CN110592210A - Sequence primer for detecting BRCA1/2 full exonic sequence and design method - Google Patents
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Abstract
A primer for detecting a BRCA1/2 full exon sequence, which comprises: an exon amplification primer pair designed for BRCA1, an exon amplification primer pair designed for BRCA 2; the exon amplification primer pair can cover the whole exon region of BRCA 1/2; the exon amplification primer pairs designed for BRCA1 and the exon amplification primer pairs designed for BRCA2 are divided into two groups respectively, and no amplification region is overlapped in each group of primers of the same gene; in each group of primer pairs, the 5 'end of the upstream primer of the first part of primers is added with SEQ ID.NO1, and the 5' end of the downstream primer of the first part of primers is added with SEQ ID.NO2; the 5 'end of the upstream primer of the second part of primers is added with SEQ ID.NO2, and the 5' end of the downstream primer is added with SEQ ID.NO1; one group of primer pairs of the BRCA1 gene and one group of primer pairs of the BRCA2 gene are randomly combined to form two primer pairs. The method has the advantages of low cost, high flux, wide detection range and short detection time; the probability of generating amplicon products between non-primer pairs in one round of PCR is low; the detection flexibility is high, and different primer groups can be set according to different requirements.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for designing primers of exon regions of BRCA1/BRCA2 genes and a primer group sequence.
Background
In 1990, researchers discovered a gene directly related to hereditary breast cancer, named as breast cancer gene No.1, which is abbreviated as BRCA 1. In 1994, another gene associated with breast cancer was discovered, designated BRCA2, and in many cases both genes were discussed together under the general designation BRCA 1/2. BRCA1 maps to chromosome 17q221.31 with a gene size of 81.19kb for a total of 24 exons, and BRCA2 maps to chromosome 13q13.1 with a gene size of 84.19kb for a total of 27 exons.
BRCA1/2 is two genes with effects of inhibiting malignant tumor, and has important effects in regulating human cell replication, repairing genetic material DNA injury, and normal cell growth. Families with this gene mutation tend to have a high incidence of breast cancer, usually at a younger age, with tumors in both breasts and concurrent ovarian cancer. The life-long risks of cancer associated with mutations in BRCA1 and BRCA2 genes were summarized, indicating that the risk for breast and ovarian cancer was 50% -85% and 15% -45%, respectively, for those with mutations in BRCA1, and 50% -85% and 10% -20%, respectively, for those with mutations in BRCA 2. Therefore, the BRCA1/2 gene mutation site detection of high-risk population has important significance for the prevention and early inspection of cancer.
In the screening of early BRCA1/2 gene mutation, Sanger sequencing is directly carried out by a conventional method, but the BRCA1/2 gene has large size and high direct sequencing cost, and methods such as a denaturing high performance liquid chromatography analysis technology, a high-resolution melting analysis technology and the like are also frequently adopted. Nowadays, the second generation sequencing (NGS) technology is increasingly high-throughput, low-cost and diversified, and has become a widely applied technology in the field of molecular diagnosis, and many laboratories currently use multiplex PCR technology to detect mutations of BRCA1/BRCA2, for example, 58 pairs of primers are designed to cover positions of full exons of BRCA1/2 and splice sites of BRCA1/2 based on multiplex PCR targeted sequencing, the master thesis at southeast university, 2017, wherein the primer pair BRCA1 and the primer pair BRCA2, which respectively cover gene lengths of 17763bp and 24383bp, and the amplicon length is between 400 and 1500bp, and 24 mutations are detected in total, and 1 BRCA1 pathogenic mutation rs80357303 is one of the pathogenic mutations, which is reported to increase the risk of breast cancer.
Second generation sequencing technology has the characteristic of high throughput, and as sequencing time is gradually shortened, the feasibility of cheap and short-period universal screening by using second generation sequencing for BRCA1 and BRCA2 is enhanced. However, the detection efficiency and accuracy of the current second-generation sequencing technology still have room for improvement.
Disclosure of Invention
Therefore, the application provides a primer for detecting a BRCA1/2 full exon sequence, a design method of the primer and application of the primer. More preferably, the application provides a primer for detecting BRCA1/2 full exon sequences by using multiplex PCR amplification, a design method of the primer and application of the primer.
In a first aspect, the present application provides a primer for detecting a full exon sequence of BRCA1/2, comprising: an exon amplification primer pair designed for BRCA1, an exon amplification primer pair designed for BRCA 2; wherein, the exon amplification primer pair designed for BRCA1 can cover the whole exon region of BRCA1, and the exon amplification primer pair designed for BRCA2 can cover the whole exon region of BRCA 2; moreover, the exon amplification primer pair designed for BRCA1 and the exon amplification primer pair designed for BRCA2 are respectively divided into two groups, and the amplification regions in each group of primers of the same gene are not overlapped; in each group of primer pairs, the 5 'end of the upstream primer of the first part of primers is added with SEQ ID.NO1, and the 5' end of the downstream primer of the first part of primers is added with SEQ ID.NO2; the 5 'end of the upstream primer of the second part of primers is added with SEQ ID.NO2, and the 5' end of the downstream primer is added with SEQ ID.NO1; one group of primer pairs of the BRCA1 gene and one group of primer pairs of the BRCA2 gene are randomly combined to form two primer pairs.
In a preferred embodiment, the primer for detecting the BRCA1/2 full exon sequence further comprises a universal library-building primer.
In a second aspect, the present application provides a method for designing a primer for detecting a full exon sequence of BRCA1/2, comprising:
step 1) designing a BRCA1 exon amplification primer pair which can cover all exon regions of BRCA1, and designing a BRCA2 exon amplification primer pair which can cover all exon regions of BRCA 2; dividing BRCA1 exon amplification primer pairs and BRCA2 exon amplification primer pairs into two groups respectively, wherein no amplification region is overlapped in each group of primers of the same gene;
step 2) adding SEQ ID.NO1 to the 5 'end of the upstream primer and SEQ ID.NO2 to the 5' end of the downstream primer of each group of primer pairs of the first part of primers; the 5 'end of the upstream primer of the second part of primers is added with SEQ ID.NO2, and the 5' end of the downstream primer is added with SEQ ID.NO1;
and 3) randomly combining a group of primer pairs of the BRCA1 gene and a group of primer pairs of the BRCA2 gene to form two primer pairs.
In a preferred embodiment, the number of the first partial primer pairs and the second partial primer pairs may be the same or different.
In a preferred embodiment, the first set of primer pairs consists of any two or more primers selected from SEQ id no3 to SEQ id no 40.
In a preferred embodiment, the second set of primer pairs consists of any two or more primers selected from SEQ id no41 to SEQ id no 72.
In a preferred embodiment, the third set of primer pairs consists of any two or more primers selected from SEQ id no73 to SEQ id no 130.
In a preferred embodiment, the fourth set of primer pairs consists of any two or more primers selected from SEQ id no131 to SEQ id no 180.
In a preferred embodiment, the universal library primer is selected from any one or more of SEQ id no181, SEQ id no 182.
The third aspect of the invention provides a kit for detecting a BRCA1/2 full exon sequence, which comprises the primer for detecting the BRCA1/2 full exon sequence.
In a preferred embodiment, the kit may further comprise distilled water.
In a preferred embodiment, the kit may further comprise a PCR amplification enzyme, such as a multiplex PCR amplification enzyme, e.g. a Vazyme multiplex PCR amplification enzyme.
In a preferred embodiment, the kit may further comprise a gDNA extraction reagent or a gDNA extraction kit.
In a fourth aspect, the invention provides a nucleotide sequence for linking to a BRCA1 exon amplification primer pair and a BRCA1 exon amplification primer pair, wherein the nucleotide sequence comprises SEQ ID.NO1 and SEQ ID.NO2.
Compared with the existing multiplex PCR technology, the application has the beneficial effects that:
1) the cost is low, and the detection range is wide;
2) the flux is high;
3) the detection time is short;
4) the probability of generating amplicon products between non-primer pairs in one round of PCR is low;
5) the detection flexibility is high, and different primer groups can be set according to different requirements.
Drawings
FIG. 1 shows the Q-Sep identification result of the product to be sequenced obtained by using the primers and the kit of the present application.
Detailed Description
For a clearer understanding of the objects, features and advantages of the present invention, reference is made to the following detailed description of the invention taken in conjunction with the accompanying drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments disclosed below.
Example 1
Primer3 is used to design primers with the target amplicon size of 220-300bp, all the primers can be ensured to cover all exon regions of BRCA1/2, and the primer pairs of BRCA1 and BRCA2 are divided into two groups respectively, so that no amplification region is overlapped in each group of primers of the same gene. Wherein the first group and the second group are BRCA1 exon amplification primers: the first group of primers is SEQ ID NO.3-SEQ ID NO.40, and the second group of primers is SEQ ID NO.41-SEQ ID NO. 72. Third and fourth sets were BRCA2 exon amplification primers: the third group of primers is SEQ ID NO.73-SEQ ID NO.130, the fourth group of primers is SEQ ID NO.131-SEQ ID NO.180, and the fifth group of primers is SEQ ID NO.181-SEQ ID NO. 182.
The 4 primer pairs of the primer groups are respectively numbered, the primer pairs are numbered according to the sequence of genome coordinates, 1, 2 and 3 … … n, SEQ ID.NO1 is added to the 5 'end of the upstream primer and SEQ ID.NO2 is added to the 5' end of the downstream primer in the odd primer pair, SEQ ID.NO2 is added to the 5 'end of the upstream primer and SEQ ID.NO1 is added to the 5' end of the downstream primer in the even primer pair.
A primer set of BRCA1 was randomly combined with a primer set of BRCA2, and the remaining BRCA1 and BRCA2 primers were combined to form primer tube 1 and primer tube 2.
A first combination of: primer tube 1(SEQ ID NO.3-SEQ ID NO.40, SEQ ID NO.73-SEQ ID NO.130), primer tube 2(SEQ ID NO.41-SEQ ID NO.72, SEQ ID NO.131-SEQ ID NO. 180).
A second combination of: primer tube 1(SEQ ID NO.3-SEQ ID NO.40, SEQ ID NO.131-SEQ ID NO.180), primer tube 2(SEQ ID NO.41-SEQ ID NO.72, SEQ ID NO.73-SEQ ID NO. 130).
In addition, for performing multiplex PCR amplification second-generation sequencing, universal library-building primers are also provided as a fifth set of primers, e.g., SEQ ID NO.181-SEQ ID NO. 182.
Example 2
The application method of the multiplex PCR detection kit comprises the following steps:
1) taking DNA of a sample to be detected as a template;
2) performing PCR reactions in two tubes using the first/third and second/fourth sets of primer pairs, respectively; preferably, the PCR program is 95 ℃ for 5 min; 95 ℃ for 30s, 60 ℃ for 30s, 58 ℃ for 30s, 56 ℃ for 30s, 70 ℃ for 1min (3 cycles); 95 ℃ for 30s, 68 ℃ for 1min (15 cycles); 5min at 72 ℃; infinity at 4 ℃; as shown in table 1:
TABLE 1 first round PCR amplification reaction System and amplification reaction conditions
3) Combining the two tubes of PCR products;
4) performing first purification on the PCR product by using Ampure beads, and then performing second purification by using beads buffer and water; specifically, 100 μ l of (Ampure beads) magnetic beads are added, mixed uniformly and kept stand for 5min, then placed on a magnetic rack, and after clarification, the supernatant is sucked and discarded; cleaning two sides with 80% ethanol, air drying, redissolving with 15 μ L distilled water for 3min, adding 18 μ L beads buffer, mixing, standing for 5min, cleaning two sides with 80% ethanol, and air drying;
5) taking the purified product as a template, re-dissolving the purified product by using 22 mu L of distilled water, and then sucking 20 mu L of supernatant to perform a second round of PCR reaction, wherein the fifth group of primers are used in the second round of PCR; the preferred PCR reaction procedure is 95 ℃ for 5 min; 95 ℃ for 30s, 65 ℃ for 1min (8 cycles); 5min at 72 ℃; infinity at 4 ℃; as shown in table 2:
TABLE 2 second round PCR amplification reaction System and amplification reaction conditions
6) Performing first purification on the PCR product by using Ampure beads, and then performing second purification by using beads buffer and water; specifically, 45 μ l of (Ampure beads) magnetic beads are added, mixed uniformly and kept stand for 5 min; then placing the mixture on a magnetic frame, and absorbing and discarding the supernatant after clarification; cleaning two sides with 80% ethanol, and air drying; redissolving with 20 μ L distilled water for 3min, adding 24 μ L beads buffer, mixing, and standing for 5 min; cleaning two sides with 80% ethanol, and air drying; after redissolving with 22. mu.L of distilled water, 20. mu.L of the supernatant was aspirated and stored for further use.
7) Detecting the concentration;
8) Q-Sep identifies the fragment size;
9) sequencing on a computer;
10) whether the mutation exists in BRAC1/2 is analyzed by a bioinformatics means.
The first to fifth sets of primers had a primer stock concentration of 50. mu.M and each primer concentration ranged from 0.05-0.40 mM.
Example 3
Blood of a healthy volunteer was collected using a 5mL EDTA blood collection tube, and the blood cell gDNA was extracted using QIAGEN kit as a PCR amplification reaction DNA template.
Multiplex PCR amplification was performed using the method described in example 2.
The sample concentration was determined to be 5.6 ng/uL.
The Q-Sep results are shown in FIG. 1, Table 3 and Table 4.
TABLE 3 off-line data quality assessment
| Sequencing original reads (bars) | Residual reads number after quality control | Ratio of remaining data after quality control |
| 1143191 | 1086806 | 95.06％ |
And (3) sequencing the sample by using an illumina sequencer, wherein the total number of reads is not 1143191, and after quality control (removing the reads with unqualified quality), 1086806 reads are remained, and the qualified reads with the quality control account for 95.06% of the total number of the reads.
TABLE 4 reference sequence alignment and depth statistics
| Parameter(s) | Numerical value |
| Average length of sequencing sequence | 287 |
| Length range of building warehouse | 370-420 |
| Comparison Rate (mapping rate) | 98％ |
| Capture area size | 29925bp |
| Actual capture area size | 29925bp |
| Coverage degree | 100％ |
| Mean depth | 9600X |
The average length of the library insert is 287bp, the size of the complete library is (370-420bp) after the library construction process and the sequencing universal sequence are added, and 98% of reads qualified by quality control can be correspondingly aligned to the human genome sequence.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ccacagtaga tgctcagtaa atatttc 27
<210> 15
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cacatacatc cctgaaccta aaataaa 27
<210> 16
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gagcagcttt cactaactaa ataagat 27
<210> 17
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
taattacagt actgtatcta cccactc 27
<210> 18
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ttagtgagga aacaaaatgt tctgcta 27
<210> 19
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ttagctcact tctataaata gactggg 27
<210> 20
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gacctgttag atgatggtga aataaag 27
<210> 21
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tatgcctagt agactgagaa ggtatat 27
<210> 21
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cacaattagc cgtaataaca ttagaga 27
<210> 22
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ctgaactact tcttcatatt cttgctt 27
<210> 23
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
aaaagtcact tttgaatgtg aacaaaa 27
<210> 24
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ttaacaaatg acttgatggg aaaaagt 27
<210> 25
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tatttcattg gtacctggta ctgatta 27
<210> 26
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gaaaccttga atgtattctg caaatac 27
<210> 27
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
aactagtagt cagtagaaat ctaagcc 27
<210> 28
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
aggattgaca aattctttaa gttcact 27
<210> 29
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
taattatagg agcatttgtt actgagc 27
<210> 30
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
agattctttt tcgagtgatt ctattgg 27
<210> 31
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gagagaaaag aatggaataa gcagaaa 27
<210> 32
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tcacttttac atattaaagc ctcatga 27
<210> 33
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
acaattcagt ttttgagtac cttgtta 27
<210> 34
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gcttttatta cagaattcag ccttttc 27
<210> 35
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
cattttgtgt gacatgaaag taaatcc 27
<210> 36
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ttaattcaaa gagatgatgt cagcaaa 27
<210> 37
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gcttctcaaa gtatttcatt ttcttgg 27
<210> 38
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gagataaagg ggaaggaaag aattttg 27
<210> 39
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gaatcttgcc cttaacttgt ttacag 26
<210> 40
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
caaacacagc tattaaaaag tcattcc 27
<210> 41
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
acggaaaagc gcgggaatta cagataa 27
<210> 42
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
cttacgcctc tcaggttccg cccctac 27
<210> 43
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gtgtgtgtat aaatttggtt tgttctt 27
<210> 44
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
ctggtttcca ctataccaaa gtaaaaa 27
<210> 45
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
tttttgaaga tctagaacca cattgtt 27
<210> 46
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
cgtctctaca gaaaacacaa aatttag 27
<210> 47
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
ttctactgtt gctgcatctt attttta 27
<210> 48
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
taacctagac taaaaggtct tatcacc 27
<210> 49
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
aagagaactt atctagtgag gatgaag 27
<210> 50
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tatttgcagt caagtcttcc aattc 25
<210> 51
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
aacctgaggt ctataaacaa agtcttc 27
<210> 52
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
ctaaaaacag cagaactttc cttaatg 27
<210> 53
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
gaaactggac tcattactcc aaataaa 27
<210> 54
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tggaacctac ttcattaata ttgcttg 27
<210> 55
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gaaacaagca tagaaatgga agaaagt 27
<210> 56
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
cctgcagtga tattaactgt ctgta 25
<210> 57
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
cctggttctt ttactaagtg ttcaaat 27
<210> 58
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
catttatttg gttctgtttt tgccttc 27
<210> 59
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tagtggtcat gagaataaaa caaaagg 27
<210> 60
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
ttatctcttc actgctagaa caactat 27
<210> 61
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ggacgttcta aatgaggtag atgaata 27
<210> 62
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
gtaggtctcc ttttacgctt taattta 27
<210> 63
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
aacagcagtt tattactcac taaagac 27
<210> 64
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ttctgaatgc tgctatttag tgttatc 27
<210> 65
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
attgaaagtt ccccaattga aagtt 25
<210> 66
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
cctacataaa actctttcca gaatgtt 27
<210> 67
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
ggggttttag aatcataaat ccagatt 27
<210> 68
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
ttaggtgtta aacgttaggt gtaaaaa 27
<210> 69
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
taaattaaac atcaactctg tctccag 27
<210> 70
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
actatatgac tgaatgaata tctctggt 28
<210> 71
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
gaatgcctta aatatgacgt gtctg 25
<210> 72
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
agcactgtgt atgtatgtaa taagtct 27
<210> 73
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
cacataagga acagtttatg gttctaa 27
<210> 74
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
gagttttacc tcagtcacat aataagg 27
<210> 75
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
tttgaatatt attggagttg aagccag 27
<210> 76
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
agtaatccat agtcaagatc ttaagca 27
<210> 77
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
gctcttagcc aaaatattag cataaaa 27
<210> 78
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
tttttaaaca cttccaaaga atgcaaa 27
<210> 79
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
caattactaa gtcataaaaa taaaccaggt 30
<210> 80
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
aataagataa actagttttt gccagtttt 29
<210> 81
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
tagagatgac aattatcaac ctcatct 27
<210> 82
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
gcaattcagt aaacgttaag tgaaata 27
<210> 83
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
aaatcacata taggaccagg tttagag 27
<210> 84
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
tcaaatagta gatgtgcttt ttgatgt 27
<210> 85
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
aaaaacctgt agttcaacta aacagag 27
<210> 86
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
ccattaaaaa tttttggacc taggttg 27
<210> 87
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
agtcttgcta gttcttactt tttgtag 27
<210> 88
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
taaactgttt ctatgagaaa ggttgtg 27
<210> 89
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
atttaatggc ttctctgatt ttggtag 27
<210> 90
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
tccattagat tcaaatgtag caaatca 27
<210> 91
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
acttatttgt tttctttttc aaagtgga 28
<210> 92
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
cttcatttca gggtatcaaa aagtcta 27
<210> 93
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
gaatcagctt ctggggtaat aaataac 27
<210> 94
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
ccaaacacta cctttttaac ttagtga 27
<210> 95
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
cttagatttg tgttttggtt gaattgt 27
<210> 96
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
cttttatatg atcatgaaaa tgccagc 27
<210> 97
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
gcccatttgt tcatgtaatc attattt 27
<210> 98
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
agctaatgaa aggaataatc ttgcttt 27
<210> 99
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
agcttggttt tctaaactga gtaaatt 27
<210> 100
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
atatcctact agtttagctt gtgttga 27
<210> 101
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
taaacattga aacaacagaa tcatgac 27
<210> 102
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
gcaatttgaa ggtacagttg aaattaa 27
<210> 103
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
tttatatttt gctccgtttt agtagca 27
<210> 104
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
ctgccagtag aaattctcat aacttag 27
<210> 105
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
tttcatcaaa aaggtttttc actttgt 27
<210> 106
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
gaaaccagaa gaattgcata acttttc 27
<210> 107
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
ctcacagaag tttttctact acaactt 27
<210> 108
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
caaagtgtaa agaaatgcag aattctc 27
<210> 109
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
ggacaattta atggctgcat ttttatt 27
<210> 110
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
aaaatcatct ctccgaaaaa caagata 27
<210> 111
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
aagtatttgc agatgagact gacttat 27
<210> 112
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
agaataaatc aaaaatttgc caaacga 27
<210> 113
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
acacgaggaa gtatttttga tacattt 27
<210> 114
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
agcaagtctt ttccaaagta ttgttta 27
<210> 115
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
gtttagaatc tgtcagttca tcatctt 27
<210> 116
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
agtttctcca tatctctctc aatttca 27
<210> 117
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
aagagtataa agaggtcctt gattagg 27
<210> 118
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
ttgagaaata aaactgatat tatttgcctt 30
<210> 119
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
aagtgttaac ttcttaacgt tagtgtc 27
<210> 120
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
acattcactg aaaattgtaa agcctat 27
<210> 121
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
tagttttaaa aggtggaaca aagactt 27
<210> 122
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
tgcaacaaag gcatattcct aaatatt 27
<210> 123
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
aaaattacac tctgtcataa aagccat 27
<210> 124
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
gggttgtgct ttttaaattt caatttt 27
<210> 125
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
tgagggaata cataaaagtt aacacac 27
<210> 126
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
tgtgtgatac atgtttactt taaattgtt 29
<210> 127
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
tactgccgta tatgattacg taatgta 27
<210> 128
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
ttgtagttgt tgaattcagt atcatcc 27
<210> 129
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
taccacccat ctgtaagttc aataatg 27
<210> 130
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
tcagtgactt gtttaaacag tggaatt 27
<210> 131
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
gtacaacttc cttggagatt ttgtc 25
<210> 132
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
tatgaaacag ttgtagatac ctctgaa 27
<210> 133
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
gaaaaacttg cattgaaagt ctcttta 27
<210> 134
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
gaaaaagacc tattagacac agagaac 27
<210> 135
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
cacagaagga atcgtcatct ataaaac 27
<210> 136
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
tagctttgaa gaatgcaggt ttaatat 27
<210> 137
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
ggctgaaaag acatagttta taacact 27
<210> 138
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
tactttgaaa cagaagcagt agaaatt 27
<210> 139
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
tccttggaag taggagttaa aataaga 27
<210> 140
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
ctctcaggat cttgattata aagaagc 27
<210> 141
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
tagagttctt gaaaatgggt tcgttta 27
<210> 142
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
tgaaaattat aaaaacgttg agctgtt 27
<210> 143
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
gatacagtat taattgactg aggcttg 27
<210> 144
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
aaatcggaca tctccttgaa tatagat 27
<210> 145
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
cacttttgtt acagtcattt ttcaaca 27
<210> 146
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
aaaaggcaga aattacagaa ctttcta 27
<210> 147
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
tatcattttt acttgaatca ctgccat 27
<210> 148
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
ttgagaatat tagtgaggaa acttctg 27
<210> 149
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
cctcataact tagaatgtcc attttgt 27
<210> 150
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
agcatgtcat ggtaatactt caaataa 27
<210> 151
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
acagtctcaa tagaaacaag gttttta 27
<210> 152
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
aagaacctac tctattgggt tttcata 27
<210> 153
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
tcctgaatca ttatatacct catcaga 27
<210> 154
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
ccttagcttt ttacacaagt tgtagta 27
<210> 155
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
gaagaatatc ctctgaatca tccaatg 27
<210> 156
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
aaactgtaaa tgaagatatt tgcgttg 27
<210> 157
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
ggatattaaa tgttctggag tacgtat 27
<210> 158
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
gtgatgttag tttggaaact tcagata 27
<210> 159
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
aacaagtgac actttggttc ctaata 26
<210> 160
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
gggagtgtta gaggaatttg atttaat 27
<210> 161
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
ttgtcataca atacctaaag gttcttc 27
<210> 162
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
caagtttctg ctacaagaaa tgaaaaa 27
<210> 163
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
atcaatgact gatttttacc aagagtg 27
<210> 164
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
cgcaaatata tctgaaactt ctagcaa 27
<210> 165
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
tgtatcaaaa gaaagaaata tatggtaagt 30
<210> 166
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
gcagttctag aagaatgaaa actctta 27
<210> 167
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
aaaatgttaa attcaaagtc tctaagac 28
<210> 168
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
ggcctgatac aattaacttg aatgtta 27
<210> 169
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
ctaaaacagc ttctcacctt gaataat 27
<210> 170
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
tcttatcttt aaatctccct tctttgg 27
<210> 171
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
taaaactgat aaaaacaaag catttaca 28
<210> 172
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
tttttgttct gattgctttt tattcca 27
<210> 173
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
ggtagctcca actaatcata agagatt 27
<210> 174
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
taagtgcttg ttagtttatg gaatctc 27
<210> 175
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
cattttgttg aatgtctctt gaaagtg 27
<210> 176
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
tcctttcttg catcttaaaa ttcatct 27
<210> 177
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
cagaatatac gatggcctcc atatata 27
<210> 178
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
tctgctttta aaggaaatac ttttgga 27
<210> 179
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
gaagcaaaag tataccaata cggaatc 27
<210> 180
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
gttgtactac atctctgatc aaagaac 27
<210> 181
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
aatgatacgg cgaccaccga gatctacact ctttccctac acgac 45
<210> 182
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
caagcagaag acggcatacg agatnnnnnn gtgactggag ttcagacgtg tgc 53
Claims (10)
1. A primer for detecting a BRCA1/2 full exon sequence, which is characterized by comprising: an exon amplification primer pair designed for BRCA1, an exon amplification primer pair designed for BRCA 2; wherein, the exon amplification primer pair designed for BRCA1 can cover the whole exon region of BRCA1, and the exon amplification primer pair designed for BRCA2 can cover the whole exon region of BRCA 2; moreover, the exon amplification primer pair designed for BRCA1 and the exon amplification primer pair designed for BRCA2 are respectively divided into two groups, and the amplification regions in each group of primers of the same gene are not overlapped; in each group of primer pairs, the 5 'end of the upstream primer of the first part of primers is added with SEQ ID.NO1, and the 5' end of the downstream primer of the first part of primers is added with SEQ ID.NO2; SEQ ID.NO2 is added to the 5 'end of the upstream primer of the second partial primer, and SEQ ID.NO1 is added to the 5' end of the downstream primer; one group of primer pairs of the BRCA1 gene and one group of primer pairs of the BRCA2 gene are randomly combined to form two primer pairs.
2. The primer for detecting the full exon sequence of BRCA1/2 according to claim 1, further comprising a universal library primer.
3. The primer for detecting the full exon sequence of BRCA1/2 according to claim 2, wherein the universal library primer is selected from any one or more of SEQ ID no181 and SEQ ID no 182.
4. The primers for detecting the full exon sequence of BRCA1/2 according to claim 1, wherein the first set of primer pairs consists of any two or more primers selected from SEQ ID no3 to SEQ ID no 40; the second set of primer pairs consists of any two or more primers selected from SEQ id no41 to SEQ id no 72; the third set of primer pairs consists of any two or more primers selected from SEQ id no73 to SEQ id no 130; the fourth set of primer pairs consists of any two or more primers selected from SEQ id no131 to SEQ id no 180.
5. The method for designing the primer for detecting the full exon sequence of BRCA1/2 according to claim 1, comprising:
step 1) designing a BRCA1 exon amplification primer pair which can cover all exon regions of BRCA1, and designing a BRCA2 exon amplification primer pair which can cover all exon regions of BRCA 2; dividing BRCA1 exon amplification primer pairs and BRCA2 exon amplification primer pairs into two groups respectively, wherein no amplification region is overlapped in each group of primers of the same gene;
step 2) adding SEQ ID.NO1 to the 5 'end of the upstream primer and SEQ ID.NO2 to the 5' end of the downstream primer of each group of primer pairs of the first part of primers; the 5 'end of the upstream primer of the second part of primers is added with SEQ ID.NO2, and the 5' end of the downstream primer is added with SEQ ID.NO1;
and 3) randomly combining a group of primer pairs of the BRCA1 gene and a group of primer pairs of the BRCA2 gene to form two primer pairs.
6. A kit for detecting the full exon sequence of BRCA1/2, which comprises the primer for detecting the full exon sequence of BRCA1/2 as claimed in claim 1.
7. The kit of full-length exons according to claim 6, wherein said kit further comprises a PCR amplification enzyme.
8. The kit of claim 6, further comprising a gDNA extraction reagent or a gDNA extraction kit.
9. The kit of full exosequences according to claim 6, characterized in that it further comprises distilled water.
10. A nucleotide sequence for connecting to BRCA1 exon amplification primer pair and BRCA1 exon amplification primer pair, comprising SEQ ID No.1 and SEQ ID No. 2.
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| CN106367481A (en) * | 2016-08-26 | 2017-02-01 | 广州永诺健康科技有限公司 | Multiplex PCR primer for amplifying BRCA1/2 gene and design method of multiplex PCR primer |
| CN106544431A (en) * | 2016-10-28 | 2017-03-29 | 林巍 | The sequencing library construction method of hyperphenylalaninemia related gene exons mutation |
| CN107400714A (en) * | 2017-08-21 | 2017-11-28 | 广州永诺生物科技有限公司 | The multiple PCR primer group and kit of colorectal cancer medication related gene detection |
| CN107446995A (en) * | 2016-05-06 | 2017-12-08 | 艾吉泰康生物科技(北京)有限公司 | For expanding the primer sets of multiple target dna sequences and its application in sample |
| CN109022559A (en) * | 2018-08-21 | 2018-12-18 | 华中农业大学 | A kind of molecular mark detection method based on two generation sequencing technologies |
| CN110029147A (en) * | 2019-01-25 | 2019-07-19 | 上海何因生物科技有限公司 | A kind of single tube realizes the non-specific PCR amplification method of continuum |
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Patent Citations (6)
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| CN107446995A (en) * | 2016-05-06 | 2017-12-08 | 艾吉泰康生物科技(北京)有限公司 | For expanding the primer sets of multiple target dna sequences and its application in sample |
| CN106367481A (en) * | 2016-08-26 | 2017-02-01 | 广州永诺健康科技有限公司 | Multiplex PCR primer for amplifying BRCA1/2 gene and design method of multiplex PCR primer |
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| CN110029147A (en) * | 2019-01-25 | 2019-07-19 | 上海何因生物科技有限公司 | A kind of single tube realizes the non-specific PCR amplification method of continuum |
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