Ancient DNA: Methods and Protocols (21 page)

14 Case Study: Using a Nondestructive DNA Extraction Method…

107

Fig. 1. Geographical distribution of chimpanzee subspecies.

extractions and amplifi cations as well as obtaining multiple clonal sequences are an absolute requirement in such studies.

Chimpanzee subspecies are divided into two geographically

and genetically defi ned groups: a central/eastern African group (
P. t. schweinfurthii
and
P. t. troglodytes
) and a western African group (
P. t. verus
and
P. t. vellerosus
) with a signifi cant phylogeographic break at the Sanaga River in central Camer
oon (Fig. 1 ).

A temporal network

( 18
) reconstructed from our historical sequences and modern chimpanzee sequences obtained from

GenBank shows that all historical haplotypes are closely related to moder
n ones (Fig. 2
), although some of them have not (yet) been found in the extant gene pool.

With 51%, the DNA extraction success rate in this study is lower than in previous studies reporting the method
( 3, 19,
20 )
, but still suffi ciently high to obtain DNA from about half of the investigated specimens. Similarly, the length of the obtained PCR

products is large enough to obtain, by using several overlapping fragments, DNA sequences suffi ciently long for phylogeographic and phylogenetic analyses. However, the high incidence of contaminating sequences found also indicates that a substantial failure rate has to be taken into account when planning a study, although there seem to be large differences among collections and species, probably depending on storage and handling.

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et al.

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Fig. 2. Temporal statistical parsimony network of modern and ancient chimp sequences. The upper layer comprises modern-day sequences obtained from GenBank, whereas the lower layer consists of ancient DNA samples generated in this study.

Haplotypes sampled in a given time layer are represented as
gray ellipses
. Those present in the overall network, but not in the individual time layer are shown as
small white ellipses
. Haplotypes shared between the two layers are connected by vertical lines . Haplotypes present in a time-horizon are connected by
solid lines
, whereas lines connecting at least one unsampled haplotype for this time-horizon are
dotted
. Those separated by more than one mutation are indicated by one small black circle for each additional mutation. Please note that for graphical reasons, not all modern sequences available were used in the network. Therefore, a larger proportion of museum sequences than shown in this fi gure are actually still present in the modern population.

Both success rate and total length of the DNA sequences that can be obtained should increase considerably when using DNA hybridization capture methods
( 22– 25
) rather than PCR for targeting specifi c DNA regions. These methods have recently been used successfully for targeting both mitochondrial (up to complete mtDNA genomes
( 25, 26
) ) and nuclear DNA
( 27
)
.
Due their ability to target very short DNA fragments, they are ideally suited for the analysis of fragmented DNA such as that recovered from museum specimens. It needs to be noted, though, that measures used to distinguish endogenous ancient DNA obtained from

Pleistocene specimens from contaminating modern DNA such as fragmentation or nucleotide substitution patterns may not be applicable to museum specimen DNA for several reasons. First, due to their younger age, museum specimens may not have accumulated DNA damage to the extent that fossil DNA dating to the Pleistocene has. Second, and perhaps even more importantly, the sequences contaminating museum specimens probably originate 14 Case Study: Using a Nondestructive DNA Extraction Method…

109

quite frequently from cross-contamination with DNA from other museum specimens, which is likely to display highly similar damage patterns. However, as our results show, this problem can be addressed at least partially by performing two consecutive extractions and by preferential use of the second extract.

Acknowledgments

We thank the lab members of the Research Group Molecular

Ecology at the Max Planck Institute for Evolutionary Anthropology, and especially Tim Heupink, for their assistance in laboratory work, museum curators for providing us with chimpanzee samples, and the Max Planck Society for fi nancial support.

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PCR Amplifi cation, Cloning, and Sequencing of Ancient DNA Tara L. Fulton and Mathias Stiller Abstract

PCR amplifi cation of DNA is routine in modern molecular biology. However, the application of PCR to ancient DNA (aDNA) experiments often requires signifi cant modifi cation to standard protocols. The degraded nature of most aDNA fragments requires targeting shorter fragments, performing replicate amplifi cations, incorporating multiple negative controls, combating PCR inhibition, using specifi c DNA polymerases to deal with damaged bases, working in a separate aDNA facility, and modifying the PCR

recipe to deal with damaged and low copy-number target DNA. In this chapter, we describe how and why these procedures are implemented, discuss aDNA-specifi c troubleshooting methodology, and suggest modifi cations to commercial cloning and sequencing procedures to reduce the expense of PCR

product cloning.

Key words:
Polymerase chain reaction , PCR optimization , BSA, inhibition , Ancient DNA , DNA polymerase

1. Introduction

The invention of the polymerase chain reaction (PCR)
( 1
) revolutionized the fi eld of ancient DNA (aDNA) research. In theory, only a single copy of the targeted DNA region is required for PCR, making it a powerful tool for amplifying aDNA from samples where only a handful of intact copies of the target region may remain.

PCR is not, by any means, a technique exclusive to aDNA research.

However, its use with aDNA requires modifi cations to the experimental design, the experiment itself, and post-experimental troubleshooting.

Ancient DNA is often highly degraded, and even exceptionally preserved permafrost specimens may contain only 5% of surviving DNA fragments longer than 300 base pairs (bp)
( 2
) . Thus, when fragments longer than 100–300 bp are targeted using PCR, it is 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_15, © Springer Science+Business Media, LLC 2012

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T.L. Fulton and M. Stiller

possible that long fragments of undamaged, modern DNA may be preferentially amplifi ed. To overcome this, a series of overlapping primer sets can be used to obtain a long stretch of continuous DNA sequence in small, stepwise fragments. This has the added advantage of identifying any nontarget amplifi cations such as numts (nuclear insertions of the mitochondrial DNA), other pseudogenes, or nonhomologous copies of the target gene, if mismatches are observed between overlapping regions of the amplifi ed fragments. It is also routine to clone at least some of the amplifi cation products of aDNA experiments, as this can identify potential contaminants or PCR artifacts and allow evaluation of the extent of postmortem damage.

The high-performing Platinum Taq High Fidelity and

AmpliTaq Gold (both from Life Technologies) are among the

most common polymerases used in aDNA experiments. The choice of polymerase is important as commercial polymerases vary widely in their effi ciency in synthesizing aDNA
( 3 )
and in the particular way they interact with damaged bases
( 4 )
. Even with high-fi delity polymerases, it is important to consider the possibility of strand jumping which can produce chimeric products. An additional benefi t of both Platinum Taq and AmpliTaq Gold is that they are hot-start polymerases, a desirable attribute as PCR amplifi cation from ancient extracts is generally set up in a facility that is spatially distant from the thermocycler.