| Abstract: |
We designed a simple and efficient protocol for the isolation of ancient DNA directly from crude tissue lysates, based on the streptavidin-coated magnetic beads capture of biotin-labelled hybrids. A key feature of our method, named `target-hooking' (T-H), is the use of the same pair of biotinylated oligonucleotides as recovery probes for the indirect hybrid capture of the target sequence, as well as PCR primers in standard amplification assays. It allows: (1) high quality templates (free from PCR-inhibitors and with an enhanced target/non-target ratio) to be rapidly isolated
(2) successful specific amplifications to occur at higher rates
(3) overall costs to be considerably reduced. The T-H protocol was optimised on crude digestions from ancient (13 000 2600BP) and aged (approximately 10 BP) cattle skeletal remains, using 5[sup ']-biotinylated oligonucleotides targeting a 173bp segment of the bovine mitochondrial DNA control region. The results from independent extractions and amplifications were compared with those obtained from standard DNA extraction procedures, showing a higher recovery efficiency and a lower background. The sensitivity, tested on a dilution series of target PCR products, was approximately a single molecule.
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