High-throughput genotyping using horizontal polyacrylamide gels with wells arranged for microplate array diagonal gel electrophoresis (MADGE).
Genotyping (typing of genetic variation) typically involves PCR followed by an allele-specific oligonucleotide-binding assay, restriction enzyme digest or direct check of the outcome of a PCR designed to distinguish genotype. Electrophoresis can resolve "bound" from "free" oligonucleotide, as well as resolve PCR fragments and digests, but it is traditionally regarded as cumbersome and laborious in comparison with solution assays. Here we describe simple horizontal polyacrylamide gels which can receive a 96-well array of samples directly, which can be stacked in tanks and which are bound to a robust support of glass. The line of electrophoresis is on a 71.6 degree diagonal relative to the columns of the array (microplate array diagonal gel electrophoresis [MADGE]). Several thousand reactions can conveniently be analyzed in a shoebox-sized apparatus in a couple of hours. High resolution is achieved in the range of 20-1000 bp, information processing is simplified and automation is possible.
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Dept. of Medicine, University College London Medical School, Rayne Institute, UK.
Humphries
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Richards
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Norton
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Reid
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D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D004996:Ethidium; D005838:Genotype; D016133:Polymerase Chain Reaction
D004996:Ethidium