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 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.
O'Dell
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Human Genetics Research Division, Southampton University School of Medicine, Southampton General Hospital, Southampton, United Kingdom. S.D.O'Dell@soton.ac.uk
Chen
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Day
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D000483:Alleles; D004591:Electrophoresis, Polyacrylamide Gel / Q000191:economics / Q000379:methods*; D005805:Genes, MHC Class I / Q000235:genetics; D005838:Genotype; D006801:Humans; D007328:Insulin / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D018598:Minisatellite Repeats / Q000235:genetics*; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D013050:Spectrometry, Fluorescence
D007328:Insulin