Allele Frequencies of Single Nucleotide Polymorphisms by Unlabelled Probe of DNA Pooling

DETERMINATION OF POPULATION ALLELE FREQUENCY USING HOMOGENEOUS, UNLABELED PROBE ANALYSIS OF POOLED DNA SAMPLES

Zhou L*, Palais R, Garrett M, Wong L, Wittwer C. Department of Pathology, University of Utah, 50 North Medical Drive, Salt Lake City Utah, USA 84132 [email protected]

Single nucleotide polymorphisms (SNPs) are the DNA marker of choice for genetic analysis of polygenic traits. High-throughput genotyping of SNPs in a large number of individuals is needed for association studies and for other studies of the relationship between genes and diseases. DNA pooling is a practical way to reduce the cost of large-scale association studies. Pooling allows the population allele frequencies to be measured using far fewer PCR reactions and genotyping assays than required when genotyping individuals one by one. We have developed a high resolution melting methodology using unlabeled probes and analysis software to determine SNP frequencies in pooled DNA samples. An asymmetric PCR is performed after which the sample is analyzed by high resolution melting in the HR-1 (Zhou et al, Clinical Chemistry 50:8, 2004). Unlabeled detection oligonucleotides (probes) complementary to excess strands in the region of the SNP and the double stranded DNA binding dye LCGreen PLUS generate a fluorescence (F) vs. temperature (T) `melting curve’. The graph of –dF/dT exhibits two peaks corresponding to the melting of the probes off of the two SNP alleles. Analysis of the relative peak heights determines the ratio of the two alleles in the sample. We have calibrated this technique by mixing known proportions of synthetic oligonucleotides, complementary and mismatched, from 0% to 100%. The mixtures were melted and quantification software was optimized using this model system. We then analyzed mixtures of genomic DNA exhibiting two genotypes homozygous for an A to G mutation in the cystic fibrosis gene. The software determined the ratio of A to G allele in the mixture to an accuracy of 1% over the range from 0% to 100% of one allele. The method was applied to a pool of ninety-six human genomic DNA samples previously genotyped individually at 8 SNP markers on chromosome Y. The software determined the allele frequencies to within 2% accuracy over a range of frequencies from 3% to 23%. This homogeneous (closed-tube) technique provides a simple, fast, and inexpensive method for the determination of SNP allele fraction without risk of contamination.