Detection of the JAK2 V617F Mutation by LightCycler PCR and Probe Dissociation Analysis

Abstract

A point mutation in the JAK2 gene, a member of the tyrosine kinase family, was recently identified and shown to be associated with several myeloproliferative disorders. Several studies identified the same JAK2 point mutation (1,849G>T), resulting in the substitution of a valine to phenylalanine at codon 617 (V617F). We developed a simple and sensitive method to detect this mutation via polymerase chain reaction and probe dissociation analysis using the LightCycler platform, and we compared this method to existing restriction fragment-length polymorphism, direct sequencing, and amplification refractory mutation system methods. We found that the LightCycler method offered advantages of speed, reliability, and more straightforward interpretation over the restriction fragment-length polymorphism and sequencing approaches.

Design and Evaluation of a Real-Time PCR Assay for Quantification of JAK2 V617F and Wild-Type JAK2 Transcript Levels in the Clinical Laboratory

“…Parallel testing of specimens submitted to our laboratory for clinical JAK2 V617F mutation testing by meltingcurve analysis 28 demonstrates that our new quantitative JAK2 V617F assay provides valuable qualitative data. All specimens that were characterized as positive or borderline positive by the melting-curve analysis were also characterized as positive by the quantitative assay (Table 3, Figure 3).…”

“…28 In brief, this assay detects the JAK2 V617F mutation by PCR and probe-dissociation analysis by using the LightCycler instrument (Roche, Indianapolis, IN), and the sensitivity of the assay was reported to be 5% by using cell line dilutions.…”

Les syndromes myéloprolifératifs

“…Elles reposent, le plus souvent, sur des PCR classiques comme la PCR allele refractory mutation system (ARMS), la PCR spécifique d'allèle, la PCR-restriction fragment length polymorphism (RFLP), la discrimination allélique ou des PCR en temps réel spéci-fiques d'allèle [93][94][95][96][97]. Le matériel étudié est soit le sang total, soit des leucocytes totaux, soit les polynucléaires …”

Estudo comparativo entre diferentes metodologias na detecção da mutação JAK2V617F em Neoplasias Mieloproliferativas Crônicas BCR-ABL1 negativo

“…O PCR-RFLP é um método de detecção da mutação JAK2V617F mais comumente utilizado, baseia-se na utilização de enzimas de restrição para detecção de mutações e polimorfismos genéticos (Lay et al, 2006).…”

“…Na tabela 1 estão as características de todos os pacientes estudados e o resultado da análise da mutação JAK2V617F através da técnica de PCR-RFLP. Esta metodologia foi escolhida com padrão ouro, pois foi a primeira a ser descrita e é a mais utilizada pelos laboratórios institucionais como também por laboratórios privados (Lay et al, 2006).…”