High resolution melting applications for clinical laboratory medicine

Abstract

Separation of the two strands of DNA with heat (melting) is a fundamental property of DNA that is conveniently monitored with fluorescence. Conventional melting is performed after PCR on any real-time instrument to monitor product purity (dsDNA dyes) and sequence (hybridization probes). Recent advances include high resolution instruments and saturating DNA dyes that distinguish many different species. For example, mutation scanning (identifying heterozygotes) by melting is closed-tube and has similar or superior sensitivity and specificity compared to methods that require physical separation. With high resolution melting, SNPs can be genotyped without probes and more complex regions can be typed with unlabeled hybridization probes. Highly polymorphic HLA loci can be melted to establish sequence identity for transplantation matching. Simultaneous genotyping with one or more unlabeled probes and mutation scanning of the entire amplicon can be performed at the same time in the same tube, vastly decreasing or eliminating the need for re-sequencing in genetic analysis. High resolution PCR product melting is homogeneous, closed-tube, rapid (1-5 min), non-destructive and does not require covalently-labeled fluorescent probes. In the clinical laboratory, it is an ideal format for in-house testing, with minimal cost and time requirements for new assay development.

Association between a p73 Gene Polymorphism and Genetic Susceptibility to Non-small Cell Lung Cancer in the South of China

“…The sequencing method is still the 'gold standard' testing of DNA mutations, but the process is relatively complex and expensive, can't be applied to clinical. High Resolution Melting Curve technology has many characteristics such as high specificity and convenient and high flux, becoming a hot technology in the detection of mutations in recent years (Erali et al, 2008). HRM is based on the physical properties of nucleic acids, a technique different from the conventional PCR, analyzing the difference of nucleic acid melting curve with monitoring saturated dye.…”

“…Shuang-Shuang Wang 1 , Hai-Yan Guo 3 , Lin-Li Dong 1 , Xiang-Qian Zhu 2 , LiangMa 1 , Wen Li 1 * Jian-Xin Tang 1 * et al, 2008;Lo et al, 2011). p73 gene encodes a protein, significant similarity with p53 throughout its DNAbinding, transactivation, and oligomerization domains (Kaghad et al, 1997).…”

Identification of non-tuberculous mycobacteria by real-time PCR coupled with a high-resolution melting system

“…High-resolution melting analysis (HRMA) is a novel PCRbased method for detection of DNA sequence variation by demonstrating fluorescence changes in the melting process of the PCR amplicon with various saturation dyes such as LC Green, SYTO9 and Eva Green (Erali et al, 2008). Previous studies have combined HRMA with real-time PCR assays for rapid detection and identification of clinically important bacteria (Ajitkumar et al, 2012;Chen et al, 2011;Cheng et al, 2006, Ricchi et al, 2011Choi et al, 2010;Douarre et al, 2012;Pang et al, 2011;Pietzka et al, 2009;Tortoli, 2009;Won et al, 2010;Yang et al, 2009).…”

Optimizing a multiplex high resolution melting curve to diagnose G6PD deficiency based on viangchan and canton mutations

“…This allows good discrimination among them. However, when the amplicon size increases, the Tm difference decreases resulting in difficulty in genotypes differentiation (Erali et al, 2008;Krypuy et al, 2006;Liew et al, 2004). For this project, the amplicon size were 80bp and 102bp which were in the optimal range for HRM analysis.…”