Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: a novel concept for mutation scanning

We report the development of a simple and inexpensive assay for the detection of DNA polymorphisms and mutations that is based on the modification of mismatched bases by potassium permanganate. Unlike the chemical cleavage of mismatch assay, which also exploits the reactivity of potassium permanganate to detect genomic variants, the assay we describe here does not require a cleavage manipulation and therefore does not require expensive or toxic chemicals or a separation step, as mismatches are detected using direct optical methods in a microplate format. Studies with individual deoxynucleotides demonstrated that the reactivity with potassium permanganate resulted in a specific colour change. Furthermore, studies with synthetic oligonucleotide heteroduplexes demonstrated that this colour change phenomenon could be applied to detect mismatched bases spectrophotometrically. A collection of plasmids carrying single point mutations in the mouse β-globin promoter region was used as a model system to develop a functional mutation detection assay. Finally, the assay was validated as 100% effective in detecting mismatches in a blinded manner using DNA from patients previously screened for mutations using established techniques, such as sequencing, SSCP and denaturing high-performance liquid chromatography (DHPLC) analysis in DNA fragments up to 300 bp in length.

我们报告了一种简单且廉价的测定法，以检测基于钾含钾不匹配评估的不匹配碱基的DNA多态性和突变，这也不同于不匹配评估的化学裂解。变体，我们在这里描述的测定不需要切割操作，因此不需要昂贵或有毒的化学物质或分离步骤，因为不匹配是使用单个脱氧核苷酸的研究使用直接的光学方法。在小鼠β-球蛋白启动子区域中携带单点突变的质粒的集合用作模型系统开发功能突变检测测定法最终，该测定方法是100％在以前使用已建立的技术筛选的患者的DNA以盲目的方式检测不匹配的方法，例如测序，SSCP在长达300 bp的DNA片段中的色谱（DHPLC）分析。