Electrophoresis for genotyping: microtiter array diagonal gel electrophoresis on horizontal polyacrylamide gels, hydrolink, or agarose.
Electrophoresis of DNA has been performed traditionally in either an agarose or acrylamide gel matrix. Considerable effort has been directed to improved quality agaroses capable of high resolution, but for small fragments, such as those from polymerase chain reaction (PCR) and post-PCR digests, acrylamide still offers the highest resolution. Although agarose gels can easily be prepared in an open-faced format to gain the conveniences of horizontal electrophoresis, acrylamide does not polymerize in the presence of air and the usual configurations for gel preparation lead to electrophoresis in the vertical dimension. We describe here a very simple device and method to prepare and manipulate horizontal polyacrylamide gels (H-PAGE). In addition, the open-faced horizontal arrangement enables loading of arrays of wells. Since many procedures are undertaken in standard 96-well microtiter plates, we have also designed a device which preserves the exact configuration of the 8 x 12 array and enables electrophoresis in tracks following a 71.6 degrees diagonal between wells (MADGE, microtiter array diagonal gel electrophoresis), using either acrylamide or agarose. This eliminates almost all of the staff time taken in setup, loading, and recordkeeping and offers high resolution for genotyping pattern recognition. The nature and size of the gels allow direct stacking of gels in one tank, so that a tank used typically to analyze 30-60 samples can readily be used to analyze 1000-2000 samples. The gels would also enable robotic loading. Electrophoresis allows analysis of size and charge, parameters inaccessible to liquid-phase methods: thus, genotyping size patterns, variable length repeats, and haplotypes is possible, as well as adaptability to typing of point variations using protocols which create a difference detectable by electrophoresis.
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Department of Medicine, University College London Medical School, Rayne Institute, United Kingdom.
Humphries
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D004247:DNA / Q000032:analysis*; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation*; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation*; D005782:Gels; D005838:Genotype; D008832:Microchemistry; D016133:Polymerase Chain Reaction; D011108:Polymers; D017186:Titrimetry
D005782:Gels; D011108:Polymers; C061589:HydroLink; D004247:DNA