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Long PCR amplification of the entire mitochondrial genome from individual helminths for direct sequencing

Abstract

Exploring mitochondrial (mt) genomes has significant implications for various fundamental research areas, including mt biochemistry and physiology, and, importantly, such genomes provide a rich source of markers for population genetics and systematic studies. Although some progress has been made, there is a paucity of information on mt genomes for many metazoan organisms, particularly invertebrates such as parasitic helminths, which relates mainly to the technical limitations associated with sequencing from tiny amounts of material. In this article, we describe a practical long PCR approach for the amplification and subsequent sequencing of the entire mt genome from individual helminths, which overcomes these limitations. The protocol includes the isolation of genomic DNA, long PCR amplification, electrophoresis and sequencing, and takes 1–3 weeks to carry out. The present user-friendly, cost-effective approach has demonstrated utility to the study of a range of parasites, and has the potential to be applied to a wide range of organisms.

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Figure 1: The long PCR to amplify the entire mitochondrial genome sequence from small amounts of total genomic DNA (gDNA) isolated from individual nematodes for subsequent direct sequencing.
Figure 2: Primer design and long PCR amplification of mt genomes from selected secernentean nematodes for subsequent sequencing.

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Acknowledgements

Thanks to colleagues who have contributed to research in our laboratory. Our research has been supported largely through grants from the Australian Research Council (including LPX0775848).

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Correspondence to Robin B Gasser.

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Hu, M., Jex, A., Campbell, B. et al. Long PCR amplification of the entire mitochondrial genome from individual helminths for direct sequencing. Nat Protoc 2, 2339–2344 (2007). https://doi.org/10.1038/nprot.2007.358

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