Determination of allele frequency in pooled DNA: comparison of three PCR-based methods.
Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However; this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47% and the lowest variation between replicates (SD = 3.18%).
Wilkening
Stefan
S
German Cancer Research Center, Molecular Genetic Epidemiology, Heidelberg, Germany. stefan_wilkening@web.de
Hemminki
Kari
K
Thirumaran
Ranjit Kumar
RK
Bermejo
Justo Lorenzo
JL
Bonn
Stefan
S
Försti
Asta
A
Kumar
Rajiv
R
D003365:Costs and Cost Analysis; D005787:Gene Frequency; D006801:Humans; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D020641:Polymorphism, Single Nucleotide