Kinetic polymerase chain reaction on pooled DNA: a high-throughput, high-efficiency alternative in genetic epidemiological studies.
The ideal technology for screening single-nucleotide polymorphisms requires high throughput with minimal cost per sample, minimal usage of valuable DNA resources, and maximal flexibility for assessment of new polymorphisms. We demonstrate here the feasibility of kinetic allele-specific PCR with DNA pooling (S. Germer et al., Genome Res., 10: 258-266, 2000) in a population study that satisfies all of the mentioned criteria and offers a powerful new tool for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage dysequilibrium scans. Three individuals prepared pooled DNA samples from 269 individuals separated into three racial/ethnic groups: Caucasians (n = 56), African-Americans (n = 86), and Hispanics (n = 127). We used kinetic allele-specific PCR to determine the allele frequencies of the common paraoxonase 1 polymorphism, PON1 Q191R, in these pools. Paraoxonase 1 is a critical enzyme for inactivating neurotoxic intermediates in the metabolism of organophosphates. In a blinded test of the technology, these nine pooled DNA samples were sent to Roche for genotyping by kinetic allele-specific PCR. The allele frequencies found were 0.266 +/- 0.011, 0.386 +/- 0.011, and 0.617 +/- 0.010, respectively, which were comparable to the frequencies of 0.269, 0.403, and 0.622 determined by PCR-restriction fragment length polymorphism analysis. These same samples were genotyped on two kinetic PCR platforms from different manufacturers, using three different DNA polymerases. The results were comparable between both platforms and among all three polymerases. The results demonstrate a powerful new technology for determining frequencies of single-nucleotide polymorphisms in an epidemiological study.
Chen
Jia
J
Department of Community and Preventive Medicine, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA.
Germer
Søren
S
Higuchi
Russell
R
Berkowitz
Gertrud
G
Godbold
James
J
Wetmur
James G
JG
D000483:Alleles; D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000235:genetics*; D005260:Female; D005787:Gene Frequency; D030342:Genetic Diseases, Inborn / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011295:Prenatal Care; D012494:Sampling Studies; D012680:Sensitivity and Specificity
D004247:DNA