O'Donnell K A, Tighe O, O'Neill C, Naughten E, Mayne P D, McCarthy T V, Vaughan P, Croke D T
Department of Pathology, The Children's Hospital, Temple Street, Dublin 1, Republic of Ireland.
Hum Mutat. 2001 May;17(5):432. doi: 10.1002/humu.1120.
Mutation detection methods based upon chemical or enzymatic cleavage of DNA offer excellent detection efficiencies coupled with high throughput and low unit cost. We describe the application of the novel technique of Glycosylase Mediated Polymorphism Detection (GMPD) to the detection of two of the most common mutations of the PAH gene in the Irish population that cause phenylketonuria (PKU), R408W and I65T, which occur at relative frequencies of 41.0% and 10.4% respectively. GMPD assays for R408W and I65T were developed permitting fluorescent detection of cleavage products on the ALFexpresstrade mark automated DNA sequencer. The method was validated by screening a panel of PKU patients whose mutant genotypes had previously been characterised by standard methods. It also proved possible to perform multiplex detection of the two mutations by co-electrophoresis of GMPD products. GMPD is a rapid and robust method for the detection of the R408W and I65T mutations, whose key advantage lies in its use of a pair of enzymes with high cleavage efficiency to detect a number of mutations as compared to the use of individual digestions with a range of specific restriction endonuclease enzymes. Hum Mutat 17:432, 2001.
基于DNA化学或酶切的突变检测方法具有出色的检测效率，同时具备高通量和低单位成本的特点。我们描述了糖基化酶介导的多态性检测（GMPD）这项新技术在检测爱尔兰人群中导致苯丙酮尿症（PKU）的PAH基因两种最常见突变R408W和I65T中的应用，这两种突变的相对发生率分别为41.0%和10.4%。针对R408W和I65T开发了GMPD检测方法，可在ALFexpresstrade mark自动DNA测序仪上对切割产物进行荧光检测。通过对一组PKU患者进行筛查验证了该方法，这些患者的突变基因型此前已通过标准方法进行了鉴定。通过对GMPD产物进行共电泳，还证明了可以对这两种突变进行多重检测。GMPD是一种快速且可靠的检测R408W和I65T突变的方法，其关键优势在于与使用一系列特定限制性内切酶进行单独消化相比，它使用了一对切割效率高的酶来检测多种突变。《人类突变》17:432，2001年。
