The sequences of PCR and sequencing primers (Thermo Electron) used for each assay are shown below. Amplicons were generated in a 50µl reaction volume with 15pmol of forward and reverse PCR primers, 0.2mM dNTPs (Promega), 1.5mM MgCl₂, 1X Buffer II (Applied Biosystems), 1U AmpliTaq Gold (Applied Biosystems) using 10ng genomic DNA. PCR conditions were 94°C for 7 min; 50 cycles with denaturation at 94°C for 30s, annealing at 60°C for 30s and elongation at 72°C for 30s; 1 cycle at 72°C for 7 min; and a final hold at 4°C. Thermocycling was performed using a PTC-0225 DNA Engine Tetrad (MJ Research).

Single-stranded biotinylated PCR products were prepared for sequencing using the Pyrosequencing™ Vacuum Prep Tool. 3µl Streptavidin Sepharose™ HP (Amersham) was added to 37µl Binding buffer (10 mM Tris-HCl pH 7.6, 2M NaCl, 1 mM EDTA, 0.1% Tween 20) and mixed with 20µl PCR product and 20µl high purity water for 10 min at room temperature using a Variomag Monoshaker (Camlab). The beads containing the immobilised templates were captured onto the filter probes after applying the vacuum and then washed with 70% ethanol for 5 sec, denaturation solution (0.2M NaOH) for 5 sec and washing buffer (10 mM Tris-Acetate pH 7.6) for 5 sec. The vacuum was then released and the beads released into a PSQ 96 Plate Low containing 45µl annealing buffer (20 mM Tris-Acetate, 2 mM MgAc₂ pH 7.6), 0.3µM sequencing primer.
Pyrosequencing reactions were performed according to the manufacturer’s instructions using the PSQ 96 SNP Reagent Kit which contained the enzyme and substrate mixture and nucleotides. Assays were performed using the nucleotide dispensation orders shown below. The sample genotype was determined using the SNP Software (Biotage AB).

Primer sequences (B= biotinylated)

Selected theoretical outcomes for multiplex assay

Sequences to analyze:

T/T T/T G/G

T/C T/C G/A

C/C C/C A/A