DETERMINATION OF POPULATION ALLELE FREQUENCE USING
HOMOGENEOUS, UNLABLELLED-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
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 an unlabelled probe/high resolution melting methodology together
with analysis software to determine SNP frequencies in pooled DNA samples. An
asymmetric PCR is performed in the presence of the double stranded DNA binding dye LC
Green Plus and an unlabelled detection oligonucleotide complementary to excess strand in
the region of the SNP. After PCR the sample is analyzed by high resolution melting in the
HR-1 (Zhou et al, Clinical Chemistry 50:8, 2004). Two peaks in the graph of –dF/dT versus
temperature are observed corresponding to the melting of the detection oligonucleotide off of
the two SNP alleles. Computer analysis of the relative peak heights determines the ratio of
the two alleles in the sample. We developed this technique using synthetic oligonucleotides
to simulate the annealed amplicon strand and detection oligonucleotide. Different
complementary and mismatched amplicon strands from 0% to 100% were mixed and melted
and software was optimized using this model system. We repeated this analysis using two
genomic DNAs homozygous for a A to G mutation in the cystic fibrosis gene. When mixed in
different ratios, and analyzed using this methodology the software was able to correctly
determine 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. This method was also applied to a pool of ninety six human
genomic DNA samples which previously had been genotyped individually at 8 SNP markers
on chromosome Y. The analysis software was able to determine the allele frequencies to
within 2% accuracy across a range of frequencies from 3% to 23%. This method is very
simple, fast and inexpensive method for the determination of SNP allele fraction.