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Int J Lab Hematol. 2011 Jun;33(3):267-71. doi: 10.1111/j.1751-553X.2010.01282.x. Epub 2010 Dec 1.

BsaXI/RFLP analysis of initial or selectively reamplified PCR product is unreliable in detecting the V617F mutation in JAK2.

Author information

1
Section on Hematology and Oncology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.

Abstract

INTRODUCTION:

Laboratory testing for the presence of the V617F mutation in JAK2 has taken on great importance in the diagnosis of myeloproliferative disorders. The availability of a facile detection method would bring this testing into greater clinical use. The polymerase chain reaction coupled with restriction fragment length polymorphisms is such a facile method. BsaXI cleaves the normal sequence but does not cleave the sequence leading to the V617F mutation.

METHODS:

We have examined the use of selective PCR reamplification with BsaXI cleavage to enrich the fraction of V617F and compared the assignment of mutation with an established qPCR method.

RESULTS:

We found that BsaXI fails to completely cleave normal sequence PCR product, leading to false positivity, particularly at low mutation levels. We also found that first-round standard PCR introduces new mutations in which subsequent reamplification and digestion cannot distinguish from the V617F mutation.

CONCLUSION:

This combination of problems effectively combines to render selective reamplification and redigestion unsuitable for detecting low fractions of the V617F mutation.

[Indexed for MEDLINE]

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