Ancient DNA Methods and Protocols
Research into ancient DNA began more than 25 years ago with the publication of short mitochondrial DNA sequence fragments from the quagga, an extinct relative of the zebra. Ancient DNA research really gained momentum following the invention of PCR, which
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1. Introduction The invention of the polymerase chain reaction (PCR) (1) revolutionized the field 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 modifications 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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possible that long fragments of undamaged, modern DNA may be preferentially amplified. 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 amplifications 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 amplified fragments. It is also routine to clone at least some of the amplification products of aDNA experiments, as this can identify potential contaminants or PCR artifacts and allow evaluation of the extent of post-mortem 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 efficiency in synthesizing aDNA (3) and in the particular way they interact with damaged bases (4). Even with high-fidelity polymerases, it is important to consider the possibility of strand jumping which can produce chimeric products. An additional benefit of both Platinum Taq and AmpliTaq Gold is that they are hot-start polymerases, a desirable attribute as PCR amplification from ancient extracts is generally set up in a facility that is spatially distant from the thermocycler. PCR inhibitors are often co-extracted with aDNA, as samples have often been exposed to environmental contaminants for tens of thousands of years. To minimize inhibition, serum albumin, and commonly bovine serum albumin (BSA), can be included in aDNA PCR. BSA binds PCR-inhibiting co-extracts and prevents target DNA from adhering to the tube rather than being amplified. Including BSA can dramatically improve PCR success (3) and is useful as a troubleshooting measure when PCRs are unsuccessful. DNA damage is also common in aDNA extracts. Several measures have been recommended to
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