Back to Search
Start Over
Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.
- Source :
-
PloS one [PLoS One] 2011; Vol. 6 (10), pp. e26534. Date of Electronic Publication: 2011 Oct 20. - Publication Year :
- 2011
-
Abstract
- Background: JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described.<br />Methodology/principal Findings: Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments.<br />Conclusions: With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.
- Subjects :
- Cell Line, Tumor
DNA Mutational Analysis instrumentation
DNA Probes genetics
Humans
Nucleic Acid Amplification Techniques
Nucleic Acid Denaturation
Polycythemia Vera enzymology
DNA Mutational Analysis methods
DNA Probes chemistry
Janus Kinase 2 genetics
Laboratories
Mutation, Missense genetics
Polycythemia Vera genetics
Transition Temperature
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 6
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 22028900
- Full Text :
- https://doi.org/10.1371/journal.pone.0026534