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High-resolution melting analysis for a reliable and two-step scanning of mutations in the tyrosine kinase domain of the chimerical bcr-abl gene.

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Title: High-resolution melting analysis for a reliable and two-step scanning of mutations in the tyrosine kinase domain of the chimerical bcr-abl gene.
Authors: Doi, Yuko / Sasaki, Daisuke / Terada, Chiharu / Mori, Sayaka / Tsuruda, Kazuto / Matsuo, Emi / Miyazaki, Yasushi / Nagai, Kazuhiro / Hasegawa, Hiroo / Yanagihara, Katsunori / Yamada, Yasuaki / Kamihira, Shimeru
Issue Date: Jul-2009
Publisher: Japanese Society of Hematology
Citation: International Journal of Hematology, 90(1), pp.37-43; 2009
Abstract: For relevant imatinib therapy against Philadelphia (Ph)-positive leukemias, it is essential to monitor mutations in the chimerical bcr-abl tyrosine kinase domain (TKD). However, there is no universally acceptable consensus on how to efficiently identify mutations in the target TKD. Recently, high-resolution melting (HRM) technology was developed, which allows gene scanning using an inexpensive generic heteroduplex-detecting dsDNA-binding dye. This study aimed to validate the introduction of HRM in a practical clinical setting for screening of mutations in sporadic sites of the chimerical bcr-abl TKD. All chimerical and wild-type abl TKD regions selectively amplified were used for HRM assays and direct sequencing. The HRM test had approximately 5-90% detection sensitivity for mutations. In contrast to mixture samples with mutant and wild-type cells, all mutant cell samples had indeterminate melting curves equivalent to those of the wild-type due to formation of only a homodulex. This issue was improved by the addition of exogenous wild-type DNA after PCR. Subsequently, HRM results gave a high accordance rate of 97.8% (44/45 samples) compared to the sequencing data. The discordant results in one appear to be due to unsuccessful amplification. Thus, HRM may be considered to be suitable for reliable scanning of mutations in the chimerical abl TKD in a clinical setting.
Keywords: Ph / bcr-abl / Mutation / Melting analysis / HRM
URI: http://hdl.handle.net/10069/21985
ISSN: 18653774
DOI: 10.1007/s12185-009-0337-y
PubMed ID: 19466505
Rights: (c)2008 Japanese Society of Hematology / The original publication is available at www.springerlink.com
Type: Journal Article
Text Version: author
Appears in Collections:Articles in academic journal

Citable URI : http://hdl.handle.net/10069/21985

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