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WO2012079490A1 - Procédé pour la construction de banque de séquençage d'adn et utilisation de celui-ci - Google Patents

Procédé pour la construction de banque de séquençage d'adn et utilisation de celui-ci 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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Prior art keywords
reagent
dna
reagent according
fragment
sample
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English (en)
Chinese (zh)
Inventor
栾合密
张俊青
程玲
孔淑娟
张秀清
杨焕明
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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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

La présente invention concerne deux réactifs, un procédé pour construire une banque de séquençage d'ADN, une banque de séquençage d'ADN, un procédé pour déterminer les informations de séquence dans un échantillon d'ADN et un kit pour construire une banque de séquençage d'ADN, un réactif comprenant 10 mM à 1000 mM d'un sel tampon, 10 mM à 100 mM d'un sel soluble, 10 mM à 300 mM de MgCl2, 1 mM à 50 mM de dithiothréitol, 1 mM à 10 mM de dATP et 1 U/ml à 40 U/ml de fragment klenow, le pH du réactif étant de 7,6 à 7,9, et l'autre réactif comprenant 10 mM à 200 mM d'un sel tampon, 1 mM à 100 mM de dithiothréitol, 10 mM à 400 mM de MgCl2, 1 mM à 40 mM d'ATP et 1 U/ml à 200 U/ml d'ADN ligase, le pH du réactif étant de 7,6 à 7,9.
PCT/CN2011/083786 2010-12-15 2011-12-09 Procédé pour la construction de banque de séquençage d'adn et utilisation de celui-ci Ceased WO2012079490A1 (fr)

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