RRML - JAK2 p.V617F mutation – tetra-primer PCR and PCR-RFLP comparative semiquantitative approaches for estimation of the mutant allele in myeloproliferative neoplasms
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Concept, Design & Programming
Dr. Adrian Man

   
 
Nr. 14(1)/2009
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JAK2 p.V617F mutation – tetra-primer PCR and PCR-RFLP comparative semiquantitative approaches for estimation of the mutant allele in myeloproliferative neoplasms

Mutaţia JAK2 p.V617F – tehnicile tetra-primer PCR şi PCR-RFLP ca metode comparative semicantitative de apreciere a alelei mutante în neoplasmele mieloproliferative

Adrian P. Trifa, Radu A. Popp, Andrei Cucuianu, Delia Dima, Mariela S. Militaru, Mariana Paţiu, Ioan V. Pop


Abstract

Polycythemia vera, essential thrombocythemia and primary myelofibrosis are the three typical BCR-ABL negative myeloproliferative neoplasms. Recent studies indicated that a somatic single-point mutation, JAK2 p.V617F is a crucial molecular event in the pathogenesis of these diseases. Taking into consideration the importance of this mutation as a diagnosis and prognosis marker, we adapted in our laboratory a tetra-primer polymerase chain reaction (tetra-primer PCR) and a polymerase chain reaction-restriction fragments length polymorphism (PCR-RFLP) previously described assays for detection of JAK2 p.V617F. Meanwhile, we built a dilution based scale of mutant p.V617F homozygous DNA in wild-type homozygous DNA which serves for a semiquantitative estimation of the mutant allele proportion. Both techniques were shown to be similar sensitive: the lower detection limit of mutant allele was around 2% (tetra-primer PCR) and around 3% (PCR-RFLP); however the tetra-primer PCR has the advantage over the PCR-RFLP of being a more rapid assay. We succesufully managed to implement tetra-primer PCR and PCR-RFLP assays for JAK2 p.V617F analysis and to provide meanwhile semiquantitative approaches for estimation of the mutant allele in myeloproliferative neoplasms.

Keywords: myeloproliferative neoplasms,JAK2 p.V617F,tetra-primer PCR,PCR-RFLP,semiquantitative approach

 
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How to cite
Trifa AP, Popp RA, Cucuianu A, Dima D, Militaru MS, Paţiu M, et al. JAK2 p.V617F mutation – tetra-primer PCR and PCR-RFLP comparative semiquantitative approaches for estimation of the mutant allele in myeloproliferative neoplasms. Rev Romana Med Lab. 2009;14(1):25-30