Ancient DNA: Methods and Protocols by Beth Shapiro Read Free Book Online Page A

microbial or ganisms ( 2 ) . Third, regardless of the environmental condition from which the sample is excavated, contaminating organic and inorganic compounds, such as humic acid and salts leaking from the surrounding soil, can accumulate in the cavities of these samples over the years. These are often coextracted along with the endogenous DNA of the sample. Therefore, ancient DNA extraction methods need not only to recover DNA molecules preserved in the samples effi ciently, but also to remove contaminating compounds that may inhibit subsequent enzymatic reactions.
    The solid matrix of bones and teeth promotes their physical preservation and the preservation of biomolecules within them.
    However, this matrix needs to be disrupted during the extraction process in order to release the DNA molecules into an aqueous solution so that it can be purifi ed. Several DNA purifi cation and concentration methods are used for ancient animal hard tissue samples. The purifi cation method described here is a two-part process, where DNA is fi rst adsorbed to the surface of silica particles and then salts and other contaminating chemicals are removed.
    The method is identical in concept and very similar in approach to methods employed in various commercially available kits.
    In previous comparative analyses, we found DNA purifi cation by adsorption to silica particles in suspension to perform best with respect to amplifi able DNA recovery from ancient bone and tooth samples when guanidinium isothiocyanate (GuSCN) was used as a chaotropic salt to drive the adsorption of DNA ( 3 ) . GuSCN seems to prevent silica particles from adsorbing potentially inhibiting coextracts that may have accumulated in the samples. One advantage to using a solid phase to pull down the DNA from an aqueous solution is that the particles can be immobilized in an appropriate device. If these device(s) allow parallel processing, salt and other chemicals can easily be washed away and DNA eluted from many samples in parallel. Single column devices are commercially available, and using a vacuum device or a microcentrifuge to remove the buffers in between the steps allows for a moderate throughput for DNA extraction of ancient and historical museum samples ( 4 ) .
    The following protocol is presented using column devices and a vacuum manifold. If no vacuum manifold is accessible, the extraction can be performed using the columns and a microcentrifuge. It is also possible to perform the extraction without the columns by using regular 1.5-or 2.0-mL tubes and resuspension of the silica particles followed by centrifugation, rather than the simpler method (vacuum-mediated washing by fl ow-through) described below.
    However, it should be noted that some DNA may be lost as it adheres to the inside surface of the pipette tips during repeated resuspension steps.
    3 DNA Extraction of Ancient Animal Hard Tissue Samples…
    23
    The presence of intact cellular structures depends on the
    degradation state of the sample. A detergent and a reducing agent are recommended for more recent samples ( 4 ) and for well-preserved ancient samples such as those from permafrost environments. However, it is usually not necessary to use these when working with ancient specimens
    ( 5 ) . Nevertheless, no negative effect has been observed when the detergents and reducing agents used below are included in the extraction buffer, even for very old, nonpermafrost specimens ( 3 ) . These are therefore included in the extraction buffer described below.
    2. Materials
     
    HPLC grade water or water with a similar purity grade is recommended to prepare all solutions and suspensions.
    2.1. DNA Release
    1. Extraction buffer: 0.45 M EDTA (pH 8.0), 1% Triton-X 100, from Bony Specimen
    50 mM
    DL -Dithiothreitol, 0.25 mg/mL proteinase K (see
    Note 1).
    2. Cutting or drilling tool with exchangeable disposable bits or discs.
    3. Mortar and pestle or freezer mill (e.g., SPEX SamplePrep 6750
    Freezer/Mill; liquid nitrogen