[Establishment and preliminary application of polymerase chain reaction with confronting two-pair primers for the single nucleotide polymorphisms of metabolic enzymes].
OBJECTIVE
To develop a simple, accurate, rapid, economic, large-scale detection method for the detection of single nucleotide polymorphisms (SNPs) metabolic enzymes, using polymerase chain reaction with confronting two-pair primers (PCR-CTPP).


METHODS
The primers of CYP1A1 (A4889G), EPHX1 (A416G) and NQO1 (C609T) were designed for PCR-CTPP, and the PCR conditions were optimized. The results of genotyping were verified by DNA sequencing. The above SNPs were detected by the PCR-CTPP detection method in a randomly selected 183 healthy individuals of Han ethnicity. The genotype frequencies were analyzed and compared with people from other ethnicities.


RESULTS
The allele-specific bands of CYP1A1 (A4889G), EPHX1 (A416G) and NQO1 (C609T) were successfully amplified by PCR-CTPP under the optimal conditions and the results of genotyping were consistent with DNA sequencing. Among 183 healthy Han individuals, the genotypic distributions of CYP1A1 (A4889G) , EPHX1 (A416G) and NQO1 (C609T) showed that the wild-type, homozygous variants, and heterozygotes were 103 (56.3%), 8 (4.4%), 72 (39.3%) and 142 (77.6%), 4 (2.2%), 37(20.2%), 60(32.8%), 32 (17.5%), 91 (49.7%) respectively. The distributions of genotypes were all in accordance with the Hardy-Weinberg equilibrium (P > 0.05), with statistical differences and with other ethnic populations (P < 0.05).


CONCLUSIONS
The SNPs of metabolic enzymes can be detected by PCR-CTPP method which is simple, accurate, rapid, economic and with large scale. PCR-CTPP can be used for large scale clinical and epidemiological screening.
Fu
Jing
J
Transplant Center, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, China.
Yang
Yi-Rong
YR
Ni
Xiao-Jie
XJ
Pan
Xiao-Dong
XD
Zheng
Jian-Jian
JJ
Zheng
Shao-Ling
SL
Lin
Ren-Yu
RY
Cai
Ming
M
Chen
Bi-Cheng
BC
D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D017931:DNA Primers; D004851:Epoxide Hydrolases / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide
D017931:DNA Primers; D003577:Cytochrome P-450 Enzyme System; D016660:NAD(P)H Dehydrogenase (Quinone); D004851:Epoxide Hydrolases