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Int J Mol Sci. 2017 Nov 3;18(11). pii: E2316. doi: 10.3390/ijms18112316.

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

Author information

1
Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pilarskiego 14/16, 90-231 Łódź, Poland. marcin.slomka@biol.uni.lodz.pl.
2
BBMRI.pl Consortium, 54-066 Wrocław, Poland. marcin.slomka@biol.uni.lodz.pl.
3
Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pilarskiego 14/16, 90-231 Łódź, Poland. marta.sobalska@biol.uni.lodz.pl.
4
BBMRI.pl Consortium, 54-066 Wrocław, Poland. marta.sobalska@biol.uni.lodz.pl.
5
Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pomorska 141/143, 90-236 Łódź, Poland. monika.caban@biol.uni.lodz.pl.
6
Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pomorska 141/143, 90-236 Łódź, Poland. gbartosz@biol.uni.lodz.pl.
7
Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pilarskiego 14/16, 90-231 Łódź, Poland. dominik.strapagiel@biol.uni.lodz.pl.
8
BBMRI.pl Consortium, 54-066 Wrocław, Poland. dominik.strapagiel@biol.uni.lodz.pl.

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 a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup.

KEYWORDS:

HRM limitations; gene scanning; genotyping; high resolution melting

PMID:
29099791
PMCID:
PMC5713285
DOI:
10.3390/ijms18112316
[Indexed for MEDLINE]
Free PMC Article

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