CN110079868A - BRCA1/2 genetic mutation detects library constructing method and kit - Google Patents
BRCA1/2 genetic mutation detects library constructing method and kit Download PDFInfo
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- CN110079868A CN110079868A CN201910211682.3A CN201910211682A CN110079868A CN 110079868 A CN110079868 A CN 110079868A CN 201910211682 A CN201910211682 A CN 201910211682A CN 110079868 A CN110079868 A CN 110079868A
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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
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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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
- 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 relates to BRCA1/2 genetic mutation detection library constructing method and kits.The present invention provides a kind of amplicon library constructing method of BRCA1/2 gene, the method includes carrying out targeting amplification comprising BRCA1/2 gene region in sample, the targeting amplification includes 1) using the sample as template, first round PCR amplification is carried out by the first primer, 2) using the product of first round PCR amplification as template, the second wheel PCR amplification is carried out by the second primer, and 3) the amplification sublibrary of BRCA1/2 gene is constructed by third round PCR amplification using the second wheel pcr amplification product as template.The present invention also provides the kits of amplification sublibrary and/or detection BRCA1/2 genetic mutation for constructing BRCA1/2 gene.Method and kit of the invention can carry out PCR reaction in a Reagent Tube, and simple and quick building expands sublibrary, significantly reduce the fussy degree of operation and the demand to detection sample, improve the total quality of sequencing data.
Description
Technical field
The present invention relates to genetic test library constructing method and products.Specifically, the present invention relates to BRCA1/2 bases
Because of the library constructing method and kit of variation detection.
Background technique
3,000,000,000 dollars of cost last the Human Genome Project (Human Genome Proiect, HGP) completed in more than 10 years
Snafu variation is brought to genomics research, by measuring the sequence of human genome DNA, gene is sought and is dyeing
The structure and function of gene is specified in position on body, interprets whole hereditary information of the mankind, mankind's first time is on a molecular scale
Full appreciation self.New-generation sequencing technology has expedited the emergence of colourful Genome Atlas.
New-generation sequencing technology is also known as large-scale parallel sequencing (massively parallel sequencing, MPS),
Refer to the principle of use " sequencing of the side Bian Hecheng ", for hundreds of thousands to millions of DNA moleculars while carrying out parallel sequencing reaction,
Then pass through the obtained raw image data of bioinformatic analysis or electrochemical signals, the nucleic acid for finally obtaining sample to be tested
The sequencing technologies of the information such as sequence or copy number, also known as high-flux sequence, deep sequencing etc..
However, some researchs do not need that full-length genome is sequenced, and specific genome area need to only be carried out
Research.Amplicon sequencing is exactly the primer by designing interested genome area, by PCR amplification, by target area domain dna
The research strategy of high-flux sequence is carried out after enrichment.
Amplicon sequencing technologies would generally use a large amount of primer, common in order to avoid the generation of a large amount of primer dimers
Method be to split reaction system to multitube from a pipe, to reduce the quantity of primer in every tube reaction, reduce primer dimerization
The generation of body.But this method, it considerably increases the fussy degree of operation and detects the demand of sample.Meanwhile it is main at present
Amplicon database technology, when being expanded to target area, it will usually use multi-cycle PCR program, due to different primers it
Preceding amplification efficiency is different, passes through two-way primer exponential type PCR amplification, after multi-cycle PCR program, the homogeneity of amplification region
It will appear marked difference, sequencing data quality may finally be caused integrally to be deteriorated.
Summary of the invention
Deficiency existing for the amplicon sequencing technologies that the present invention is used for current new-generation sequencing process carries out specific aim
Research proposes new BRCA1/2 genetic mutation detection library constructing method, substantially reduces entire library structure when variation detection
Workload needed for building process, while reducing the demand to sample size.
In some embodiments, the present invention provides a kind of amplicon library constructing method of BRCA1/2 gene, the side
Method includes carrying out targeting amplification comprising BRCA1/2 gene region in sample, and the targeting amplification includes 1) being with the sample
Template, by the first primer carry out first round PCR amplification, 2) using the product of first round PCR amplification as template, pass through the second primer
It carries out the second wheel PCR amplification and 3) using the second wheel pcr amplification product as template, BRCA1/ is constructed by third round PCR amplification
The amplification sublibrary of 2 genes.In some embodiments, the sample may include the genomic DNA or logical from subject
Cross the cDNA of reverse transcription acquisition.In some embodiments, the sample may include the BRCA1/2 gene from subject.
In some embodiments, the BRCA1/2 gene from subject may include wild type BRCA1/2 gene or variation BRCA1/
2 genes.
In some embodiments, the first round PCR amplification in method of the invention and/or the second wheel PCR amplification can be with
Recurring number appropriate is carried out, such as carries out 1-10 circulation, such as 1,2,3,4,5,6,7,8,9,10 circulations.In some implementations
In scheme, first round PCR amplification and/or the second wheel PCR amplification in method of the invention only carry out 1 circulation, i.e., one change
Property, annealing, extend circulation.In some embodiments, a denaturation, annealing in method of the invention, extend can be in circulation
Including one or many annealing, such as 1,2,3,4,5 annealing.It has been found that first round PCR amplification and/or the second wheel PCR expand
Energization, which enough passes through, only carries out the expansion that 1 circulation (preferably repeatedly being annealed in 1 circulation) constructs high quality BRCA1/2 gene
Increase sublibrary.In some embodiments, the sequencing depth of the amplification sublibrary of the BRCA1/2 gene of method of the invention building
It is distributed more uniform.In some embodiments, be denaturalized, the temperature annealing, extend circulation can according to the first of use and/or
The selection appropriate of second primer.In some embodiments, first round PCR amplification and/or the second wheel PCR in method of the invention
The annealing temperature of amplification is not particularly limited, such as can use any annealing temperature between 55 DEG C -70 DEG C, such as 55 DEG C,
56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C, 65 DEG C, 66 DEG C, 67 DEG C, 68 DEG C, 69 DEG C, 70 DEG C or
Temperature between any.In some embodiments, first round PCR amplification and/or the second wheel PCR amplification in method of the invention
Annealing time be not particularly limited, such as can anneal 30-180 seconds, such as 30 seconds, 45 seconds, 60 seconds, 75 seconds, 90 seconds, 105
Second, the time between 120 seconds, 135 seconds, 150 seconds, 165 seconds, 180 seconds or any.In some embodiments, the annealing packet
Include the different temperatures annealing between 55 DEG C -70 DEG C.In some embodiments, such as the annealing may include moving back at 57 DEG C
Fire 30 seconds, 59 DEG C are annealed 30 seconds, and 61 DEG C are annealed 30 seconds for annealing 30 seconds and 62 DEG C, and total annealing time is between 30-180 seconds.It lifts
For example, the annealing may include annealing 15 seconds at 55 DEG C, and 56 DEG C are annealed 30 seconds, 61 DEG C of annealing annealing 15 in 45 seconds and 62 DEG C
Second, total annealing time is between 30-180 seconds.
In some embodiments, the first primer in the method for the present invention and the second primer can be respectively forward primer and
Reverse primer, or vice versa, i.e., the first primer and the second primer can be respectively reverse primer and forward primer.
In some embodiments, the first primer in the method for the present invention and the second primer include targeting BRCA1/2 gene
Specific sequence.In some embodiments, as it is known to the person skilled in the art, can to amplified production add connector or
Index sequence.In some embodiments, the first primer in the method for the present invention and the second primer can also comprising connector and/
Or index sequence.In some embodiments, connector or index sequence can add in third round PCR amplification.Some
In embodiment, connector and/or index sequence in the method for the present invention are not particularly limited, and those skilled in the art can adopt
Use sequence appropriate as connector and/or index sequence.
In some embodiments, the first round PCR in the method for the present invention, the second wheel PCR and/or third round PCR include
Purification step.The method purified to pcr amplification product is well known to those skilled in the art, for example, can by magnetic bead into
The row purification step.In some embodiments, first round PCR of the invention, the second wheel PCR and/or third round PCR (packet
Include purification step) it is carried out in a Reagent Tube.As described above, amplicon is surveyed in order to avoid the generation of a large amount of primer dimers
It usually requires to split reaction system into multitube the quantity for reducing primer in every tube reaction from a pipe in sequence technical process, reduce
The generation of primer dimer.In contrast, method of the invention can carry out a small number of circulations using single primer and (such as only carry out one
A circulation), PCR can be carried out in a Reagent Tube without splitting PCR reaction system from a pipe to multitube.Some
In embodiment, method of the invention significantly reduces the fussy degree of operation.More importantly method of the invention passes through
It is carried out in single Reagent Tube, the demand to detection sample can be reduced, so that in the prior art not due to sample size
Foot is possibly realized without the detection that can be carried out.By reducing operating procedure and reducing sample requirement, method of the invention can also
Error caused by many more manipulations is reduced, the sensitivity and reliability of detection is improved, improves the total quality of sequencing data.
In some embodiments, the first primer in the method for the present invention and/or the second primer include following one or more
(connector and/or index sequence that following sequences include can be replaced the specific sequence of targeting BRCA1/2 gene in a primer
For any connector appropriate and/or index sequence, as long as the specific sequence for wherein targeting BRCA1/2 gene is constant):
In some embodiments, the method for the present invention includes the specific sequences of the listed targeting BRCA1/2 gene of this paper
One or more.In some embodiments, the specific sequence of listed targeting BRCA1/2 gene can be mutual group herein
It closes, it is preferable to employ the combinations that forward primer and reverse primer match.In some embodiments, the method for the present invention includes this
Any pair in the specific sequence of literary listed targeting BRCA1/2 gene or multipair forward primer and reverse primer are (i.e. listed
The matched primer pair of primer numbers, such as the primer pair of BRCA1_17_F1 and BRCA1_17_R1).
In some embodiments, other than the specific sequence of targeting BRCA1/2 gene, sequence of the invention can be with
Comprising any connector and/or primer appropriate for high-flux sequence analysis, such sequence is analyzed according to high-flux sequence
Platform it is different and can be different, and be known to the skilled in the art.For example, in some embodiments, it is appropriate
Sequence may include the connector and/or primer for being suitble to Illumina sequencing analysis.In some embodiments, sequence packet appropriate
Include one or more of for example following sequences:
TruSeq Universal Adapter:
5′AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 3′；
TruSeq Indexed Adapter:5 ' GATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCT
CGTATGCCGTCTTCTGCTTG3′；
PCR Primer 1.0:5 ' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA 3 '；
PCR Primer20:5 ' CAAGCAGAAGACGGCATACGAGAT 3 '；
Flow cell is anchored TruSeq Universal Adapter:5 ' AATGATACGGCGACCACCGA3 '；
Flow cell is anchored TruSeq Indexed Adapter:5 ' CAAGCAGAAGACGGCATACG 3 '；
Read 2Sequencing Primer:5 ' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 3 '；
Multiplexing Index Read Sequencing Primer:5 ' GATCGGAAGAGCACACGTCTGAAC
TCCAGTCAC 3′；Read1Sequencing Primer:5 ' ACACTCTTTCCCTACACGACGCTCTTCCGATCT 3 '.
In some embodiments, it has been found that the parameter of existing design of primers Primary Reference is that the Tm value of primer and GC contain
Amount and primer length etc., but the amplification that such design of primers principle based on Tm can not eliminate archaeal dna polymerase introducing is inclined
Good property causes amplification Preference more serious, may significantly affect the accuracy of testing result, therefore preferably tie according to experiment
Fruit is adjusted the initiation site and termination site of BRCA1/2 primer specificity target area, selects advantageous primer sequence.
In some embodiments, inventor has found the BRCA1/2 specific primer sequence and existing primer sequence of above-mentioned adjustment
The amplification Preference that can significantly reduce archaeal dna polymerase is compared in building library, improves the overall accuracy of testing result.In some realities
It applies in scheme, first round PCR amplification and/or the second wheel PCR amplification are included in 55 DEG C of -70 DEG C of annealing 30- in method of the invention
180 seconds, the optionally described annealing included that the different temperatures between 55 DEG C -70 DEG C is annealed 30-180 seconds.In some embodiments,
First round PCR amplification and/or the second wheel PCR amplification are included in 57 DEG C, 59 DEG C, 61 DEG C and 62 DEG C each annealing in method of the invention
30 seconds.In some embodiments, first round PCR amplification and/or the second wheel PCR amplification are included in 52 in method of the invention
DEG C, 55 DEG C, each annealing 30 seconds of 58 DEG C and 61 DEG C.
In some embodiments, the present invention provides a kind of kit, and the kit includes container, the container difference
PCR reagent comprising being appropriate for the method for the invention, such as archaeal dna polymerase, primer and PCR buffer.In some implementations
In scheme, the container includes one or more primers of the specific sequence of listed targeting BRCA1/2 gene herein.Some
In embodiment, the kit can be used for constructing the amplification sublibrary and/or detection BRCA1/2 gene of BRCA1/2 gene
Variation.
In some embodiments, the present invention provides the method for detection BRCA1/2 genetic mutation, and the method includes passing through
Method described herein prepares high-throughput sequencing library, then carries out high-flux sequence, to detect whether BRCA1/2 gene is deposited
It is making a variation.In some embodiments, the present invention provides the kit of detection BRCA1/2 genetic mutation, and the kit includes
Container, the container separately include the PCR reagent for being appropriate for the method for the invention, such as archaeal dna polymerase, primer and PCR
Buffer.In some embodiments, the container includes one of the specific sequence of listed targeting BRCA1/2 gene herein
Or multiple primers.In some embodiments, the method can be obtained to the genomic DNA from subject or by reverse transcription
It is detected in the cDNA obtained with the presence or absence of BRCA1/2 genetic mutation.In some embodiments, from the BRCA1/ of subject
2 genes may include wild type BRCA1/2 gene or variation BRCA1/2 gene.In some embodiments, the present invention provides this
The kit of invention is preparing the purposes in the product (such as detection kit) for detecting BRCA1/2 genetic mutation.
In some embodiments, method of the invention greatly reduces primer dimerization using single primer PCR Library development flow
The possibility that body is formed and the influence to Library development flow may finally realize that the detection of BRCA1/2 genetic mutation is completed in a reaction.
In some embodiments, method of the invention is used sets suitable for the improved primer of the method for the present invention
Meter, improves the total quality of sequencing data.
In some embodiments, method of the invention use improved primer, during multiplex PCR, can reduce by
The amplification Preference that archaeal dna polymerase introduces, to improve the overall accuracy of testing result.
In some embodiments, method of the invention carries out building library using single primer PCR.Amplicon sequencing is carried out at present
The upstream and downstream primer of pairing is added in reaction system by the library construction process of use simultaneously simultaneously when carrying out multiplex PCR.It should
Process exists significantly staggeredly when primer quantity is more or between primer location, easily formation primer dimer.And it is different
The difference of primer amplification efficiency is gradually amplified with the exponential amplification of PCR, and sequencing data quality may finally be caused whole
It is deteriorated.The single primer amplification method that the present invention uses, by forward primer and reverse primer be allocated as being put into twice in reaction system into
Row reaction, reduces the possibility of dimer formation, simultaneously because only single primer participates in PCR process, the growth of amplified production
Mode is linear increase, greatly reduces the difference of PCR efficiency between different primers, to improve the whole matter of sequencing data
Amount.
In some embodiments, invention broadly provides a kind of novel detect in people's BRCA1/2 genetic mutation to try
Agent box.Deficiency existing for the amplicon sequencing technologies that the present invention is used for current new-generation sequencing process optimizes, significantly
Workload needed for shortening entire library construction process, while reducing the demand to sample size.
Term explanation:
Amplicon builds library: amplicon library construction method, is used to be enriched with targeting region of DNA domain using multiple PCR technique,
Method to construct new-generation sequencing library.
Tm value: DNA melting temperature, ultraviolet absorption value reaches maximum value during referring to the double-spiral structure thermal denaturation DNA
1/2 when temperature.
Genomic library: the full gene group DNA of certain biology is cut into the collection of the DNA fragmentation Clone formation of certain length
It closes, can be used for the amplification, Sanger sequencing or new-generation sequencing in downstream.
New-generation sequencing technology: also known as large-scale parallel sequencing (massively parallel sequencing
(MPS)), refer to the principle of use " sequencing of the side Bian Hecheng ", for hundreds of thousands to millions of DNA moleculars while carrying out parallel survey
Then sequence reaction passes through the obtained raw image data of bioinformatic analysis or electrochemical signals, finally obtains to test sample
The sequencing technologies of the information such as the nucleic acid sequence of product or copy number, also known as high-flux sequence, deep sequencing, next-generation sequencing etc..
Detailed description of the invention
Fig. 1 shows the library construction process that the sequencing of existing amplicon uses, when carrying out multiplex PCR simultaneously will pairing up and down
Trip primer is added in reaction system simultaneously.
The exemplary flow for the single primer amplification method that Fig. 2 display present invention uses.
Fig. 3 shows method and existing method (CleanPlex BRCA1&2panel, PARAGON-916005) of the invention
Exemplary comparative.
Fig. 4 show existing method (CleanPlex BRCA1&2panel, PARAGON-916005) product in primer or
The presence of primer dimer.
Fig. 5 shows primer free and dimer residual in the method for the present invention product.
Fig. 6 shows the library constructed using PARAGON-916005 and the present invention, and the homogeneity point of depth is sequenced after sequencing
Analysis.Depth peak is sequenced in the present invention and minimum difference is smaller, and whole sequencing depth distribution is more uniform.
Specific embodiment
BRCA1/2 library construction
1 main agents and instrument
2 experimental implementations
2.1 Forward Primer-PCR
2.1.1 Hot-Start is usedDNA P0lymerase in a Ready-to-Use Master
Mix, in PCR pipe, according to the form below configures F-PCR mix:
2.1.2 it is simply centrifuged after the slight mixing that is vortexed, PCR pipe is placed in PCR instrument, following procedure is run:
The purifying of 2.2 F primer PCR products
2.2.1 agencourt ampu0re XP-PCR purification beads is taken out from 4 DEG C of refrigerators in advance,
Equilibrium at room temperature 30min；
2.2.2 ampure beads after equilibrium at room temperature, which is vortexed, mixes, and takes 48ul ampure beads (1.2X) to new
1.5mL centrifuge tube, and previous step 40ul PCR product is all transferred to；
2.2.3 PCR product and magnetic bead are vortexed and are mixed, be incubated at room temperature 5min；
2.2.4 after the completion of being incubated for, centrifuge tube is placed on magnetic frame and stands 2-3min, after solution clarification, inhaled and abandon solution,
Period never contacts magnetic bead；
2.2.5 80% fresh ethyl alcohol 200ul is added in centrifuge tube, stands 30s, after, it inhales and abandons ethyl alcohol, repeated washing one
Time；
2.2.6 it inhales after abandoning ethyl alcohol, magnetic bead is dried to surface in frosted shape, after magnetic bead dries, 11ul is added
Nuclease-free H2O is incubated at room temperature 3min；
2.2.7 after the completion of being incubated for, centrifuge tube is placed on magnetic frame, stands 2-3min, after solution clarification, draws 10ul
Solution is transferred in new PCR pipe, and being drawn to a small amount of magnetic bead will not influence subsequent experimental；
23 Reverse Primer-PCR
2.3.1 Hot-Start is usedDNA Polymerase in a Ready-to-Use Master
Mix, in PCR pipe, according to the form below configures R-PCR mix:
2.3.2 it is simply centrifuged after the slight mixing that is vortexed, PCR pipe is placed in PCR instrument, following procedure is run:
2.4 Reverse Primer-PCR product purifications
2.4.1 the ampure beads after equilibrium at room temperature, which is vortexed, mixes；
2.4.2 it takes 48ul ampure beads (1.2X) to new 1.5mL centrifuge tube, and previous step 40ul PCR is produced
Object is all transferred to；
2.4.3 PCR product and magnetic bead are vortexed and are mixed, be incubated at room temperature 5min；
2.4.4 after the completion of being incubated for, centrifuge tube is placed on magnetic frame and stands 2-3min, after solution clarification, inhaled and abandon solution,
Period never contacts magnetic bead；
2.4.5 80% fresh ethyl alcohol 200ul is added in centrifuge tube, after standing 30s, inhales and abandons ethyl alcohol, repeated washing one
Time；
2.4.6 it inhales after abandoning ethyl alcohol, magnetic bead is dried to surface in frosted shape, after magnetic bead dries, 21ul is added
Nuclease-free H2O is incubated at room temperature 3min；
2.4.7 after the completion of being incubated for, centrifuge tube is placed on magnetic frame, stands 2-3min, after solution clarification, draws 20ul
Solution is transferred in new PCR pipe；
2.5 Index-PCR
2.5.1 following reaction system is prepared in the 0.2ml PCR Tube of 2.4.7；
2.5.2 it is vortexed and mixes, following reaction is carried out after centrifugation:
The recycling of 2.6 PCR products
2.6.1 previous step 50ul reaction product is transferred completely into 1.5ml centrifuge tube, adds 60ul to be vortexed uniform
AMpure Beads (1.2x) is vortexed and mixes, is statically placed on magnetic frame after being placed at room temperature for 5min, AMpure Beads is kept completely separate
After abandon supernatant.
2.6.2 it keeps sample to be placed on magnetic frame, adds 80% ethyl alcohol of 200ul, remove supernatant after 30s, then repeat washing
Once；
2.6.3 it dries to dripless and remains；
2.6.4 plus 41ul Nuclease-free water is vortexed and mixes, and is statically placed on magnetic frame after being placed at room temperature for 5min,
Transfer 40ul supernatant is into 1.5ml centrifuge tube after AMpure Beads is kept completely separate；
2.6.5 it is uniform that 48ul AMpure Beads (1.2X) mixed is added into above-mentioned centrifuge tube, after being placed at room temperature for 5min
It is statically placed on magnetic frame, AMpure Beads abandons supernatant after being kept completely separate；
2.6.6 2.6.2-2.6.3 step 1 time is repeated；
2.6.7 plus 21ul Nuclease-free water is vortexed and mixes, and is statically placed on magnetic frame after being placed at room temperature for 5min,
Transfer 20ul supernatant is into 1.5ml centrifuge tube after AMpure Beads is kept completely separate；
2.7 product QC
2.7.1 reagent and instrument operation instruction are referred to, using Qubit dsDNA HS Assay Kit in 3.0 instrument of Qubit
The enterprising style of writing library Concentration Testing of device.
2.7.2 reagent and instrument operation instruction are referred to, 24DNA Extended Range Labchip and HT DNA are used
High Sens Reagent Kit, Dual Protocol, in the enterprising style of writing library clip size of Labchip GX Touch instrument
Detection.
3. method of the invention is compared with existing method (see, for example, Fig. 3)
Advantage of the present invention:
1. reducing cumbersome degree and the need to initial sample size it is not necessary that initial sample is divided into two parts carry out library construction
It asks.
2. two-way primer is carried out in two steps amplification when targeting amplification, and 1 PCR cycle is only needed, reduces primer two
A possibility that aggressiveness formation, reduce PCR duration, the variation of PCR exponential amplification bring homogeneity will not be introduced.
3. integrated operation duration substantially reduces.
With existing method (such as commercial prod CleanPlex BRCA1&2panel,
PARAGON-916005 comparison):
1. integrated operation duration shortens about half an hour:
2. Library Quality:
A. picture is derived from CleanPlex BRCA1&2panel, Quality Control in PARAGON-916005 product description in Fig. 4
Part illustrates, it is observed that still having a small amount of primer or primer dimer in product:
B. it is product Quality Control of the present invention in Fig. 5, it can be seen that primer free and dimer residual:
3. sequencing result is carried out homogeneity analysis (Fig. 6), depth peak is sequenced in the present invention and minimum difference is smaller,
Whole sequencing depth distribution is more uniform:
List of primers
Claims (10)
1. a kind of amplicon library constructing method of BRCA1/2 gene, the method includes to including BRCA1/2 gene in sample
Region carries out targeting amplification, and the targeting amplification includes carrying out first round PCR by the first primer 1) using the sample as template
Amplification, 2) using the product of first round PCR amplification as template, the second wheel PCR amplification is carried out by the second primer and 3) with second
Wheel pcr amplification product is template, and the amplification sublibrary of BRCA1/2 gene is constructed by third round PCR amplification.
2. method described in claim 1, wherein first round PCR amplification and/or the second wheel PCR amplification only carry out 1 circulation.
3. the described in any item methods of claim 1-2, wherein the first primer and the second primer are respectively forward primer and reversed
Primer, or respectively reverse primer and forward primer.
4. the described in any item methods of claim 1-3, wherein the first primer and the second primer include targeting BRCA1/2 gene
Specific sequence optionally also includes connector and/or index sequence.
5. the described in any item methods of claim 1-4, wherein the sample includes wild type or variation BRCA1/2 gene sequence
Column.
6. the described in any item methods of claim 1-5, wherein first round PCR, the second wheel PCR and/or third round PCR include pure
Change step, such as purification step is carried out by magnetic bead, the optionally described first round PCR, the second wheel PCR and/or third round PCR exist
It is carried out in one Reagent Tube.
7. method described in any one of claims 1-6, wherein the first primer and/or the second primer include following one or more
The specific sequence of BRCA1/2 gene is targeted in primer:
8. the described in any item methods of claim 1-7, wherein first round PCR amplification and/or the second wheel PCR amplification are included in 55
DEG C -70 DEG C annealing 30-180 seconds, the optionally described annealing includes that the different temperatures between 55 DEG C -70 DEG C is annealed 30-180 seconds.
9. a kind of kit, the kit includes container, and the container, which separately includes, is appropriate for any one of claim 1-8
The PCR reagent of the method, such as archaeal dna polymerase, primer and PCR buffer.
10. kit as claimed in claim 9 is used to construct the amplification sublibrary and/or detection BRCA1/ of BRCA1/2 gene
2 genetic mutations.
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Citations (10)
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