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Robert P. Loewe GeneWake GmbH, Germany |
Abstract
High resolution melting (HRM) has become a prominent feature in qPCR instrument and chemistry line up. The ordeal however of designing a valid HRM experiment from the primer sequence to amplicon size and designating the proper controls for each unique question remains. Some obstacles are of cause greatly debated in literature and these will be summarized, but additionally a short guide to achieve a successful result is needed and given here. Touching the topics of primer design for sufficient amplicon length, the great focus will be experimental design and genotype calling by curve discussion. Furthermore the non-trivial situation underlining somatic mutations is debated, where frequency of single nucleotide exchanges can range from none to present in all cells. Aim is to give people an insight for this highly relevant method.
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