AUTOMATIC DETECTION SYSTEM OF GENETIC POLYMORPHISMS

我们开发了两种新的点突变检测方法，TaqMan-ASA和SYBR Green-ASA。在TaqMan探针或SYBR Green染料存在的情况下，用荧光检测器（PRISM 7700序列检测系统）实时监测两组等位基因特异性引物的APCR扩增子。两种PCR反应的扩增效率差异是通过“阈值”循环来区分突变型和正常等位基因。这些方法通过直接检测阳性反应，消除了后续凝胶电泳或额外杂交步骤的要求。我们应用这些方法检测了苯丙酮尿患者中的R111X、IVS-4、Y204C、R241C、R243Q、R245V、R252W、R278W、IVS-9、Y356X和R413P突变，日本la型糖原储存病患者中普遍存在的727g>t突变，以及高加索中链酰基辅酶a脱氢酶缺乏症患者中常见的K329E突变。这些技术可以自动化，对各种单核苷酸多态性和突变的基因型分析很有用。

We have developed two novel methods for the detection of point mutations, TaqMan-ASA and SYBR Green-ASA. APCR amplicon using two sets of allele-specific primers in the presence of a TaqMan probe or SYBR Green dye was monitored in real time with a fluorescence detector (PRISM 7700 Sequence Detection System). The difference in amplification efficiency between the two PCR reactions was determined by "threshold" cycles to differentiate mutant and normal alleles. These methods eliminate the requirement for subsequent gel electrophoresis or additional hybridization steps by directly detecting positive reactions. We applied these methods to detect R111X, IVS-4, Y204C, R241C, R243Q, R245V, R252W, R278W, IVS-9, Y356X and R413P mutations in patients with phenylketonuria, a prevalent 727g>t mutation in Japanese patients with glycogen storage disease type la, and a common K329E mutation in Caucasian patients with medium-chain acyl-CoA dehydrogenase deficiency. These techniques can be automated and are useful for the genotype analysis of a variety of single nucleotide polymorphisms and mutations.