![]() For the HFE p.H63D, genotypes were also easily distinguished in the region of 76–82☌, but using the addition of known wild-type genotype DNA in all unknown samples plus a reaction without addition. ![]() Genotypes for the HFE p.C282Y mutation were easily distinguished in the region of 80–86☌. Results: The melting assay was developed in a unique procedure and to ensure the result in approximately 112 min (31 min for sample preparation and 81 min for the PCR-HRM step). ![]() Validation studies were conducted in samples bi-directionally sequenced. Methods: Genotypes for the HFE p.C282Y (c.G845A rs1800562) and p.H63D (c.C187G, rs1799945) mutations were assessed by polymerase chain reaction (PCR) followed by high resolution melting (HRM) analysis with the Rotor-Gene 6000 ® instrument. In this scenario, the main aim was to develop a genotyping assay that has advantages compared to other methods. Background: The genotyping of HFE p.C282Y and p.H63D mutations is one of the most requested molecular analyses in the laboratorial routine.
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