Kneppers A L, Deutz-Terlouw P P, den Dunnen J T, van Ommen G J, Bakker E
MGC Department of Human Genetics, Leiden University, University Hospital Leiden, The Netherlands.
Hum Mutat. 1995;5(3):235-42. doi: 10.1002/humu.1380050308.
We have developed a rapid and nonradioactive method to screen for point mutations using the Pharmacia PhastSystem. In an SSCP analysis, we applied the two multiplex exon PCR kits, commonly used for the detection of deletions in Duchenne and Becker muscular dystrophy patients. The different exon bands in the multiplex SSCP pattern could be identified by running well-characterised deletion patients in this system. Two common polymorphisms were easily identifiable and are helpful in the haplotype analysis in families. Screening of 70 patients in which no gross rearrangement was detectable with the multiplex PCR and Southern blot, resulted in the identification of 6 patients with a band shift after SSCP analysis. Of these 6 band shifts, 5 were the result of a frame shift or termination mutation. The other band shift was found to be a rare polymorphism unlikely to be the cause of the patient's phenotype. Application of this technique enabled us to improve diagnosis in the families involved and will allow us to extend the search for point mutations in the remaining exons of the dystrophin gene.
我们开发了一种快速且非放射性的方法，利用Pharmacia PhastSystem筛选点突变。在单链构象多态性（SSCP）分析中，我们应用了两种常用于检测杜兴氏和贝克氏肌营养不良患者缺失情况的多重外显子PCR试剂盒。通过在该系统中检测特征明确的缺失患者，可以识别多重SSCP模式中的不同外显子条带。两种常见的多态性易于识别，有助于家族中的单倍型分析。对70例通过多重PCR和Southern印迹未检测到明显重排的患者进行筛查，结果在SSCP分析后发现6例患者出现条带移位。在这6例条带移位中，5例是移码或终止突变的结果。另一个条带移位被发现是一种罕见的多态性，不太可能是患者表型的原因。应用这项技术使我们能够改善相关家族的诊断，并将有助于我们在肌营养不良蛋白基因的其余外显子中进一步寻找点突变。
