Free Ancient DNA: Methods and Protocols by Beth Shapiro

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Chapter 5
Extraction of DNA from Paleofeces
    Melanie Kuch and Hendrik Poinar
    Abstract
    Paleofeces are the nonmineralized remains of dung from extant and extinct fauna. They represent a surprisingly large proportion of fossil remains recovered from cave sites across the world. Paleofeces contain the DNA of the defecator as well as the DNA of ingested plant and animal remains. To successfully extract DNA from paleofeces, a balance must be achieved between the minimization of DNA loss during extraction and the removal of coeluates that would otherwise inhibit the Taq DNA polymerase during downstream applications. Here we present a simplifi ed version of a protocol to extract DNA from paleofecal remains.
    Key words: DNA extraction , Feces , Ancient DNA , Silica DNA-purifi cation 1. Introduction
     
    Paleofeces, or as they are sometimes labeled, coprolites, are the nonmineralized remains of feces. As paleofeces are nonhardened fossils, they are typically assumed to be rare in the fossil record.
    However, deposits of megafaunal dung, namely that of the extinct ground sloth
    Nothrotheriops shastensis found in caves of the
    American southwest, rival in extent the vast deposits of mammoth bone and teeth in the permafrost ( 1 ) .
    Paleofeces are most often found within caves and rock shelters ( 2 ) , although some have been found at open-air sites. Paleofeces likely make up a large percentage of the sediment found within cave fl oors, and to some degree, that found within permafrost soils as well. This may explain the success in retrieving the DNA of past inhabitants of caves ( 3 ) and megafauna of the high arctic ( 4 ) .
    The fi rst successful extraction of DNA from paleofeces involved the use of PTB, a thiazolium salt shown to be successful in the reversal of Maillard-induced crosslinks from r educing sugars
    ( 5– 8 ) .
    While it is unclear to what degree the PTB unlinks these nucleic Beth Shapiro and Michael Hofreiter (eds.), Ancient DNA: Methods and Protocols , Methods in Molecular Biology, vol. 840, DOI 10.1007/978-1-61779-516-9_5, © Springer Science+Business Media, LLC 2012
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    M. Kuch and H. Poinar
    acid/protein complexes, we have noted distinct improvements in total DNA yields when including PTB in ancient DNA extractions.
    Since the fi rst publication reporting the successful extraction of DNA from paleofeces, subsequent reports have used these techniques to isolate not only the DNA of the defecator, but also DNA from the ingested contents ( 9, 10 ) ; please see r ef. ( 11 ) for a more comprehensive review.
    Below, we present the most up-to-date extraction protocol for the recovery of DNA from paleofecal remains. Since the original publication ( 12 ) , we have assessed the relative success of various modifi cations using quantitative PCR assays and by measuring inhibition as gauged by the delaying of quantifi cation cycles ( 13 ) .
    The protocol can be optimized for the content of the dung (plant material versus a more meat-based diet). As with many fossil and subfossil samples, feces contain potent inhibitors with similar molecular