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Extraction of Ancient DNA 57
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Extraction of Ancient DNA
Wera M. Schmerer
1. Introduction
The DNA extraction process represents one of the critical stages in the analysis
of degraded or ancient DNA. If polymerase chain reaction (PCR) amplification starts
from a poor extract containing low template quantities, stochastic variation in the
amplification of individual alleles may lead to allelic dropout (1), resulting in a high
risk of false-homozygous typing of a heterozygous sample (2).
In analyzing repetitive sequences, such as STR loci, besides quantity, the quality
of the extracted DNA used as template is of particular importance. PCR amplification
of STR loci is generally accompanied by the generation of so-called shadow bands,
byproducts that are shortened in length by one repeat unit compared with the allelic
product (3). Because the accumulation of this artifact is increased with degradation of
the DNA template (4) when amplifying ancient DNA, the intensity of a shadow band
can exceed the intensity of the allelic product. As “artifact alleles” (2,5), these products
may be mistaken for a true allele of the sample, complicating the determination of a
genotype or even resulting in false genotyping. Because a PCR may not be affected
each time, independent amplifications of the same sample may result in differing
genotyping results (e.g., see ref. 6) for the same sample (2,7,8).
The degree at which these two artifacts occur is related to quantity and quality
of extracted DNA amplified within a PCR (1,4,8,9). Therefore, an optimized DNA
extraction, to get the highest possible amount of target DNA with the best possible
quality (which means with the highest possible reduction of additional degradation
during the extraction procedure), is an essential precondition for optimal reproducibility
of STR genotyping in the case of samples containing ancient DNA.
The protocols presented here are the results of a study with the aim to optimize the
extraction of ancient DNA from historical skeletal material described by Schmerer et al.
(10,11). (For further detailed information on this study, also refer to ref. 12.)
The protocol described in detail represents a consensus protocol developed for the
extraction of DNA with a wide range in degree of degradation (cf. ref. 10) and may
be used regardless of the state of DNA preservation present in the skeletal material
intended for analysis. Additional information on special protocols designed for three
different degrees of DNA degradation is given in the table (see Note 1) and can be used
to adapt the protocol in case of known DNA preservation.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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