Abstract
MeltMADGE reconfigures the mutation scanning process of denaturing gradient gel electrophoresis so that the independent variable is time rather than space and the dependent (denaturing) variable is temperature rather than concentration of chemical denaturant. Use of a thermal ramp enables the use of a homogeneous gel and therefore of high-density arrays of wells such as those of microplate array diagonal gel electrophoresis (MADGE). In this configuration, electrophoresis of products on 10–12 96-well meltMADGE gels can be conducted in a 1- to 2-liter tank in a 1- to 2-h run, enabling the scanning of a target amplicon in over 1,000 subjects simultaneously. Gels are read by imaging the fluorescence of UV-excited ethidium bromide, giving a simple, economical system for identifying rarer sequence variants in target genes; it is suitable for large-scale case-control or population studies and other comparable applications. Different amplicons with similar melting characteristics can also be combined in the same run.
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Acknowledgements
Support for meltMADGE development came from the UK Medical Research Council and from the Department of Health—UK National Genetics Reference Laboratory (Wessex). T.R.G. was a British Heart Foundation Intermediate Fellow. C.R.B. is funded by a Wellcome Trust 4-year Ph.D. studentship in Molecular, Genetic and Lifecourse Epidemiology (WT083431MA).
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Alharbi, K., Aldahmesh, M., Gaunt, T. et al. MeltMADGE for mutation scanning of specific genes in population studies. Nat Protoc 5, 1800–1812 (2010). https://doi.org/10.1038/nprot.2010.136
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DOI: https://doi.org/10.1038/nprot.2010.136
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