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).
Psq hs 96 gold snp reagents
The PSQ HS 96 Gold SNP Reagents are a set of reagents designed for use with the PyroSequencing technology. The reagents enable the analysis of single nucleotide polymorphisms (SNPs) in a high-throughput manner.
Lab products found in correlation
2 protocols using psq hs 96 gold snp reagents
Pyrosequencing-based JAK2 V617F Genotyping
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).
KRAS Mutation and MMR Analysis
Expression of MMR proteins (MLH1, PMS2, MSH2, and MSH6) was analyzed in formalin-fixed, paraffin-embedded tumor sections using immunohistochemistry. Monoclonal antibodies included anti-MLH1 (clone G168-728), anti-PMS2 (clone MRQ-28), anti-MSH2 (clone G219-1129), and anti-MSH6 (clone 44). MMR protein loss was defined as the absence of nuclear staining in tumor cells in the presence of positive nuclear staining in normal colonic epithelium and stromal cells. Tumors were defined as MMR-deficient (dMMR) if one or more MMR proteins was lost, and MMR-proficient (pMMR) if all MMR proteins were detected.
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