The sample preparation and pyrosequencing for JAK2 V617F genotyping and quantification were performed according to a previously published protocol [18 (link)]. After PCR, the biotinylated strand was captured on streptavidin Sepharose beads (Amersham Biosciences, Uppsala, Sweden) and annealed with sequencing primers for the strand. Pyrosequencing was performed separately for the strands using PSQ HS 96 Gold SNP Reagents and a PSQ HS 96 pyrosequencing machine (Biotage, Uppsala, Sweden).
Genomic DNA was extracted from the peripheral blood or bone marrow using Chemagic DNA blood kit (PerkinElmer, Baesweiler, Germany), according to the manufacturer's instructions. The JAK2 V617F mutation and allele burdens were determined by pyrosequencing. Extracted DNA was amplified using the following biotin-labeled primers: forward 5′-GAAGCAGCAAGTATGATGAGCA-3′; reverse 5′-TGCTCTGAGAAAGGCATTAGAA-3′. Single-stranded biotinylated templates were then isolated and sequenced using the sequencing primer 5′-TCTCGTCTCCACAGA-3′. Percentages of JAK2 V617F mutant alleles were determined using the Allele Frequency Quantification function in PyroMark Q24 Software 2.0, according to the manufacturer's specifications (Qiagen).
Genomic DNA was extracted from the peripheral blood or bone marrow using Chemagic DNA blood kit (PerkinElmer, Baesweiler, Germany), according to the manufacturer's instructions. The JAK2 V617F mutation and allele burdens were determined by pyrosequencing. Extracted DNA was amplified using the following biotin-labeled primers: forward 5′-GAAGCAGCAAGTATGATGAGCA-3′; reverse 5′-TGCTCTGAGAAAGGCATTAGAA-3′. Single-stranded biotinylated templates were then isolated and sequenced using the sequencing primer 5′-TCTCGTCTCCACAGA-3′. Percentages of JAK2 V617F mutant alleles were determined using the Allele Frequency Quantification function in PyroMark Q24 Software 2.0, according to the manufacturer's specifications (Qiagen).
