title: Quantitative determination of allele frequency in pooled DNA by using sequencing method.
abstract: Quantitative determination of the allele frequency of single-nucleotide polymorphism (SNP) in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present such a simple, accurate, and inexpensive method for quantitative determining the allele frequency in pooled DNA samples. Three steps of DNA pooling, PCR amplification and sequencing are involved in this assay. Although direct determination of the allele frequency from the two allele-specific fluorescence intensities is possible, correction for differential response of alleles is important. We explored the effect of differential response of alleles on test statistics and provide a solution to this problem based on heterozygous fluorescence intensities. We demonstrate the accuracy and reliability of this assay on pooled DNA samples with pre-determined allele frequencies from 7.1% to 53.9%. The accuracy of allele frequency measurements is high, with a correlation coefficient of r² = 0.997 between measured and known frequencies. We believe that by providing a means for SNP genotyping up to hundreds of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies.
authors.affiliation: School of Pharmacy, Medical College, Soochow University, No. 199 Ren'ai Road, Dushu Lake Higher Education Town, Suzhou 215123, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Cao
authors.forename: Qiu-Jun
authors.initials: QJ
authors.lastname: Wang
authors.forename: Xu-Ting
authors.initials: XT
authors.lastname: Zhu
authors.forename: Huan
authors.initials: H
authors.lastname: Zhou
authors.forename: Rui
authors.initials: R
authors.lastname: Li
authors.forename: Wei-Peng
authors.initials: WP
authors.lastname: Wang
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016022:Case-Control Studies; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D005260:Female; D005787:Gene Frequency / Q000235:genetics*; D056726:Genetic Association Studies / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D013274:Stomach Neoplasms / Q000235:genetics
chemical_list: D004247:DNA