Realtime exonuclease-mediated allelic discrimination (READ): a simple homogeneous genotyping assay for SNPs at the ABC gene loci.
OBJECTIVE
Members of the ATP-binding-cassette transporter family are implicated in the traffic of drugs/xenobiotics. Several SNPs in these ATP-binding-cassette genes were previously identified to show evidence of recent positive selection. These recent positive selection SNPs may confer functional effects and account for variation in drug response. To facilitate association studies between these SNPs and drug response, we report the development of a homogeneous (realtime exonuclease-mediated allelic discrimination) assay to genotype these SNPs.


METHODS
Realtime exonuclease-mediated allelic discrimination involves real-time PCR using a proof-reading enzyme and simultaneous genotype determination by product presence/absence as detected using SYBR Green I stain.


RESULTS
A total of 29 recent positive selection SNPs from 17 ATP-binding-cassette transporter genes were evaluated. Of the 777 eealtime exonuclease-mediated allelic discrimination assays, 773 genotypes (approximately 99.5%) were concordant with the Perlegen data and other genotyping methods.


CONCLUSIONS
Therefore, this simple, robust, rapid, cost-effective single-step, closed-tube assay with a scalable and automatable platform has potential applications in population genetic screening and association studies.
Wang
Zihua
Z
Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Sew
Pui-Hoon
PH
Chong
Samuel S
SS
Lee
Caroline G L
CG
D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000483:Alleles; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D004364:Pharmaceutical Preparations / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D004364:Pharmaceutical Preparations; D004247:DNA; D005090:Exodeoxyribonucleases