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


CONCLUSIONS
This combination of problems effectively combines to render selective reamplification and redigestion unsuitable for detecting low fractions of the V617F mutation.
Section on Hematology and Oncology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
G C
GC
Shepard
H L
HL
Lawson
G A
GA
Hawkins
J
J
Owen
D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D009196:Myeloproliferative Disorders / Q000175:diagnosis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000592:standards; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA
D053614:Janus Kinase 2