It is almost impossible, especially where human aDNA is concerned, to be 100% sure that your aDNA is authentic; however, we can build an argument for authenticity by following these criteria:
• Multiple extraction and PCR controls. Each set of extractions should include at least three extraction controls that contain no sample material but are otherwise treated identically. For each set of PCRs, multiple negative PCR controls should be added to identify and source contamination that may occur during the extraction or preparation of the PCR.
• Repeated amplifications from the same or several extracts. This allows detection of sporadic contaminants and consistent sequence changes due to damage or PCR error.
• Inverse correlation between amplification efficiency and length of amplification product. aDNA is fragmented, therefore the longer the fragment of aDNA you are trying to amplify, the more difficult it is to achieve amplification. Most aDNA sequences are between 100 and 500 nucleotides long unless preservation is unusually good.
• Bacterial cloning of PCR products and sequencing of multiple clones. This allows quantification of sequence mixture in the amplification products. Sequence mixture results from damage, PCR error, or contamination with living DNA.
• Quantitation of the number of amplifiable DNA molecules. This shows how many template molecules any given PCR begins from and gives a probability of whether consistent base changes are likely to occur or not.
• Biochemical assays of macromolecular preservation. Good biochemical preservation can support the authenticity of an ancient DNA sequence. Poor biochemical preservation indicates that a sample is unlikely to contain DNA.
• Reproduction of results by an independent laboratory. This can detect if there is contamination of chemicals or samples during handling in the laboratory. However, contaminants that are already on a sample before it arrives in the laboratory will be faithfully reproduced in a second laboratory.
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