SYBR green dye-based probe-free SNP genotyping: Introduction of T-Plex real-time PCR assay

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 need...

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Bibliographic Details
Published in:Analytical biochemistry 2013-10, Vol.441 (2), p.225-231
Main Authors: Baris, Ibrahim, Etlik, Ozdal, Koksal, Vedat, Ocak, Zeynep, Baris, Saniye Tugba
Format: Article
Language:English
Subjects:
Alleles
Base Sequence
DNA Primers - genetics
epidemiological studies
fibroblast growth factor receptor 3
FVL
genes
Genotype
genotyping
Humans
melting point
MTHFR
Organic Chemicals - analysis
Polymorphism, Single Nucleotide
quantitative polymerase chain reaction
Real-Time Polymerase Chain Reaction - instrumentation
Sensitivity and Specificity
single nucleotide polymorphism
SNP
SYBR green
T-Plex real-time PCR
Tetra-primer ARMS PCR
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Summary: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 Tm 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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2013.07.007