Sensitive Detection and Quantification of the JAK2V617F Allele by Real-Time PCR: Blocking Wild-Type Amplification by Using a Peptide Nucleic Acid Oligonucleotide

摘要: A single G-to-T missense mutation in the gene for JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found several myeloproliferative neoplasms. Reliable quantification of this mutant allele increasing clinical and therapeutic interest predicting diagnosing group Because JAK2V617F somatically acquired may be followed by loss heterozygosity, percentage versus wild-type DNA blood can vary between 0% almost 100%. Therefore, we developed real-time PCR assay detection low-to-high range burden. To allow meet these criteria, amplification was blocked with peptide nucleic oligonucleotide. patient diluted could amplified linearly from 0.05% 100%, acceptable reproducibility quantification. The sensitivity (n = 3 3). In 9 100 healthy donors, weak positive/background signal observed isolated blood, corresponding approximately 0.01% allele. one individual, duplicate. relevance finding not clear. By inhibiting allele, sensitive linear detect quantify JAK2V617F.