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Single-strand conformation polymorphism (SSCP) for the analysis of genetic variation

Nature Protocols volume 1, pages 3121–3128 (2006)Cite this article

Abstract

The accurate analysis of genetic variation has major implications in many areas of biomedical research, including the identification of infectious agents (such as parasites), the diagnosis of infections, and the detection of unknown or known disease-causing mutations. Mutation scanning methods, including PCR-coupled single-strand conformation polymorphism (SSCP), have significant advantages over many other nucleic acid techniques for the accurate analysis of allelic and mutational sequence variation. The present protocol describes the SSCP method of analysis, including all steps from the small-scale isolation of genomic DNA and PCR amplification of target sequences, through to the gel-based separation of amplicons and scanning for mutations by SSCP (either by the analysis of radiolabeled amplicons in mutation detection enhancement (MDE) gels or by non-isotopic SSCP using precast GMA gels). The subsequent sequence analysis of polymorphic bands isolated from gels is also detailed. The SSCP protocol can readily detect point mutations for amplicon sizes of up to 450–500 bp, and usually takes 1–2 days to carry out. This user-friendly, low-cost, potentially high-throughput platform has demonstrated the utility to study a wide range of pathogens and diseases, and has the potential to be applied to any gene of any organism.

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Figure 1: The principle of PCR-based SSCP analysis.
Figure 2: Selected examples of isotopic and non-isotopic SSCP analyses of amplicons (∼230–450 bp) derived from nuclear ribosomal DNA regions from parasites or yeast.

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Acknowledgements

We would like to thank colleagues who have contributed to research in our laboratory. Our research has been supported largely through grants from the Australian Research Council, Genetic Technologies Limited, the Australian Academy of Science, Meat and Livestock Australia and, more recently, Elchrom Scientific AG. Approval has been granted by Wiley Press to reproduce partial images (presented in Fig. 2) from previous publications23,30.

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Authors and Affiliations

  1. Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, 3030, Victoria, Australia

    Robin B Gasser, Min Hu, Bronwyn E Campbell, Aaron J Jex, Ian Beveridge & Xingquan Zhu

  2. Department of Biology, University of Saskatchewan, Saskatchewan, S7N 5E2, Canada

    Neil B Chilton

  3. Dipartimento di Sanità e Benessere Animale, Facoltà di Medicina Veterinaria, Valenzano, 70010, Bari, Italy

    Domenico Otranto & Claudia Cafarchia

  4. Laboratory of Parasitology, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, Guangdong Province, The People's Republic of China

    Xingquan Zhu

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  1. Robin B Gasser
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Correspondence to Robin B Gasser.

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Gasser, R., Hu, M., Chilton, N. et al. Single-strand conformation polymorphism (SSCP) for the analysis of genetic variation. Nat Protoc 1, 3121–3128 (2006). https://doi.org/10.1038/nprot.2006.485

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