Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pilarskiego 14/16, 90-231 Łódź, Poland.
BBMRI.pl Consortium, 54-066 Wrocław, Poland.
Int J Mol Sci. 2017 Nov 3;18(11):2316. doi: 10.3390/ijms18112316.
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.
高分辨率熔解（HRM）是一种方便的基因扫描方法，也可用于个体和多个单核苷酸多态性（SNP）的基因分型。这种快速、简单、闭管、同质且具有成本效益的方法具有高特异性和灵敏度的能力，同时允许轻松过渡到高通量规模。在本文中，我们提供了一些来自我们实验室实践的例子，这些例子说明了可能影响 HRM 结果的性能和数据分析的问题，特别是在基于参考曲线的靶向基因分型方面。我们按照典型的实验工作流程呈现这些例子，并讨论了各自的实验误差和局限性对结果的质量和分析的关键意义。对正确执行 HRM 基因分型实验有决定性影响的实验细节包括 DNA 源材料的类型和质量、分离方法和模板 DNA 制备的可重复性、引物和扩增子设计、自动化衍生的制备和移液不一致性，以及替代变体的熔解曲线区分的物理限制以及对测序验证的样本的仔细选择。我们提供了实际遇到的问题的案例分析和讨论，并提供了新尝试 HRM 基因分型的研究人员应考虑的解决方案，特别是在高通量设置中。
