A Real Time PCR Assay for the Detection of the GSTP1 Single Nucleotide Polymorphism A313G
ML Baker, AL Marchetti, CL Bartels, JM Pipas, CE Fadul, GJ Tsongalis. Dartmouth Medical School, Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH
Background: GSTP1 is a member of the glutathione-S-transferase multigene family, and is a cytosolic enzyme responsible for catalyzing the metabolism and detoxification of xenobiotic agents via conjugation with glutathione. The single-nucleotide polymorphism A313G (Ile105Val) is known to alter substrate affinity of GSTP1 via steric restriction, and is associated with variations in GSTP1 metabolic activity. This study evaluates a real-time PCR assay for detection of the A313G SNP.
Design: In this study we utilized DNA samples from 40 de-identified Caucasian patients who had been previously tested for other genetic disorders to assess the specificity of an allelic discrimination assay (TAQman SNP Genotyping Assay C_3237198, ABI Assays-on-Demand) using the ABI 7500 FAST real time PCR system. DNA from these samples was isolated using the Qiagen EZ1 Robotic workstation. In addition, DNA was extracted from 24 formalin-fixed, paraffin-embedded tissue sections from colon cancer specimens to determine if this assay would perform on archived materials. This assay utilized the following: ABI 2X FAST universal master mix, 10-20 ng of genomic DNA in a total reaction volume of 10 l using the default fast cycling conditions. A post amplification plate read was used for allelic discrimination according to the ABI 7500 user manual.
Results: Of 40 Caucasian DNA samples, 13 (32.5%) were homozygous for the A allele, 19 (47.5%) were heterozygous for A/G, and 8 (20%) were homozygous for the G allele. This corresponds to the frequency data presented in the NCI SNP500 database (http://snp500cancer.nci.nih.gov). Of 24 colon cancer cases, the following distribution was observed: 11/24 (45.8%) were homozygous for the A allele, 12/24 (50%) were heterozygous for A/G, and 1/24 (4%) was homozygous for the G allele.
Conclusions: Our study suggests that the ABI 7500 FAST TAQman SNP Genotyping Assay is suitable for differentiating the A from the G alleles in clinical DNA samples arising from whole blood or archived tissues. Real time PCR thermocycling capabilities have resulted in a very robust TAQman assay that has the advantage of improved turn-around-time and throughput.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 231, Wednesday Afternoon