Publication: Two-round allele specific-polymerase chain reaction: A simple and highly sensitive method for JAK2V617F mutation detection
Issued Date
2009-03-01
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ISSN
00098981
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2-s2.0-58849163658
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Mahidol University
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SCOPUS
Bibliographic Citation
Clinica Chimica Acta. Vol.401, No.1-2 (2009), 148-151
Suggested Citation
Supattra Kannim, Wanna Thongnoppakhun, Chirayu U. Auewarakul Two-round allele specific-polymerase chain reaction: A simple and highly sensitive method for JAK2V617F mutation detection. Clinica Chimica Acta. Vol.401, No.1-2 (2009), 148-151. doi:10.1016/j.cca.2008.12.010 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/27274
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Title
Two-round allele specific-polymerase chain reaction: A simple and highly sensitive method for JAK2V617F mutation detection
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Abstract
Background: Philadelphia chromosome (Ph1)-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 Ph1-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 Ph1-negative MPD patients should be feasible with this highly sensitive method. © 2008 Elsevier B.V. All rights reserved.