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Multiplex amplification of ancient DNA

Abstract

This method is designed to assemble long, continuous DNA sequences using minimal amounts of fragmented ancient DNA as template. This is achieved by a two-step approach. In the first step, multiple fragments are simultaneously amplified in a single multiplex reaction. Subsequently, each of the generated fragments is amplified individually using a single primer pair, in a standard simplex (monoplex) PCR. The ability to amplify multiple fragments simultaneously in the first step allows the generation of large amounts of sequence from rare template DNA, whereas the second nested step increases specificity and decreases amplification of contaminating DNA. In contrast to current protocols using many template-consuming simplex PCRs, the method described allows amplification of several kilobases of sequence in just one reaction. It thus combines optimal template usage with a high specificity and can be performed within a day.

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Figure 1: Primer design strategy for multiplex amplification of ancient DNA.
Figure 2
Figure 3: Anticipated results.

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Acknowledgements

We thank our lab members, especially C. Stäubert and I. Böselt, for comments that improved the manuscript. This work was funded by the Max Planck Society, the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung und Forschung.

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Correspondence to Michael Hofreiter.

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The authors declare no competing financial interests.

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Römpler, H., Dear, P., Krause, J. et al. Multiplex amplification of ancient DNA. Nat Protoc 1, 720–728 (2006). https://doi.org/10.1038/nprot.2006.84

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