Kinetic FP-TDI assay for SNP allele frequency determination.
Strategies for identifying genetic risk factors in complex diseases by association studies require the comparison of allele frequencies of numerous SNPs between affected and control populations. Theoretically, hundreds of thousands of SNP markers across the genome will have to be genotyped in these studies. Genotyping SNPs one sample at a time is extremely costly and time consuming. To streamline whole genome association studies, some have proposed to screen SNPs by pooling the DNA samples initially for allele frequency determination and perform individual genotyping only when there is a significant discrepancy in allele frequencies between the affected and control populations. Here we describe a new method for determining the allele frequency of SNPs in pooled DNA samples using a two-color primer extension assay with real-time monitoring of fluorescence polarization (named kinetic FP-TDI assay). By comparing the ratio of the rate of incorporation of the two allele-specific dye-terminators, one can calculate the relative amounts of each allele in the pooled sample. The accuracy of allele frequency determination with pooled samples is within 3.3 +/- 0.8% of that determined by genotyping individual samples that make up the pool.
Xiao
Ming
M
Washington University School of Medicine, St. Louis, MO, USA.
Latif
Sherif Medhat
SM
Kwok
Pui-Yan
PY
D000465:Algorithms; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods; D005787:Gene Frequency; D006801:Humans; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods
D005456:Fluorescent Dyes; D004247:DNA