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

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