The rapid detection of PCR-SSCP analysis using fluorescently labeled primers.

Abstract

A non-radioisotopic PCR-SSCP method using fluorescently labeled primers was developed for use in a clinical setting. In order to establish PCR-SSCP method for the clinical use, we developed a non-radioisotopic PCR-SSCP method using fluorescence-labeled primers. Electrophoresis in buffer containing with MgCl2 provided good separation of the bands and use of an image analyser gave more sensitive detection than conventional method. The mutations of K-ras gene and androgen receptor gene were confirmed by DNA sequencing analysis of the known mutations of K-ras gene and the unknown mutations of androgen receptor gene. The fluorescence-mediated PCR-SSCP system made it possible to detect mutations more rapidly and with more sensitivity than conventional PCR. Thus, the improved PCR-SSCP method will be useful for the clinical screening of unknown mutatios.