Whole genome amplification

DNA was extracted from archived dried blood spots stored on Schleicher and Schuell #907 filter paper stored at −70C. Samples were collected from 1994-1997 and DNA extraction was performed in 2002. Prior work has demonstrated sufficient yield from dried blood samples when stored at −20C for over twenty years; samples in this study were stored for at most eight years.^{29 (link)} Samples were processed as follows. In brief, one 1/16” hole punch (MC Mieth Manufacturing, Port Orange, FL) from the dried blood spot was collected for each case and control. To prevent cross-contamination, the punch was flame-sterilized between samples. Filter paper punches without blood spots served as negative controls. In order to have sufficient DNA for multiple reactions, each sample was PCR-amplified using a multi-strand displacement reaction resulting in whole genome amplification (Qiagen, Valencia, CA) and yielded 20 μl of 1μg/μl DNA. The amplified DNA was diluted 300-fold with reagent grade water and used in all genotyping reactions. DNA amplified using the multi-strand displacement reaction has been found to have better than 99% concordance with traditionally extracted DNA in subsequent Taqman genotyping reactions.^{30 (link)}