CN110129414A - A kind of BRCA high-throughput sequencing library and its construction method and application - Google Patents
A kind of BRCA high-throughput sequencing library and its construction method and application Download PDFInfo
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- CN110129414A CN110129414A CN201910349374.7A CN201910349374A CN110129414A CN 110129414 A CN110129414 A CN 110129414A CN 201910349374 A CN201910349374 A CN 201910349374A CN 110129414 A CN110129414 A CN 110129414A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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- 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
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Abstract
The present invention provides a kind of construction methods of BRCA high-throughput sequencing library, belong to high-flux sequence field.The construction method of BRCA high-throughput sequencing library provided by the invention includes the generation of initial libraries, the purifying of initial libraries, the generation of sequencing library and the purifying of sequencing library.A possibility that BRCA high-throughput sequencing library construction method whole process provided by the invention carries out in a vessel, avoids cross contamination.Method provided by the invention utilizes magnetic bead reaction bonded washing operation, utmostly ensure that the efficient and convenient of purifying.Using method provided by the invention, the utilization rate of template can be made to greatly improve.
Description
Technical field
The invention belongs to high throughput sequencing technologies fields, and in particular to a kind of BRCA high-throughput sequencing library and its building side
Method and application.
Background technique
Class breast cancer susceptibility gene (BRCA1/2) is found in mammary gland cancer family first, for the cream with genetic predisposition
The tumor susceptibility gene of gland cancer and oophoroma.BRCA1 is positioned at No. 17 chromosome q21 of the mankind, is lost in a manner of autosomal dominant inheritance
It passes, there is very high genepenetrance.BRCA1 is about 100kb, contains 24 exons, gene product is that 1863 amino acid are constituted
Phosphorylating protein.BRCA2 is positioned at No. 13 chromosome q12 of the mankind, and complete genome DNA is about 70kb, wherein encoding the head of district
About 10kp.The gene order and BRCA1 of BRCA2 is made of without obvious relation 27 exons, wherein the 11st exon is long
About 4932bp, mRNA are about 10.2kb, and the BRCA2 albumen of coding contains 3418 amino acid.BRCA1/2 gene structure and function
It is abnormal closely related with the generation of breast cancer and oophoroma, as a kind of tumor suppressor gene, cell can not only be inhibited to grow, also joined
With cell cycle regulating, genetic transcription is adjusted, a variety of important cells activities such as DNA damage reparation and apoptosis, is maintaining gene
It plays an important role in group stability.
Currently, high throughput sequencing technologies have been widely used medical research and clinical diagnosis field, it is the inspection in carcinogenic site
Survey provides new detection means.High throughput sequencing technologies survey that is comprehensive, and constantly declining because its result coverage area
Sequence cost shows higher and higher cost performance in medical research and clinical application.Limited sample size and examining report are defeated
More stringent requirements are proposed to the technology for timeliness out.Simplify library construction process, improve low initial amount sample build Kucheng
Power is imperative.By optimizing and improve library constructing method, shortening the enzyme reaction time and building library step, improve sample conversion
Become the big key point that high throughput sequencing technologies expand application field at the efficiency in library.
Classical high-throughput library construction step include: 1. genomes interrupt (including physics interrupts method and chemistry interrupts method,
As covaris ultrasound interrupts, fragmentase digestion interrupts)；2. double-stranded DNA (Deoxyribonucleic acid, deoxidation core
Ribosomal ribonucleic acid) end reparation；3.3 ' ends add dATP (deoxyadenosine triphosphate, deoxyadenosine triphosphate)；4.
Add and the matched connector of microarray dataset；5.PCR (Polymerase Chain Reaction, polymerase chain reaction) amplification.Often
Require to carry out purifying reaction between a step, also need to carry out after this enzymatic reaction sometimes a Piece Selection (step 1,4,5 its
One of), whole flow process needs general 8~9 hours that could complete.
Classical high-throughput library constructing method be current acceptance is most wide, Data Representation is most stable, data skewed popularity most
Few banking process, but it is built library process and takes a long time, step is excessive and purification process in target fragment loss so that library
Template utilization rate it is not high, thereby increases and it is possible to cause cross contamination.Classical high-throughput library constructing method is increasingly unable to satisfy fastly
The market demand for building library and fast report period micro to low initial amount of speed development.
Summary of the invention
In view of above-mentioned technical problem, the present invention provides a kind of BRCA high-throughput sequencing library and its construction method and answer
With.BRCA high-throughput sequencing library is constructed using method provided by the invention, the used time is short, and all operationss carry out in a pipe,
Cross contamination will not be caused.
The present invention provides a kind of construction methods of BRCA high-throughput sequencing library, include the following steps:
(1) using include class mammary gland tumor susceptibility gene DNA fragmentation as template, carry out multiplexed PCR amplification in a reservoir, obtain
BRCA initial libraries；
(2) the magnetic bead liquid of 0.8~1.2 times of PCR reaction system volume is added in Xiang Suoshu container, after reacting 1~30min,
Supernatant is abandoned, the first magnetic bead is obtained；
(3) cleaning purifying is carried out to the BRCA initial libraries adsorbed on step (2) first magnetic bead；
(4) the BRCA initial libraries of the cleaning after purification are successively carried out plus A reacts and connection is reacted, obtain BRCA survey
Preface library；
(5) magnetic of 0.8~1.2 extraordinarily A reaction system volume is added into the former container equipped with the BRCA sequencing library
Pearl liquid after reacting 1~30min, abandons supernatant, obtains the second magnetic bead；
(6) cleaning purifying is carried out to step (5) second magnetic bead, obtains purifying magnetic ball；
(7) DNA of the purifying magnetic bead surfaces is eluted, the eluent obtained after connection is that text is sequenced in the BRCA purified
Library；
Step (1)~(7) operate in a same vessel.
Preferably, the template is B150A_1~60 or B150B_1~60；When the template is B150A_1~60,
The nucleotide sequence of the primer of B150A_1~60 is expanded as shown in No.1~120 SEQ ID.
Preferably, PCR reaction system of the multiplexed PCR amplification using 25 μ l, the PCR reaction system in the step (1)
Component including following content:
| Component | Volume |
| Polymerase Mix | 12.5μl |
| Primer library | 7μl |
| DNA profiling | 0.5μl |
| Water | 5μl |
Wherein, Suzhou is preferably purchased from containing buffer system needed for polymerase, dNTP and reaction in the polymerase Mix
New marine growth Science and Technology Co., Ltd., article No. NH9333.
Preferably, multiplexed PCR amplification program in the step (1) are as follows: 98 DEG C, 3min；98 DEG C, 30s, 58 DEG C, 15s, 22
A circulation；72 DEG C, 15s, 72 DEG C, 4min.
Preferably, the solvent for cleaning in the step (3) includes the ethanol water of magnetic bead washing lotion and volume ratio 68~72%
Solution.
Preferably, the step (4) plus A reaction are carried out with 20 μ l's plus A system, and described plus A system includes 1 μ L 5U/ μ L
Klenow Fragment, 0.5 μ L 10U/ μ L T4 PNK, 2 μ 10 × Custsmart of L Buffer, 0.25 μ L 10mM
DATP, 0.25 μ L 100mM magnesium acetate, the purified PCR product of 8 μ L water and 8 μ L, the total amount of the PCR product are 100ng.
Preferably, described plus A response procedures are as follows: 37 DEG C, 30min, 75 DEG C, 20min.Wherein 37 DEG C are to add A reaction, 75 DEG C
It is to be inactivated to the bioactive substance in 25 μ l systems.
Preferably, step (4) the connection reaction is carried out with the linked system of 25 μ L, and the linked system is to complete to add A
0.5 μ 10 × Custsmart of L Buffer, the ligase of 2 μ L 600U/ μ L, 2.5 μ L 10uM are added in 20 μ L systems of reaction
Sequence measuring joints.
Preferably, the connection response procedures are as follows: 25 DEG C, 2h, 65 DEG C, 10min.
The present invention also provides the BRCA high-throughput sequencing libraries that above-mentioned construction method obtains.
The utility model has the advantages that
The present invention provides a kind of construction method of BRCA high-throughput sequencing library, all operationss of this method hold at one
It is carried out in device, not only reduces the loss of target fragment, it is thus also avoided that a possibility that cross contamination.
Method provided by the invention utilizes magnetic bead reaction bonded washing operation, utmostly ensure that the efficient of purifying and just
It is prompt.Using method provided by the invention, the utilization rate of template can be made to greatly improve.
Since BRCA high throughput library constructing method provided by the invention is easy to operate, replacement container is not had to, in conjunction with magnetic bead
The method of purifying is, it can be achieved that the automation in BRCA high throughput library constructs.
Detailed description of the invention
Fig. 1-A is the testing result before converting in library Quality Control described in the embodiment of the present invention 1；
Fig. 1-B is the testing result after converting in library Quality Control described in the embodiment of the present invention 1；
Fig. 1-C is the testing result in library Quality Control described in the embodiment of the present invention 1 before purification；
Fig. 1-D is the testing result in library Quality Control described in the embodiment of the present invention 1 after purification；
Fig. 2 is automatic Building library facilities conceptual schematic drawing described in the embodiment of the present invention 2.
Specific embodiment
The present invention provides a kind of construction methods of BRCA high-throughput sequencing library, include the following steps:
(1) using include class mammary gland tumor susceptibility gene DNA fragmentation as template, carry out multiplexed PCR amplification in a reservoir, obtain
BRCA initial libraries；
(2) the magnetic bead liquid of 0.8~1.2 times of PCR reaction system volume is added in Xiang Suoshu container, after reacting 1~30min,
Supernatant is abandoned, the first magnetic bead is obtained；
(3) cleaning purifying is carried out to the BRCA initial libraries adsorbed on step (2) first magnetic bead；
(4) the BRCA initial libraries of the cleaning after purification are successively carried out plus A reacts and connection is reacted, obtain BRCA survey
Preface library；
(5) magnetic of 0.8~1.2 extraordinarily A reaction system volume is added into the former container equipped with the BRCA sequencing library
Pearl liquid after reacting 1~30min, abandons supernatant, obtains the second magnetic bead；
(6) cleaning purifying is carried out to step (5) second magnetic bead, obtains purifying magnetic ball；
(7) DNA of the purifying magnetic bead surfaces is eluted, the eluent obtained after connection is that text is sequenced in the BRCA purified
Library；
Step (1)~(7) operate in a same vessel.
BRCA high throughput library constructing method whole process provided by the invention operates in a vessel, can effectively reduce purpose
The loss of segment, while a possibility that be avoided that cross contamination.
The present invention using include class mammary gland tumor susceptibility gene DNA fragmentation as template, carry out multiplexed PCR amplification in a reservoir, obtain
To BRCA initial libraries.In the present invention, the template is preferably B150A_1~60 or B150B_1~60.In reality of the invention
It applies in example, the template source is the tumor cell gene group of people, is bought from card riel Institute for Medical Research, the U.S. (Coriell
institute).In the present invention, when being template with B150A_1~60, the nucleotides sequence of the primer of B150A_1~60 is expanded
Column are as shown in No.1~120 SEQ ID.
In the present invention, the container of the multiplexed PCR amplification is preferably the PCR pipe of 0.2ml.The multi-PRC reaction body
System is preferably 25 μ L systems, more preferably includes the component of following content:
| Component | Volume |
| Polymerase Mix | 12.5μl |
| Primer library | 7μl |
| DNA profiling | 0.5μl |
| Water | 5μl |
Polymerase Mix is purchased from the new marine growth Science and Technology Co., Ltd. in Suzhou, article No. NH9333.
Preferably, multiplexed PCR amplification program in the step (1) are as follows: 98 DEG C, 3min；98 DEG C, 30s, 58 DEG C, 15s, 22
A circulation；72 DEG C, 15s, 72 DEG C, 4min.
After obtaining BRCA initial libraries, magnetic bead liquid is added into the container equipped with BRCA initial libraries by the present invention, uses magnetic bead
Adsorption reaction is carried out to initial libraries DNA.The source of the magnetic bead liquid is not particularly limited in the present invention, this field conventional commercial
Product.In an embodiment of the present invention, the magnetic bead liquid is selected from Beckman Agencourt AMPure XP (specification
5ml is purchased from Beckman Coulter Inc., the U.S.,XP Beckman Coulter,
cat.no.A63881).In the present invention, preferred before the magnetic bead liquid reaction to mix, the mode of the mixing is preferably pressure-vaccum.
In an embodiment of the present invention, it is adjusted at 70~80% taken amounts of total liquid volume using 200 μ l liquid-transfering guns, is blown up and down repeatedly
It inhales 6~10 times, bubble generation should be avoided in the process of the pressure-vaccum.In this step, the addition volume of the magnetic bead liquid is preferably
0.8~1.2 times of PCR reaction system volume, preferably 0.9~1.1 times of PCR reaction system volume, more preferably 1 times of PCR reaction
System volume.In the present invention, the time of the adsorption reaction be 1~30min, preferably 1.5~5min, more preferably
2min.After reaction, the present invention abandons supernatant, obtains magnetic bead.In the present invention, the separation method of supernatant and magnetic bead is preferred
Are as follows: PCR pipe is placed on magnetic frame and is stood.The time of the standing is preferably 1~3min, more preferably 2min.
After obtaining magnetic bead, the present invention carries out cleaning purifying to the BRCA initial libraries adsorbed on the magnetic bead.In the present invention
In, the cleaning preferably includes the ethanol water of magnetic bead washing lotion and volume ratio 68~72% with solution.
When magnetic bead washing lotion cleaning purifying initial libraries, B&W washing lotion is added in the present invention preferably into the pipe containing magnetic bead, makes magnetic
Pearl suspension reaction 1~3min, more preferable suspension reaction 2min.After reaction was completed, the separation method of magnetic bead and B&W washing lotion is preferred
Are as follows: with strong magnets or magnetic frame absorption magnetic bead in tube wall side, supernatant is drawn, magnetic bead is retained.In the present invention, the magnetic bead
Washing lotion preferably includes 20% (w/v) PEG8000,2M NaCl, 25mM EDTA, 0.9mM KAc.The cleaning of the magnetic bead washing lotion is used
Amount is preferably 30~50 μ l, is optimized according to target fragment size, wherein 30 μ L are suitable for the nucleic acid small pieces of 100-150bp
Section, purifying of the 40 μ L suitable for the nucleic acid fragment of 200-600bp.In this step, the effect of the magnetic bead washing lotion is anti-in PCR
Ying Hou enhances magnetic bead to the adsorption efficiency of large fragment, to achieve the purpose that remove large fragment.
When carrying out cleaning purifying to magnetic bead using ethanol water, the present invention is added centainly preferably into the pipe for have magnetic bead
The ethanol water that volumetric concentration is 68~72%, then adsorbing magnetic bead back and forth on the different two sides of tube wall repeatedly with magnetic frame makes
Magnetic bead is obtained sufficiently to suspend.After reaction, Beads enrichment method is preferred are as follows: with strong magnets or magnetic frame absorption magnetic bead in tube wall
Side to solution is clarified, then draws supernatant, retains magnetic bead.In the present invention, the volumetric concentration of the ethanol water is preferably
70%.The dosage of the ethanol water is preferably 90~110 μ l, more preferably 100 μ l.In the present invention, the ethyl alcohol is washed
The effect of liquid is the extra salt ion of removal and other non-purpose nucleic acid segments.
In the present invention, when cleaning includes two kinds with solution, preferably first magnetic bead is cleaned with magnetic bead washing lotion, then uses
Ethanol water cleans magnetic bead.The cleaning sequence may make the magnetic bead impurity residual after cleaning minimum.Ethanol water
After magnetic bead cleaning, magnetic bead is preferably placed in 3~10min of room temperature by the present invention, keeps residual ethanol volatilization complete.
After purification, the present invention is successively carried out to the BRCA initial libraries of the cleaning after purification plus A reacts and connection for cleaning
Reaction, obtains BRCA sequencing library.
In the present invention, described plus A reaction preferably adds A system to carry out in 20 μ l, and described plus A system preferably includes 1 μ L
The Klenow Fragment, the T4PNK of 0.5 μ L 10U/ μ L, 2 μ L10 × Custsmart Buffer, 0.25 μ L of 5U/ μ L
10mM dATP, 0.25 μ L 100mM magnesium acetate, the purified PCR product of 8 μ L water and 8 μ L, the total amount of the PCR product are
100ng.In the present invention, the program of described plus A reaction is preferably 37 DEG C, 30min, and 75 DEG C, 20min.Wherein 37 DEG C are to add A anti-
It answers, 75 DEG C are inactivated to the bioactive substance in 25 μ l systems.It is carried out using system provided by the invention plus A is operated,
Add A compared to traditional Taq enzyme, it is more efficient and can carry out being more advantageous to automatic operation at a constant temperature.
Add A after reaction, the present invention preferably to add A react after PCR pipe in be added magnetic bead liquid carry out adsorption reaction, so
Cleaning purifying is carried out to the DNA fragmentation of magnetic bead absorption afterwards.In the present invention, the addition volume of the magnetic bead liquid be preferably 0.8~
1.2 extraordinarily A system volumes, preferably 0.9~1.1 extraordinarily A system volume, more preferably 1 extraordinarily A system volume.In the present invention
In, the time of the adsorption reaction is 1~30min, preferably 1.5~5min, more preferably 2min.After adsorption reaction,
The present invention abandons supernatant, obtains magnetic bead.In the present invention, the separation method of supernatant and magnetic bead is preferred are as follows: PCR pipe is placed in magnetic
It is stood on power frame.The time of the standing is preferably 1~3min, more preferably 2min.
The present invention carries out cleaning purifying to the DNA fragmentation that magnetic bead adsorbs.In the present invention, the cleaning is preferably wrapped with solution
Include the ethanol water of B&W washing lotion and volume ratio 68~72%.
When carrying out cleaning purifying with B&W, B&W washing lotion is added in the present invention preferably into the pipe containing magnetic bead, keeps magnetic bead outstanding
Floating reaction 1~3min, preferably 2min.After reaction, magnetic bead and the separation method of B&W washing lotion are preferred are as follows: use strong magnets
Or magnetic frame absorption magnetic bead to solution is clarified, then draws supernatant with pipettor, retains magnetic bead.In the present invention, the magnetic bead is washed
Liquid preferably includes 20% (w/v) PEG8000,2M NaCl, 25mM EDTA, 0.9mM KAc.The cleaning dosage of the B&W washing lotion
Preferably 30~50 μ l, more preferably 40 μ l.In this step, the effect of the B&W washing lotion is to enhance magnetic after PCR reaction
Pearl is to the adsorption efficiency of large fragment, to achieve the purpose that remove large fragment.
When carrying out cleaning purifying with ethanol water, volumetric concentration 68 is added in the present invention preferably into the pipe containing magnetic bead
Then~72% ethanol water adsorbs magnetic bead back and forth on different two sides repeatedly with magnetic frame sufficiently to suspend and move magnetic
Pearl is sufficiently to react.After reaction, the separation method of magnetic bead and ethanol water is preferred are as follows: is inhaled with strong magnets or magnetic frame
Attached magnetic bead to solution is clarified, then draws supernatant with pipettor, retains magnetic bead.In the present invention, the volume of the ethanol water
Concentration is preferably 70%.The dosage of the ethanol water is preferably 90~110 μ l, more preferably 100 μ l.In the present invention,
The effect of the ethyl alcohol washing lotion is to remove the salt ion not adsorbed by magnetic bead and other non-purpose nucleic acid segments.
In the present invention, when cleaning includes two kinds with solution, magnetic bead first preferably is cleaned with B&W washing lotion, then use ethanol water
Solution cleans magnetic bead.Impurity residual is minimum in the magnetic bead that cleaning sequence can make.After ethanol water cleaning, the present invention is preferred
By magnetic bead in being placed at room temperature for 3~10min, ethyl alcohol in solution is made to volatilize completely.
It is attached reaction after adding A, obtains BRCA sequencing library.In the present invention, the connection reaction is preferably with 25 μ L
Linked system carry out, the linked system be complete plus A react 20 μ L systems in add 0.5 10 × Custsmart of μ L
Buffer (10mM ATP&100mM DTT), the ligase (luxuriant and rich with fragrance roc 600u/ul) of 2 μ L600U/ μ L, 2.5 μ L 10uM sequencing connect
Head.In the present invention, the program of the connection reaction is preferred are as follows: and 25 DEG C, 2h, 65 DEG C, 10min.
After obtaining BRCA sequencing library, magnetic bead liquid is preferably added into the pipe equipped with BRCA sequencing library and inhales by the present invention
Then reaction enclosure carries out cleaning purifying to the DNA fragmentation of magnetic bead absorption.In the present invention, the additive amount of the magnetic bead liquid is 0.8
~1.2 times of coupled reaction system volumes, preferably 0.9~1.1 times of coupled reaction system volume, more preferably 1.0 times connections are anti-
Answer system volume.The time of the reaction is 1~30min, preferably 1.5~5min, more preferably 2min.After reaction,
The present invention abandons supernatant, obtains magnetic bead.In this step, the separation method of supernatant and magnetic bead is preferred are as follows: PCR pipe is placed in magnetic
It is stood on power frame, the time of the standing is preferably 1~3min, more preferably 2min.
The present invention carries out cleaning purifying to the DNA fragmentation that magnetic bead adsorbs.In the present invention, the cleaning is preferably wrapped with solution
Include the ethanol water of magnetic bead washing lotion and/or 68~72.
When carrying out cleaning purifying with magnetic bead, B&W washing lotion is added in the present invention preferably into the pipe containing magnetic bead, keeps magnetic bead outstanding
Floating reaction 1~3min, preferably 2min.After reaction, magnetic bead and the separation method of B&W washing lotion are preferred are as follows: use strong magnets
Or magnetic frame absorption magnetic bead to solution is clarified, then draws supernatant with pipettor, retains magnetic bead.In this step, the magnetic bead is washed
Liquid includes 20% (w/v) PEG8000,2MNaCl, 25mM EDTA, 0.9mM KAc.The cleaning dosage of the magnetic bead washing lotion is preferred
The building of current high-throughput sequencing library is used for for 30~50 μ l, more preferably 40 μ l.In this step, the B&W washing lotion
Effect is to enhance magnetic bead to the adsorption efficiency of large fragment, to achieve the purpose that remove large fragment after PCR reaction.
When carrying out cleaning purifying with ethanol water, it is water-soluble that ethyl alcohol is added in the present invention preferably into the pipe containing magnetic bead
Then liquid adsorbs magnetic bead on different two sides repeatedly with magnetic frame back and forth and suspends and move magnetic bead sufficiently sufficiently to react.Reaction
After, the separation method of magnetic bead and ethanol water is preferred are as follows: and magnetic bead to solution, which is adsorbed, with strong magnets or magnetic frame clarifies,
Supernatant is drawn with pipettor again, retains magnetic bead.In this step, the volume ratio of the ethanol water is 68~72%, preferably
It is 70%.The dosage of the ethanol water is preferably 90~110 μ l, more preferably 100 μ l.In this step, the ethyl alcohol
The effect of washing lotion is to remove the salt ion not adsorbed by magnetic bead and other non-purpose nucleic acid segments.
The present invention preferably first cleans magnetic bead with B&W washing lotion, then cleans magnetic bead with ethanol water.The cleaning sequence can make
To magnetic bead in impurity residual it is minimum.After ethyl alcohol cleans, the present invention makes second preferably by magnetic bead in being placed at room temperature for 3~10min
Alcohol volatilizees completely.
After purification through over cleaning, the present invention elutes the DNA of magnetic bead surfaces to magnetic bead, in the present invention, the elution
Liquid is preferably Elution Buffer.The additive amount of the Elution Buffer is preferably 15~25 μ l, more preferably 20 μ l.
The Elution Buffer selects 10mM Tris-EDTA buffer solution (pH=8.0).The present invention to the DNA of magnetic bead surfaces into
After row elution, obtained eluent is the BRCA sequencing library purified.
The present invention preferably to obtain BRCA sequencing library carry out library Quality Control.The library Quality Control includes quantitative Quality Control and piece
Section Quality Control.The quantitative Quality Control is preferably measured using fluorescence quantitative PCR method, in an embodiment of the present invention, the fluorescence
Quantitative PCR commodity in use kit carry out (kit it is excellent using health be century DNA library quantification kit, commodity article No.:
CW2684M), specific steps are the specification of the DNA library quantification kit kit in century referring to health.The segment Quality Control is excellent
Choosing is measured using Agilent 2100, in an embodiment of the present invention, is carried out on 2100 biological analyser of Agilent, is had
Body step is referring to product description.
BRCA high throughput library constructing method operating procedure provided by the invention is easy, complete in 24 hours overall process used times
At more existing known to build library kit (such asTumor 170 is sequenced requirements of process 48 hours, Thermo Fish
Requirements of process 48 hours is sequenced) used time is shorter.BRCA high throughput library constructing method provided by the invention utilizes magnetic bead reaction knot
Washing operation is closed, the efficient and convenient of purifying is utmostly ensure that, the conversion ratio in library is finally made to commonly reach 95% or more,
So that the template of 10ng builds library template amount enough and can satisfy the requirement of detection as minimum, the removal of non-target fragment is also
To being obviously improved.
The present invention provides the BRCA high-throughput sequencing library that above-mentioned construction method obtains, which protects in building process
Each step in high-throughput library construction process has been stayed, has used and changes magnetic bead local environment to reach to different fragments size
The change of nucleic acid absorption ability.Simultaneously because all operationss carry out in a pipe, the utilization to template utmostly ensure that
Rate, ensure that the high conversion in library, conversion ratio generally the template quantity of 95% or more and the repeatable starting of height can be low
To 10ng；PCR enrichment process avoids base Preference to the greatest extent, so that sequencing uniformity is good, is sequenced every time
The Ratio control of most deep reading times and minimum reading times is within 5 times；Compared with the prior art, library structure of the invention
Construction method improves the utilization to template to the greatest extent, reduces the loss in library construction process, and since whole process exists
It is carried out in one purification vessel, largely avoids the cross contamination of sample room.
Below with reference to embodiment, to a kind of construction method and products thereof of BRCA high-throughput sequencing library provided by the invention
It is clearly and completely described with application.Based on the embodiments of the present invention, those of ordinary skill in the art are not making wound
All other embodiment obtained under the premise of the property made labour, shall fall within the protection scope of the present invention
Embodiment 1
A kind of construction method of BRCA high-throughput sequencing library, the generation including initial libraries, the purifying of initial libraries are surveyed
The generation in preface library, the purifying of sequencing library.
Step 1, the generation of initial libraries: the scheme that targeting target fragment is captured and expanded using multiplex PCR.
The reaction system of 0.025ml is added in 0.2ml PCR pipe, is expanded according to specific response procedures for detailed process, specific anti-
Answer system and program such as table 1, table 2.
The reaction system of table 1, initial libraries
| Ingredient | Volume |
| Polymerase Mix | 12.5μl |
| Primer library | 7μl |
| Template | 0.5 μ l (total 10ng) |
| Water | 5μl |
Table 2, initial libraries amplification program
Step 2: the purifying of initial libraries: removing primer using magnetic beads for purifying and replace reaction solution, to carry out adding A anti-
It answers.It is specific as follows:
1. it takes in the former pipe after PCR reaction amplification and 1 times of volume AMPure XP Beads (25 μ L systems add 25 μ L) is added,
Pressure-vaccum mixes.PCR pipe, which is placed on magnetic frame, abandons supernatant after being stored at room temperature 2 minutes.
2. being added 40 μ L washing lotions (20% (w/v) PEG8000,2M NaCl, 25mM EDTA, 0.9mM KAc), suspension magnetic
Pearl is stored at room temperature 2min.Magnetic bead is adsorbed with strong magnets or magnetic frame, until solution clarification.Supernatant is drawn with pipettor,
Supernatant is abandoned, magnetic bead is retained.
3. 100 μ L, 70% ethyl alcohol is added, magnetic bead is adsorbed back and forth on different two sides repeatedly with magnetic frame with the magnetic that sufficiently suspends
Pearl is just washed, and supernatant is abandoned, and retains magnetic bead.
4. being placed at room temperature for 5 minutes, ethyl alcohol is made to volatilize completely.
Step 3: plus A: the present invention using achievees the effect that using T4PNK and Klenow Fragment add A, add A anti-
It answers system as shown in table 3, adds A response procedures are as follows: 37 DEG C, 30min, 75 DEG C, 20min.
Table 3 plus A reaction system
| Component | Volume | Unit | Brand |
| Klenow Fragment | 1μL | 5U/μl | Fei Peng Biological Co., Ltd. |
| T4 PNK | 0.5μL | 10U/μL | Fei Peng Biological Co., Ltd. |
| dATP | 0.25μL | 10mM | Fei Peng Biological Co., Ltd. |
| Custsmart Buffer | 2μL | 10× | NEB biotech firm, the U.S. |
| PCR product after purification | 8μL | ||
| 100mM magnesium acetate | 0.25μL | Sigma aldrich company, the U.S. | |
| Water | 8μL | ||
| Total volume | 20μL |
It is connected after adding A:
After adding A to react, take plus the reaction solution original pipe of A, to complete plus 20 μ L systems that A reacts in add 0.5 μ L 10 ×
Custsmart Buffer (10mM ATP&100mM DTT), the ligase (luxuriant and rich with fragrance roc 600u/ul) of 2 μ L 600U/ μ L, 2.5 μ L
10uM sequence measuring joints form 25 μ L linked systems.Reaction is attached by following procedure: 25 DEG C, 2h, 65 DEG C, 10min.
Step 4 purifies product after connection, to remove extra connector and substitutional solution.
1 times of volume AMPure XP Beads (25 μ L systems add 25 μ L), pressure-vaccum are added in the reaction solution original pipe for adding A 1. taking
It mixes.PCR pipe is placed on magnetic frame in being stored at room temperature 2 minutes abandoning supernatants.
2. being added 40 μ L washing lotions (20% (w/v) PEG8000,2M NaCl, 25mM EDTA, 0.9mM KAc), suspension magnetic
Pearl is stored at room temperature 2min.Magnetic bead is adsorbed with strong magnets or magnetic frame, until solution clarification.Supernatant is drawn with pipettor,
Supernatant is abandoned, magnetic bead is retained.
3. 100 μ L, 70% ethyl alcohol is added, magnetic bead is adsorbed back and forth on different two sides repeatedly with magnetic frame with the magnetic that sufficiently suspends
Pearl is just washed, and supernatant is abandoned, and retains magnetic bead.
4. being placed at room temperature for 5 minutes, ethyl alcohol is made to volatilize completely.
5. 20 μ L Elution Buffer, sufficiently suspension magnetic bead are added, 2min is stored at room temperature with eluted dna.Magnetic bead is used
Magnet absorption, supernatant DNA solution is sequencing library.
Step 5, library Quality Control.Its Chinese library quantitatively uses quantitative fluorescent PCR.The segment Quality Control in library uses Agilent
2100 carry out, and specific steps are referring to Related product specification.
Quality Control the result is shown in Figure 1.As shown in Figure 1, by the sequencing library that method provided by the invention obtains, conversion ratio reaches
Originally 95% or more, 90% non-purpose band is removed.Wherein, Fig. 1-A, Fig. 1-B are the testing result of conversion front and back, can
See that conversion ratio is 98.61；Fig. 1-C, Fig. 1-D be before purification after result, it is seen that be to non-purpose band removal rate afterwards before purification
96.21%.
The construction method of the BRCA high-throughput sequencing library provided in this embodiment whole used time, library turned less than 24 hours
Rate is big by 95% or more, and the removal degree of non-target fragment is up to 90% or more.
Embodiment 2
Automate constructing plan
The first step injects 1 μ l template into the target hole bottom of 96 orifice plates, after 96 orifice plates are placed on 96 grillages, pop one's head in
96 orifice plate tops are protruded into, switching channel to pipeline A channel to be opened, to target hole full of first round PCR reaction solution in pipeline
The first round PCR reaction solution of 29 μ l of middle injection.96 orifice plates are removed, sealing film is sticked, concussion mixes, and centrifugation is placed in PCR instrument
It is reacted.Reaction solution component such as table 1, reaction condition such as table 2.
Second step, switching pipeline to pipeline B to take out first round PCR from PCR instrument full of magnetic bead mixed liquor in pipeline
96 orifice plates terminated are placed on grillage, and probe will be pierced at the top of PCR pipe and be stopped at pipe top, inject 15 μ L magnetic beads, are mixed and are stood 5 points
After clock comes into full contact with it, being placed on magnetic frame after 2 minutes makes magnetic bead be adsorbed in bottom, draws supernatant and is transferred to the purifying of 96 holes
Plate.
96 orifice plates are placed on grillage by third step, and probe will be pierced at the top of PCR pipe and be stopped at pipe top, inject 27 μ L magnetic beads,
It opens magnetic frame power supply and switches positive and negative anodes repeatedly, so that magnetic bead and reaction solution mix, close magnetic frame and stand 3 minutes.Again
It opens on magnetic frame power supply, so that magnetic bead is adsorbed in bottom side, draws probe and go deep into the other side, draw supernatant.
4th step switches to pipeline C, so that being full of BW13 in pipeline, pops one's head in and injects 40 μ L solution into duct, suspends mixed
It is even and stand 2min.Opening magnetic frame magnetic force makes magnetic bead be adsorbed on tube wall side, adsorbs 3min, opens and draws probe, in
Draw supernatant in the other side.
5th step switches to pipeline D, so that being full of 70% ethyl alcohol in pipeline, opens injection probe and injects into purifying pipe
100 μ L70% ethyl alcohol, 10 switch in magnetic force frame positive and negative anodes repeatedly, so that it is sufficiently outstanding to guarantee to adsorb magnetic bead back and forth in tube wall two sides
Floating magnetic bead.Last magnetic force stops at side, guarantees that magnetic bead is sufficiently adsorbed in tube wall, opens and draw probe, supernatant is drawn.It repeats
Once.
6th step switches pipeline E, so that being full of air in pipeline, opens injection probe so that air constantly injects purifying
Pipe, so that magnetic bead is dry.
7th step switches pipeline F, so that full of the reagent for adding A in pipeline, mainly by T4 PNK and Klenow
Fragment completes the step of adding A jointly, and wherein reaction system is as shown in table 3, and reaction condition is 37 DEG C, 30min, 75 DEG C,
20min。
8th step switches to pipeline B, so that being full of bead suspension in pipeline, probe is pierced into sealing film, opens injection and visits
Head injects 30 μ L bead suspensions, switch in magnetic force frame positive and negative anodes, so that magnetic bead and liquid close magnetic force power supply after mixing well, it is quiet
Setting makes the abundant adsorbed target nucleic acid fragment of magnetic bead for 3 minutes.Magnetic force power supply is opened, so that magnetic bead is adsorbed in side tube wall, opens and inhales
Probe is taken, supernatant is sucked.
9th step switches to pipeline F, opens probe, injects 40 μ L BW10, switches positive and negative anodes, so that magnetic bead is filled with BW10
Divide and mix, close magnetic frame power supply, stands 3min.Magnetic frame power supply is opened, so that magnetic bead is adsorbed in side tube wall, opens to draw and visit
Head sucks supernatant.
Tenth step switches to pipeline D, injects 70% ethanol solution, 100 μ L, and opening magnetic frame power supply and switching positive and negative anodes makes
Magnetic bead and ethanol solution mix, and magnetic bead is finally made to be adsorbed in side tube wall, open absorption probe and suck supernatant.
11st step switches to pipeline H, opens injection probe, and 15 μ L elutions of injection dissolve magnetic bead with water.
12nd step switches to pipeline F, opens injection probe, injects street corner and connection reaction solution and enzyme.25 DEG C of connections
2h。
13rd step repeats Step 8: nine.
14th step switches to pipeline G, opens injection probe, so that air enters in pipe, magnetic bead is dry.
12nd step switches to pipeline H, opens injection probe, and injection 20 μ L elution water is opened magnetic frame power supply, made
It obtains elution to be mixed well with water and magnetic bead, closes magnetic frame power supply standing after 3 minutes, magnetic frame power supply is again turned on, so that magnetic bead
It is adsorbed in tube wall side.Sealed membrane is opened, supernatant is drawn, supernatant is the sequencing library built at this time.
Note: as do not used electromagnetism, magnetic switching can be completed by the distance of mobile strong magnets.
Have been described in detail above embodiments of the present invention, but this is only to facilitate the example for understanding and lifting, it should not be by
It is considered as and limits the scope of the present invention.Equally, any person of ordinary skill in the field can technology according to the present invention
Various possible equivalent changes or replacement are made in the description of scheme and its preferred embodiment, but all these changes or replacement are all
It should belong to scope of protection of the claims of the invention.
Sequence table
<110>Anhui Ding Jing Biotechnology Co., Ltd
Shanghai Ding Jing biological medicine Science and Technology Co., Ltd.
<120>a kind of BRCA high-throughput sequencing library and its construction method and application
<160> 120
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ttttacctca gtcacataat aaggaatgca 30
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggttctaagc aacactgtga cgta 24
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ctttcttcag aagctccacc ct 22
<210> 4
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aggtttgcct aaattcctag tttgtagt 28
<210> 5
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tttcaggaag gaatgttccc aatagtag 28
<210> 6
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ccaggctctt agccaaaata ttagca 26
<210> 7
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
caccacaaag agataagtca ggtatgat 28
<210> 8
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
agatttgtaa atctcagggc aaaggt 26
<210> 9
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
agtagatgtg ctttttgatg tctgacaa 28
<210> 10
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gaccaggttt agagactttc tcaaagg 27
<210> 11
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tgactgaccg aaaaataaaa tgccaa 26
<210> 12
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
caatgtggtc tttgcagcta tttactt 27
<210> 13
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gcaaacgctg atgaatgtga aaaatcta 28
<210> 14
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
tggtcacatg aagaaatatg caataggg 28
<210> 15
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
tgaggaaaca gtggtaaata agagagatga 30
<210> 16
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
tcctccttct gtgagcaaac ag 22
<210> 17
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
aaaaataccg aaagaccaaa aatcagaact 30
<210> 18
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
cctaaacaat catgtataca gatgatgcct 30
<210> 19
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gtttattgca ttcttctgtg aaaagaagc 29
<210> 20
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
tgctttttgg atcattttca cactgtc 27
<210> 21
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
aaaagtggaa tacagtgata ctgactttca 30
<210> 22
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
tggcaacagc tcaacgtttt tataattt 28
<210> 23
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
tccagactct gaagaacttt tctcaga 27
<210> 24
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
cctctgcaag aacataaacc aaatctttt 29
<210> 25
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
caaatgggca ggactcttag gt 22
<210> 26
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
ctgaggcttg ctcagtttct tttg 24
<210> 27
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
cctcagatgt tattttccaa gcaggat 27
<210> 28
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
gcattcatta tgacatgaag atcagcatct 30
<210> 29
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
gcttttattc tgctcatggc acaa 24
<210> 30
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
caacaaaagt gccagtagtc atttcaatat 30
<210> 31
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
gaagataaca aatatactgc tgccagtaga 30
<210> 32
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
ttgagctttc gcaacttcca aaaa 24
<210> 33
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
gaaaaatatt agtgtcgcca aagagtcatt 30
<210> 34
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
ccctggaagg tcactagttg atttc 25
<210> 35
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
ggttttcata cagctagcgg gaaa 24
<210> 36
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
accacagtct caatagaaac aaggttttta 30
<210> 37
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
agtacatgaa aatgtagaaa aagaaacagc a 31
<210> 38
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
gaccatcaaa tattccttct ctaagcca 28
<210> 39
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
atgacaaaaa tcatctctcc gaaaaacaag 30
<210> 40
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
caagttcctc aacgcaaata tcttcatt 28
<210> 41
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
ggtagggcca cctgcattta 20
<210> 42
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
catccaatgc ctcgtaacaa cc 22
<210> 43
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
acataaggtt tttgctgaca ttcagagt 28
<210> 44
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
tgatacctgg acagattttc cacttg 26
<210> 45
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
gaccagctca caagagaaga aaatact 27
<210> 46
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
ctgggtttct cttatcaaca cgaggaagta ttt 33
<210> 47
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
caacaagaca aacaacagtt ggtattagg 29
<210> 48
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
tgtcagttca tcatcttcca taaaagctt 29
<210> 49
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
aaagagtcaa tactttagct ttaaaaaaat ggt 33
<210> 50
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
gtggatcacc tgaggtcaga at 22
<210> 51
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
gctgatttct gttgtatgct tgtactg 27
<210> 52
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
gtaatcggct ctaaagaaac atgatgc 27
<210> 53
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
gtgaggtaga ttgtaaagtc aaaggct 27
<210> 54
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
aggtgcggta aaatttggat tctgta 26
<210> 55
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
caaccaaagt ctttgttcca ccttttaa 28
<210> 56
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
caacggaaat atctaactga aaggcaaa 28
<210> 57
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
aaaggtacag cagactgtgg aatgt 25
<210> 58
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
aaagactctg catttttgct gttaattttt 30
<210> 59
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
gttgttgaat tcagtatcat cctatgtggt 30
<210> 60
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
tgccgtatat gattacgtaa tgtaatgct 29
<210> 61
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
gcaataaaac tagtagtgca gataccca 28
<210> 62
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
caatgactga tttttaccaa gagtgcaa 28
<210> 63
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
ttcagtgatg gaggaaatgt tggtt 25
<210> 64
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
gtggtggctc agctacttga g 21
<210> 65
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
caaaatatgt ggaggcccaa caa 23
<210> 66
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
tgttgctatt ctttgtctaa caccaaaaa 29
<210> 67
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
tgagctctaa ttttgttgta tttgtcctgt 30
<210> 68
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
acttccacac ggttgtgaca tc 22
<210> 69
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
tgataatcac ttcttccatt gcatctttct 30
<210> 70
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
atggagattc cataaactaa caagcactt 29
<210> 71
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
gagccccttc acttcagcaa 20
<210> 72
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
actggtagct ccaactaatc ataagagat 29
<210> 73
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
ccattctagg acttgcccct ttc 23
<210> 74
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
atgtgtggtg atgctgaaaa gtaac 25
<210> 75
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
gggagggaga ctgtgtgtaa tattt 25
<210> 76
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 76
agttcttttg gtcatcaatc tctttctcc 29
<210> 77
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 77
aagagaagag ccttggattt cttgag 26
<210> 78
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 78
cctgttgaac cagacaaaag agc 23
<210> 79
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 79
ggtcctgtgg ctctgtacct gt 22
<210> 80
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 80
aggaccctgg agtcgattga tt 22
<210> 81
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 81
ggcagagaag acttctgagg cta 23
<210> 82
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 82
cttcatccgg agagtgtagg gta 23
<210> 83
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 83
gtgaaagtat ctagcactgt gtatgtatgt 30
<210> 84
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 84
acttccattg aaggaagctt ctctt 25
<210> 85
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 85
gtaactcaga ctcagcatca gca 23
<210> 86
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 86
aaaactgagg ctctttagct tcttagg 27
<210> 87
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 87
attaaattac acagaactgt gattgttttc tag 33
<210> 88
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 88
gctcatacta ctgatactgc tgggta 26
<210> 89
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 89
aaaatcaaag tgtttgttcc aatacagca 29
<210> 90
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 90
caattggtgg cgatggtttt ctc 23
<210> 91
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 91
aaggctcaga tacaaacaca gctatt 26
<210> 92
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 92
gaacagtacc cgttcccttg a 21
<210> 93
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 93
agatgatgtc agcaaaccta agaatgt 27
<210> 94
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 94
ccagtcctgc caatgagaag aa 22
<210> 95
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 95
tccaatacct aagtttgaat ccatgctt 28
<210> 96
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 96
caaaggcatc tcaggaacat cac 23
<210> 97
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 97
ggaagcaggg aagctcttca tc 22
<210> 98
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 98
tcatgcatct caggtttgtt ctga 24
<210> 99
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 99
tccaggaaga ctttgtttat agacctca 28
<210> 100
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 100
ctgaaagaga aatgggaaat gagaacattc 30
<210> 101
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 101
atttggagta atgagtccag tttcgtt 27
<210> 102
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 102
agaagaggaa tgtgcaacat tctctg 26
<210> 103
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 103
tggatactta aagccttctg tgtcatttc 29
<210> 104
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 104
cccaaagatc tcatgttaag tggagaaa 28
<210> 105
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 105
catttgttaa cttcagctct gggaaa 26
<210> 106
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 106
caaattgata gttgttctag cagtgaagag 30
<210> 107
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 107
attcgagttc catattgctt atactgct 28
<210> 108
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 108
tcctgaggat tttatcaaga aagcagattt 30
<210> 109
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 109
ccttcttccg ataggttttc ccaaatatt 29
<210> 110
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 110
agaaagttaa tgagtggttt tccagaagt 29
<210> 111
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 111
gctgggagtc cgcctatcat ta 22
<210> 112
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 112
gttctgtttc aaacttgcat gtgga 25
<210> 113
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 113
aggtcccaaa tggtcttcag aataatc 27
<210> 114
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 114
gctttctgta atcgaaagag ctaaaatgtt 30
<210> 115
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 115
gcctaccaca aatacaaatt atgaccaag 29
<210> 116
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 116
ttctcttcag gaggaaaagc acag 24
<210> 117
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 117
accacgtcat agaaagtaat tgtgcaa 27
<210> 119
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 119
aaggttgata atcacttgct gagtgt 26
<210> 119
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 119
cttctataaa gttaggtgtt tcctgggtt 29
<210> 120
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 120
tcagtcataa cagctcaaag ttgaacttat 30
Claims (10)
1. a kind of construction method of BRCA high-throughput sequencing library, which comprises the steps of:
(1) using include class mammary gland tumor susceptibility gene DNA fragmentation as template, multiplexed PCR amplification is carried out in a reservoir, at the beginning of obtaining BRCA
Beginning library；
(2) the magnetic bead liquid of 0.8~1.2 times of PCR reaction system volume is added in Xiang Suoshu container, after reacting 1~30min, in abandoning
Clear liquid obtains the first magnetic bead；
(3) cleaning purifying is carried out to the BRCA initial libraries adsorbed on step (2) first magnetic bead；
(4) the BRCA initial libraries of the cleaning after purification are successively carried out plus A reacts and connection is reacted, obtain BRCA sequencing text
Library；
(5) the magnetic bead liquid of 0.8~1.2 extraordinarily A reaction system volume is added into the former container equipped with the BRCA sequencing library,
After reacting 1~30min, supernatant is abandoned, the second magnetic bead is obtained；
(6) cleaning purifying is carried out to step (5) second magnetic bead, obtains purifying magnetic ball；
(7) DNA of the purifying magnetic bead surfaces is eluted, the eluent obtained after connection is the BRCA sequencing library purified；
Step (1)~(7) operate in a same vessel.
2. construction method according to claim 1, which is characterized in that the template be B150A_1~60 or B150B_1~
60；When the template is B150A_1~60, amplification B150A_1~60 with the nucleotide sequence such as SEQ ID No.1 of primer~
Shown in 120.
3. construction method according to claim 1, which is characterized in that multiplexed PCR amplification uses 25 μ l in the step (1)
PCR reaction system, the PCR reaction system includes the component of following content: 12.5 μ L polymerase Mix, 5 μ L water, 7 μ L primers
Library, 0.5 μ L are nucleic acid-templated.
4. construction method according to claim 3, which is characterized in that the multiplexed PCR amplification program are as follows: 98 DEG C, 3min；
98 DEG C, 30s, 58 DEG C, 15s, 22 circulations；72 DEG C, 15s, 72 DEG C, 4min.
5. construction method according to claim 1, which is characterized in that the solvent for cleaning in the step (3) includes magnetic
The ethanol water of pearl washing lotion and volume ratio 68~72%.
6. construction method according to claim 1, which is characterized in that the step (4) plus A react and add A system with 20 μ l
It carrying out, described plus A system includes the T4 PNK of the Klenow Fragment, 0.5 μ L 10U/ μ L of 1 μ L 5U/ μ L, 2 μ L 10 ×
Custsmart Buffer, 0.25 μ L 10mM dATP, 0.25 μ L 100mM magnesium acetate, the purified PCR of 8 μ L water and 8 μ L
Product, the total amount of the PCR product are 100ng.
7. construction method according to claim 6, which is characterized in that described plus A response procedures are as follows: 37 DEG C, 30min, 75
DEG C, 20min.
8. construction method according to claim 1, which is characterized in that the connector of 25 μ L of step (4) the connection reaction
System carries out, and the linked system is to add 0.5 μ 10 × Custsmart of L Buffer, 2 μ in the 20 μ L systems for completing that A is added to react
The ligase of L 600U/ μ L, 2.5 μ L 10uM sequence measuring joints.
9. construction method according to claim 8, which is characterized in that the connection response procedures are as follows: 25 DEG C, 2h, 65 DEG C,
10min。
10. the BRCA high-throughput sequencing library that construction method described in claim 1~9 any one obtains.
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