Psq hs 96 pyrosequencer

10 μl PCR product, 2 μl streptavidin-Sepharose beads (GE Healthcare), 38 μl binding buffer (Biotage) and 30 μl H₂O were combined in each well of a 96-well plate and mixed vigorously on a plate shaker for 5 min. The PCR products were then prepared using a vacuum work table (Biotage AB). The biotinylated PCR products, attached to the filter probes of the vacuum apparatus, were immersed in 70% (v/v) ethanol for 5 seconds, denatured in PyroMark Denaturation solution (Biotage AB) for 5 sec (allowing only the biotin labeled strand of the PCR product to remain attached to the filter probes) and immersed in 1X PyroMark Wash buffer (Biotage AB) for 5 sec. The single-stranded PCR products were then re-suspended in a PSQ HS 96-well plate containing 0.5 μl sequencing primer (at 10 pmol/μl) and 11.5 μl annealing buffer (Biotage AB) per well. The plate was incubated at 80°C for 2 minutes to allow the sequencing primer to anneal to the single-stranded PCR product. The PSQ 96-well plate and a PSQ HS 96 capillary dispensing tip holder (Biotage AB) containing enzyme, substrate and dNTPs (PyroGold reagent kit Biotage AB), were placed into a PSQ HS 96 Pyrosequencer (Biotage AB). The data were analysed using the SNP software (Biotage AB).

Genomic DNA was PCR amplified and subjected to mutation analysis for codons 12, 13 and 61 of KRAS and NRAS by pyrosequencing using a PSQ HS 96 Pyrosequencer (Biotage, Uppsala, Sweden). CEBPA was assessed by direct Sanger sequencing on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) [26 (link)]. A fluorescence-based multiplex PCR was used to detect internal tandem duplication (ITD) and D835 point mutation of the FLT3 gene. The PCR products were then subjected to capillary electrophoresis on an ABI Prism 3100 Genetic Analyzer to distinguish wild and mutant genotypes. For the third patient KIT, NPM1, KRAS and NRAS mutation were assessed using a next generation sequencing (NGS)-based assay [27 (link)].

Bisulfite treatment of genomic DNA was performed using the EpiTect bisulfite kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Pyrosequencing was carried out for 8 of the SRAMs, reflecting both promoter or first exon CpGi’s (CBLC, MST1R, LAD1, ESRP1, HOXB4) and non-CpGi genes (PRSS8, RAB25, AXL) (Additional file 16: Table S9). One microliter of bisulfite-treated DNA was used for each polymerase chain reaction (PCR). After an initial hot-start at 95°C for 5 minutes, all PCR reactions ran at 95°C for 30 seconds, annealing at various temperatures for 30 seconds, and underwent an extension step at 72°C for 30 seconds. All reactions were carried out with a nested PCR step during which a biotinylated universal primer was added. After PCR, the biotinylated strand was captured on streptavidin-coated beads (Amersham Bioscience, Uppsala, Sweden) and incubated with sequencing primers. Pyrosequencing was performed with PSQ HS 96 Gold reagents on a PS QHS 96 pyrosequencer (Biotage, Uppsala, Sweden) as published previously [48 (link)].