Detection of Single Nucleotide Polymorphism by Real-time Polymerase Chain Reaction Coupled with High Specific Invader Assay in Single Tube
10.11895/j.issn.0253-3820.150051
- VernacularTitle:实时荧光聚合酶链式反应偶联高特异性核酸侵入反应检测单核苷酸多态性
- Author:
Menglin ZHENG
;
Xiemin QI
;
Huan TONG
;
Yunlong LIU
;
Bingjie ZOU
;
Qinxin SONG
;
Guohua ZHOU
- Publication Type:Journal Article
- Keywords:
Real time polymerase chain reaction;
Invader assay;
Single nucleotide polymorphism;
Aldehyde dehydrogenase-2 gene;
Cytochrome p450 2 C19*2 gene
- From:
Chinese Journal of Analytical Chemistry
2015;(7):1001-1008
- CountryChina
- Language:Chinese
-
Abstract:
A method for the real-time polymerase chain reaction ( PCR ) coupled with high specific invader assay to detect single nucleotide polymorphism ( SNP) was established. To reduce the background signal, the amount of flap endonuclease 1 ( FEN1 enzyme ) and wild-type detection probe was optimized. Under the optimum conditions including 0. 05 μmo/L invasive oligonucleotide probe, 0. 125 μmol/L wild-type detection probe, 0. 5 μmol/L mutation detection probe, 0. 25 μmol/L each fluorescence resonance energy transfer (FRET) probe and 1. 5 U FEN1, the background signal of wild-type sample and mutation sample was dramatically decreased and the background interference to the detecting results was thus eliminated. A total of 21 cases of aldehyde dehydrogenase-2*2 ( ALDH2*2 ) , 19 cases of cytochrome p450 2 C19*2 ( CYP2 C19*2 ) and 19 cases of CYP2C19*3 were analyzed with the established method, and the genotypes of ALDH2*2 were 10 cases of GG homozygote, 8 cases of GA heterozygote and 3 cases of AA homozygote; the genotypes of CYP2C19*2 were 9 cases of GG homozygote, 8 cases of GA heterozygote and 2 cases of AA homozygote;and the genotypes of CYP2C19*3 were 18 cases of GG homozygote and 1 case of GA heterozygote. These results were consistent with those by pyrosequencing. The established method was specific, simple, short time-consuming and low cost, and could be used for the detection of SNP genotyping with non-polluting in single closed tube.
