CN105696089A - DNA (deoxyribonucleic acid) library construction method for breast cancer genes BRCA on basis of Ion Torrent sequencing platform - Google Patents
DNA (deoxyribonucleic acid) library construction method for breast cancer genes BRCA on basis of Ion Torrent sequencing platform Download PDFInfo
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Abstract
The invention discloses a DNA (deoxyribonucleic acid) library construction method for breast cancer genes BRCA on the basis of an Ion Torrent sequencing platform. The construction method comprises steps such as enrichment of target fragments through multiple PCRs (polymerase chain reactions), primer clearing, linker connection, library amplification, library detection and the like. According to the construction method, multiple pairs of PCR primers are adopted for enrichment and amplification of the target fragments, the operation is simple, the cost is low, loss of a library is reduced, and the success rate of the library construction is increased. The library construction method is suitable for DNA library sequencing of BRCA1 and BRCA2 on the Ion Torrent sequencing platform.
Description
Technical field
The present invention relates to gene sequencing field, particularly high-flux sequence field, specifically the DNA library construction method of a kind of mastocarcinoma gene BRCA1 and BRCA2 based on IonTorrent order-checking platform。
Background technology
A new generation's high flux technique of gene detection, the current main serial IonTorrent with Life company of Hiseq, Miseq with Illumina companyTMSeries sequenator is representative, has the advantages such as the incomparable high flux of other technologies, low cost。Have been widely used for different kind organism research and medical science detection。Species but without reference sequences are carried out accent order-checking by genomic level, it is thus achieved that the reference sequences of these species, lay the foundation for follow-up study。To the species having reference sequences, carry out full-length genome and resurvey sequence, full-length genome level detects mutational site, it has been found that the molecular basis of individual variation。New-generation sequencing technology constructs the Genome Atlas of wide variety of conventional species by genome sequencing, also promotes the high speed development of sequencing technologies。But full-length genome structure is complicated, and the cycle is long, costly, limit its development。
Meanwhile, high throughput sequencing technologies also plays an important role in gene diagnosis and gene therapy。The tumor susceptibility gene of some disease generally only accounts for genomic very small part, is not required to full-length genome is checked order to the research of these tumor susceptibility genes, and has only to specific several genes are studied。Target sequence catches the appearance of sequencing technologies so that the research of specific genome area is possibly realized。First this technology synthesizes the oligonucleotide sequences being combined in a large number with specific genome area complementation, is enriched with target zone by pcr amplification, then with high throughput sequencing technologies, these sections is checked order。Compared with genome sequencing, specified disease tumor susceptibility gene order-checking expense is lower, and the order-checking degree of depth is higher, and sequencing result is more accurate, it is also possible to detect that low frequency suddenlys change。
According to existing report: BRCA1/2 gene mutation occupies very big ratio (40%～45%) in hereditary breast cancer patient, and is present in the patient of breast carcinoma HIF of 80%。The people carrying BRCA1 or BRCA2 gene mutation has the higher risk suffering from hereditary breast cancer and ovarian cancer syndrome。Breast cancer related gene BRCA1 and BRCA2 order-checking is through the primer of design BRCA1 and BRCA2 gene region, by multiplexed PCR amplification, carries out high-flux sequence after being enriched with by target area domain dna。Adopting BRCA1 and BRCA2 gene sequencing, breast cancer related gene BRCA1 and BRCA2 can be carried out primary study by researcher。This high method targetedly can find efficiently, verifies and screening BRCA1 and BRCA2 genovariation。Comparing genome sequencing, target area BRCA1 and BRCA2 gene are had by breast cancer related gene order-checking to be studied more quickly and accurately, it is adaptable to for the detection of great amount of samples。
Summary of the invention
It is an object of the invention to provide the DNA library construction method of a kind of mastocarcinoma gene BRCA based on IonTorrent order-checking platform, with the problem solving to propose in above-mentioned background technology。
For achieving the above object, the present invention provides following technical scheme:
The DNA library construction method of a kind of mastocarcinoma gene BRCA based on IonTorrent order-checking platform, specifically comprises the following steps that
(1) target area amplification: by the DNA of sample to be tested and multiple PCR primer, through multi-PRC reaction, obtain the first sample；
(2) primer is removed: takes the first sample and mixes with primer scavenger reagent and react, obtains the second sample；
(3) joint connects: with joint P1, the second sample is carried out joint coupled reaction, obtains the 3rd sample；
(4) magnetic beads for purifying: the 3rd sample is carried out magnetic beads for purifying, obtains the 4th sample；
(5) amplified library: the 4th sample is added amplification enzyme Mix and amplimer carries out pcr amplification, and carry out magnetic beads for purifying, obtain DNA library of interest；
(6) library detection: the library obtained is adopted the polyacrylamide gel electrophoresis detection library concentration of Qubit and 11～13%, library fragments size and Library Quality, to obtain final product；
The PAGE formula of described 11～13% is: water, 3～4mL；30% acrylamide, 2022～2062 μ L；5*TBE, 1.2～1.8mL；9～11%AP, 100～120 μ L；TEMED, 9～11 μ L。
As the further scheme of the present invention: the multiple PCR primer in step (1) is IonAmpliSeqTMBRCA1andBRCA2Panel。
As the further scheme of the present invention: the reaction system in step (1) is 10 μ L systems, the constituent of reaction system particularly as follows:
As the further scheme of the present invention: in step (1), the period of PCR reaction is 18。
As the further scheme of the present invention: the primer scavenger reagent in step (2) is FuPaReagentTM, addition is that each above-mentioned 10 μ LPCR systems add 0.9～1.1 μ L primer scavenger reagent。
As the further scheme of the present invention: the joint P1 of step (3) is:
5'CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT3'
5'ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGTT3'。
As the further scheme of the present invention: the label joint sequence in step (3) is prepared by the A of positive and negative two sequences compositionXAdapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNNNGAT3'
5'ATCNNNNNNNNNNCTGAGTCGGAGACACGC3',
A thereinXAdapter is selected from:
A1Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTATTCGTCGAT3'
5'ATCGACGAATAGACTGAGTCGGAGACACGC3'；
A2Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGAGGCAATTGCGAT3'
5'ATCGCAATTGCCTCTGAGTCGGAGACACGC3'；
A3Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTTAGTCGGACGAT3'
5'ATCGTCCGACTAACTGAGTCGGAGACACGC3'；
A4Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGCAGATCCATCGAT3'
5'ATCGATGGATCTGCTGAGTCGGAGACACGC3'；
A5Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCGCAATTACGAT3'
5'ATCGTAATTGCGACTGAGTCGGAGACACGC3'；
A6Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCGAGACGCGAT3'
5'ATCGCGTCTCGAACTGAGTCGGAGACACGC3'；
A7Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTGCCACGAACGAT3'
5'ATCGTTCGTGGCACTGAGTCGGAGACACGC3'；
A8Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGCCTGAGATACGAT3'
5'ATCGTATCTCAGGCTGAGTCGGAGACACGC3'；
A9Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGAACCTCATTCGAT3'
5'ATCGAATGAGGTTCTGAGTCGGAGACACGC3'；
A10Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTTACAACCTCGAT3'
5'ATCGAGGTTGTAACTGAGTCGGAGACACGC3'；
A11Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGAACCATCCGCGAT3'
5'ATCGCGGATGGTTCTGAGTCGGAGACACGC3'；
A12Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGATCCGGAATCGAT3'
5'ATCGATTCCGGATCTGAGTCGGAGACACGC3'；
A13Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCGACCACTCGAT3'
5'ATCGAGTGGTCGACTGAGTCGGAGACACGC3'；
A14Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGCGAGGTTATCGAT3'
5'ATCGATAACCTCGCTGAGTCGGAGACACGC3'；
A15Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCCAAGCTGCGAT3'
5'ATCGCAGCTTGGACTGAGTCGGAGACACGC3'；
A16Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTTACACACGAT3'
5'ATCGTGTGTAAGACTGAGTCGGAGACACGC3'。
As the further scheme of the present invention: the system of described joint coupled reaction is 14.9～16.1 μ L, the system of joint coupled reaction particularly as follows:
As the further scheme of the present invention: in step (4), magnetic bead used isXP magnetic bead。
As the further scheme of the present invention: step (5) Chinese library amplification enzyme Mix isPCRSuperMixHighFidelity, primer is LibraryAmplificationPrimerMix。
As the further scheme of the present invention: step (5) Chinese library amplification system is 25～27 μ L, particularly as follows: add 24～26 μ L in the 4th sampleThe LibraryAmplificationPrimerMix of PCRSuperMixHighFidelity and 0.5～1.5 μ L。
As the present invention further scheme: the PCR cycle of step (5) Chinese library amplification is 4～6。
Compared with prior art, the invention has the beneficial effects as follows: the method for the present invention is compared with the banking process of the conventional DNA library based on breast cancer related gene BRCA1 and BRCA2, step is simple, by pcr amplification once after the enrichment of target area, then adopt magnetic beads for purifying product, namely obtain sequencing library；Reduce the cost building storehouse simultaneously, decrease the probability introducing sudden change in pcr amplification so that sequencing result is more accurate。
Accompanying drawing explanation
Fig. 1 is that the present invention is based on IonTorrentTMThe flow chart of the DNA library construction method of breast cancer related gene BRCA1 and the BRCA2 of order-checking platform。
Fig. 2 be BRCA1 and the BRCA2 gene that embodiment 1 provides DNA library construction method in 12% polyacrylamide gel figure of PCR primer, wherein swimming lane 1,5 is DNA molecular standard (Takara, 20bpDNAladder), swimming lane 2,3,4,6,7,8,9,10,11,12 is build behind storehouse the product after the circulation of PCR5, sample。
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments。Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain under not making creative work premise, broadly fall into the scope of protection of the invention。
The invention discloses the DNA library construction method of breast cancer susceptibility gene BRCA1 and BRCA2, agents useful for same all can be buied by market: wherein, IonAmpliSeqTMBRCA1andBRCA2Panel (Life company), 5*IonAmpliSeqTMHiFiMix (Life company), FuPaReagentTM(Life company),XP magnetic bead (Beckman company), joint P1 and Adapter (BiooScientific),PCRSuperMixHighFidelity (Life company), LibraryAmplificationPrimerMix (Life company)。
Below in conjunction with embodiment, the present invention is expanded on further:
Embodiment 1: based on IonTorrentTMThe DNA library construction method of breast cancer related gene BRCA1 and the BRCA2 of order-checking platform
The present embodiment adopts following reagent: IonAmpliSeqTMBRCA1andBRCA2Panel (Life company), 5*IonAmpliSeqTMHiFiMix (Life company), FuPaReagentTM(Life company),XP magnetic bead (Beckman company), joint P1 and Adapter (BiooScientific),PCRSuperMixHighFidelity (Life company), LibraryAmplificationPrimerMix (Life company), dehydrated alcohol, Nuclease-freewater, 30% acrylamide, 5*TBE, 10% Ammonium persulfate., TEMED。
A kind of reagent of fresh configuration is needed: 70% ethanol before this method experiment。
The instrument and equipment that this experiment need to be used: centrifuge, magnetic frame, liquid-transfering gun, PCR instrument, concussion instrument, exometer2.0, vertical electrophoresis apparatus。
Major experimental step
(1) DNA of sample to be tested gathers
In the present embodiment, DNA sample comes autoblood or mouth epithelial cells。Adopt poba gene group DNA extraction kit (sky root) extract genomic DNA or adopt buccal swab genomic DNA rapid extraction test kit (Yuanping City is white), extract according to test kit description operating procedure。
(2) DNA of sample to be tested is carried out2.0 concentration measure
1) in sample tube, 1 μ LQubitdsDNAHSReagent and 199 μ LQubitdsDNAHSbuffer is added, whirlpool mixing 4s；
2) in the sample cell add working solution, 1 μ L working solution is abandoned in suction, adds the DNA sample (guaranteeing that DNA is added in working solution) of 1 μ L, and whirlpool mixing 4s, the final volume of each pipe is 200 μ L；
3) all of pipe at room temperature lucifuge hatches 2min；
4) in the main screen of Qubit2.0 exometer, press " DNA " key, then select dsDNAHighSensitivity analytical model；
5) sample cell being put into Qubit2.0 fluorophotometer, cover lid, by " Read "；
6) selecting CalculateInitial initial concentration, then select VolumeofSampleUsed:1 μ L, the result now shown is sample initial concentration, unit ng/ μ L；
7) next sample is read: take out sample in Qubit2.0 fluorophotometer, put into new sample, by " ReadNextSample "；
8) repeated sample reading, until all samples runs through。
(3) multiplexed PCR amplification enrichment target fragment。Add each reagent according to following system and carry out pcr amplification:
PCR reaction condition:
(4) primer is removed
Adding 1 μ LFuPaReagent (totally 11 μ L) in PCR primer, vortex fully mixes, and wink, from 2s, reacts according to following program:
(5) joint connects and purification
1) dilution mixture joint
2) removing and add following components in the PCR product after primer one step up, whirlpool fully mixes, and wink is from 2s。
Above-mentioned system is put into PCR instrument, reacts by follow procedure:
3) magnetic beads for purifying connects product, adoptsXP magnetic beads for purifying product:
A: PCR primer be transferred in 1.5mL centrifuge tube, adds 45 μ LXP magnetic bead, fully blows and beats mixing；
B: incubated at room 5min；
C: sample tube placed on magnetic frame, waits that 3min clarifies completely to solution in pipe；
D: careful Aspirate supernatant also discards；
E: keep centrifuge tube on magnetic frame, the gentle alcoholic solution 30 adding 200 μ L freshly prepared 70%, carefully remove ethanol；
F: repeat step e and clean once。
G: the air-dry magnetic bead 5min of room temperature, not over-drying (air-dry, not cracking)；
Joint P1 is:
5'CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT3'
5'ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGTT3'。
The AXAdapter of sample is as follows respectively:
A1Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTATTCGTCGAT3'
5'ATCGACGAATAGACTGAGTCGGAGACACGC3'；
A2Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGAGGCAATTGCGAT3'
5'ATCGCAATTGCCTCTGAGTCGGAGACACGC3'；
A3Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTTAGTCGGACGAT3'
5'ATCGTCCGACTAACTGAGTCGGAGACACGC3'；
A4Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGCAGATCCATCGAT3'
5'ATCGATGGATCTGCTGAGTCGGAGACACGC3'；
A5Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCGCAATTACGAT3'
5'ATCGTAATTGCGACTGAGTCGGAGACACGC3'；
A6Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCGAGACGCGAT3'
5'ATCGCGTCTCGAACTGAGTCGGAGACACGC3'；
A7Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTGCCACGAACGAT3'
5'ATCGTTCGTGGCACTGAGTCGGAGACACGC3'；
A8Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGCCTGAGATACGAT3'
5'ATCGTATCTCAGGCTGAGTCGGAGACACGC3'；
A9Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGAACCTCATTCGAT3'
5'ATCGAATGAGGTTCTGAGTCGGAGACACGC3'；
A10Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTTACAACCTCGAT3'
5'ATCGAGGTTGTAACTGAGTCGGAGACACGC3'。
(6) amplified library, adding the LibraryAmplificationPrimerMix of the amplification enzyme Mix and 1 μ L of 25 μ L in magnetic bead air-dry after purification, resuspended magnetic bead mixes, and magnetic frame is placed 3min, taking supernatant to PCR pipe, vortex mixing carries out pcr amplification reaction:
PCR reaction condition:
(7) library carries out magnetic beads for purifying, adoptsXP magnetic beads for purifying product
1) PCR primer is transferred in 1.5mL centrifuge tube, adds 25 μ L'sXP magnetic bead, whirlpool mixing or piping and druming mixing；
2) incubated at room 5min；
3) it is placed on magnetic frame by sample pipe static at least 5min or until solution clarification；
4) carefully supernatant is transferred in new 1.5mL centrifuge tube；
5) add 60 μ L'sXP magnetic bead, piping and druming mixing；
6) incubated at room 5min；
7) it is placed on magnetic frame by sample pipe static 3min or clarifies to solution, abandoning supernatant；
8) keep centrifuge tube on magnetic frame, the gentle alcoholic solution adding 200 μ L freshly prepared 70%, rotating centrifugal pipe, carefully remove ethanol；
9) step 8 is repeated) clean again once；
10) the air-dry 5min of room temperature, it is impossible to over-drying (air-dry, not cracking)；
11) taking off centrifuge tube, add the LowTE solution of 30 μ L in sample tube, blow and beat resuspended magnetic bead, vortex mixes；
12) sample tube is placed on magnetic frame, stands 2min, the supernatant (28 μ L) of eluting is transferred in new centrifuge tube；
(8) use2.0 detection library concentration
1) in sample tube, 1 μ LQubitdsDNAHSReagent and 199 μ LQubitdsDNAHSbuffer is added, whirlpool mixing 4s；
2) in the sample cell add working solution, 3 μ L working solutions are abandoned in suction, add the DNA sample (guaranteeing that DNA is added in working solution) of 3 μ L, and whirlpool mixing 4s, the final volume of each pipe is 200 μ L。
3) all of pipe at room temperature lucifuge hatches 2min；
4) existIn the main screen of 2.0 exometers, press " DNA " key, then select dsDNAHighSensitivity analytical model；
5) sample cell is put into2.0 fluorophotometers, cover lid, by " Read "；
6) selecting CalculateInitial initial concentration, then select VolumeofSampleUsed:3 μ L, the result now shown is sample initial concentration, unit ng/ μ L；
7) next sample is read: take outSample in 2.0 fluorophotometers, puts into new sample, by " ReadNextSample "；
8) repeated sample reading, until all samples runs through；
(9) detecting library fragments size with 12%PAGE, the DNA library that purification is good respectively takes 5 μ L sample detections, and result is shown in accompanying drawing 2。
By the sequencing result of IonTorrentTM platform is analyzed, the coverage of all samples all meets or exceeds 97%, and homogeneity on average reaches 100%, and the data volume of each sequencing reaction sample is evenly distributed。
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when without departing substantially from the spirit of the present invention or basic feature, it is possible to realize the present invention in other specific forms。Therefore, no matter from which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the invention rather than described above limits, it is intended that all changes in the implication of the equivalency dropping on claim and scope included in the present invention。Any accompanying drawing labelling in claim should be considered as the claim that restriction is involved。
Claims (12)
1. the DNA library construction method based on the mastocarcinoma gene BRCA of IonTorren order-checking platform, it is characterised in that specifically comprise the following steps that
(1) target area amplification: by the DNA of sample to be tested and multiple PCR primer, through multi-PRC reaction, obtain the first sample；
(2) primer is removed: takes the first sample and mixes with primer scavenger reagent and react, obtains the second sample；
(3) joint connects: with joint P1, the second sample is carried out joint coupled reaction, obtains the 3rd sample；
(4) magnetic beads for purifying: the 3rd sample is carried out magnetic beads for purifying, obtains the 4th sample；
(5) amplified library: the 4th sample is added amplification enzyme Mix and amplimer carries out pcr amplification, and carry out magnetic beads for purifying, obtain DNA library of interest；
(6) library detection: the library obtained is adopted the polyacrylamide gel electrophoresis detection library concentration of Qubit and 11～13%, library fragments size and Library Quality, to obtain final product；
The PAGE formula of described 11～13% is: water, 3～4mL；30% acrylamide, 2022～2062 μ L；5*TBE, 1.2～1.8mL；9～11%AP, 100～120 μ L；TEMED, 9～11 μ L。
2. the DNA library construction method of the mastocarcinoma gene BRCA based on IonTorren order-checking platform according to claim 1, it is characterised in that the multiple PCR primer in step (1) is IonAmpliSeqTMBRCA1andBRCA2Panel。
3. the DNA library construction method of mastocarcinoma gene BRCA of the platform that checks order based on IonTorren according to claim 1, it is characterised in that the reaction system in step (1) is 10 μ L systems, the constituent of reaction system particularly as follows:
4. the DNA library construction method of the mastocarcinoma gene BRCA based on IonTorren order-checking platform according to claim 1, it is characterised in that in step (1), the period of PCR reaction is 18。
5. the DNA library construction method of the mastocarcinoma gene BRCA based on IonTorren order-checking platform according to claim 1, it is characterised in that the primer scavenger reagent in step (2) is FuPaReagentTM, addition is that each above-mentioned 10 μ LPCR systems add 0.9～1.1 μ L primer scavenger reagent。
6. the DNA library construction method of the mastocarcinoma gene BRCA based on IonTorren order-checking platform according to claim 1, it is characterised in that the joint P1 of step (3) is:
5'CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT3'
5'ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGTT3'。
7. the DNA library construction method of the mastocarcinoma gene BRCA based on IonTorren order-checking platform according to claim 1, it is characterised in that the label joint sequence in step (3) is prepared by the A of positive and negative two sequences compositionXAdapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNNNGAT3'
5'ATCNNNNNNNNNNCTGAGTCGGAGACACGC3',
A thereinXAdapter is selected from:
A1Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTATTCGTCGAT3'
5'ATCGACGAATAGACTGAGTCGGAGACACGC3'；
A2Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGAGGCAATTGCGAT3'
5'ATCGCAATTGCCTCTGAGTCGGAGACACGC3'；
A3Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTTAGTCGGACGAT3'
5'ATCGTCCGACTAACTGAGTCGGAGACACGC3'；
A4Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGCAGATCCATCGAT3'
5'ATCGATGGATCTGCTGAGTCGGAGACACGC3'；
A5Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCGCAATTACGAT3'
5'ATCGTAATTGCGACTGAGTCGGAGACACGC3'；
A6Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCGAGACGCGAT3'
5'ATCGCGTCTCGAACTGAGTCGGAGACACGC3'；
A7Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTGCCACGAACGAT3'
5'ATCGTTCGTGGCACTGAGTCGGAGACACGC3'；
A8Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGCCTGAGATACGAT3'
5'ATCGTATCTCAGGCTGAGTCGGAGACACGC3'；
A9Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGAACCTCATTCGAT3'
5'ATCGAATGAGGTTCTGAGTCGGAGACACGC3'；
A10Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTTACAACCTCGAT3'
5'ATCGAGGTTGTAACTGAGTCGGAGACACGC3'；
A11Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGAACCATCCGCGAT3'
5'ATCGCGGATGGTTCTGAGTCGGAGACACGC3'；
A12Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGATCCGGAATCGAT3'
5'ATCGATTCCGGATCTGAGTCGGAGACACGC3'；
A13Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCGACCACTCGAT3'
5'ATCGAGTGGTCGACTGAGTCGGAGACACGC3'；
A14Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGCGAGGTTATCGAT3'
5'ATCGATAACCTCGCTGAGTCGGAGACACGC3'；
A15Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCCAAGCTGCGAT3'
5'ATCGCAGCTTGGACTGAGTCGGAGACACGC3'；
A16Adapter:
5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTTACACACGAT3'
5'ATCGTGTGTAAGACTGAGTCGGAGACACGC3'。
8. the DNA library construction method of mastocarcinoma gene BRCA of the platform that checks order based on IonTorren according to claim 5, it is characterised in that the system of described joint coupled reaction is 14.9～16.1 μ L, the system of joint coupled reaction particularly as follows:
9. the DNA library construction method method of the mastocarcinoma gene BRCA based on IonTorren order-checking platform according to claim 1, it is characterised in that in step (4), magnetic bead used isXP magnetic bead。
10. the DNA library construction method of the mastocarcinoma gene BRCA based on IonTorren order-checking platform according to claim 1, it is characterised in that step (5) Chinese library amplification enzyme Mix isPCRSuperMixHighFidelity, primer is LibraryAmplificationPrimerMix。
11. the DNA library construction method of the mastocarcinoma gene BRCA based on IonTorren order-checking platform according to claim 1, it is characterized in that, step (5) Chinese library amplification system is 25～27 μ L, particularly as follows: add 24～26 μ L in the 4th sampleThe LibraryAmplificationPrimerMix of PCRSuperMixHighFidelity and 0.5～1.5 μ L。
12. the DNA library construction method of the mastocarcinoma gene BRCA based on IonTorren order-checking platform according to claim 1, it is characterised in that the PCR cycle of step (5) Chinese library amplification is 4～6。
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| CN107904666A (en) * | 2017-12-15 | 2018-04-13 | 中源协和基因科技有限公司 | A kind of library construction and quantitative approach applied to tumour driving genetic test |
| CN108753924A (en) * | 2018-06-08 | 2018-11-06 | 安徽鼎晶生物科技有限公司 | A kind of lung cancer high-throughput sequencing library and its construction method and application |
| CN109629007A (en) * | 2018-12-07 | 2019-04-16 | 北京安智因生物技术有限公司 | A kind of heredity goes out the construction of gene library method and kit of blood coagulation |
| CN109680038A (en) * | 2018-12-07 | 2019-04-26 | 北京安智因生物技术有限公司 | A kind of the construction of gene library method and kit of heredity gynecological tumor |
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Cited By (4)
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|---|---|---|---|---|
| CN107904666A (en) * | 2017-12-15 | 2018-04-13 | 中源协和基因科技有限公司 | A kind of library construction and quantitative approach applied to tumour driving genetic test |
| CN108753924A (en) * | 2018-06-08 | 2018-11-06 | 安徽鼎晶生物科技有限公司 | A kind of lung cancer high-throughput sequencing library and its construction method and application |
| CN109629007A (en) * | 2018-12-07 | 2019-04-16 | 北京安智因生物技术有限公司 | A kind of heredity goes out the construction of gene library method and kit of blood coagulation |
| CN109680038A (en) * | 2018-12-07 | 2019-04-26 | 北京安智因生物技术有限公司 | A kind of the construction of gene library method and kit of heredity gynecological tumor |
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