5′ Nuclease Assays for the Loci CCR5-+/Δ32, CCR2-V64I, and SDF1-G801A Related to Pathogenesis of AIDS

Abstract

Abstract Background: Variations within the human genome play important roles in human disease. To study variations related to susceptibility to AIDS, we have developed 5′ nuclease assays that eliminate post-PCR molecular biology steps. Methods: TaqMan assays based on the 5′ nuclease activity of Taq polymerase and fluorescent resonance energy transfer were developed to score alleles at the biallelic loci CCR5-+/Δ32, CCR2-V64I and SDF1-G801A. For each assay, 72 samples were analyzed. Data collection and analysis were performed on the Prism 7700 Sequence Detection System. For comparison with gel electrophoresis methods, each locus was also scored on a subset of 24 samples, using restriction enzymes or single-strand conformational polymorphism (SSCP). Results: Clear allelic discrimination was obtained on each of the 72 samples for all three TaqMan assays. The TaqMan scores for the subset of 24 samples were concordant with the restriction enzyme and SSCP scores. Conclusions: Because of its simplicity, speed, and potential for automation and miniaturization, TaqMan is an excellent candidate for investigation of genetic variation in clinical, research, and forensic settings.