EVALUATION OF THE AUTOMATED FLUORESCENT ANALYSIS OF THE H-RAS MINISATELLITE AFTER OPTIMIZED PCR AMPLIFICATION IN COMPARISON WITH A STANDARDIZED SOUTHERN BLOT TECHNIQUE

Determination of allele sizes? loss of heterozygosity or genetic insta bility at minisatellite VNTR loci, are routinely performed by the conv entional Southern technique. We have investigated the potential use of automated DNA sequencer for the analysis of the H-ras minisatellite. We report the modifications of amplification parameters and electropho resis conditions on the sequencer. Seventy-one colorectal carcinomas a nd the corresponding normal tissues were amplified with fluorescent-la beled primers, analyzed on sequencer, and concurrently controlled by S outhern blotting. The results on sequencer showed that a Hydrolink mat rix used in non-denaturing conditions and a specific analysis software facilitate a more accurate fragment size calculation.