DNA analysis by fluorescence quenching detection.
The analysis of human genetic variations such as single nucleotide polymorphisms (SNPs) has great applications in genome-wide association studies of complex genetic traits. We have developed an SNP genotyping method based on the primer extension assay with fluorescence quenching as the detection. The template-directed dye-terminator incorporation with fluorescence quenching detection (FQ-TDI) assay is based on the observation that the intensity of fluorescent dye R110- and R6G-labeled acycloterminators is universally quenched once they are incorporated onto a DNA oligonucleotide primer. By comparing the rate of fluorescence quenching of the two allelic dyes in real time, we have extended this method for allele frequency estimation of SNPs in pooled DNA samples. The kinetic FQ-TDI assay is highly accurate and reproducible both in genotyping and in allele frequency estimation. Allele frequencies estimated by the kinetic FQ-TDI assay correlated well with known allele frequencies, with an r(2) value of 0.993. Applying this strategy to large-scale studies will greatly reduce the time and cost for genotyping hundreds and thousands of SNP markers between affected and control populations.
Xiao
Ming
M
Cardiovascular Research Institute and the Department of Dermatology, University of California, San Francisco, California 94143, USA.
Kwok
Pui-Yan
PY
D000483:Alleles; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D005787:Gene Frequency / Q000235:genetics; D005838:Genotype; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA