CN106676169B - Hybridization capture kit for detecting breast cancer susceptibility genes BRCA1 and BRCA2 mutation and method thereof - Google Patents
Hybridization capture kit for detecting breast cancer susceptibility genes BRCA1 and BRCA2 mutation and method thereof Download PDFInfo
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Abstract
The invention discloses a hybridization capture kit for detecting breast cancer susceptibility genes BRCA1 and BRCA2 mutation, which comprises a hybridization reagent, a PCR amplification reagent and an enrichment degree detection reagent; wherein the reagent for hybridization comprises the following probe mixture of SEQ NO.1-SEQ NO.173, and the molar ratio is SEQ NO.1: SEQ NO. (2-172) and SEQ NO. 173: 1: 1: 1, the above probe mixture was used in an amount of 1. mu.l at a concentration of 2nM each upon detection. The kit can be used for detecting full exon mutation of BRCA1 and BRCA2 genes with high sensitivity, high flux, low cost and easy operation, and assists a clinician in screening out individuals with breast cancer susceptibility risk, thereby achieving the purpose of preventing breast cancer.
Description
Technical Field
The invention relates to the field of gene mutation detection, in particular to a hybridization capture kit for detecting breast cancer susceptibility genes BRCA1 and BRCA2 mutation and a detection method thereof.
Background
Breast cancer is one of the common malignant tumors in women, called female killers, with incidence second only to lung cancer. Wherein the hereditary breast cancer accounts for about 5 to 7 percent of the incidence rate of the breast cancer. The biggest risk factor for breast and ovarian cancer is the genetic mutation of the breast cancer susceptibility gene BRCA1 or BRCA 2. BRCA1 and BRCA2 are two tumor suppressor genes, the structural and functional abnormalities of which are closely related to the occurrence of breast cancer and ovarian cancer, can not only suppress cell growth, but also participate in various important cell activities such as cell cycle regulation, gene transcription regulation, DNA damage repair and apoptosis, and play an important role in maintaining genome stability. Because mutation of BRCA1 and BRCA2 genes has certain relation with genetic breast cancer, detection of BRCA genes and search of pathogenic mutation sites of BRCA genes can help to screen out high risk groups and contribute to risk assessment and early diagnosis.
At present, methods for BRCA gene mutation detection mainly include a high resolution melting curve method (HRM), a multiplex PCR amplification capture technology, a restriction fragment length polymorphism analysis method (RFLP) and a DNA direct sequencing method.
The high resolution melting curve method is a novel PCR-based technology, which is characterized in that fragments are amplified by PCR under the condition of fluorescent chimeric dye, and the amplified products are melted by a rapid and controllable heating treatment. The fluorescence level is monitored in real time in the process of temperature rise, and the fluorescence signal is gradually reduced along with the melting of the double-stranded DNA. The difference in melting temperature of the DNA duplex reflects genetic variation. The method has the following disadvantages: the specific variant cannot be judged through the graph of the melting curve, and the detection in downstream analysis still needs to be supplemented by sequencing and the like.
The multiple PCR amplification capture technology utilizes multiple PCR amplification to enrich DNA fragments of target regions in sample genomes, and then performs library construction and high-throughput sequencing, and has the following defects: the flux is small, the length of the longest genomic DNA fragment obtained by one PCR at present should not exceed 50kb, and special enzyme and special PCR conditions are needed, so the cost is high and the stability is poor. Of course, fragments of less than 100kb are also feasible by means of multiple PCR, and the cost performance of each amplified fragment is generally the best around 500 bp.
The RFLP method is a method in which PCR is combined with restriction enzyme cleavage. This method has the following disadvantages: RFLP experiment operation is tedious, detection cycle is long, cost is high, false positive caused by incomplete enzyme digestion in the first round exists, non-closed tube operation is easy to pollute, and clinical detection requirements are difficult to meet.
The direct DNA sequencing method has the following disadvantages: the detection period is long, the cost is high, the tube closing operation is not performed, the cross contamination is difficult to avoid, and the flux is not high. In addition, the method has low sensitivity, and the problems of heterozygosity mutation, gel compression, GC enrichment area and the like make it difficult to obtain accurate data through one-time sequencing, and false positive and the like can be avoided only by repeating the sequencing for many times, so that the direct sequencing method is difficult to be applied to clinical detection.
Disclosure of Invention
The invention aims to provide a kit for detecting BRCA1 and BRCA2 gene mutation with high flux, high sensitivity, high stability and low cost aiming at the defects of the existing method for detecting BRCA gene mutation. The invention mainly utilizes a liquid phase probe hybridization capture technology to capture and enrich the full exon areas of BRCA1 and BRCA2 genes, then determines the enriched full exon sequences through a high-throughput sequencing technology, and finally analyzes high-throughput sequencing data through bioinformatics, thereby discovering the mutation condition and the mutation frequency of the full exon areas.
Another objective of the invention is to provide a method for detecting mutation hybridization enrichment of breast cancer susceptibility genes BRCA1 and BRCA 2.
In order to realize the purpose of the invention, the invention adopts the technical scheme that:
a hybridization capture kit for detecting breast cancer susceptibility genes BRCA1 and BRCA2 mutation comprises a hybridization reagent, a PCR amplification reagent and an enrichment degree detection reagent; wherein the reagent for hybridization comprises the following probe mixture of SEQ NO.1-SEQ NO.173, and the molar ratio is SEQ NO.1: SEQ NO. (2-172) and SEQ NO. 173: 1: 1: 1, when in detection, the dosage of the probe mixture with the concentration of 2nM each is 1 mul;
the PCR amplification reagent comprises the following primer pair consisting of SEQ NO.176 and SEQ NO. 177:
| SEQ NO.176 | AATGATACGGCGACCACCGA |
| SEQ NO.177 | CAAGCAGAAGACGGCATACGA |
the enrichment degree detection reagent comprises the following primer pair consisting of SEQ NO.156 and SEQ NO. 157:
| SEQ NO.156 | ACCATCACGTGCACTAACAAGACAG |
| SEQ NO.157 | GTCCTCTTACTCTCTCACCTCAAG |
in a preferred embodiment of the present invention, the hybrid capture kit for detecting mutations in breast cancer susceptibility genes BRCA1 and BRCA2 further comprises reagents for a hybrid system: block3.1/3.2, Human Cot-1
PerfectHybTMPlus hybridization buffer; wherein, Block3.1/3.2 comprises the following primers consisting of SEQ NO.174 and SEQ NO. 175:
| SEQ NO.174 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT |
| SEQ NO.175 | CAAGCAGAAGACGGCATACGAGATIIIIIIGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT |
and the molar ratio is SEQ NO. 174: SEQ No.175 ═ 1: 1, the amount of the mixture solution was 1. mu.l at a concentration of 300. mu.M each for detection.
In a preferred embodiment of the present invention, the reagents used in the hybridization system consist of the following volumes of components:
in a preferred embodiment of the present invention, the hybrid capture kit for detecting mutations in breast cancer susceptibility genes BRCA1 and BRCA2 further comprises Streptavidin beads carrying avidin for anchoring target DNA fragments by biotin avidin reaction after hybridization, and 1 × washing buffer, 0.1 × washing buffer for washing off non-target DNA, and FE buffer for eluting captured DNA.
In a preferred embodiment of the present invention, the 1 xwashing buffer comprises sodium chloride at a concentration of 0.15M, sodium citrate at 15mM, and SDS at 0.1%.
In a preferred embodiment of the present invention, the 0.1 × washing buffer comprises 0.015M sodium chloride, 1.5mM sodium citrate, 0.1% SDS.
In a preferred embodiment of the invention, the FE buffer is a solution of 10mM EDTA containing 95% formamide at pH 8.2.
In a preferred embodiment of the present invention, the hybrid capture kit for mutation detection of breast cancer susceptibility genes BRCA1 and BRCA2 further comprises BW buffer for washing Streptavidin beads after hybridization.
In a preferred embodiment of the invention, the BW buffer comprises 5mM Tris. Cl, 0.5mM EDTA, 1.0M sodium chloride and 0.01% Tween-20.
In a preferred embodiment of the present invention, the PCR amplification reagent further comprises a PCR pre-mix and deionized water.
In a preferred embodiment of the present invention, the PCR premix is a2 Xpremix, and the final concentration is 1X.
In a preferred embodiment of the invention, the 2 Xpremix comprises 2 XQ 5buffer, 1 XGC enhancer buffer, dNTP with 0.4mM concentration of each component, and Q5 high fidelity DNA polymerase with 2000U/ml concentration in 0.2. mu.l.
In a preferred embodiment of the present invention, the enrichment detecting reagent further comprises 2 × SYBR Green Master Mix (vazyme), Primer Mix (10uM each), dH2O and input/R1/R2.
A method for detecting mutation hybridization enrichment of breast cancer susceptibility genes BRCA1 and BRCA2 specifically comprises the following steps:
1) and (5) constructing a library.
2) And (3) hybridization and capture: after the constructed libraries are mixed in equal proportion, the mixture of the probes of SEQ NO.1-SEQ NO.173 is used for carrying out liquid phase probe capture hybridization, and DNA is obtained by enrichment; wherein the mole ratio of the probe mixture of SEQ NO.1-SEQ NO.173 is SEQ NO.1: SEQ NO. (2-172) SEQ NO. 173: 1: 1: 1, when in detection, the dosage of the probe mixture with the concentration of 2nM each is 1 mul;
the hybridization system adopted by the hybridization capture consists of the following components in volume:
the hybridization procedure was as follows:
h1 and H2 are mixed evenly and centrifuged and then are respectively put into PCR, and the denaturation annealing program is as follows:
after the program was run, H1 was carefully added to H2 in one portion using a pipette due to PerfectHybTMBlowing and uniformly mixing the Plus hybridization buffer with a gun head when the Plus hybridization buffer is relatively viscous; placing the mixed solution on a vortex oscillator, oscillating, mixing uniformly, centrifuging, placing into a PCR instrument, and hybridizing at 50 ℃ overnight;
resuspend Streptavidin beads with 1ml BW, wash twice;
carefully transferring the hybridized mixed solution into a centrifuge tube containing SA beads by using a pipette gun, slowly flushing the SA beads on the tube wall to the bottom of the centrifuge tube at one time, whirling for 10s, then throwing, and incubating for 45min at 50 ℃ and 1200 rpm;
washing SA beads twice with 500. mu.l of preheated 1 × washing buffer/each sample, and incubating at 50 ℃ and 1200rpm for 1min each time;
then washing the SA beads twice with 500. mu.l of 0.1 × washing buffer/each sample, and incubating at 1200rpm for 1min at 50 ℃;
adding 20 mul FE buffer into the washed magnetic beads, carrying out vortex oscillation, uniform mixing and short centrifugation, and then incubating at the temperature of 95 ℃ and the speed of 1200rpm for 10 min;
finally use
Purifying the supernatant by a Nucleotide Removal Kit;
3) and (3) PCR amplification: and performing PCR amplification on DNA obtained by one round of enrichment, and purifying by using 1X AMPur XP beads after amplification by using a primer pair (SEQ NO.176 and SEQ NO.177) used for amplification.
The amplification system comprises: 20. mu.l of the hybridization product, 5. mu.l of primers (SEQ NO.176 and SEQ NO.177), 50. mu.l of PCR premix, and deionized water in a total volume of 100. mu.l.
The PCR amplification procedure was as follows:
4) two rounds of hybridization enrichment: 20ng of the purified PCR product in one round is reserved for enrichment degree detection, and the rest is used for two rounds of hybridization. Wherein, the amount of the input in the second round of hybridization is 100ng-500ng, the hybridization time is 4h, and the rest conditions are the same as the conditions in the same round of hybridization;
5) and (3) PCR amplification: performing PCR amplification on DNA obtained by the two rounds of enrichment, wherein the amplification primer pair SEQ NO.176 and SEQ NO.177 has the cycle number of 10, and the amplification primer pair is purified by using 1 multiplied by AMPur XP beads;
6) and (3) detecting the enrichment degree: respectively using StepOne to the multiplex DNA sample library pool obtained in the step 2), the PCR amplified one-round DNA and two-round DNA obtained in the step 3) and the PCR amplified two-round DNA obtained in the step 5)TM and StepOnePlusTMRealtime PCR system carries out Realtime PCR enrichment detection, each sample is subjected to three times of repetition, ultrapure water is used as a negative control, and any one library before mixing is used as a positive control. The using amount of the templates is 3.44 ng; calculating the average value of Ct of each sample, and subtracting the average value of Ct of the DNA sample after enrichment from the average value of Ct of the DNA sample before enrichment to obtain delta Ct; the enrichment degree is calculated, and the enrichment degree calculation method comprises the following steps: fold attribute EΔCpWherein E is the PCR amplification efficiency of the primer pair SEQ NO.156 and SEQ NO.157, the amplification efficiency of the primer pair is 2.0 by detection, R2Is 0.991;
7) sequencing: sequencing the PCR amplification product obtained in the step 2) with certain enrichment after the detection in the step 6).
8) And (3) analysis: analyzing the sequencing result obtained in the step 7) to complete mutation detection of the whole exon region.
Compared with the prior art, the detection method and the kit have the following advantages:
1) the design of the tiled interval probes, 173 probes are uniformly arranged in the whole exon region at intervals, so that the mutation detection of the whole exon region can be realized, known mutations can be analyzed, and new mutations can be found;
2) the cost is low, the stability is high, the repeatability is good, the flux is high, and a plurality of sample libraries can be detected simultaneously;
3) by capturing and focusing the key sequence through the sequence, the size of an area needing to be covered by subsequent sequencing is reduced, the sequencing depth is increased, the data processing difficulty is effectively reduced, the sensitivity is improved, and the detection rate of mutation in a mixed sample is increased.
4) The sample source is wide, genomic DNA from various sources such as FFPE tissue, FF tissue, blood cells, saliva and plasma-derived free DNA fragments can be used, and the requirement on the sample to be detected is low.
In general, the kit can be used for detecting the mutation of the full exons of BRCA1 and BRCA2 genes with high sensitivity, high flux, low cost and easy operation, and assists a clinician in screening out individuals with susceptibility risks of breast cancer, so as to achieve the purpose of preventing the occurrence of the breast cancer.
Drawings
FIG. 1 is a round of enrichment detection of the hybrid capture kit for detecting mutations in BRCA1 and BRCA2 of breast cancer susceptibility genes according to the present invention;
FIG. 2 is a diagram of two rounds of hybridization enrichment detection of the hybridization capture kit for detecting mutations of breast cancer susceptibility genes BRCA1 and BRCA2 according to the present invention;
FIG. 3 is a diagram of Agilent Bioanalyzer2100 assay for enriching libraries by hybrid capture kit for mutation detection of BRCA1 and BRCA2 of breast cancer susceptibility genes according to the present invention;
FIG. 4 is a diagram showing the data analysis results of the hybrid capture kit for detecting mutations in BRCA1 and BRCA2 of breast cancer susceptibility genes.
Detailed Description
The following describes in detail a hybrid capture kit and method for detecting mutations in BRCA1 and BRCA2 of the present invention with reference to the accompanying drawings and specific examples.
The kit for detecting the mutation of the breast cancer susceptibility genes BRCA1 and BRCA2 comprises a reagent for hybridization, a PCR amplification reagent and an enrichment degree detection reagent.
1) And (3) hybridization and capture: the hybridization reagent comprises a probe mixture (SEQ NO.1-SEQ NO.173), and the molar ratio is SEQ NO.1: SEQ NO. (2-172): SEQ NO. 173: 1: 1: 1. for detection, the above probe mixture was used in an amount of 1. mu.l at a concentration of 2nM each. The hybridization capture kit for detecting the mutation of the breast cancer susceptibility genes BRCA1 and BRCA2 also comprises reagents used by a hybridization system: block3.1/3.2(SEQ NO.174 and SEQ NO.175), Human Cot-1
PerfectHybTMPlus hybridization buffer. The mole ratio of Block3.1/3.2 is SEQ NO. 174: SEQ No.175 ═ 1: 1, the amount of the mixture solution was 1. mu.l at a concentration of 300. mu.M each for detection. In addition, Human Cot-1
The standard of (1) is 1mg/ml, and the dosage is 1 mul during detection. PerfectHybTMThe amount of Plus hybridization buffer was 50. mu.l. The hybridization system also comprises a Multiplex DNA sample library pool which is used for detecting each library to be enriched by using an Agilent Bioanalyzer2100 high-sensitivity DNA chip to obtain the distribution range of the library fragments and the average fragment length and concentration of the main peak. Then, mixing according to equal mass and equal proportion to obtain 520ng of library, wherein 500ng is used for hybridization, and the rest 20ng is used for detection of enrichment after hybridization.
Preferably, the hybridization system consists of the following volumes of the components:
the hybridization procedure was as follows:
h1 and H2 are mixed evenly and centrifuged and then are respectively put into PCR, and the denaturation annealing program is as follows:
after the program was run, H1 was carefully added to H2 in one portion using a pipette due to PerfectHybTMThe Plus hybridization buffer was relatively viscous and was then blown up and mixed well with a spearhead. The mixed solution is put on a vortex oscillator to be evenly mixed and centrifuged, and then put in a PCR instrument to be hybridized overnight at 50 ℃. The Streptavidin beads were resuspended in 1ml BW and washed twice. Carefully transferring the hybridized mixed solution into a centrifuge tube containing SA beads by using a pipette gun, slowly flushing the SA beads on the tube wall to the bottom of the centrifuge tube at one time, shaking by vortex for 10s, then throwing off, and incubating at 50 ℃ and 1200rpm for 45 min. The SA beads were washed twice with pre-warmed 500. mu.l of 1 × washing buffer/each sample, and incubated at 50 ℃ for 1min at 1200 rpm. The SA beads were then washed twice with 500. mu.l of 0.1 × washing buffer/each sample, and incubated for 1min at 1200rpm at 50 ℃. Mu.l FE buffer was added to the washed beads, vortexed, briefly centrifuged, and incubated at 95 ℃ and 1200rpm for 10 min. Finally use
The supernatant was purified using the Nucleotide Removal Kit.
2) And (3) PCR amplification: and performing PCR amplification on DNA obtained by one round of enrichment, and purifying by using 1X AMPur XP beads after amplification by using a primer pair (SEQ NO.176 and SEQ NO.177) used for amplification.
The amplification system comprises: 20. mu.l of the hybridization product, 5. mu.l of primers (SEQ NO.176 and SEQ NO.177), 50. mu.l of PCR premix, and deionized water in a total volume of 100. mu.l.
PCR amplification procedure:
3) two rounds of hybridization enrichment: 20ng of the purified PCR product in one round is reserved for enrichment degree detection, and the rest is used for two rounds of hybridization. Wherein, the amount of the input in the two rounds of hybridization is 100ng-500ng, the hybridization time is 4h, and the rest conditions are the same as the hybridization in the same round.
4) And (3) PCR amplification: and performing PCR amplification on the DNA obtained by the two rounds of enrichment, wherein the amplification uses a primer pair (SEQ NO.176 and SEQ NO.177), the cycle number used for amplification is 10, and the DNA is purified by using 1 x AMPur XP beads after amplification.
5) And (3) detecting the enrichment degree: respectively using StepOne to the multiplex DNA sample library pool obtained in the step 1), the PCR amplified one-round DNA and two-round DNA obtained in the step 2) and the step 4) after the PCR amplification respectivelyTM and StepOnePlusTMAnd performing Realtime PCR enrichment detection on the Realtime PCR system, performing three repetitions on each sample, and performing negative control on ultrapure water and positive control on any library before mixing. The amount of template used was 3.44ng each. Calculating the average value of Ct of each sample, and subtracting the average value of Ct of the DNA sample after enrichment from the average value of Ct of the DNA sample before enrichment to obtain delta Ct. The enrichment degree is calculated, and the enrichment degree calculation method comprises the following steps: fold attribute EΔCpWherein E is the PCR amplification efficiency of the primer pair (SEQ NO.156 and SEQ NO.157), the amplification efficiency of the primer pair is 2.0 by detection, and R is2Is 0.991.
Description of the effects: as can be seen in FIG. 1, after one round of hybridization, the enrichment degree of DNA in which the detection primer is located is 210. As can be seen in FIG. 2, after two rounds of hybridization, the enrichment degree of the DNA where the detection primer is located is 216. The result shows that the target fragment is indeed enriched to a certain degree after two rounds of hybridization, and the enrichment effect is obvious. FIG. 3 is the mass analysis of two rounds of hybridization enriched products, from which it can be seen that the library has a single main peak and the size is consistent with the size of the library before hybridization, and can be used for further sequencing analysis. The information analysis of the 4 hybridized libraries in FIG. 4 shows that the hybridization kit realizes effective enrichment of BRCA whole exons and can be effectively used for detecting BRCA1 and BRCA2 gene whole exon mutation. It should be noted that although in some cases the detection of the gene is of great auxiliary significance for the diagnosis, prevention and treatment of the relevant cancer, the mutation of these genes does not necessarily lead to the occurrence of the disease, and therefore the detection of these genes does not relate to a method for the diagnosis or treatment of the disease.
The kit has the characteristics of high sensitivity, high flux, low cost, easy operation, high stability and the like, and can assist a clinician in screening out individuals with the risk of susceptibility to breast cancer, thereby achieving the purpose of preventing the occurrence of breast cancer. Compared with the high-resolution melting curve method, HRM cannot judge a specific variant through a melting curve graph, and the detection in downstream analysis still needs additional description such as sequencing and the like. The multiple PCR amplification capture technology has small flux, the length of the longest genomic DNA fragment obtained by one PCR at present should not exceed 50kb, and special enzyme and special PCR conditions are needed, so the cost is high, and the stability is poor. RFLP experiment operation is tedious, detection cycle is long, cost is high, false positive caused by incomplete enzyme digestion in the first round exists, non-closed tube operation is easy to pollute, and clinical detection requirements are difficult to meet. The direct DNA sequencing method has the disadvantages of long detection period, high cost, non-closed tube operation, difficulty in avoiding cross contamination, low flux, low sensitivity, and the like, and is difficult to obtain accurate data through one-time sequencing due to the problems of heterozygosity mutation, gel compression, GC enrichment area and the like, and is difficult to be applied to clinical detection because false positive can be avoided only by repeated sequencing for many times.
While the preferred embodiments of the present invention have been illustrated and described in detail, it should be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings without inventive faculty. Therefore, the technical solutions available to those skilled in the art through logical analysis, reasoning or limited experiments based on the present inventive concept should be within the scope of protection defined by the claims.
Claims (5)
1. A hybridization capture kit for detecting breast cancer susceptibility genes BRCA1 and BRCA2 mutation is characterized by comprising a hybridization reagent, a PCR amplification reagent and an enrichment degree detection reagent; wherein the reagent for hybridization comprises a probe mixture shown as SEQ NO.1-SEQ NO.173, and the molar ratio of the three of SEQ NO.1 to SEQ NO.2 to 172 to SEQ NO.173 is 1: 1: 1, when in detection, the dosage of the probe mixture with the concentration of 2nM is 1 mul;
the PCR amplification reagent comprises a primer pair consisting of SEQ NO.176 and SEQ NO. 177;
the enrichment degree detection reagent comprises a primer pair consisting of SEQ NO.156 and SEQ NO. 157;
also included are reagents for a hybridization system comprising:
Block3.1/3.2、Human Cot-1
PerfectHybTM Plus hybridization buffer；
wherein, Block3.1/3.2 comprises a primer composed of SEQ NO.174 and SEQ NO. 175;
and the molar ratio is SEQ NO. 174: SEQ No.175 ═ 1: 1, the amount of the mixture solution was 1. mu.l, each of which had a concentration of 300. mu.M in the detection.
2. The hybrid capture kit for the detection of mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 of claim 1, wherein the reagents used in the hybrid system consist of the following volumes of components:
h1: 500ng of a multiplex DNA sample library with the volume less than or equal to 7 mu l;
1. mu.l of Cot-1 DNA human;
1. mu.l of Block 3.1/3.2;
H2：
50 μ l PerfectHybTM Plus hybridization buffer；
Mu.l of the probe mixture were each present at a concentration of 2 nM.
3. The hybrid capture kit for detecting mutations in breast cancer susceptibility genes BRCA1 and BRCA2 of claim 2, further comprising Streptavidin beads carrying avidin to anchor target DNA fragments by biotin avidin reaction after hybridization, and 1 xwashing buffer for washing off non-target DNA, 0.1 xwashing buffer, and FE buffer for eluting captured DNA
(ii) a The 1 xwashing buffer comprises sodium chloride at a concentration of 0.15M, sodium citrate at a concentration of 15mM, and 0.1% SDS; the 0.1 xwashing buffer comprises 0.015M sodium chloride, 1.5mM sodium citrate, 0.1% SDS; the FE buffer solution was a 10mM EDTA solution containing 95% formamide at pH 8.2.
4. The hybrid capture kit for the detection of mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 of claim 3, further comprising BW buffer for washing streptavidin beads after hybridization; the BW buffer included 5mM Tris. Cl, 0.5mM EDTA, 1.0M sodium chloride and 0.01% Tween-20.
5. The hybrid capture kit for the detection of mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 of claim 1, wherein said PCR amplification reagents further comprise PCR pre-mix and deionized water; the PCR premix is 2 multiplied by the premix, and the final concentration is 1 multiplied by the premix when in use.
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| CN107201401A (en) * | 2017-05-23 | 2017-09-26 | 深圳市第二人民医院 | A kind of Multiple-Factor Model and its method for building up for pathogenesis of breast carcinoma risk profile |
| CN109097443B (en) * | 2017-06-21 | 2022-03-22 | 苏州吉赛基因测序科技有限公司 | Method for capturing genome target sequence for high-throughput sequencing |
| CN107858426B (en) * | 2017-08-22 | 2021-04-09 | 上海派森诺生物科技股份有限公司 | Hybridization capture kit for hot spot detection of 16 genes for tumor individualized medication and method thereof |
| CN107760771A (en) * | 2017-11-16 | 2018-03-06 | 杭州迪安医学检验中心有限公司 | A kind of breast cancer susceptibility gene BRCA1 and BRCA2 full genomes capture primer, kit and its method |
| CN108085387B (en) * | 2017-11-27 | 2020-03-27 | 天津诺禾致源生物信息科技有限公司 | Specific capture probe, kit and sequencing library for detecting human BRCA1/2 gene mutation and construction method thereof |
| CN108179177A (en) * | 2017-12-29 | 2018-06-19 | 博迪泰（厦门）生物科技有限公司 | A kind of kit and detection method of quick detection nucleic acid mutation |
| CN108624686B (en) * | 2018-03-30 | 2019-06-04 | 南京世和基因生物技术有限公司 | A kind of probe library, detection method and the kit of detection BRCA1/2 mutation |
| CN108753954B (en) * | 2018-06-26 | 2022-11-18 | 中南大学湘雅医院 | Capture probe set, kit, library construction method and application of dementia-related genes |
| CN109234396B (en) * | 2018-10-10 | 2019-07-16 | 无锡市第五人民医院 | A breast cancer susceptibility gene BRCA2 locus g.32336534T>C mutant and its application |
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| CN102409047B (en) * | 2010-09-21 | 2014-07-23 | 深圳华大基因科技服务有限公司 | Method for building sequencing library by hybridization |
| CN103667254B (en) * | 2012-09-18 | 2017-01-11 | 南京世和基因生物技术有限公司 | Enrichment and detection method of target gene fragment |
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| CN105063208B (en) * | 2015-08-10 | 2018-03-06 | 北京吉因加科技有限公司 | A kind of target dna low frequency mutation enrichment sequence measurement to dissociate in blood plasma |
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