High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies.
High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies.
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DOI：
10.3390/ijms18112316
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发表时间：
2017-11-03
影响因子：
5.6
通讯作者：
Strapagiel D
中科院分区：
文献类型：
--
作者：
Słomka M;Sobalska-Kwapis M;Wachulec M;Bartosz G;Strapagiel D
高分辨率熔解（HRM）是一种方便的基因扫描以及单个和多个单核苷酸多态性（SNP）基因分型的方法。这种快速、简单、闭管、均质和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，我们提供了一些有问题的问题，可能会影响HRM结果的性能和数据分析，特别是在参考曲线为基础的靶向基因分型，从我们的实验室实践的例子。我们提出了这些例子，以典型的实验工作流程，并讨论了各自的实验误差和限制的质量和分析结果的重要意义。对正确执行HRM基因分型实验具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制备的重现性、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和仔细选择用于测序验证的样品。我们提供了一个逐案分析和讨论我们遇到的实际问题和解决方案，应该考虑到新尝试HRM基因分型的研究人员，特别是在高通量设置。
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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作者：
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通讯作者：
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作者：
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通讯作者：
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