A real-time polymerase chain reaction assay for rapid, sensitive, and specific quantification of the JAK2V617F mutation using a locked nucleic acid-modified oligonucleotide.
J Mol Diagn
; 12(4): 512-9, 2010 Jul.
Article
em En
| MEDLINE
| ID: mdl-20431031
ABSTRACT
The JAK2V617F mutation has emerged as an essential molecular determinant of myeloproliferative neoplasms (MPNs). The aim of this study was to evaluate the analytical and clinical performances of a real-time PCR (qPCR) assay using a combination of hydrolysis probes and a wild-type blocking oligonucleotide, all containing locked nucleic acid (LNA) bases. Moreover, we validated a procedure for precise quantification of the JAK2V617F allele burden. We used DNA samples from patients suspected to suffer from MPN and dilutions of HEL cells, carrying the mutation, to compare the LNA-qPCR assay to two previously published methods. All assays detected the same 36 JAK2V617F positive patients of 116 suspected MPN diagnostic samples. No amplification of normal donor DNA was observed in the LNA-qPCR, and the assay was able to detect and reproducibly quantify as few as 0.4% of the JAK2V617F allele in wild-type alleles. Quantification of the JAK2V617F allele burden showed similar proportion levels among the different MPN entities as described by other groups. In conclusion, the LNA-qPCR is a rapid, robust, sensitive, and highly specific assay for quantitative JAK2V617F determination that can be easily implemented in clinical molecular diagnostic laboratories. Moreover, precise quantification allows determination of JAK2V617F burden at diagnosis as well as the evaluation of response to JAK2 inhibitors.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oligonucleotídeos
/
Bioensaio
/
Reação em Cadeia da Polimerase
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Janus Quinase 2
/
Mutação
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article