Higher resolution microplate array diagonal gel electrophoresis: Application to a multiallelic minisatellite
- 31 May 2000
- journal article
- research article
- Published by Hindawi Limited in Human Mutation
- Vol. 15 (6) , 565-576
- https://doi.org/10.1002/1098-1004(200006)15:6<565::aid-humu8>3.0.co;2-7
Abstract
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 repeats (VNTR) locus). After PCR amplification, we achieved precise sizing of class I alleles (range 641 to 843 bp) on 96-well open-face polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels, obtaining resolution of the 2% mobility difference which represents one tandem repeat. PCR products were run double-stranded, but no additional bands were generated except in the case of differences of three, two, and one repeat between alleles; none compromised allele identification, and in the latter case the heteroduplex was a useful confirmation signal. No end labelling of primers was required, as the sensitive Vistra Green™ intercalating dye for double strands was used for visualization of bands from diluted samples. Duracryl™, 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 gels in a thermostatically controlled tank allowed up to 1,000 samples to be run in 90 min. Gels were analyzed using a FluorImager® 595 fluorescent scanning system, and alleles identified using a combination of Phoretix™ software for band migration measurement and Microsoft® Excel® to compute allele sizes. Unlike other systems for minisatellite allele sizing, throughput was not limited (in time or cost) by electrophoresis. Hum Mutat 15:565–576, 2000.Keywords
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