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Heteroduplex analysis by capillary array electrophoresis for rapid mutation detection in large multiexon genes

Nature Protocols volume 2, pages 237–246 (2007)Cite this article

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Abstract

Heteroduplex analysis (HA) has proven to be a robust tool for mutation detection. HA by capillary array electrophoresis (HA-CAE) was developed to increase throughput and allow the scanning of large multiexon genes in multicapillary DNA sequencers. HA-CAE is a straightforward and high-throughput technique to detect both known and novel DNA variants with a high level of sensitivity and specificity. It consists of only three steps: multiplex-PCR using fluorescently labeled primers, heteroduplex formation and electrophoresis in a multicapillary DNA sequencer. It allows, e.g., the complete coding and flanking intronic sequences of BRCA1 and BRCA2 genes from two patients (approximately 25 kb each) to be scanned in a single run of a 16-capillary sequencer, and has enabled us to detect 150 different mutations to date (both single nucleotide substitutions, or SNSs, and small insertions/deletions). Here, we describe the protocol developed in our laboratory to scan BRCA1, BRCA2, MLH1, MSH2 and MSH6 genes using an ABI3130XL sequencer. This protocol could be adapted to other instruments or to the study of other large multiexon genes and can be completed in 7–8 h.

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Figure 1: Outline of fluorescent heteroduplex analysis by capillary array electrophoresis (HA-CAE).
Figure 2: Spatial calibrations with (a) POP-7 polymer and (b) 5.5% CAP, 5% glycerol, 1 × TBE.
Figure 3: Examples of multiplex-PCR of the BRCA1 gene.
Figure 4: GeneMapper screenshots of several heteroduplex analysis by capillary array electrophoresis (HA-CAE) electropherograms.
Figure 5: Effect of a common single nucleotide substitution (SNS) on the detection of another SNS by heteroduplex analysis by capillary array electrophoresis.

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  • 21 August 2008

    In the version of this article initially published, "Universidad de Valladolid" in the first affiliation should have read "Universidad de Valladolid-CSIC". The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work has been supported by the Junta de Castilla y León through the regional Cancer Prevention Program. We are grateful to the patients and the clinicians, especially Dr. García Girón and Dr. Lastra (Hospital General Yagüe, Burgos, Spain), who collaborated in this study. Finally, we also thank Lara Hernández Sanz for her excellent technical support.

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Authors and Affiliations

  1. Laboratorio de Genética del Cáncer, Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid-CSIC, Valladolid, Spain

    Eladio Velasco, Mar Infante, Mercedes Durán, Lucía Pérez-Cabornero, David J Sanz & Cristina Miner

  2. Departamento de Biopatología Clínica, Laboratorio de Biología Molecular, Hospital La Fé, Valencia, Spain

    Eva Esteban-Cardeñosa

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  1. Eladio Velasco
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  6. Eva Esteban-Cardeñosa
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  7. Cristina Miner
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E.V., M.I. and M.D. contributed equally to this work.

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Correspondence to Eladio Velasco.

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Velasco, E., Infante, M., Durán, M. et al. Heteroduplex analysis by capillary array electrophoresis for rapid mutation detection in large multiexon genes. Nat Protoc 2, 237–246 (2007). https://doi.org/10.1038/nprot.2006.482

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