Estimation of allele frequency in pooled DNA by using PCR-RFLP combined with microchip electrophoresis.
Biallelic marker, most commonly single nucleotide polymorphism (SNP), is widely utilized in genetic association analysis, which can be speeded up by estimating allele frequency in pooled DNA instead of individual genotyping. Several methods have shown high accuracy and precision for allele frequency estimation in pools. Here, we explored PCR restriction fragment length polymorphism (PCR-RFLP) combined with microchip electrophoresis as a possible strategy for allele frequency estimation in DNA pools. We have used the commercial available Agilent 2100 microchip electrophoresis analysis system for quantifying the enzymatically digested DNA fragments and the fluorescence intensities to estimate the allele frequencies in the DNA pools. In this study, we have estimated the allele frequencies of five SNPs in a DNA pool composed of 141 previously genotyped health controls and a DNA pool composed of 96 previously genotyped gastric cancer patients with a frequency representation of 10-90% for the variant allele. Our studies show that accurate, quantitative data on allele frequencies, suitable for investigating the association of SNPs with complex disorders, can be estimated from pooled DNA samples by using this assay. This approach, being independent of the number of samples, promises to drastically reduce the labor and cost of genotyping in the initial association analysis.
Wang
Wei-peng
WP
School of Pharmacy, Soochow University, Dushu Lake Higher Education Town, Suzhou, China. wangweipeng@suda.edu.cn
Zhang
Rui-hong
RH
Wu
Ping
P
Wang
Sen
S
Li
Rong
R
D016022:Case-Control Studies; D004247:DNA / Q000032:analysis / Q000235:genetics*; D046209:Electrophoresis, Microchip / Q000379:methods*; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide
D004247:DNA