A fast SSP-PCR method for genotyping the ATP-binding cassette subfamily B member 1 gene C3435T and G2677T polymorphisms in Chinese transplant recipients.
OBJECTIVE
P-glycoprotein, the product of the ATP-binding cassette subfamily B member 1 (ABCB1) gene (or the so-called multidrug resistance 1 gene), is an ATP-driven efflux pump contributing to the pharmacokinetics as well as the pharmacokinetics of drugs that are P-glycoprotein substrates, such as tacrolimus. This paper describes the development of a new method for detection of the 3435C/T and 2677G/T/A single nucleotide polymorphisms of the ABCB1 gene. The method is a simple sequence-specific primer polymerase chain reaction (SSP-PCR).


METHODS
158 Chinese health checkup examinees and 214 transplant recipients were included in the study. Genomic DNA was extracted from peripheral blood and amplified with SSP-PCR to detect the 3435C/T and 2677G/T/A mutations in ABCB1. The SSP-PCR condition was optimized, and the PCR results were compared with those of DNA sequencing.


RESULTS
In the optimized condition, the two polymorphisms could be clearly distinguished after one-step PCR and electrophoresis. The ABCB1 3435C/T and 2677G/T/A genotypes of the subjects were scanned, and allele-specific bands were successfully amplified by SSP-PCR, which were in full accordance with the results of sequencing.


CONCLUSIONS
As a fast, simple and inexpensive genotyping tool, the method would be practicable in large clinical studies on interindividual pharmacokinetics.
Wu
Cun-Zao
CZ
Transplantation Center, First Affiliate Hospital of Wenzhou Medical College, Wenzhou, China.
Ni
Xiao-Jie
XJ
Zheng
Shao-Ling
SL
Yang
Yi-Rong
YR
Xia
Peng
P
Zeng
Yan-Jun
YJ
Chen
Bi-Cheng
BC
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D000328:Adult; D000368:Aged; D044466:Asian People / Q000235:genetics*; D002681:China; D017931:DNA Primers; D005260:Female; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D016030:Kidney Transplantation; D016031:Liver Transplantation; D008297:Male; D008875:Middle Aged; D016377:Organ Transplantation; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods; D013997:Time Factors
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017931:DNA Primers