Microplate Array Diagonal Gel Electrophoresis for Cohort Studies of Microsatellite Loci

Abstract

After PCR amplification, we have achieved precise sizing of trinucleotide and tetranucleotide microsatellite alleles on 96-well open-faced polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels: two tetranucleotide repeats, HUMTH01 (five alleles 248—263 bp) and DYS390 (eight alleles 200—228 bp), and DYS392, a trinucleotide repeat (eight alleles 210—231 bp). A gel matrix of DuracrylTM, a high mechanical strength polyacrylamide derivative, and appropriate ionic conditions provide the 1.3%—1.5% band resolution required. No end-labeling of primers is needed, as the sensitive Vistra GreenTM intercalating dye is used for the visualization of bands. Co-run markers bracketing the PCR fragments ensure accurate sizing without inter-lane variability. Electrophoresis of multiple gels in a thermostatically controlled tank allows up to 1000 samples to be run in 90 min. Gel images were analyzed using a FluorImager® 595 fluorescent scanning system, and alleles were identified using PhoretixTM software for band migration measurement and Microsoft® Excel® to compute fragment sizes. Estimated sizes were interpolated precisely to achieve accurate binning. Microsatellite-MADGE represents a utilitarian method for high-throughput genotyping in cohort studies, using standard laboratory equipment.