Somatic mutation detection efficiency in EGFR : a comparison between high resolution melting analysis and Sanger sequenc
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RESEARCH ARTICLE
Open Access
Somatic mutation detection efficiency in EGFR: a comparison between high resolution melting analysis and Sanger sequencing Reenu Anne Joy1, Sukrishna Kamalasanan Thelakkattusserry1, Narendranath Vikkath1, Renjitha Bhaskaran2, Sajitha Krishnan3, Damodaran Vasudevan1,3 and Prasanth S. Ariyannur1,3*
Abstract Background: High resolution melting curve analysis is a cost-effective rapid screening method for detection of somatic gene mutation. The performance characteristics of this technique has been explored previously, however, analytical parameters such as limit of detection of mutant allele fraction and total concentration of DNA, have not been addressed. The current study focuses on comparing the mutation detection efficiency of High-Resolution Melt Analysis (HRM) with Sanger Sequencing in somatic mutations of the EGFR gene in non-small cell lung cancer. Methods: The minor allele fraction of somatic mutations was titrated against total DNA concentration using Sanger sequencing and HRM to determine the limit of detection. The mutant and wildtype allele fractions were validated by multiplex allele-specific real-time PCR. Somatic mutation detection efficiency, for exons 19 & 21 of the EGFR gene, was compared in 116 formalin fixed paraffin embedded tumor tissues, after screening 275 tumor tissues by Sanger sequencing. Results: The limit of detection of minor allele fraction of exon 19 mutation was 1% with sequencing, and 0.25% with HRM, whereas for exon 21 mutation, 0.25% MAF was detected using both methods. Multiplex allele-specific real-time PCR revealed that the wildtype DNA did not impede the amplification of mutant allele in mixed DNA assays. All mutation positive samples detected by Sanger sequencing, were also detected by HRM. About 28% cases in exon 19 and 40% in exon 21, detected as mutated in HRM, were not detected by sequencing. Overall, sensitivity and specificity of HRM were found to be 100 and 67% respectively, and the negative predictive value was 100%, while positive predictive value was 80%. (Continued on next page)
* Correspondence: [email protected] 1 Molecular Oncology Diagnostics Laboratory, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Kochi 682041, India 3 Department of Biochemistry, Amrita School of Medicine, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Ponekkara P.O., Kochi, Kerala 682041, India Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If
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