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Mutation screening using fluorescence multiplex denaturing gradient gel electrophoresis (FMD): detecting mutations in the BRCA1 gene

Nature Protocols volume 1, pages 3101–3110 (2006)Cite this article

Abstract

Fluorescent multiplex denaturing gradient gel electrophoresis (FMD) is a mutation screening technique designed to detect unknown as well as previously identified mutations. FMD constitutes a recent modification of the standard denaturing gradient gel electrophoresis (DGGE) technique, which combines multiplex PCR amplification of target DNA using fluorescently labeled primers with DGGE separation of the amplicon mixture, allowing immediate identification of sequence variants by wet gel scanning. FMD permits the simultaneous detection of small insertions, deletions and single nucleotide substitutions among multiple DNA fragments (up to 480 fragments) from 96 samples in parallel for each run. It increases output and reduces cost dramatically compared with classical DGGE, without sacrificing sensitivity and accuracy in detecting mutations. This protocol details an accurate, fast, nonradioactive and cost-effective way to screen the BRCA1 gene for mutations with high sensitivity, providing easily interpreted results. It may also be adapted to screen other target genes and/or used in large-scale epidemiological studies.

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Figure 1
Figure 2
Figure 3: The schematic illustration of the setup of the gradient maker and gel casting apparatus.
Figure 4: Image of FMD detection for BRCA1 nested multiplex short PCR number 6 to amplify target exons 10, 13, 14 and 23 (see Table 4) of the BRCA1 gene.

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Acknowledgements

The information for the primer sequences was kindly provided by Dr. Jan Vijg.

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  1. The Centre for Research in Women's Health, University of Toronto, 790 Bay Street, Toronto, M5G IN8, Ontario, Canada

    Shiyu Zhang, Graciela Kuperstein & Steven A Narod

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  1. Shiyu Zhang
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Correspondence to Steven A Narod.

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Zhang, S., Kuperstein, G. & Narod, S. Mutation screening using fluorescence multiplex denaturing gradient gel electrophoresis (FMD): detecting mutations in the BRCA1 gene. Nat Protoc 1, 3101–3110 (2006). https://doi.org/10.1038/nprot.2006.445

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