Higher resolution microplate array diagonal gel electrophoresis: Application to a multiallelic minisatellite

The 5' polymorphic region of the insulin (INS, MIM# 176730) gene contains a variable tandem repetition of 14-15 bp (a variable number of tandem repeat s (VNTR) locus). After PCR amplification, we achieved precise sizing of cla ss I alleles (range 641 to 843 bp) on 96-well open-face polyacrylamide micr oplate array diagonal gel electrophoresis (MADGE) gels, obtaining resolutio n of the 2% mobility difference which represents one tandem repeat. PCR pro ducts were run double-stranded, but no additional bands were generated exce pt in the case of differences of three, two, and one repeat between alleles ; none compromised allele identification, and in the latter case the hetero duplex was a useful confirmation signal. No end labelling of primers was re quired, as the sensitive Vistra Green(TM) intercalating dye for double stra nds was used for visualization of bands from diluted samples. Duracryl(TM), a high mechanical-strength polyacrylamide derivative, proved to have good resolution properties for electrophoresis. A co-run ladder ensured precise binning without inter-lane variability. Simultaneous electrophoresis of gel s in a thermostatically controlled tank allowed up to 1,000 samples to be r un in 90 min. Gels were analyzed using a FluorImager(R) 595 fluorescent sca nning system, and alleles identified using a combination of Phoretix(TM) so ftware for band migration measurement and Microsoft(R) Excel(R) to compute allele sizes. Unlike other systems for minisatellite allele sizing, through put was not limited tin time or cost) by electrophoresis. Hum Mutat 15:565- 576, 2000. (C) 2000 Wiley-Liss, Inc.