Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of Sao Paulo Medical School, SP, Brazil.
Clin Chem Lab Med. 2011 Oct;49(10):1633-6. doi: 10.1515/CCLM.2011.654. Epub 2011 Jun 17.
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.
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.
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.
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.
HFE 基因 p.C282Y 和 p.H63D 突变的基因分型是实验室常规中最常要求的分子分析之一。在这种情况下，主要目的是开发一种与其他方法相比具有优势的基因分型检测方法。
采用聚合酶链反应（PCR）结合 Rotor-Gene 6000（®）仪器的高分辨率熔解（HRM）分析对 HFE 基因 p.C282Y（c.G845A；rs1800562）和 p.H63D（c.C187G，rs1799945）突变进行基因分型。在双向测序的样本中进行验证研究。
熔解分析是在独特的程序中开发的，以确保大约 112 分钟（31 分钟用于样本制备，81 分钟用于 PCR-HRM 步骤）内得到结果。在 80-86°C 区域内，HFE 基因 p.C282Y 突变的基因型很容易区分。对于 HFE 基因 p.H63D，基因型也很容易在 76-82°C 区域内区分，但在所有未知样本中加入已知野生型基因型 DNA，并在没有添加的情况下进行反应。在验证中，两种方法的基因型完全一致。
我们使用 Rotor-Gene 6000（®）仪器的基因分型检测方法适用于实验室常规，具有许多优势，尤其是在大规模需求方面。主要优点是不依赖凝胶电泳和突变试剂来可视化片段，减少因样本制备而导致的污染机会，无需使用探针方法且具有成本效益。
