Remoção de citosinas desaminadas e detecção de metilação in vivo em DNA antigo

Nucleic Acids Research Advance Access published online on December 22, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp1163

Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA

Adrian W. Briggs*, Udo Stenzel, Matthias Meyer, Johannes Krause, Martin Kircher and Svante Pääbo

Max-Planck-Institute for Evolutionary Anthropology, D-04103 Leipzig, Germany

*To whom correspondence should be addressed. Tel: +49 (0) 341 3550 539; Fax: 49 (0) 341 3550 550; Email: briggs@eva.mpg.de

Received September 28, 2009. Revised October 30, 2009. Accepted November 24, 2009.

DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil–DNA–glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms.

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