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MeltMADGE for mutation scanning of specific genes in population studies

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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Figure 1: Microplate array diagonal gel electrophoresis.
Figure 2: Observed versus predicted Tm.
Figure 3: MeltMADGE apparatus.
Figure 4: MeltMADGE gel preparation and loading.
Figure 5: Artificial positive control in meltMADGE.
Figure 6: Images from two gels from a large-scale meltMADGE run.
Figure 7: Band patterns from a thermal ramp analysis of a SNP (using tracks in an H-PAGE gel).
Figure 8: A set of individual track images excised from a full meltMADGE gel image.
Figure 9: Phoretix software analysis of meltMADGE images.

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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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All authors have seen, commented on and contributed to the finalization of the paper, and have contributed to the development of the approach.

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Correspondence to Ian N M Day.

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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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