Lovisa Lovmar, Mona Fredriksson, Ulrika Liljedahl, Snaevar Sigurdsson, Ann-Christine Syvšnen
Molecular Medicine, Department of Medical Sciences, Uppsala University, Entrance 70, 3rd floor, Research dept. II, University Hospital, SE-751 85 Uppsala, Sweden
Whole genome amplification (WGA), such as primer extension preamplification (PEP) and multiple displacement amplification (MDA), have the potential to provide an unlimited source of DNA for large scale genetic studies. We have performed a quantitative evaluation of PEP and MDA prior to PCR and multiplex genotyping of single nucleotide polymorphisms (SNPs) using our in-house four-color fluorescence tag-array minisequencing system.
The applied 'array of arrays' format enabled analysis of 80 separate samples for up to 200 SNPs per standard microscope slide and reaction. Thus we were able to simultaneously analyze 15 CEPH family members in all three template types on a single microarray slide, facilitating accurate, quantitative comparison of the results. Forty-five SNPs distributed over the whole genome were genotyped and in total more than 5400 genotypes were generated per reaction and microscope slide.
The WGA methods were evaluated with respect to genotyping success, signal to noise ratios, power of genotype discrimination, yield, and imbalanced amplification of alleles in the MDA product. Cluster analysis was applied for assessing the quality of the genotyping clusters.
Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA (88.7% and 99.7% respectively). For PEP products the power of genotype discrimination was lower than for MDA due to a two-fold lower signal to noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied.
We also showed that the variation in amplification of the two SNP alleles depends on the amount of genomic DNA subjected to MDA. To obtain faithful representation of the alleles 3 ng DNA, equivalent to approximately 1000 genome copies, should be used per WGA reaction. Assuming an average multiplexing level of ten SNPs per PCR, a genome wide association study using 300 000 SNP markers would thus require less than one microgram of genomic DNA.
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