title: SYBR green dye-based probe-free SNP genotyping: introduction of T-Plex real-time PCR assay.
abstract: Single-nucleotide polymorphism (SNP) genotyping is widely used in genetic association studies to characterize genetic factors underlying inherited traits. Despite many recent advances in high-throughput SNP genotyping, inexpensive and flexible methods with reasonable throughput levels are still needed. Real-time PCR methods for discovering and genotyping SNPs are becoming increasingly important in various fields of biology. In this study, we introduce a new, single-tube strategy that combines the tetra-primer ARMS PCR assay, SYBR Green I-based real-time PCR, and melting-point analysis with primer design strategies to detect the SNP of interest. This assay, T-Plex real-time PCR, is based on the T(m) discrimination of the amplified allele-specific amplicons in a single tube. The specificity, sensitivity, and robustness of the assay were evaluated for common mutations in the FV, PII, MTHFR, and FGFR3 genes. We believe that T-Plex real-time PCR would be a useful alternative for either individual genotyping requests or large epidemiological studies.
authors.affiliation: Department of Molecular Biology and Genetics, Koc University, 34450 Istanbul, Turkey. Electronic address: ibaris@ku.edu.tr.
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Baris
authors.forename: Ozdal
authors.initials: O
authors.lastname: Etlik
authors.forename: Vedat
authors.initials: V
authors.lastname: Koksal
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Ocak
authors.forename: Saniye Tugba
authors.initials: ST
authors.lastname: Baris
mesh_terms: D000483:Alleles; D001483:Base Sequence; D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D005838:Genotype; D006801:Humans; D009930:Organic Chemicals / Q000032:analysis*; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation*; D012680:Sensitivity and Specificity
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I
keywords: FVL; MTHFR; SNP; SYBR green; T-Plex real-time PCR; Tetra-primer ARMS PCR