Title: Mycobacterium tuberculosis DNA extraction from mummified and skeletal human remains using Linear Polyacrylamide (LPA)

Authors: Frank Maixner¹, Heidi Y. Jäger¹, Mohamed S. Sarhan¹, Guido Valverde¹, Albert Zink¹

Affiliation: ¹Institute for Mummy Studies, Eurac Research, Viale Druso 1, 39100 Bolzano, Italy

Abstract: Nucleic acid extraction from ancient tissue material is prone to co-extract inhibitory substances that make further molecular analysis difficult or even impossible. In addition, ancient endogenous DNA of pathogens (e.g., Mycobacterium tuberculosis) and of the human host are in most cases only present in minute amounts.

In this study we introduce an innovative new method using linear polyacrylamide (LPA) to efficiently precipitate and purify nucleic acids extracted from ancient tissue samples in one working step. The LPA method replaces the precipitation step in classic liquid-phase/organic extraction protocols or can be easily applied as an additional post-extraction step on impure DNA extracts. As a proof of concept, we experimented this method on different ancient human mummy samples (bones, soft tissues, and gut contents) from different time periods (5000 BC - 1900 AD) known to contain inhibitory compounds.

We could demonstrate that LPA precipitates nucleic acids even in aqueous ethanol solution without the addition of chaotropic salts, resulting in the recovery of highly pure DNA from all tested samples that displayed inhibition with previously published extraction protocols. Compared to the currently most widely used silica-based extraction method for ancient DNA, our LPA method resulted in comparable DNA qualities and overall DNA compositions (human endogenous content, microbial diversity). First results indicate that the M. tuberculosis DNA seem to aggregate in the interphase during organic DNA extraction possibly allowing a chemical enrichment of pathogen DNA from ancient tissue material.