).
3.2 实验步骤  3.2 Experimental steps
具体实验方法可参见 Agilent Technologies. Sureselect target enrichment system for illumina paired-end sequencing library. G3360-90020, 通过参照将其全文并入本文。  Specific experimental methods can be found in Agilent Technologies. Sureselect target enrichment system for illumina paired-end sequencing library. G3360-90020, which is incorporated herein in its entirety by reference.
分别吸取两份 50 ng/μΐ Fosmid样品（深圳华大基因研究院提供）各 40 μΐ置于 2个 Covaris 样品管中， 分别命名为样品 X和样品 Y, 然后按下列步骤进行实验。  Two 50 μg/μΐ Fosmid samples (supplied by Shenzhen Huada Gene Research Institute) were taken in two Covaris sample tubes, named sample X and sample Y, respectively, and then tested according to the following steps.
3.2.1片段化  3.2.1 Fragmentation
利用 Covaris公司生产 E210型号打断仪， 将样品 X和样品 Y的 DNA进行片段化， 以 便获得 DNA片段， 其中打断仪的参数设置如下：
Figure imgf000017_0001
Using the E210 model interrupter manufactured by Covaris, the DNA of sample X and sample Y was fragmented to obtain a DNA fragment, and the parameters of the interrupter were set as follows:
Figure imgf000017_0001
3.2.2 末端修复  3.2.2 End Repair
根据安捷伦 SureSelect platform的操作手册 G3360-90020中的说明，分别将样品 X和样 品 Y的 DNA片段进行末端修复， 以便获得末端修复产物。  The DNA fragments of sample X and sample Y were end-repaired according to the instructions in the Agilent SureSelect platform manual G3360-90020 to obtain the end repair product.
3.2.2 3'端添加碱基 A  3.2.2 Adding bases at the 3' end A
按照下表中的配比分别准备样品 X和样品 Y的 3'端添加碱基 A反应体系：  Prepare Samples X and Y at the 3' end of the sample Y according to the ratios in the table below.
样品 X的反应体系  Sample X reaction system
Figure imgf000017_0002
Figure imgf000017_0002
样品 γ 的反应体系  Sample γ reaction system
Figure imgf000017_0003
总体积 35μ1 将准备好的样品 X和样品 Υ的 3 '端添加碱基 Α反应体系，分别置于 37 °C下反应 30min, 以便获得 3 '端添加碱基 A的产物， 然后， 样品 X的 3 '端添加碱基 A的产物不经纯化， 而将 样品 Y的 3 '端添加碱基 A的产物经过柱纯化后溶于 37μ1 ΕΒ洗脱液中， 备用。
Figure imgf000017_0003
The total volume of 35μ1 is to add the base Α reaction system of the prepared sample X and the 3′ end of the sample ,, and respectively react at 37 ° C for 30 min to obtain the product of adding the base A at the 3′ end, and then, the sample X The product of the base A added at the 3' end was not purified, and the product of the base A added to the 3' end of the sample Y was purified by column and dissolved in 37 μl of the eluate for use.
3.2.3添加接头  3.2.3 Adding a connector
样品 X的反应体系  Sample X reaction system
Figure imgf000018_0001
Figure imgf000018_0001
样品 γ的反应体系  Sample γ reaction system
Figure imgf000018_0002
Figure imgf000018_0002
其中， 接头的序列同实施例 1。  The sequence of the linker is the same as in the first embodiment.
按照上表中的配比分别准备样品 X和样品 Υ的添加接头反应体系， 然后将准备好的样 品 X和样品 Υ的添加接头反应体系， 分别置于 16 °C下过夜进行反应 18h, 以便获得连接产 物， 备用。  Prepare the joint reaction system for the sample X and the sample 按照 according to the ratios in the above table, and then add the prepared sample reaction system of the sample X and the sample , to the reaction system at 16 ° C for 18 hours to obtain the reaction. Connect the product, spare.
3.2.4 PCR扩增，  3.2.4 PCR amplification,
按照实施例 1中 1.2.6项所示的方法， 分别将样品 X和样品 Y的连接产物配制成 PCR 扩增反应体系， 然后分别将样品 X和样品 Y的 PCR扩增反应体系于 PCR仪中进行 PCR扩 增反应， 以便获得扩增产物， 然后利用 2 %琼脂糖凝胶电泳分别将 2种样品的扩增产物进行 分离， 切胶回收 200bp的扩增产物， 分别溶于 30μ1的超纯水中， 由此， 获得样品 X的 DNA 测序文库和样品 Υ的 DNA测序文库。  According to the method shown in item 1.2.6 of Example 1, the ligation products of sample X and sample Y were respectively formulated into a PCR amplification reaction system, and then the PCR amplification reaction systems of sample X and sample Y were respectively subjected to a PCR instrument. The PCR amplification reaction was carried out to obtain an amplification product, and then the amplification products of the two samples were separately separated by 2% agarose gel electrophoresis, and the 200 bp amplification product was recovered by gelation and dissolved in 30 μl of ultrapure water. Thereby, a DNA sequencing library of the sample X and a DNA sequencing library of the sample were obtained.
3.2.5 Q-PCR检测 分别将获得的样品 X和样品 Y的 DNA测序文库进行 Q-PCR检测， 结果如下表：
Figure imgf000019_0001
3.2.5 Q-PCR detection The obtained DNA sequencing libraries of Sample X and Sample Y were subjected to Q-PCR detection, and the results are as follows:
Figure imgf000019_0001
由上表可知，样品 X和样品 Y的 DNA测序文库的 CT值没有显著差别，表明利用根据 本发明实施例的构建 DNA测序文库的方法和 Illumina测序平台提供的构建 DNA测序文库 的方法， 构建的样品 DNA的测序文库的浓度相当。 工业实用性  As can be seen from the above table, the CT values of the DNA sequencing libraries of Sample X and Sample Y are not significantly different, indicating that the method for constructing a DNA sequencing library according to an embodiment of the present invention and the method for constructing a DNA sequencing library provided by the Illumina sequencing platform are constructed. The concentration of the sequencing library of sample DNA is comparable. Industrial applicability
本发明的两种试剂、 构建 DNA测序文库的方法、 DNA测序文库、 确定 DNA样品 的序列信息的方法以及用于构建 DNA测序文库的试剂盒， 能够有效地应用于样品 DNA 的 DNA测序文库的构建以及测序， 并且获得的文库质量好， 测序结果准确。  The two reagents of the present invention, the method for constructing the DNA sequencing library, the DNA sequencing library, the method for determining the sequence information of the DNA sample, and the kit for constructing the DNA sequencing library can be effectively applied to the construction of the DNA sequencing library of the sample DNA. As well as sequencing, and the obtained library is of good quality and the sequencing results are accurate.
尽管本发明的具体实施方式已经得到详细的描述， 本领域技术人员将会理解。 根据已 经公开的所有教导， 可以对那些细节进行各种修改和替换， 这些改变均在本发明的保护范 围之内。 本发明的全部范围由所附权利要求及其任何等同物给出。  Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of those details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
在本说明书的描述中， 参考术语 "一个实施例"、 "一些实施例"、 "示意性实施例"、 "示 例"、 "具体示例"、 或 "一些示例" 等的描述意指结合该实施例或示例描述的具体特征、 结 构、 材料或者特点包含于本发明的至少一个实施例或示例中。 在本说明书中， 对上述术语 的示意性表述不一定指的是相同的实施例或示例。 而且， 描述的具体特征、 结构、 材料或 者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。  In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Claims

权利要求书 Claim
1、 一种试剂， 其特征在于， 包含： 緩冲盐、 二硫苏糖醇、 MgCl2。 What is claimed is: 1. A reagent comprising: a buffer salt, dithiothreitol, MgCl 2 .
2、 根据权利要求 1所述的试剂， 其特征在于， 包含：  2. The reagent according to claim 1, comprising:
lOmM-lOOOmM的緩冲盐；  lOmM-lOOOOmM buffer salt;
lOmM-lOOmM的可溶性盐；  lOmM-100 mM soluble salt;
10mM~300mM的 MgCl2; 10 mM to 300 mM MgCl 2 ;
lmM〜50mM的二^ ¾苏糖醇；  LmM~50 mM di-threo-threitol;
lmM〜10mM的 dATP; 以及  lmM to 10 mM dATP;
lU/ml〜 40U/ml的 Klenow片段（ 3 '→ 5 ' exo ),  KLow fragment (3 '→ 5 ' exo ) of lU/ml~ 40U/ml,
其巾，  Its towel,
所述试剂的 pH为 7.6〜7.9。  The pH of the reagent is 7.6 to 7.9.
3、 根据权利要求 2所述的试剂， 其特征在于 包含 50mM〜500mM的緩冲盐。  The reagent according to claim 2, which comprises 50 mM to 500 mM of a buffer salt.
4、 根据权利要求 2所述的试剂， 其特征在于 包含 lOOmM的緩冲盐。  4. The reagent according to claim 2, characterized in that it comprises 100 mM of a buffer salt.
5、 根据权利要求 2所述的试剂， 其特征在于 包含 25mM〜75mM的可溶性盐  The reagent according to claim 2, which comprises a soluble salt of 25 mM to 75 mM.
6、 根据权利要求 2所述的试剂， 其特征在于 包含 50mM的可溶性盐。  6. The reagent according to claim 2, characterized in that it comprises 50 mM of a soluble salt.
7、 根据权利要求 2所述的试剂， 其特征在于 包含 50mM〜200mM的 MgCl2。 7. The reagent according to claim 2, characterized in that it comprises 50 mM to 200 mM of MgCl 2 .
8、 根据权利要求 2所述的试剂， 其特征在于 包含 lOOmM的 MgCl2。 8. The reagent according to claim 2, characterized in that it comprises 100 mM of MgCl 2 .
9、 根据权利要求 2所述的试剂 其特征在于， 包含 2.5mM〜7.5mM的 dATP。  The reagent according to claim 2, which comprises 2.5 mM to 7.5 mM of dATP.
10、 根据权利要求 2所述的试剂， 其特征在于， 包含 5mM的 dATP。  The reagent according to claim 2, which comprises 5 mM of dATP.
11、 根据权利要求 2所述的试剂， 其特征在于， 包含 10U/ml〜 30U/ml的 Klenow片段 ( 3'→5' exo λ The reagent according to claim 2, comprising a Klenow fragment of 10 U/ml to 30 U/ml (3' → 5' exo λ
12、根据权利要求 2所述的试剂，其特征在于，包含 20U/ml的 Klenow片段（ 3'→5' exo )。  The reagent according to claim 2, which comprises a 20 U/ml Klenow fragment (3'→5' exo).
13、 根据权利要求 2所述的试剂， 其特征在于， 所述试剂的 pH为 7.9。  The reagent according to claim 2, wherein the reagent has a pH of 7.9.
14、 根据权利要求 2所述的试剂， 其特征在于， 所述緩冲盐为选自 Tris-HCl和磷酸盐 的至少一种。  The reagent according to claim 2, wherein the buffer salt is at least one selected from the group consisting of Tris-HCl and phosphate.
15、 根据权利要求 2所述的试剂， 其特征在于， 所述緩冲盐为 Tris-HCl。  The reagent according to claim 2, wherein the buffer salt is Tris-HCl.
16、 根据权利要求 2 所述的试剂， 其特征在于， 所述可溶性盐为选自氯化钠和氯化钾 的至少一种。  The reagent according to claim 2, wherein the soluble salt is at least one selected from the group consisting of sodium chloride and potassium chloride.
17、 根据权利要求 2所述的试剂， 其特征在于， 所述可溶性盐为氯化钠。  The reagent according to claim 2, wherein the soluble salt is sodium chloride.
18、 根据权利要求 1所述的试剂， 其特征在于， 包含： 10mM〜200mM的緩冲盐； 18. The reagent according to claim 1, comprising: 10 mM to 200 mM buffer salt;
ImM-lOOmM的二^ ¾苏糖醇；  ImM-100 mM bis3⁄4 threitol;
10mM~400mM的 MgCl2; 10 mM to 400 mM MgCl 2 ;
1 mM〜40mM的 ATP； 以及  1 mM to 40 mM ATP;
lU/ml〜 200U/ml的 DNA连接酶,  lU/ml~200U/ml DNA ligase,
其巾，  Its towel,
所述试剂的 pH为 7.6 ~ 7.9。  The pH of the reagent is from 7.6 to 7.9.
19 根据权利要求 8所述的试剂， 其特征在于 包含 50mM〜150mM的緩冲盐。  The reagent according to claim 8, which comprises 50 mM to 150 mM of a buffer salt.
20 根据权利要求 8所述的试剂， 其特征在于 包含 lOOmM的緩冲盐。 The reagent according to claim 8, which comprises 100 mM of a buffer salt.
21 根据权利要求 8所述的试剂， 其特征在于 包含 10mM〜90mM 的二硫苏糖醇。  The reagent according to claim 8, which comprises 10 mM to 90 mM of dithiothreitol.
22 根据权利要求 8所述的试剂， 其特征在于 包含 50mM的二硫苏糖醇。 The reagent according to claim 8, which comprises 50 mM of dithiothreitol.
23 根据权利要求 8所述的试剂， 其特征在于 包含 50mM〜200mM的 MgCl2。 The reagent according to claim 8, characterized in that it contains 50 mM to 200 mM of MgCl 2 .
24 根据权利要求 8所述的试剂， 其特征在于 包含 lOOmM的 MgCl2。 The reagent according to claim 8, characterized in that it contains 100 mM of MgCl 2 .
25 根据权利要求 8所述的试剂， 其特征在于 包含 5mM〜20mM的 ATP。  The reagent according to claim 8, which comprises 5 mM to 20 mM of ATP.
26 根据权利要求 8所述的试剂， 其特征在于 包含 10mM的 ATP。  The reagent according to claim 8, which comprises 10 mM of ATP.
27 根据权利要求 8所述的试剂 其特征在于 包含 50U/ml〜 150U/ml的 DNA连接 醉。  The reagent according to claim 8, characterized in that it contains 50 U/ml to 150 U/ml of DNA linked to drunk.
28 根据权利要求 18所述的试剂 其特征在于 包含 72U/ml的 DNA连接酶。  The reagent according to claim 18, which comprises 72 U/ml of DNA ligase.
29 根据权利要求 18所述的试剂 其特征在于 所述试剂的 pH为 7.6。  The reagent according to claim 18, wherein the reagent has a pH of 7.6.
30 根据权利要求 18所述的试剂 . 其特征在于 所述緩冲盐为选自 Tris-HCl和磷酸盐 的至少一种。  The reagent according to claim 18, wherein the buffer salt is at least one selected from the group consisting of Tris-HCl and phosphate.
31、 根据权利要求 18所述的试剂 其特征在于， 所述緩冲盐为 Tris-HCl。  The reagent according to claim 18, wherein the buffer salt is Tris-HCl.
32、 一种构建 DNA测序文库的方法， 其特征在于， 包含以下步骤：  32. A method of constructing a DNA sequencing library, comprising the steps of:
将 DNA片段化， 以便获得 DNA片段；  Fragmenting DNA to obtain a DNA fragment;
将所述 DNA片段进行末端修复， 以便获得经过末端修复的 DNA片段；  End-repairing the DNA fragment to obtain a DNA fragment that has been repaired at the end;
使用权利要求 1-17任一项所述的试剂， 对所述经过末端修复的 DNA片段进行处理， 以便获得 3'端添加碱基 A的 DNA片段；  Using the reagent according to any one of claims 1-17, the end-repaired DNA fragment is treated to obtain a DNA fragment having a base A added at the 3' end;
使用权利要求 18-31任一项所述的试剂，将所述 3'端添加碱基 A的 DNA片段与接头进 行连接， 以便获得连接产物；  Using the reagent according to any one of claims 18 to 31, the DNA fragment to which the base A is added at the 3' end is linked to a linker to obtain a ligation product;
将所述连接产物进行片段选择， 以便获得目的片段；  The ligation product is subjected to fragment selection to obtain a fragment of interest;
将所述目的片段进行 PCR扩增， 以便获得扩增产物； 以及 分离纯化所述扩增产物， 所述扩增产物构成所述 DNA测序文库。 The target fragment is subjected to PCR amplification to obtain an amplification product; The amplification product is isolated and purified, and the amplification product constitutes the DNA sequencing library.
33、 根据权利要求 32所述的方法， 其特征在于， 所述片段化是通过选自超声打断法和 酶切处理的至少一种进行的。  33. The method of claim 32, wherein the fragmentation is performed by at least one selected from the group consisting of ultrasonic disruption and digestion.
34、 根据权利要求 33所述的方法， 其特征在于， 所述超声打断法是利用 Covaris超声 打断仪进行的。  34. The method of claim 33, wherein the ultrasonic breaking method is performed using a Covaris ultrasonic interrupter.
35、 根据权利要求 32所述的方法， 其特征在于， 所述 DNA片段的长度为 200-800bp。  35. The method according to claim 32, wherein the DNA fragment has a length of 200 to 800 bp.
36、根据权利要求 32所述的方法，其特征在于，使用权利要求 1-16任一项所述的试剂， 对所述经过末端修复的 DNA片段进行处理是在 12°C〜40°C下进行的。  The method according to claim 32, wherein the end-repaired DNA fragment is treated at 12 ° C to 40 ° C using the reagent according to any one of claims 1-16. ongoing.
37、根据权利要求 32所述的方法，其特征在于，使用权利要求 1-16任一项所述的试剂， 对所述经过末端修复的 DNA片段进行处理是在 37 °C下进行的。  The method according to claim 32, wherein the end-repaired DNA fragment is treated at 37 ° C using the reagent according to any one of claims 1-16.
38、根据权利要求 32所述的方法， 其特征在于， 所述接头包含第一链和第二链， 其中， 所述第一链为：  38. The method of claim 32, wherein the linker comprises a first chain and a second chain, wherein the first chain is:
5-Phos/TACAC  5-Phos/TACAC
所述第二链为：  The second chain is:
5-Phos/TTGGl  5-Phos/TTGGl
39、 根据权利要求 32所述的方法， 其特征在于， 使用权利要求 17-30任一项所述的试 剂， 将所述 3'端添加碱基 A的 DNA片段与接头进行连接是在 4°C〜22°C下进行的。  39. The method according to claim 32, wherein the DNA fragment of the base A added base A is linked to the linker at 4° using the reagent according to any one of claims 17-30. C~22 °C.
40、 根据权利要求 32所述的方法， 其特征在于， 使用权利要求 17-30任一项所述的试 剂， 将所述 3'端添加碱基 A的 DNA片段与接头进行连接是在 16°C下进行的。  40. The method according to claim 32, wherein the DNA fragment of the base A added base A is ligated to the linker at 16° using the reagent according to any one of claims 17-30. Performed under C.
41、 根据权利要求 32所述的方法， 其特征在于， 利用琼脂糖凝胶电泳对所述连接产物 进行片段选择。  41. The method according to claim 32, wherein the ligation product is subjected to fragment selection by agarose gel electrophoresis.
42、 根据权利要求 32所述的方法， 其特征在于， 所述目的片段的长度为 100-1000bp。  42. The method according to claim 32, wherein the segment of interest has a length of 100-1000 bp.
43、 根据权利要求 32所述的方法， 其特征在于， 所述目的片段的长度为 200-800bp。  43. The method according to claim 32, wherein the target segment has a length of 200-800 bp.
44、根据权利要求 32所述的方法， 其特征在于， 使用第一 PCR引物和第二 PCR引物， 将所述目的片段进行 PCR扩增，  44. The method of claim 32, wherein the target fragment is PCR amplified using a first PCR primer and a second PCR primer,
其中， 所述第一 PCR引物的序列为：  Wherein the sequence of the first PCR primer is:
5*CAAGCAGAAG.  5*CAAGCAGAAG.
GTGCTCTTCCGATCT GTGCTCTTCCGATCT
所述第二 PCR引物的序列为：  The sequence of the second PCR primer is:
GATCT GATCT
45、 一种 DNA测序文库， 其是通过根据权利要求 32-44中任一项所述的方法构建的。45. A DNA sequencing library constructed by the method of any one of claims 32-44.
46、 一种确定 DNA样品的序列信息的方法， 其特征在于， 包括下列步骤： 46. A method of determining sequence information of a DNA sample, comprising the steps of:
才艮据权利要求 32-44任一项所述的方法， 构建所述 DNA样品的 DNA测序文库； 以及 对所述 DNA测序文库进行测序， 以便确定所述 DNA样品的序列信息。  The DNA sequencing library of the DNA sample is constructed according to the method of any one of claims 32-44; and the DNA sequencing library is sequenced to determine sequence information of the DNA sample.
47、 根据权利要求 46所述的方法， 其特征在于， 对所述 DNA测序文库进行测序前， 进一步包括利用 Agilent Bioanalyzer 2100和 Q-PCR的至少一种对所述 DNA测序文库进行检 测。  47. The method of claim 46, wherein prior to sequencing the DNA sequencing library, further comprising detecting the DNA sequencing library using at least one of Agilent Bioanalyzer 2100 and Q-PCR.
48、 根据权利要求 46所述的方法， 其特征在于， 所述测序是利用 Illumina测序平台进 行的。  48. The method of claim 46, wherein the sequencing is performed using an Illumina sequencing platform.
49、 一种用于构建 DNA测序文库的试剂盒， 其特征在于， 包含：  49. A kit for constructing a DNA sequencing library, comprising:
第一试剂， 所述第一试剂为权利要求 1-17任一项所述的试剂； 以及  a first reagent, the first reagent being the reagent of any one of claims 1-17;
第二试剂， 所述第二试剂为权利要求 18-31任一项所述的试剂。  A second reagent, which is the reagent of any one of claims 18-31.
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