CN105200154A - Multiplex-PCR (polymerase chain reaction) detection method and kit for BRCA1 and BRCA2 gene mutation - Google Patents
Multiplex-PCR (polymerase chain reaction) detection method and kit for BRCA1 and BRCA2 gene mutation Download PDFInfo
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Abstract
The invention provides a method and a kit for detecting BRCA1 and BRCA2 gene mutation. The method and the kit utilize two sets of PCR (polymerase chain reaction) primers, wherein a first set of PCR primers comprises 189 pairs of PCR primers and is divided into two groups, a first group comprises 95 pairs of primers with sequences represented as SEQ ID NO.5-SEQ ID NO.194, a second group comprises 94 pairs of primers with sequences represented as SEQ ID NO.195-SEQ ID NO.382, a sequence represented as SEQ ID NO.1 is added to the 5' end of each upstream primer, and a sequence represented as SEQ ID NO.2 is added to the 5' end of each downstream primer; sequences of upstream and downstream primers of a second set of PCR primers are represented as SEQ ID NO.3 and SEQ ID NO.4.
Description
Technical field
The present invention relates to detection in Gene Mutation field, particularly relate to transgenation multiplex PCR and detect.
Background technology
BRCA1 and BRCA2 is two and has the genes suppressing malignant tumour to occur, and somaticly to copy, plays an important role in the normal growth of hereditary material DNA injury repairing, cell mediator.The sudden change of BRCA1 and BRCA2 found have hundreds of more than, some is relevant with hereditary breast cancer and ovarian cancer.Sum up the lifetime risk display of the relevant cancer of BRCA1 with BRCA2 transgenation, having BRCA1 transgenation person to suffer from breast cancer with the risk of ovarian cancer is 50%-85% and 15%-45% respectively, and having BRCA2 transgenation person to suffer from breast cancer with the risk of ovarian cancer is 50%-85% and 10%-20% respectively.Compared with common women, these are all very high trouble cancer probabilities.
Due to BRCA1 and BRCA2 gene sudden change to familial breast cancer, ovarian cancer have certain contact, so the mutator gene detecting BRCA1 and BRCA2 has important clinical meaning to the diagnosis of associated cancer, prevention and therapy.In addition, because the sudden change of BRCA1 and BRCA2 is numerous, detect these sudden changes very meaningful for the research of genes involved, such as, for Polymorphism Analysis and family's evolutionary history research.
At present, the BRCA detection method of gene mutation generally applied is mainly restriction small segment length polymorphism analysis method (RFLP method) and DNA direct sequencing.RFLP method is the method combined with restriction enzyme digestion by PCR, and the method exists following shortcoming: experimental implementation is loaded down with trivial details, sense cycle is long, cost is high, there is first round enzyme cuts the false positive not exclusively caused, the operation of non-stopped pipe, is easy to pollute, be difficult to meet clinical detection requirement.There is following shortcoming in DNA direct sequencing: sense cycle is longer, cost is high, non-stopped pipe operates, is difficult to avoid crossed contamination, flux not high.In addition, the sensitivity of DNA direct Sequencing is lower, the problem such as existence of the contracting of heterozygous mutant, glue laminated, GC enrichment region makes to be difficult to obtain accurate data by once sequencing, usually needing repeatedly to repeat order-checking and just may avoid false positive etc., therefore direct Sequencing method also difficulty be applicable to clinical detection.
Also have publication to use PCR method to detect breast cancer susceptibility gene mutation, such as patent " detects multiple PCR reagent kit of breast cancer susceptibility gene mutation and preparation method thereof " (Authorization Notice No.: CN101200766B).But, this patent multiplex PCR used is only limited on the fractional mutations site on BRCA1 and BRCA2 Gene Partial exon, and this obviously can not cover BRCA1 and BRCA2 gene spread on multiple exon hundreds of sudden change, therefore this patent use on have larger limitation.
Therefore, this area needs to can be used for highly sensitive, high-throughput, low cost detect BRCA1 and BRCA2 transgenation method and test kit.
Summary of the invention
The object of the invention is in order to provide can be used for highly sensitive, high-throughput, low cost detect BRCA1 and BRCA2 sudden change method and test kit.The present invention mainly utilizes multiple PCR technique to catch enrichment to BRCA1 and BRCA2 gene coding region, then the coding region sequence after enrichment is measured by high throughput sequencing technologies, eventually through bioinformatic analysis high-flux sequence data, thus find catastrophe and the frequency thereof of coding region.
Therefore, the invention provides a kind of method detecting BRCA1 and BRCA2 sudden change, described method comprises
1) catch: carry out first round specificity multi-PRC reaction, 189 pairs of primer pair sample DNAs are used to increase, described 189 pairs of PCR primer divide two test tubes to react, first test tube primer 95 is to (SEQIDNO.5-SEQIDNO.194), second test tube primer 94 is to (SEQIDNO.195-SEQIDNO.382), and 5 ' end of each upstream primer adds sequence SEQIDNO.1:
" ACACTCTTTCCCTACACGACGCTCTTCCGATCTTAC ", 5 ' end of each downstream primer adds sequence SEQIDNO.2: " GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTA ";
2) storehouse is built: carry out second and take turns PCR reaction, use PCR primer pair: upstream primer SEQIDNO.3: " AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC ", downstream primer SEQIDNO.4:
" CAAGCAGAAGACGGCATACGAGATATCACGTTGTGACTGGAGTTCAGACGT " is to step 1) amplified production that obtains increases;
3) checking order, to step 2) amplified production that obtains checks order;
4) analyze, to step 3) sequencing result that obtains analyzes, and completes the abrupt climatic change of full coding region.
Present invention also offers a kind of BRCA1 and BRCA2 transgenation multiple PCR detection kit, described test kit comprises: two cover PCR primer and pcr amplification premix solution (such as, from KAPA2GFast warm start multiplex amplification test kit);
Two described cover PCR primer, first set PCR primer comprises 189 pairs of PCR primer, divide two groups, first group comprises primer 95 to (SEQIDNO.5-SEQIDNO.194), second group comprises primer 94 to (SEQIDNO.195-SEQIDNO.382), and 5 ' end of each upstream primer adds SEQIDNO.1:
" ACACTCTTTCCCTACACGACGCTCTTCCGATCTTAC " sequence, 5 ' end of each downstream primer adds SEQIDNO.2: " GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTA " sequence;
The upstream primer sequence SEQIDNO.3 of the second cover PCR primer is:
" AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC ", downstream primer sequence SEQIDNO.4 is
“CAAGCAGAAGACGGCATACGAGAT
ATCACGTTGTGACTGGAGTTCAGACGT”。
In test kit of the present invention, described first set is specificity multiple PCR primer, and described second cover PCR primer is a pair Standard PCR primer, has sequencing sequence in its 5 ' end band.
In a preferred embodiment, described test kit also comprises distilled water.
In a preferred embodiment, in described test kit, the reaction system of the PCR reaction of first set primer is by following proportions: first set PCR primer 10 parts, pcr amplification premix solution (such as pcr amplification premix solution) 12.5 parts, DNA profiling 3 parts, 4.5 parts, water.
In a preferred embodiment, the reaction system of the second cover PCR reaction is by following proportions: the second cover PCR primer 2.5 parts, pcr amplification premix solution (such as pcr amplification premix solution) 7.5 parts, first round PCR reclaim product 5 parts, 4.5 parts, water.
Compared to the prior art, detection method of the present invention and test kit tool have the following advantages:
1) with low cost;
2) low to sample requirement amount to be detected, can the pole low DNA sample in blood plasma and serum be analyzed;
3) blanket type design of primers, can realize full coding region abrupt climatic change, not only can analyze known mutations, can also find new sudden change;
4) highly sensitive;
5) detection time is short, in conjunction with high throughput sequencing technologies, can realize full coding region abrupt climatic change in the short period of time.
Test kit of the present invention can be used for highly sensitive, high-throughput, low cost detection BRCA sudden change, assists clinician to realize the individualized treatment of tumour patient, reduces Operative risk and patient burden.
Should be appreciated that description and the follow-up detailed description of aforementioned cardinal principle are exemplary illustration and explanation, should not be used as the restriction to the claimed content of the present invention.
Accompanying drawing explanation
With reference to the accompanying drawing of enclosing, the following description by embodiment of the present invention is illustrated by the more object of the present invention, function and advantage, wherein:
Fig. 1: the second takes turns PCR reaction through 2% agarose electrophoresis qualification result.
Embodiment
By reference to one exemplary embodiment, object of the present invention and function and the method for realizing these objects and function will be illustrated.But the present invention is not limited to following disclosed one exemplary embodiment; Can be realized it by multi-form.The essence of specification sheets is only help various equivalent modifications Integrated Understanding detail of the present invention.
The invention provides a kind of method detecting BRCA1 and BRCA2 sudden change, although in some cases, important Auxiliary Significance is had for the diagnosis of associated cancer, prevention and therapy to the detection of these genes, but the sudden change of these genes is not the generation that must cause described disease, therefore the detection of these genes is not related to diagnosis or the methods for the treatment of of disease.In addition, the method detecting sudden change of the present invention can also have other purposes many, such as, carry out Polymorphism Analysis and family's evolutionary history research.Method of the present invention can also be used for the sample research of nonspecific object, such as, detect mixing blood products.
In the present invention, sequence SEQIDNO.5-SEQIDNO.382 matches formation 189 pairs of first set PCR primer between two, and namely SEQIDNO.5 and SEQIDNO.6, SEQIDNO.7 and SEQIDNO.8......SEQIDNO.381 and SEQIDNO.382 match formation 189 primer pairs respectively.
In the present invention, described 189 pairs of first set PCR primer divide two groups, first group comprises primer 95 to (SEQIDNO.5-SEQIDNO.194), second group comprises primer 94 to (SEQIDNO.195-SEQIDNO.382), this is through contriver by computer simulation and carries out testing the result carrying out adjusting, make the cross reaction avoided between primer like this, make the efficiency and the quality that improve PCR.
In the present invention, 5 ' end of each upstream primer adds sequence SEQIDNO.1 " ACACTCTTTCCCTACACGACGCTCTTCCGATCTTAC ", 5 ' end of each downstream primer adds sequence SEQIDNO.2 " GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTA ", and the consensus sequence that upstream and downstream primer adds is the second binding site of taking turns that PCR reacts primer.
In the present invention, the upstream primer sequence of the second cover PCR primer is: SEQIDNO.3 " AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC ",
Downstream primer sequence is SEQIDNO.4 " CAAGCAGAAGACGGCATACGAGAT
aTCACGTtGTGACTGGAGTTCAGACGT ", its middle and lower reaches underscore partial sequence is barcode sequence, for distinguishing multiple sample.
In the present invention, pcr amplification premix solution, the solution namely containing the required component of the pcr amplification such as Taq DNA polymerase, dNTPs, damping fluid (template and primer except), such as pcr amplification premix solution.
In a specific embodiments of the present invention, the application method of multiple PCR detection kit of the present invention is as follows:
1) DNA (such as conventionally) of detected sample is extracted as template;
2) carry out first round multiplexed PCR amplification reaction with first set primer, first group and second group of primer pair carry out respectively in two test tubes;
3) pcr amplification product is carried out purifying recovery by magnetic bead;
4) carry out second as template with the second cover primer with the product that first round PCR reclaims and take turns PCR reaction;
5) pcr amplification product is carried out purifying recovery by magnetic bead;
6) electroresis appraisal;
7) collect product and carry out upper machine order-checking (such as s-generation high-flux sequence);
8) whether there occurs sudden change by the gene of bioinformatic analysis qualification BRCA1 and BRCA2.
Wherein, described multiplexed PCR amplification reaction conditions is preferably as follows:
First round PCR reacts amplification system:
First round PCR reacts amplification condition:
First round pcr amplification after product purifying:
1) (Tris-Hcl weighs 121.1gTris-Hcl and is placed in 1L beaker, adds in the deionized water of more 800mL, fully stir PCR primer to be added isopyknic recovery damping fluid.Add the Hcl of 42mL, be settled to 1L.)
With 10ul magnetic bead (Beckman-B46053), concussion mixing 30s;
2) mixture is positioned over 30s on magnetic frame;
3) sucking-off supernatant liquor, dries 30s, leaves magnetic frame;
4) washing lotion W1 (70% ethanol) is added, concussion mixing 30s;
5) mixture is positioned over 30s on magnetic frame;
6) sucking-off supernatant liquor, dries 30s, leaves magnetic frame;
7) washing lotion W2 (H is added
2o), concussion mixing 30s;
8) mixture is positioned over 30s on magnetic frame;
9) sucking-off supernatant liquor, dries 2min, leaves magnetic frame;
10) (ATE, 48.4gTris-Hcl are dissolved in 150mL deionized water, and heat 45 DEG C and be stirred to dissolving, 14.61gEDTA adds above-mentioned solution, continue to stir to add 20ul elutriant.Add 11.42mLAC, add while stirring, become clarification after adding AC, EDTA thoroughly dissolves.Surveying PH is 8.6, adds deionized water and is settled to 200mL), concussion mixing 30s;
11) mixture is positioned over 30s on magnetic frame;
12) supernatant is collected for subsequent use.
Second takes turns PCR amplification system:
Second takes turns PCR reacts amplification condition:
Second takes turns the same first round pcr amplification after product purifying above of pcr amplification after product purification step.
Embodiment
One, the preparation of multiple PCR reagent kit of the present invention and assembling
1. the design of two-wheeled PCR primer and preparation
The first round 189 pairs of PCR primer,
First round PCR primer, points two groups, first group to comprise primer 95 right, and second group to comprise primer 94 right: often organize and get 1ul (single primer concentration 1uM), mixing;
Second takes turns PCR primer (overall primer concentration 0.1uM): get 1.25ul;
All primers all synthesize through automatic dna synthesizer.
2. the assembling of test kit
Pcr amplification premix solution 1 (KAPA, No:KK5801): 12.5ul;
Pcr amplification premix solution 2 (KAPA, No:KK2601): 7.5ul;
First round PCR primer first group: 5ul, first round PCR primer second group: 5ul
Second takes turns PCR primer test tube: 1.25ul.
Two, the application experiment of multiple PCR reagent kit of the present invention
1. detect sample
The sample source detected is in the blood of certain volunteer.
2. detection method
DNA extraction: conventionally extract the DNA of detected sample as template.
Pcr amplification:
First round PCR reacts amplification system and reacts amplification system with first round PCR mentioned above.
First round PCR reacts amplification condition and reacts amplification condition with first round PCR mentioned above.
First round pcr amplification after product purification step is with first round pcr amplification after product purifying mentioned above.
Second takes turns PCR amplification system takes turns PCR reaction amplification system with mentioned above second.
Second takes turns PCR reaction amplification condition takes turns PCR reaction amplification condition with mentioned above second.
Second takes turns pcr amplification after product purification step with first round pcr amplification after product purifying mentioned above.
Measuring A pipe 260/280 is 1.79, corresponding DNA concentration 5.02ng/ul.
Measuring B pipe 260/280 is 1.81, corresponding DNA concentration 6.87ng/ul.
Electroresis appraisal the results are shown in Figure 1.
3. detected result
By machine order-checking in the result of two-wheeled PCR, and carry out bioinformatic analysis.
1) first quality evaluation is carried out to sequencing data.
2) the sequencing data amount after assessment is added up, as shown in the table.
3) reference sequences comparison and depth data statistics
Adopt Burrows-WheelerAligner (BWA) software previous step to be generated the data after Quality Control (cleandata) to compare with full-length genome, BWA is the comparison software that a analysis is accurate, analysis efficiency is high.SequenceAlignmentMap (SAM) document result is generated after comparison completes.Then the tumor-necrosis factor glycoproteins (the unnecessary information produced in PCR process) in picard software removing SAM is adopted, finally, we utilize samtools software that the destination file of SAM is changed into BAM file, BAM form is exactly the compression result of SAM form, and SAM file layout illustrates and refers to http://samtools.sourceforge.net/SAM1.pdf.
| Project | Value |
| Sequencing sequence mean length (Average read length) | 139 |
| Order-checking base average mass values (Average base quality) | 32 |
| On average build storehouse length (Average lab size) | 193.8 |
| Repetition rate (Duplication rate) (%) | Nothing |
| Comparison rate (Align rate) (%) | 82.56 |
| The target area size (Total target base) of catching | 15915 |
| The size (Covered target base) in actual sequencing result coverage goal region | 15662 |
| Fraction of coverage (Coverage rate) (%) | 98.41 |
| Total valid data amount (Total effective base) (Mb) | 84.51 |
| The valid data amount (Effective base on target) (Mb) of target area | 14.89 |
| Capture rate (Capture rate) (%) | 17.62 |
| The mean depth (Target average depth) of target area | 935.54 |
| The target area degree of depth is greater than the ratio (Target 4X rate) (%) shared by the region of 4 layers | 95.97 |
| The target area degree of depth is greater than the ratio (Target 10X rate) (%) shared by the region of 10 layers | 95.27 |
| (the ratio Target 20X rate shared by region that the target area degree of depth is greater than 20 layers) (%) | 92.57 |
| Capture region title (Panel name) | BRCA1/2 |
Comparison rate: the sequence-tumor-necrosis factor glycoproteins/cleandata of human genome in comparison
Fraction of coverage: the target area size of the size in actual sequencing result coverage goal region/catch
Total valid data amount: the sequence-tumor-necrosis factor glycoproteins of human genome in comparison
Capture rate: the valid data amount of target area/
4) the sudden change distribution found in this example:
| Exon same sense mutation (exonic_synonymous) | 9 |
| Exon nonsense mutation (exonic_nonsynonymous) | 12 |
| Exon 1 sudden change sum (exonic) | 21 |
| Intron region mutation sum (intronic) | 4 |
| Variable brief introduction region mutation sum (splicing) | 1 |
| 5 ' UTR region mutation sum (5-UTR) | 1 |
Although invention has been described in conjunction with the preferred embodiments, be to be understood that protection scope of the present invention is not limited to embodiment as described herein.In conjunction with the explanation of the present invention disclosed here and practice, other embodiments of the present invention are all easy to expect and understand for those skilled in the art.Illustrate and embodiment be only considered to exemplary, true scope of the present invention and purport limited by claim.
Claims (6)
1. detect a method for BRCA1 and BRCA2 sudden change, described method comprises
1) catch: carry out first round specificity multi-PRC reaction, 189 pairs of primer pair sample DNAs are used to increase, described 189 pairs of PCR primer divide two test tubes to react, first test tube primer, 95 couples of SEQIDNO.5-SEQIDNO.194, second test tube primer, 94 couples of SEQIDNO.195-SEQIDNO.382,5 ' end of each upstream primer adds sequence SEQIDNO.1, and 5 ' end of each downstream primer adds sequence SEQIDNO.2;
2) storehouse is built: carry out second and take turns PCR reaction, use PCR primer pair: upstream and downstream primer SEQIDNO.3 and SEQIDNO.4 is to step 1) amplified production that obtains increases;
3) checking order, to step 2) amplified production that obtains checks order;
4) analyze, to step 3) sequencing result that obtains analyzes, and completes the abrupt climatic change of full coding region.
2. a BRCA1 and BRCA2 transgenation multiple PCR detection kit, described test kit comprises: two cover PCR primer;
Two described cover PCR primer, first set PCR primer comprises 189 pairs of PCR primer, divide two groups, first group comprises primer 95 couples of SEQIDNO.5-SEQIDNO.194, second group comprises primer 94 couples of SEQIDNO.195-SEQIDNO.382,5 ' end of each upstream primer adds sequence SEQIDNO.1, and 5 ' end of each downstream primer adds sequence SEQIDNO.2;
The upstream and downstream primer sequence of the second cover PCR primer is SEQIDNO.3 and SEQIDNO.4 respectively.
3. the test kit of claim 2, described test kit also comprises pcr amplification premix solution (such as, from the pcr amplification premix solution of KAPA2GFast warm start multiplex amplification test kit).
4. the test kit of claim 2, described test kit also comprises water.
5. the test kit of claim 3, in described test kit, the reaction system of the PCR reaction of first set primer is by following proportions: first set PCR primer 10 parts, pcr amplification premix solution 12.5 parts, DNA profiling 3 parts, 4.5 parts, water.
6. the test kit of claim 2, in described test kit, the reaction system of the second cover PCR reaction is by following proportions: PCR primer 2.5 parts, pcr amplification premix solution 7.5 parts, first round PCR reclaim product 5 parts, 4.5 parts, water.
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