Multiplex PCR amplification of ancient DNA.

Multiplex PCR allows the simultaneous amplification of up to dozens of target fragments in a single PCR. It is therefore a powerful tool to obtain many kilobases of continuous sequence from minute amounts of ancient DNA (aDNA), which usually must be amplified in multiple short and overlapping fragments. Because significantly less template is required compared to amplifying each fragment separately, multiplex PCR is particularly beneficial when the fossil material itself, or access to the fossil material, is limited. The recently refined two-step multiplex PCR protocol consists of a first-step reaction (the actual multiplex PCR) that then acts as the template for the second-step PCR. During the second step, nested primers are used in individual amplification reactions. Although the same set of primers can be used in both steps, using a nested set in the second step adds an additional level of selectivity and specificity, minimizing PCR artifacts. This is particularly important when complex mixtures of template DNA, such as aDNA extracts, are amplified.