Tawata M, Iwase E, Aida K, Onaya T
Third Department of Internal Medicine, University of Yamanashi Medical School, Japan.
Genet Anal. 1996 Jan;12(3-4):125-7. doi: 10.1016/1050-3862(95)00120-4.
We have developed a new genome screening method and named it as polymerase chain reaction-restriction fragment-single strand conformation polymorphism (PCR-RF-SSCP) analysis. This method consists of three steps: (1) amplification of long DNA by PCR; (2) digestion of the amplified PCR products by restriction enzyme(s) and labelling the restriction sites; (3) polyacrylamide gel electrophoresis under non-denaturating conditions (SSCP analysis) and under denaturating conditions containing 8M urea. Theoretically, this method enables us to detect even a base substitution, deletion or insertion in up to 22,000 base pairs amplified from genomic DNA by PCR in one analysis. The procedures are very simple, reproducible and require no special apparatus. This method is applicable to any genes already known and we believe that this method is very useful for mass screening of the genome.
我们开发了一种新的基因组筛选方法，并将其命名为聚合酶链反应-限制性片段-单链构象多态性（PCR-RF-SSCP）分析。该方法包括三个步骤：（1）通过PCR扩增长DNA；（2）用限制性内切酶消化扩增的PCR产物并标记限制性位点；（3）在非变性条件下（SSCP分析）和含有8M尿素的变性条件下进行聚丙烯酰胺凝胶电泳。理论上，该方法能够在一次分析中检测出通过PCR从基因组DNA扩增的长达22,000个碱基对中的碱基置换、缺失或插入。该程序非常简单、可重复，且不需要特殊设备。该方法适用于任何已知基因，我们认为该方法对于基因组的大规模筛选非常有用。
