CN110592210B - Sequence primer for detecting BRCA1/2 total exon and design method - Google Patents
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Abstract
A primer for detecting BRCA1/2 whole-exon, comprising: an exon amplification primer pair designed for BRCA1, an exon amplification primer pair designed for BRCA 2; the exon amplification primer pair can cover all exon areas of BRCA 1/2; the exon amplification primer pair designed for BRCA1 is divided into two groups, and the interior of each group of primers of the same gene is not overlapped with an amplification region; in each primer pair, SEQ ID NO1 is added to the 5 'end of the upstream primer of the first part, and SEQ ID NO2 is added to the downstream 5' end of the first part; SEQ ID NO2 is added to the 5 'end of the upstream primer of the second part primer, and SEQ ID NO1 is added to the 5' end of the downstream primer; a set of primer pairs of the BRCA1 gene and a set of primer pairs of the BRCA2 gene are randomly combined to form two primer pairs. The application has low cost, high flux, wide detection range and short detection time; the probability of generating amplicon products between non-primer pairs in a 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 application relates to the technical field of biology, in particular to a method for designing primers in exon regions of BRCA1/BRCA2 genes and a primer group sequence.
Background
In 1990, researchers found a gene directly related to hereditary breast cancer, named breast cancer number 1 gene, abbreviated BRCA1 in english. In 1994, another gene related to breast cancer was found and called BRCA2, and in many cases two genes are discussed together as BRCA 1/2. BRCA1 is located on chromosome 17q221.31, the gene size is 81.19kb, 24 exons are shared, BRCA2 is located on chromosome 13q13.1, the gene size is 84.19kb, and 27 exons are shared.
BRCA1/2 is two genes with the function of inhibiting malignant tumor occurrence, and plays an important role in regulating the replication of human cells, the repair of genetic material DNA damage and the normal growth of cells. Families possessing this genetic mutation tend to have a high incidence of breast cancer, usually occurring at a younger age, with tumors on both breasts of the patient, and with ovarian cancer at the same time. The life-long risk of cancers related to BRCA1 and BRCA2 gene mutation is summarized, and the risks of breast cancer and ovarian cancer of patients with BRCA1 gene mutation are 50% -85% and 15% -45%, and the risks of breast cancer and ovarian cancer of patients with BRCA2 gene mutation are 50% -85% and 10% -20%, respectively. Therefore, the detection of the BRCA1/2 gene mutation site of the high-risk population is of great significance to the prevention and early detection of cancers.
Screening of early BRCA1/2 gene mutation is carried out by directly carrying out Sanger sequencing by a conventional method, but the BRCA1/2 gene has large size, the direct sequencing cost is high, and the methods such as a denaturation high performance liquid chromatography analysis technology, a high resolution melting analysis and the like are also frequently adopted. Today, 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 a plurality of laboratories currently detect mutations of BRCA1/BRCA2 by adopting a multiplex PCR technology, for example Yan Rong (based on detection of mutation at the whole exon locus of BRCA1/2 of a breast cancer susceptible gene by multiplex PCR targeting sequencing, university of southeast university's paper, 2017) research and design 58 pairs of primers, covering the regions of BRCA1/2 exons and splice sites, including 25 pairs of BRCA1 primers and 33 pairs of BRCA2 primers, respectively covering 17763bp and 24383bp of gene length, and detecting 24 mutations in total, wherein 1 BRCA1 pathogenic mutation rs80357303 is reported to increase the risk of developing breast cancer.
The second generation sequencing technology has the characteristic of high flux, and the feasibility of common screening with low price and short period is enhanced by using the second generation sequencing to carry out BRCA1 and BRCA2 along with the gradual shortening of the sequencing time. However, the detection efficiency and accuracy of the current second generation sequencing technology still have room for improvement.
Disclosure of Invention
For this purpose, the application provides a primer for detecting BRCA1/2 total exon, a design method of the primer and application of the primer. More preferably, the application provides a primer for detecting the BRCA1/2 full-exon sequence by multiplex PCR amplification, a design method of the primer and application of the primer.
In a first aspect, the application provides a primer for detecting BRCA1/2 full-exon, comprising: an exon amplification primer pair designed for BRCA1 and an exon amplification primer pair designed for BRCA 2; wherein, the exon amplification primer pair designed for BRCA1 can cover all exon areas of BRCA1, and the exon amplification primer pair designed for BRCA2 can cover all exon areas of BRCA 2; the pair of the exon amplification primers designed for BRCA1 and the pair of the exon amplification primers designed for BRCA2 are respectively divided into two groups, and no amplification region is overlapped in each group of primers of the same gene; in each primer pair, SEQ ID NO1 is added to the 5 'end of the upstream primer of the first part, and SEQ ID NO2 is added to the downstream 5' end of the first part; SEQ ID NO2 is added to the 5 'end of the upstream primer of the second part primer, and SEQ ID NO1 is added to the 5' end of the downstream primer; a set of primer pairs of the BRCA1 gene and a set of primer pairs of the BRCA2 gene are randomly combined to form two primer pairs.
In a preferred embodiment, the primers for detecting BRCA1/2 full-exon sequences further comprise universal pool-building primers.
The second aspect of the application provides a method for designing a primer for detecting a BRCA1/2 full-exon, comprising the following steps:
step 1) designing a BRCA1 exon amplification primer pair capable of covering all exon regions of BRCA1, and designing a BRCA2 exon amplification primer pair capable of covering all exon regions of BRCA 2; the BRCA1 exon amplification primer pair and the BRCA2 exon amplification primer pair are respectively divided into two groups, and no amplification region is overlapped in each group of primers of the same gene;
step 2) adding SEQ ID NO1 at the 5 'end of the upstream primer and SEQ ID NO2 at the downstream 5' end of the first partial primer in each primer pair; SEQ ID NO2 is added to the 5 'end of the upstream primer of the second part primer, and SEQ ID NO1 is added to the 5' end of the downstream primer;
step 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 first partial primer pairs and second partial primer pairs may be the same or different.
In a preferred embodiment, the first primer pair consists of any two or more primers selected from the group consisting of SEQ id.no3 to SEQ id.no40.
In a preferred embodiment, the second primer pair consists of any two or more primers selected from the group consisting of SEQ id.no41 to SEQ id.no72.
In a preferred embodiment, the third primer pair consists of any two or more primers selected from the group consisting of SEQ id.no73 to SEQ id.no130.
In a preferred embodiment, the fourth primer pair consists of any two or more primers selected from the group consisting of SEQ id.no131 to SEQ id.no180.
In a preferred embodiment, the universal pool-building primer is selected from any one or more of SEQ id.noc181, SEQ id.noc182.
In a third aspect, the application provides a kit for detecting the whole BRCA1/2 exon, which comprises the primer for detecting the whole BRCA1/2 exon.
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 gDNA extraction kit.
In a fourth aspect, the application provides a nucleotide sequence for ligation to a BRCA1 exon amplification primer pair, comprising SEQ ID. NOs 1, SEQ ID. NOs 2.
Compared with the existing multiplex PCR technology, the method 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 a 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 results of Q-Sep identification of the products to be sequenced using the primers and kit of the application.
Detailed Description
For a clearer understanding of the objects, features and advantages of the present application, reference is made to the following detailed description of the application taken in conjunction with the accompanying drawings and detailed description.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced in other ways than those described herein, and therefore the present application is not limited to the specific embodiments disclosed below.
Example 1
Primer3 is used for designing primers with the target amplicon size of 220-300bp, so that all the primers can cover all exon areas of BRCA1/2, and the primer pairs of BRCA1 and BRCA2 are respectively divided into two groups, so that no amplification area is overlapped in each group of primers of the same gene. Wherein the first and second sets are BRCA1 exon amplification primers: the first set of primers is SEQ ID NO.3-SEQ ID NO.40, and the second set of primers is SEQ ID NO.41-SEQ ID NO.72. The third and fourth sets are BRCA2 exon amplification primers: the third set of primers is SEQ ID NO.73-SEQ ID NO.130, the fourth set of primers is SEQ ID NO.131-SEQ ID NO.180, and the fifth set of primers is SEQ ID NO.181-SEQ ID NO.182.
The 4 primer sets are respectively numbered, 1,2 and 3 … … n are sequentially numbered according to genome coordinates, SEQ ID NO1 is added to the 5 'end of the upstream primer set and SEQ ID NO2 is added to the 5' end of the downstream primer set, and SEQ ID NO2 is added to the 5 'end of the upstream primer set and SEQ ID NO1 is added to the 5' end of the downstream primer set.
A set of primers for BRCA1 and a set of primers for BRCA2 are randomly combined, and the remaining BRCA1 and BRCA2 primers are combined to form a primer tube 1 and a primer tube 2.
First combination: primer set 1 (SEQ ID NO.3-SEQ ID NO.40, SEQ ID NO.73-SEQ ID NO. 130), primer set 2 (SEQ ID NO.41-SEQ ID NO.72, SEQ ID NO.131-SEQ ID NO. 180).
A second combination: primer set 1 (SEQ ID NO.3-SEQ ID NO.40, SEQ ID NO.131-SEQ ID NO. 180), primer set 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 pool-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 provided by the application comprises the following steps:
1) Taking DNA of a sample to be detected as a template;
2) Performing a PCR reaction in the two tubes using the first/third primer pairs and the second/fourth primer pairs, respectively; the preferred PCR procedure is 95℃for 5min;95℃30s,60℃30s,58℃30s,56℃30s,70℃1min (3 cycles); 95℃for 30s and 68℃for 1min (15 cycles); 72 ℃ for 5min;4 ℃ infinity; as shown in table 1:
TABLE 1 first round PCR amplification reaction System and amplification reaction conditions
3) Combining the two PCR products;
4) The PCR product was purified for the first time using Ampure bead, then purified for the second time using beads buffer and water; specifically, 100 mu L (Ampure beads) magnetic beads are added, mixed uniformly and stood for 5min, then placed on a magnetic rack, and supernatant is sucked and removed after clarification; washing two sides with 80% ethanol, air drying, re-dissolving with 15 μl distilled water for 3min, adding 18 μl beads buffer, mixing, standing for 5min, washing two sides with 80% ethanol, and air drying;
5) Taking the purified product as a template, using 22 mu L of distilled water for redissolving, and then sucking 20 mu L of supernatant for carrying out a second round of PCR reaction, wherein a fifth set of primers are used for the second round of PCR reaction; the preferred PCR reaction procedure is 95℃for 5min;95 ℃ for 30s,65 ℃ for 1min (8 cycles); 72 ℃ for 5min;4 ℃ infinity; as shown in table 2:
TABLE 2 second round PCR amplification reaction System and amplification reaction conditions
6) The PCR product was purified for the first time using Ampure bead, then purified for the second time using beads buffer and water; specifically, 45 mu L (Ampure beads) magnetic beads are added to be uniformly mixed and kept stand for 5min; then placing the mixture on a magnetic rack, and sucking and discarding supernatant after clarification; cleaning two sides with 80% ethanol, and air drying; dissolving in 20 μL distilled water for 3min, adding 24 μL beads buffer, mixing, and standing for 5min; 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 later use.
7) Detecting the concentration;
8) Q-Sep identification fragment size;
9) Sequencing on a machine;
10 Analysis of BRAC1/2 for the presence of mutations by bioinformatics means.
The primer mother liquor concentration of the first to fifth sets of primers is 50. Mu.M and the concentration of each primer is in the range of 0.05-0.40mM.
Example 3
Blood of a healthy volunteer was collected using a 5mL EDTA-tube, and gDNA of blood cells was extracted using a QIAGEN kit as a DNA template for PCR amplification reaction.
Multiplex PCR amplifications were performed using the method described in example 2.
The concentration of the sample was determined to be 5.6ng/uL.
The Q-Sep results are shown in FIG. 1, table 3 and Table 4.
Table 3, off-the-shelf data quality assessment
| Sequencing original reads (bars) | Number of reads remaining after quality control (bar) | Ratio of remaining data after quality control |
| 1143191 | 1086806 | 95.06％ |
The samples were sequenced using an illuminea sequencer, and total numbers of reads were measured as no 1143191, with 1086806 reads remaining after quality control (removal of unqualified reads), and qualified reads for quality control accounting for 95.06% of total reads.
TABLE 4 alignment of reference sequences and depth data statistics
| Parameters (parameters) | Numerical value |
| Average length of sequenced sequence | 287 |
| Length range of warehouse building | 370-420 |
| Alignment rate (mapping rate) | 98％ |
| Capture area size | 29925bp |
| Actual capture area size | 29925bp |
| Coverage degree | 100％ |
| Average depth | 9600X |
The average length of library insert is 287bp, the size of the complete library is 370-420bp after the library building process and the sequence general sequence are added, and 98% of ready-quality reads can be correspondingly aligned to human genome sequence.
The above description of the specific embodiments of the present application has been given by way of example only, and the present application is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present application will occur to those skilled in the art, and are also within the scope of the present application. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present application without departing from the spirit and scope thereof.
SEQUENCE LISTING
<110> Shanghai Artemisia Gene technology Co., ltd
<120> sequence primer for detecting BRCA1/2 full-exon and design method
<130> IPI20190148
<140> 2019107456253
<141> 2019-08-13
<160> 182
<170> PatentIn version 3.5
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<400> 12
ctgatgatgg tcaatttatt ttgtcca 27
<210> 13
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 13
gaccaagatt tttggcaaaa ctataag 27
<210> 14
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 14
ccacagtaga tgctcagtaa atatttc 27
<210> 15
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 15
cacatacatc cctgaaccta aaataaa 27
<210> 16
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 16
gagcagcttt cactaactaa ataagat 27
<210> 17
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 17
taattacagt actgtatcta cccactc 27
<210> 18
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 18
ttagtgagga aacaaaatgt tctgcta 27
<210> 19
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 19
ttagctcact tctataaata gactggg 27
<210> 20
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 20
gacctgttag atgatggtga aataaag 27
<210> 21
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 21
tatgcctagt agactgagaa ggtatat 27
<210> 22
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 22
ctgaactact tcttcatatt cttgctt 27
<210> 23
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 23
aaaagtcact tttgaatgtg aacaaaa 27
<210> 24
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 24
ttaacaaatg acttgatggg aaaaagt 27
<210> 25
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 25
tatttcattg gtacctggta ctgatta 27
<210> 26
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 26
gaaaccttga atgtattctg caaatac 27
<210> 27
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 27
aactagtagt cagtagaaat ctaagcc 27
<210> 28
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 28
aggattgaca aattctttaa gttcact 27
<210> 29
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 29
taattatagg agcatttgtt actgagc 27
<210> 30
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 30
agattctttt tcgagtgatt ctattgg 27
<210> 31
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 31
gagagaaaag aatggaataa gcagaaa 27
<210> 32
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 32
tcacttttac atattaaagc ctcatga 27
<210> 33
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 33
acaattcagt ttttgagtac cttgtta 27
<210> 34
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 34
gcttttatta cagaattcag ccttttc 27
<210> 35
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 35
cattttgtgt gacatgaaag taaatcc 27
<210> 36
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 36
ttaattcaaa gagatgatgt cagcaaa 27
<210> 37
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 37
gcttctcaaa gtatttcatt ttcttgg 27
<210> 38
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 38
gagataaagg ggaaggaaag aattttg 27
<210> 39
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 39
gaatcttgcc cttaacttgt ttacag 26
<210> 40
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 40
caaacacagc tattaaaaag tcattcc 27
<210> 41
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 41
acggaaaagc gcgggaatta cagataa 27
<210> 42
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 42
cttacgcctc tcaggttccg cccctac 27
<210> 43
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 43
gtgtgtgtat aaatttggtt tgttctt 27
<210> 44
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 44
ctggtttcca ctataccaaa gtaaaaa 27
<210> 45
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 45
tttttgaaga tctagaacca cattgtt 27
<210> 46
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 46
cgtctctaca gaaaacacaa aatttag 27
<210> 47
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 47
ttctactgtt gctgcatctt attttta 27
<210> 48
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 48
taacctagac taaaaggtct tatcacc 27
<210> 49
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 49
aagagaactt atctagtgag gatgaag 27
<210> 50
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 50
tatttgcagt caagtcttcc aattc 25
<210> 51
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 51
aacctgaggt ctataaacaa agtcttc 27
<210> 52
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 52
ctaaaaacag cagaactttc cttaatg 27
<210> 53
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 53
gaaactggac tcattactcc aaataaa 27
<210> 54
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 54
tggaacctac ttcattaata ttgcttg 27
<210> 55
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 55
gaaacaagca tagaaatgga agaaagt 27
<210> 56
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 56
cctgcagtga tattaactgt ctgta 25
<210> 57
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 57
cctggttctt ttactaagtg ttcaaat 27
<210> 58
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 58
catttatttg gttctgtttt tgccttc 27
<210> 59
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 59
tagtggtcat gagaataaaa caaaagg 27
<210> 60
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 60
ttatctcttc actgctagaa caactat 27
<210> 61
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 61
ggacgttcta aatgaggtag atgaata 27
<210> 62
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 62
gtaggtctcc ttttacgctt taattta 27
<210> 63
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 63
aacagcagtt tattactcac taaagac 27
<210> 64
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 64
ttctgaatgc tgctatttag tgttatc 27
<210> 65
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 65
attgaaagtt ccccaattga aagtt 25
<210> 66
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 66
cctacataaa actctttcca gaatgtt 27
<210> 67
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 67
ggggttttag aatcataaat ccagatt 27
<210> 68
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 68
ttaggtgtta aacgttaggt gtaaaaa 27
<210> 69
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 69
taaattaaac atcaactctg tctccag 27
<210> 70
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 70
actatatgac tgaatgaata tctctggt 28
<210> 71
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 71
gaatgcctta aatatgacgt gtctg 25
<210> 72
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 72
agcactgtgt atgtatgtaa taagtct 27
<210> 73
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 73
cacataagga acagtttatg gttctaa 27
<210> 74
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 74
gagttttacc tcagtcacat aataagg 27
<210> 75
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 75
tttgaatatt attggagttg aagccag 27
<210> 76
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 76
agtaatccat agtcaagatc ttaagca 27
<210> 77
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 77
gctcttagcc aaaatattag cataaaa 27
<210> 78
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 78
tttttaaaca cttccaaaga atgcaaa 27
<210> 79
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 79
caattactaa gtcataaaaa taaaccaggt 30
<210> 80
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 80
aataagataa actagttttt gccagtttt 29
<210> 81
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 81
tagagatgac aattatcaac ctcatct 27
<210> 82
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 82
gcaattcagt aaacgttaag tgaaata 27
<210> 83
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 83
aaatcacata taggaccagg tttagag 27
<210> 84
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 84
tcaaatagta gatgtgcttt ttgatgt 27
<210> 85
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 85
aaaaacctgt agttcaacta aacagag 27
<210> 86
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 86
ccattaaaaa tttttggacc taggttg 27
<210> 87
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 87
agtcttgcta gttcttactt tttgtag 27
<210> 88
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 88
taaactgttt ctatgagaaa ggttgtg 27
<210> 89
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 89
atttaatggc ttctctgatt ttggtag 27
<210> 90
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 90
tccattagat tcaaatgtag caaatca 27
<210> 91
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 91
acttatttgt tttctttttc aaagtgga 28
<210> 92
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 92
cttcatttca gggtatcaaa aagtcta 27
<210> 93
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 93
gaatcagctt ctggggtaat aaataac 27
<210> 94
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 94
ccaaacacta cctttttaac ttagtga 27
<210> 95
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 95
cttagatttg tgttttggtt gaattgt 27
<210> 96
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 96
cttttatatg atcatgaaaa tgccagc 27
<210> 97
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 97
gcccatttgt tcatgtaatc attattt 27
<210> 98
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 98
agctaatgaa aggaataatc ttgcttt 27
<210> 99
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 99
agcttggttt tctaaactga gtaaatt 27
<210> 100
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 100
atatcctact agtttagctt gtgttga 27
<210> 101
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 101
taaacattga aacaacagaa tcatgac 27
<210> 102
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 102
gcaatttgaa ggtacagttg aaattaa 27
<210> 103
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 103
tttatatttt gctccgtttt agtagca 27
<210> 104
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 104
ctgccagtag aaattctcat aacttag 27
<210> 105
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 105
tttcatcaaa aaggtttttc actttgt 27
<210> 106
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 106
gaaaccagaa gaattgcata acttttc 27
<210> 107
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 107
ctcacagaag tttttctact acaactt 27
<210> 108
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 108
caaagtgtaa agaaatgcag aattctc 27
<210> 109
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 109
ggacaattta atggctgcat ttttatt 27
<210> 110
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 110
aaaatcatct ctccgaaaaa caagata 27
<210> 111
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 111
aagtatttgc agatgagact gacttat 27
<210> 112
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 112
agaataaatc aaaaatttgc caaacga 27
<210> 113
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 113
acacgaggaa gtatttttga tacattt 27
<210> 114
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 114
agcaagtctt ttccaaagta ttgttta 27
<210> 115
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 115
gtttagaatc tgtcagttca tcatctt 27
<210> 116
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 116
agtttctcca tatctctctc aatttca 27
<210> 117
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 117
aagagtataa agaggtcctt gattagg 27
<210> 118
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 118
ttgagaaata aaactgatat tatttgcctt 30
<210> 119
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 119
aagtgttaac ttcttaacgt tagtgtc 27
<210> 120
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 120
acattcactg aaaattgtaa agcctat 27
<210> 121
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 121
tagttttaaa aggtggaaca aagactt 27
<210> 122
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 122
tgcaacaaag gcatattcct aaatatt 27
<210> 123
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 123
aaaattacac tctgtcataa aagccat 27
<210> 124
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 124
gggttgtgct ttttaaattt caatttt 27
<210> 125
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 125
tgagggaata cataaaagtt aacacac 27
<210> 126
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 126
tgtgtgatac atgtttactt taaattgtt 29
<210> 127
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 127
tactgccgta tatgattacg taatgta 27
<210> 128
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 128
ttgtagttgt tgaattcagt atcatcc 27
<210> 129
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 129
taccacccat ctgtaagttc aataatg 27
<210> 130
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 130
tcagtgactt gtttaaacag tggaatt 27
<210> 131
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 131
gtacaacttc cttggagatt ttgtc 25
<210> 132
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 132
tatgaaacag ttgtagatac ctctgaa 27
<210> 133
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 133
gaaaaacttg cattgaaagt ctcttta 27
<210> 134
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 134
gaaaaagacc tattagacac agagaac 27
<210> 135
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 135
cacagaagga atcgtcatct ataaaac 27
<210> 136
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 136
tagctttgaa gaatgcaggt ttaatat 27
<210> 137
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 137
ggctgaaaag acatagttta taacact 27
<210> 138
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 138
tactttgaaa cagaagcagt agaaatt 27
<210> 139
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 139
tccttggaag taggagttaa aataaga 27
<210> 140
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 140
ctctcaggat cttgattata aagaagc 27
<210> 141
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 141
tagagttctt gaaaatgggt tcgttta 27
<210> 142
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 142
tgaaaattat aaaaacgttg agctgtt 27
<210> 143
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 143
gatacagtat taattgactg aggcttg 27
<210> 144
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 144
aaatcggaca tctccttgaa tatagat 27
<210> 145
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 145
cacttttgtt acagtcattt ttcaaca 27
<210> 146
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 146
aaaaggcaga aattacagaa ctttcta 27
<210> 147
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 147
tatcattttt acttgaatca ctgccat 27
<210> 148
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 148
ttgagaatat tagtgaggaa acttctg 27
<210> 149
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 149
cctcataact tagaatgtcc attttgt 27
<210> 150
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 150
agcatgtcat ggtaatactt caaataa 27
<210> 151
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 151
acagtctcaa tagaaacaag gttttta 27
<210> 152
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 152
aagaacctac tctattgggt tttcata 27
<210> 153
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 153
tcctgaatca ttatatacct catcaga 27
<210> 154
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 154
ccttagcttt ttacacaagt tgtagta 27
<210> 155
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 155
gaagaatatc ctctgaatca tccaatg 27
<210> 156
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 156
aaactgtaaa tgaagatatt tgcgttg 27
<210> 157
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 157
ggatattaaa tgttctggag tacgtat 27
<210> 158
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 158
gtgatgttag tttggaaact tcagata 27
<210> 159
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 159
aacaagtgac actttggttc ctaata 26
<210> 160
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 160
gggagtgtta gaggaatttg atttaat 27
<210> 161
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 161
ttgtcataca atacctaaag gttcttc 27
<210> 162
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 162
caagtttctg ctacaagaaa tgaaaaa 27
<210> 163
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 163
atcaatgact gatttttacc aagagtg 27
<210> 164
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 164
cgcaaatata tctgaaactt ctagcaa 27
<210> 165
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 165
tgtatcaaaa gaaagaaata tatggtaagt 30
<210> 166
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 166
gcagttctag aagaatgaaa actctta 27
<210> 167
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 167
aaaatgttaa attcaaagtc tctaagac 28
<210> 168
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 168
ggcctgatac aattaacttg aatgtta 27
<210> 169
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 169
ctaaaacagc ttctcacctt gaataat 27
<210> 170
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 170
tcttatcttt aaatctccct tctttgg 27
<210> 171
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 171
taaaactgat aaaaacaaag catttaca 28
<210> 172
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 172
tttttgttct gattgctttt tattcca 27
<210> 173
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 173
ggtagctcca actaatcata agagatt 27
<210> 174
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 174
taagtgcttg ttagtttatg gaatctc 27
<210> 175
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 175
cattttgttg aatgtctctt gaaagtg 27
<210> 176
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 176
tcctttcttg catcttaaaa ttcatct 27
<210> 177
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 177
cagaatatac gatggcctcc atatata 27
<210> 178
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 178
tctgctttta aaggaaatac ttttgga 27
<210> 179
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 179
gaagcaaaag tataccaata cggaatc 27
<210> 180
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 180
gttgtactac atctctgatc aaagaac 27
<210> 181
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 181
aatgatacgg cgaccaccga gatctacact ctttccctac acgac 45
<210> 182
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<220>
<221> misc_feature
<222> (25)..(30)
<223> n is a, c, g, or t
<400> 182
caagcagaag acggcatacg agatnnnnnn gtgactggag ttcagacgtg tgc 53
Claims (7)
1. A primer for detecting BRCA1/2 whole-exon sequence, comprising: an exon amplification primer pair designed for BRCA1 and an exon amplification primer pair designed for BRCA 2; wherein, the exon amplification primer pair designed for BRCA1 can cover all exon areas of BRCA1, and the exon amplification primer pair designed for BRCA2 can cover all exon areas of BRCA 2; the pair of the exon amplification primers designed for BRCA1 and the pair of the exon amplification primers designed for BRCA2 are respectively divided into two groups, and no amplification region is overlapped in each group of primers of the same gene; the first primer pair consists of SEQ ID. NO3 to SEQ ID. NO40 primers; the second primer pair consists of SEQ ID. NO41 to SEQ ID. NO72 primers; the third primer pair consists of SEQ ID. NO73 to SEQ ID. NO130 primers; the fourth primer pair consists of SEQ ID.NO131 to SEQ ID.NO180 primers; in each primer pair, SEQ ID NO1 is added to the 5 'end of the upstream primer of the first part, and SEQ ID NO2 is added to the downstream 5' end of the first part; SEQ ID NO2 is added to the 5 'end of the upstream primer of the second part primer, and SEQ ID NO1 is added to the 5' end of the downstream primer; wherein the first group of primers and the second group of primers are primers of BRCA1 genes, and the third group of primers and the fourth group of primers are primers of BRCA2 genes; the first set of primers and the third set of primers form a primer pair, and the second set of primers and the fourth set of primers form a primer pair.
2. The primer for detecting the whole exon sequence of BRCA1/2 according to claim 1, further comprising a universal library-building primer.
3. The primer for detecting BRCA1/2 full-exon sequence according to claim 2, wherein the universal library-building primer is selected from any one or more of SEQ id no181, SEQ id no 182.
4. A kit for detecting the whole BRCA1/2 exon sequence, which comprises the primer for detecting the whole BRCA1/2 exon sequence according to claim 1.
5. The kit of whole exonuclease according to claim 4, further comprising a PCR amplification enzyme.
6. The kit of claim 4, wherein the kit further comprises a gDNA extraction reagent or gDNA extraction kit.
7. The kit of whole exonic sequence according to claim 4, wherein the kit further comprises distilled water.
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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 |
-
2019
- 2019-08-13 CN CN201910745625.3A patent/CN110592210B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| 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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