Multiple fluorescence-based PCR-SSCP analysis.

Abstract

Multiple fluorescence-based polymerase chain reaction single-strand conformation polymorphism (MF-PCR-SSCP) was developed. The target sequence was amplified by PCR using forward and reverse primers labeled with two different fluorescent dyes at their 5' ends. The amplified products were then heat-denatured, mixed with internal standard DNA markers labeled with a third fluorescent dye and applied to a temperature-controlled gel in an automated DNA sequencer, with a gel-temperature-controlling system. Mutations were detected as positional shifts of two-colored peaks in the electrophoretogram. The image data were analyzed by the computer program GENESCAN 672. The peak positions were standardized to internal DNA size markers. MF-PCR-SSCP analysis of 7 human tumor cell lines with 7 different single base mutations of the human K-ras oncogene detected all mutations even under the same electrophoresis conditions. Complete loss of heterozygosity was detected in two cell lines simultaneously. A gel temperature at 20 degrees C and polyacrylamide concentration of 10% gave the best separation. MF-PCR-SSCP is superior to the current PCR-SSCP in several ways: it does not involve radioactivity, migration patterns are standardized to internal standard DNA markers, there is a strict temperature-controlling system and the higher percentage of the gel enables better separation with resultant 100% detection of mutations most likely under one set of electrophoresis conditions.