Psq hs 96a instrument

A 5μl aliquot of biotinylated PCR product was incubated with 4μl Sepharose beads (Amersham Biosciences, Piscataway, NJ) in a 100μl final volume at room temperature with agitation at 1100rpm for 15 minutes. To remove unattached PCR products, the beads were washed with 70% ethanol, DNA denatured with 0.2M NaOH, then washed again with 10mM Tris-Acetate (pH 7.6) and suspended in 12μl of annealing buffer (20mM Tris-Acetate (pH 7.6), 2mM Mg-Acetate) containing 30μM sequencing primers. To anneal the primer and template, the mixture was incubated at 75°C for 2 minutes followed by slow (10–15 minutes) cooling to room temperature. Pyrosequencing was then performed using PSQ 96 Gold reagents, and a PSQ HS 96A instrument (Qiagen, Valencia, CA).

Candidate single nucleotide polymorphisms (SNPs) were genotyped as previously described.^{10 (link)} Briefly, DNA was extracted from blood using the Gentra Puregene blood kit (Qiagen, Valencia, CA). DNA sequences containing SNPs of interest were amplified from 37.5 ng genomic DNA with the PyroMark PCR Kit (Qiagen), using a primer with an M13 tail and a universal biotinylated M13 primer. PCR product was captured on a PyroMark Vacuum Prep Tool (Qiagen) by aspiration and washed in 70% ethanol, Pyromark Denaturation Solution (Qiagen), and PyroMark Wash Buffer (Qiagen). PCR product was released into 15 uL of 0.3 uM sequencing primer diluted in PyroMark Annealing Buffer (Qiagen), heated at 70°C for 15 minutes, and cooled at room temperature for 10 minutes before running on a PSQ hs 96A Instrument (Qiagen).