Pyromark q24 vacuum prep workstation

For pyrosequencing genotyping, PCR reactions were performed in a total of 25 µl containing 10 ng of gDNA, 200 µM of each dNTP, 1× PCR buffer, 2.0 mM MgCl_2, 0.6 units of HotStartTaq DNA polymerase (Qiagen) and 0.4 µM of primers pyr-Vgsc-F and pyr-Vgsc-R (Table 1) [21 (link)]. After initial denaturation at 95 °C for 15 min, PCR amplification was performed for 40 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, followed by a final extension step at 72 °C for 10 min.
The pyrosequencing genotyping was performed using single-stranded PCR products obtained using the PyroMark Q24 Vacuum Prep Workstation then used in pyrosequencing reactions performed using the PyroMark Gold Q96 reagent kit (Qiagen). The sequencing primer and dispensation order are described in Table 1.

Detection of mutations of BRAF gene was performed using pyrosequencing (PSQ), which is based on ‘sequencing by synthesis’ principle [29 (link)]. PCR reaction using the PyroMark Q24 BRAF Kit (Qiagen, CA, USA) allowed the amplification of exons 11 and 15 of the gene. The biotinylated PCR products was immobilized onto streptavidin-coated sepharose beads (Amersham Biosciences, NJ, USA) and processed to obtain a single strand DNA through the PyroMark Q24 Vacuum Prep Workstation (Qiagen, CA, USA). The strands were separated using 0.1 mol/l NaOH. The supernatant was then discarded and 0.3 μΜ of the PSQ primer added annealed to the captured strand and incubated at 80°C for 2 minutes on the PSQ plate. The biotinylated DNA with annealed sequence primer was released from the streptavidin surface according to the manufacturer’s instructions (Qiagen, CA, USA). The primed single-stranded DNA templates were subjected to real-time sequencing of the region surrounding codons 464–469 in exon 11 and codon 600 in exon 15. The optimal nucleotide dispensation order was determined by assessing the theoretical outcome of a number of dispensation orders. Identified mutations were confirmed by PSQ of an independent PCR [30 (link)].