To prevent contamination, sample preparation, DNA extractions, and PCR setup were carried out in a dedicated ancient DNA facility physically isolated from other biological laboratories (including post-PCR facilities) with positive air pressure, daily exposure of surfaces to ultraviolet (UV)-irradiation, and where full body suits, face masks, and disposable gloves are worn. The PCR products were purified prior to DNA sequencing using the Invisorb® Vacuum Manifold and Invisorb PCR HTS 96 kit (both from Invitek GmbH, Berlin, Germany). Cycling was performed in an Eppendorf® Mastercycler® gradient thermal cycler (Eppendorf Nordic, Horsholm, Denmark) using denaturation at 94☌ for 2 min, followed by 45–50 cycles of 94☌ for 30 s, 50°–55☌ for 30 s, and 68°–72☌ for 30 s, and a final cycle for 7–10 min at 68°–72☌. PCR amplification was performed using conventional primers (see Supplementary Table S2 for primer details) in 25-µL reactions containing 1× PCR High Fidelity PCR buffer, 2.5 mM magnesium sulfate solution (Invitrogen, Carlsbad, CA, USA), 0.4 mM dNTP mix, 1 U Platinum® Taq DNA Polymerase High Fidelity (Invitrogen), 1 µM each primer, and 1–5 µL DNA extract. To address this problem, we introduce a simple technique that in most cases halves the number of sequencing errors in recovered sequences from short PCR products using conventional capillary electrophoresis sequencing equipment through the addition of a nonspecific nucleotide tail to the 5′ end of the sequencing primers.ĭNA was extracted from two bone specimens of the extinct Pleistocene woolly rhinoceros ( Coelodonta antiqutatis) and from three bone specimens of Pleistocene musk oxen ( Ovibos sp.) (sample details in Supplementary Table S1) using conventional silica-based methods ( 7, 8). However, the use of such methods requires investment in expensive new equipment, which is not always a practical solution for research groups that already have access to conventional gel and capillary sequencing equipment. An alternative approach is the application of new sequencing technologies, such as pyrosequencing, that are tailored for use on small DNA fragments ( 5, 6). Two methods have commonly been used to overcome this problem: ( i) molecular cloning of the PCR products prior to sequencing, enabling sequencing primers located in the flanking vector DNA to increase the length and quality of the sequenced fragment and ( ii) sequencing both strands in opposite directions, which produces two complementary sequences with good quality dual coverage in the middle and single coverage at the extremes. Specifically, these methods produce poor quality electropherograms at the 5′ end of the sequences, probably due to the irregular behavior and poor separation of the shortest DNA fragments during subsequent electrophoresis.
Unfortunately, conventional dye-labeled Sanger sequencing platforms perform poorly on short DNA fragments such as these when standard sequencing methodologies are applied. The degraded nature of DNA in many subfossil, archival, and forensic specimens often prevents the PCR amplification of fragments that are >100–200 bp in length ( 1–4).
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