Establishment of female genital cancer relevant SNP sites multiple detection method
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A cancer-related, genital technology, applied in the field of biotechnology applications, can solve problems such as difficulty in further increasing the number of sites and limitations
Inactive Publication Date: 2013-03-13
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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[0004] At present, T-ARMS-PCR is still based on single detection, and a few laboratories can also achieve multiple detection (Multiplex-T-ARMS-PCR, M-T-ARMS-PCR), but the number of sites that can be detected at the same time is difficult to further improve. The increase is mainly limited by two limitations: the bias of multiplex amplification and the resolution of agarose electrophoresis
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Embodiment approach 1
[0014] Embodiment 1: multiplex PCR amplification
[0015] A Multiplex PCR Kit (QIAGEN) kit was used. The PCR reaction system was 20 μl, including 0.5 μl of whole blood DNA template, 2 μl of buccal swab DNA template, 500 nM of universal primers, and the concentrations of other primers are shown in Table 1. The PCR reaction is divided into 6 steps to amplify the target fragment: Step 1: Pre-denaturation at 95°C for 10 minutes; Step 2: Denaturation at 95°C for 30 seconds, renaturation at 60°C for 30 seconds, extension at 72°C for 45 seconds, cycle 3 times; Step 3: 95°C Denaturation for 30s, 58°C for 30s, 72°C for 45s, cycle 10 times; Step 4: 95°C for 30s, 68°C for 30s, 72°C for 45s, cycle 20 times; Step 5: 95°C for 30s , renatured at 55°C for 30s, extended at 72°C for 45s, and cycled 12 times; step 6: extended at 72°C for 5min, and stored at 4°C. Two replicates were made for each specimen.
[0017] use system (Qiagen) capillary electrophoresis system, DNA high-resolution cartridge (Qiagen) and OH700 method were used as separation conditions, used in conjunction with QX Alignment Marker 15bp / 500bp, QX DNA Size Marker 25-450bp, and analyzed by Biocalculator software (Qiagen) to determine whether there were products of corresponding length. SNP typing.
Embodiment approach 3
[0018] Embodiment 3: Sequencing Verification
[0019] PCR reaction system 25 μl, including 0.5 μl whole blood DNA template, 2 μl buccal swab DNA template, Premix Ex Version 2.0 (TakKaRa) 12.5μl, take 500nM of outer primer without Tag for each site. The PCR reaction was: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 45 s, and 45 cycles; extension at 72°C for 5 min, and storage at 4°C. The products were sequenced, and the sequencing results were compared with the results of M-T-ARMS-PCR electrophoresis.
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Abstract
The invention belongs to the biological technology application field, and relates to simultaneous detection and typing of six female genital cancer relevant SNP sites (comprising rs4784227, rs12196489, rs3803662, rs889312, rs750749 and rs749292) of specimens such as whole blood and buccal swabs of normal health check-up population. The flanking nucleotide sequences of SNP sites rs4784227(C>T), rs12196489(G>A), rs3803662(T>C), rs889312(C>A), rs750749 (T>C) and rs749292(G>A) downloaded from http: / / www.ncbi.nlm.nih.gov / snp / , internal and external specific primers of each site suitable for a T-ARMS-PCR technology, and the single-tube multiple PCR typing is carried out on six SNP sites. According to the invention, a multiple chimeric primer PCR technology and capillary electrophoresis are improved, the disadvantage that a conventional single-tube four primers amplification refractory mutation system PCR can not be preformed multiple detection, the characteristics of high throughput, simplicity and rapidity provide new means for rapidly typing of female genital cancer relevant SNP sites, and the establishment of the female genital cancer relevant SNP sites multiple detection method has important meaning for genotype distribution of the female genital cancer relevant SNP sites and prevention of the female genital cancer in our country.
Description
field of invention [0001] The invention belongs to the field of biotechnology applications, and relates to the simultaneous detection and analysis of six female genital cancer-related SNP sites (including rs4784227, rs12196489, rs3803662, rs889312, rs750749 and rs749292) in samples such as whole blood and oral swabs of normal physical examination populations. type. Specifically in http: / / www.ncbi.nlm.nih.gov / snp / Download the nucleotides flanking SNP sites rs4784227 (C>T), rs12196489 (G>A), rs3803662 (T>C), rs889312 (C>A), rs750749 (T>C) and rs749292 (G>A) Sequence, design internal and external specific primers suitable for T-ARMS-PCR technology for each site, and perform single-tube multiplex PCR typing on 6 SNP sites. This patent overcomes the shortcomings of the conventional single-tube four-primer amplification hindered mutation system PCR that cannot be detected multiple times through the two improvements of multiple chimeric primer PCR technology and ...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 马学军刘颖张晨
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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