Association analysis using pooled samples is based on the addition of equal amounts of DNA from each relevant individual to either Case or Control pools, followed by quantitative measurement of the signals from each allele at each typed SNP, as a measure of the case or control allele frequency (Macgregor et al., 2008). Genomic DNA was extracted from peripheral venous blood samples. DNA concentrations in each sample were measured using PicoGreen (Molecular Probes) for the quantitation of double-stranded DNA in solution on a Fluoroskan Ascent CF plate reader (Labsystems, Chicago, IL). DNA samples were initially diluted to 70 ng/μL using 1X TE before another quantification measurement using the PicoGreen method. Each sample was then diluted to 45 ng/μL, re-quantitified using Pico Green and diluted to a final concentration of 30 ng/μL. Pools were constructed by combining equal volumes (10 μL) of each DNA sample. Variance introduced by different pool sizes was taken into account during data analysis. All pipetting steps requiring volumes greater than 1 μL were performed on an Eppendorf EpMotion 5070 or EpMotion 5075 robot (Eppendorf AG, Hamburg, Germany).
