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Higher resolution microplate array diagonal gel electrophoresis: Application to a multiallelic minisatellite
Author(s) -
O'Dell Sandra D.,
Chen Xiaohe,
Day Ian N.M.
Publication year - 2000
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/1098-1004(200006)15:6<565::aid-humu8>3.0.co;2-7
Subject(s) - minisatellite , biology , diagonal , genetics , resolution (logic) , gel electrophoresis , electrophoresis , microbiology and biotechnology , computational biology , microsatellite , artificial intelligence , gene , computer science , allele , geometry , mathematics
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. © 2000 Wiley‐Liss, Inc.