Development of primer-special TaqMan PCR: a novel SNP detection method to detect CYP2C9 3 in South Chinese.
BACKGROUND
CYP2C9 3 (1075A/C) is an inherited single nuclear polymorphism (SNP) of cytochrome P450 (CYP) 2C9, which affects the activity of the enzyme. In vitro studies with several drugs have indicated that the CYP2C9 3 variant has an impaired capacity for drug metabolism. Therefore an efficient detection assay for this mutation may be important for clinical dose adjustment.


OBJECTIVE
The aim of this work was to develop an appropriate tool for detection of the CYP2C9 3 polymorphism in the clinical laboratory.


METHODS
The previously described TaqMan mismatch amplification mutation assay (TaqMAMA) was modified to a primer-special (PS)-TaqMan PCR to satisfy the high-throughput requirements of a clinical laboratory. 404 genomic DNA samples from South Chinese individuals were genotyped to test the detection system. The results were checked by bi-directional sequencing.


RESULTS
PS-TaqMan PCR could correctly genotype the CYP2C9 allele from a genomic template at a concentration of 1 x 104 to 1 x 1011 copies/PCR. Among the 404 genomic DNA samples, 24 heterozygotes and 380 wild-type homozygotes were detected and confirmed by bi-directional sequencing.


CONCLUSIONS
PS-TaqMan PCR was successfully developed for CYP2C9 3 detection. This efficient, reliable, high-throughput tool could satisfy the requirements of a clinical laboratory test.
Qiu
Geng
G
Da An Gene Diagnosis Center, Sun Yat-sen College of Medical Science, Sun Yat-sen University, Guangzhou, China. rongfu_s@163.com
Wang
Ying
Y
Fu
Rong
R
He
Yunshao
Y
Chen
Zirong
Z
Chen
Jiachang
J
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D044466:Asian People / Q000235:genetics*; D001483:Base Sequence; D002681:China; D065729:Cytochrome P-450 CYP2C9; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000378:metabolism*; D015894:Genome, Human / Q000235:genetics; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011785:Quality Assurance, Health Care; D019914:Taq Polymerase / Q000378:metabolism*
D017931:DNA Primers; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D019914:Taq Polymerase