High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies

International Journal of Molecular Sciences
Marcin SłomkaDominik Strapagiel

Abstract

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide...Continue Reading

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Jul 28, 2019·Acta Neurologica Scandinavica·Giovanna PannuzzoVenera Cardile
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Dec 11, 2019·Analytical Chemistry·Yuxia ZhanGuanghua Luo

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Methods Mentioned

BETA
genotyping
PCR
X-ray

Software Mentioned

HRM
Bio
TESTOPLEK
Rad Precision Melt Analysis Software
SNaPshot
CodonCode Aligner

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