Two-round allele specific-polymerase chain reaction: A simple and highly sensitive method for JAK2V617F mutation detection

Abstract

Background Philadelphia chromosome (Ph 1)-negative myeloproliferative disorders (MPD) are hematopoietic stem cell disorders characterized by extensive proliferation of myeloid blood cells. JAK2V617F has recently been identified in the majority of Ph 1-negative MPD and becomes an essential diagnostic marker. Methods To screen for JAK2V617F, a two-round allele specific-polymerase chain reaction (AS-PCR) was developed and compared to PCR–restriction fragment length polymorphism (PCR–RFLP), denaturing high performance liquid chromatography (DHPLC), and DNA sequencing. A primary AS-PCR was performed followed by a secondary AS-PCR, which was an amplification of the primary AS-PCR products using the same set of primers under alternative conditions. Results By primary AS-PCR, a strong mutant-DNA band was seen in the DNA mixture containing as low as 2.5% of mutant allele. An ambiguous band was seen in 1% dilution while being totally absent in 0.1% dilution. After secondary AS-PCR, a mutant DNA band was clearly detected at 0.01% dilution. The detection sensitivity of PCR–RFLP and DHPLC was 2.5% while sequencing analysis was unable to detect below 5% dilution. Conclusion Two-round AS-PCR is simple and inexpensive, making it a suitable method for JAK2V617F mutation screening. Moreover, monitoring of minimal residual disease after specific treatment of Ph 1-negative MPD patients should be feasible with this highly sensitive method.