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

Overview

Journal Int J Mol Sci

Publisher MDPI

Specialties Biochemistry
Chemistry
Molecular Biology

Date 2017 Nov 4

PMID 29099791

Citations 49

Authors

Marcin Slomka

Marta Sobalska-Kwapis

Monika Wachulec

Grzegorz Bartosz

Dominik Strapagiel

Affiliations

Soon will be listed here.

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.

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References

De Leeneer K, Coene I, Poppe B, De Paepe A, Claes K . Rapid and sensitive detection of BRCA1/2 mutations in a diagnostic setting: comparison of two high-resolution melting platforms. Clin Chem. 2008; 54(6):982-9. DOI: 10.1373/clinchem.2007.098764. View

Ebili H, Ilyas M . High resolution melt analysis, DNA template quantity disparities and result reliability. Clin Lab. 2015; 61(1-2):155-9. DOI: 10.7754/clin.lab.2014.140821. View

Iacumin L, Ginaldi F, Manzano M, Anastasi V, Reale A, Zotta T . High resolution melting analysis (HRM) as a new tool for the identification of species belonging to the Lactobacillus casei group and comparison with species-specific PCRs and multiplex PCR. Food Microbiol. 2014; 46:357-367. DOI: 10.1016/j.fm.2014.08.007. View

Martino A, Mancuso T, Rossi A . Application of high-resolution melting to large-scale, high-throughput SNP genotyping: a comparison with the TaqMan method. J Biomol Screen. 2010; 15(6):623-9. DOI: 10.1177/1087057110365900. View

Daugaard I, Kjeldsen T, Hager H, Hansen L, Wojdacz T . The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM. Exp Mol Pathol. 2015; 99(3):632-40. DOI: 10.1016/j.yexmp.2015.11.007. View

Poulou M, Destouni A, Kakourou G, Kanavakis E, Tzetis M . Prenatal diagnosis for CF using High Resolution Melting Analysis and simultaneous haplotype analysis through QF-PCR. J Cyst Fibros. 2014; 13(6):617-22. DOI: 10.1016/j.jcf.2014.04.002. View

Valisheva I, Harris R, Zhu-Shimoni J . A sensitive mutation screening method supporting cell line development for biotherapeutics. Anal Biochem. 2016; 505:73-5. DOI: 10.1016/j.ab.2016.04.007. View

Tong S, Giffard P . Microbiological applications of high-resolution melting analysis. J Clin Microbiol. 2012; 50(11):3418-21. PMC: 3486245. DOI: 10.1128/JCM.01709-12. View

Erali M, Voelkerding K, Wittwer C . High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol. 2008; 85(1):50-8. PMC: 2606052. DOI: 10.1016/j.yexmp.2008.03.012. View

10.

Cousins M, Donnell D, Eshleman S . Impact of mutation type and amplicon characteristics on genetic diversity measures generated using a high-resolution melting diversity assay. J Mol Diagn. 2012; 15(1):130-7. PMC: 3532711. DOI: 10.1016/j.jmoldx.2012.08.008. View

11.

Seipp M, Durtschi J, Liew M, Williams J, Damjanovich K, Pont-Kingdon G . Unlabeled oligonucleotides as internal temperature controls for genotyping by amplicon melting. J Mol Diagn. 2007; 9(3):284-9. PMC: 1899416. DOI: 10.2353/jmoldx.2007.060136. View

12.

Er T, Chang J . High-resolution melting: applications in genetic disorders. Clin Chim Acta. 2012; 414:197-201. DOI: 10.1016/j.cca.2012.09.012. View

13.

Wittwer C . High-resolution DNA melting analysis: advancements and limitations. Hum Mutat. 2009; 30(6):857-9. DOI: 10.1002/humu.20951. View

14.

Cheng J, Huang C, Lin C, Chen C, Chang Y, Chang S . Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR. Clin Chem. 2006; 52(11):1997-2004. DOI: 10.1373/clinchem.2006.069286. View

15.

Erali M, Wittwer C . High resolution melting analysis for gene scanning. Methods. 2010; 50(4):250-61. PMC: 2836412. DOI: 10.1016/j.ymeth.2010.01.013. View

16.

Vossen R, Aten E, Roos A, den Dunnen J . High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum Mutat. 2009; 30(6):860-6. DOI: 10.1002/humu.21019. View

17.

Gorniak P, Ejduk A, Borg K, Makuch-Lasica H, Nowak G, Lech-Maranda E . Comparison of high-resolution melting analysis with direct sequencing for the detection of recurrent mutations in DNA methyltransferase 3A and isocitrate dehydrogenase 1 and 2 genes in acute myeloid leukemia patients. Eur J Haematol. 2015; 96(2):181-7. DOI: 10.1111/ejh.12566. View

18.

Pompanon F, Bonin A, Bellemain E, Taberlet P . Genotyping errors: causes, consequences and solutions. Nat Rev Genet. 2005; 6(11):847-59. DOI: 10.1038/nrg1707. View

19.

Wojdacz T, Dobrovic A, Hansen L . Methylation-sensitive high-resolution melting. Nat Protoc. 2009; 3(12):1903-8. DOI: 10.1038/nprot.2008.191. View

20.

Caban M, Strapagiel D, Dziadek J, Korycka-Machala M, Grzelak A . Principles of a New Protocol for Prediction of Azole Resistance in Candida albicans Infections on the Basis of ERG11 Polymorphisms. Curr Microbiol. 2016; 73(2):172-82. PMC: 4923106. DOI: 10.1007/s00284-016-1039-3. View