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Genotyping of the hemochromatosis HFE p.H63D and p.C282Y mutations by high-resolution melting with the Rotor-Gene 6000® instrument
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Paulo Caleb Junior Lima Santos
Published/Copyright:
June 17, 2011
Abstract
Background: The genotyping of HFE p.C282Y and p.H63D mutations is one of the most requested molecular analyses in the laboratorial routine. In this scenario, the main aim was to develop a genotyping assay that has advantages compared to other methods.
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. Validation studies were conducted in samples bi-directionally sequenced.
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). Genotypes for the HFE p.C282Y mutation were easily distinguished in the region of 80–86°C. For the HFE p.H63D, genotypes were also easily distinguished in the region of 76–82°C, but using the addition of known wild-type genotype DNA in all unknown samples plus a reaction without addition. In validation, genotypes were 100% concordant between methods.
Conclusions: Our genotyping assay with the Rotor-Gene 6000® instrument applies to the laboratorial routine with several advantages, especially in large-scale demand. The main advantages were the non-dependence on gel electrophoresis and on mutagenic reagents for visualization of fragments, reduction of the chances for contamination due to sample preparation, the lack of use of probe-based methods and cost-effectiveness.
Received: 2011-3-9
Accepted: 2011-5-26
Published Online: 2011-06-17
Published in Print: 2011-10-01
©2011 by Walter de Gruyter Berlin Boston
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