Ancient DNA: Methods and Protocols (6 page)

15. The eluate is also likely to contain DNA and can be retained for processing, either in parallel or at a later date
( 8 )
.

16. The temperature setting for digestion can be modifi ed depending on the sample. Recent work suggests that lower temperatures may have a benefi cial effect on DNA recovery. If the temperature is lowered, increase the length of time the samples are left to rotate until complete digestion is achieved
( 8 )
.

17. Very occasionally, the phase-lock tubes may not separate properly between aqueous and hydrophobic phases. In this case, care should be taken when manually removing the aqueous

phase by pipette.

18. Adding a small volume of ultrapure water to the fi lters prior to adding the extract may aid in absorption of DNA to the membrane. Depending on the volume of extract to be processed, this step may have to be repeated multiple times until the entire sample has passed through the membrane.

19. Flushing water through the membrane after the entire sample has been passed through may help to further remove any

potential inhibitors from the fi nal extract.

20. After extraction, DNA can be roughly quantifi ed by measurement on a spectrophotometric platform. Note that this does 2 A Phenol–Chloroform Protocol for Extracting DNA from Ancient Samples 19

not give an indication of how much of the DNA in the extract is derived from the sample vs. from coextracted environmental contaminants.

21. It may be useful to subdivide the fi nal extracts into aliquots of 20–50 m L and to use these as necessary. DNA is susceptible to damage from repeat fr
eeze–thaw cycles ( 13
) and should be defrosted as infrequently as possible.

Acknowledgments

Thanks to Beth Shapiro for the opportunity to contribute this chapter.

References

1. Hagelberg E, Clegg JB (1991) Isolation and 8. Rohland N, Hofreiter M (2007) Comparison characterization of DNA from archaeological

and optimization of ancient DNA extraction.

bone. Proc Biol Sci 244:45–50

Biotechniques 42:343–352

2. Hagelberg E, Sykes B, Hedges R (1989) 9. Poinar HN (2002) The genetic secrets some Ancient bone DNA amplifi ed. Nature

fossils hold. Acc Chem Res 35:676–684

342:485

10. Vasan S, Zhang X, Kapurnitou A, Bernhagen J,

3. Hofreiter M, Serre D, Poinar HN, Kuch M,

Teichberg S, Basgen J, Wagle D, Shih D, Terlecky

Pääbo S (2001) Ancient DNA. Nat Rev Genet

I, Bucala R, Cerami A, Egan J, Uhlrich P (1996)

2:353–359

An agent cleaving glucose-derived protein cross—

4. Cooper A, Poinar HN (2000) Ancient DNA:

links in vitro and in vivo. Nature 382:275–278

do it right or not at all. Science 289:1139

11. Adler CJ, Haak W, Donlon D, Cooper A

5. Leonard JA, Shanks O, Hofreiter M, Kreuz E,

(2010) Survival and recovery of DNA from

Hodges L, Ream W, Wayne RK, Fleischer RC

ancient teeth and bones. J Archaeol Sci

(2007) Animal DNA in PCR reagents plagues

38(5):956–964

ancient DNA research. J Archaeol Sci 12. Gilbert MTP, Wilson AS, Bunce M, Hansen AJ, 34:1361–1366

Willerslev E, Shapiro B, Higham TFG, Richards

6. Rohland N, Hofreiter M (2007) Ancient DNA

MP, O’Connell TC, Tobin DJ, Janaway RC,

extraction from bones and teeth. Nat Protoc

Cooper A (2004) Ancient mitochondrial DNA

2:1756–1762

from hair. Curr Biol 14:R463–R464

7. Handt O, Höss M, Krings M, Pääbo S (1994)

13. Lindahl T (1993) Instability and decay of the

Ancient DNA: methodological challenges.

primary structure of DNA. Nature 362:

Experientia 50:524–527

709–715

sdfsdf

DNA Extraction of Ancient Animal Hard Tissue

Samples via Adsorption to Silica Particles

Nadin Rohland

Abstract

A large number of subfossil and more recent skeletal remains, many of which are stored in museums and private collections, are potentially accessible for DNA sequence analysis. In order to extract the small amount of DNA preserved in these specimens, an effi cient DNA release and purifi cation method is required. In this chapter, I describe an effi cient and straightforward purifi cation and concentration method that uses DNA adsorption to a solid surface of silica particles. Comparative analysis of extraction methods has shown that this method works reliably for ancient as well as younger, museum-preserved specimens.

Key words:
Ancient DNA , DNA extraction , Bones , Teeth , Museum-specimen , Silica , Column 1. Introduction

The most abundant faunal remains are partial skeletons. Bones and teeth are the hardest tissues of vertebrates and can persist for hundreds of thousands of years without fossilization if sediments or permafrost shield them from unstable environmental conditions.

When environmental conditions are unfavorable for microbial life that would otherwise metabolize the hard tissue, this can lead to the preservation of DNA molecules within these ancient skeletons.

Such conditions are common to permafrost regions, where large numbers of preserved faunal remains have been found. In more moderate climatic ecosystems, well-preserved skeletal remains can be found within sediment deposits in natural shelters such as caves.

Three major obstacles impede DNA analyses of ancient skeletal remains. First, the total amount of DNA preserved in very old bones and teeth is likely to be very small, and often the DNA fragments that do remain are highly damaged
( 1
) . The same may be Beth Shapiro and Michael Hofreiter (eds.),
Ancient DNA: Methods and Protocols
, Methods in Molecular Biology, vol. 840, DOI 10.1007/978-1-61779-516-9_3, © Springer Science+Business Media, LLC 2012

21

22

N. Rohland

true for modern specimens that have been treated with chemical preservatives to prepare them for long-term storage in museums.

Second, if DNA is preserved in ancient bones or teeth, it is often contaminated with DNA from bacteria, fungi, or other 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 is needed) for grinding sample pieces into fi ne powder.

4. 15-mL tubes.

5. Rotary mixer, wheel, or similar device to keep samples constantly in motion during incubation steps.

2.2. DNA Purifi cation

1. Silicon dioxide (see Note 2).

and Concentration

2. 30% HCl.

3. Binding buffer: 5 M Guanidinium thiocyanate, 0.3 M sodium acetate (pH 5.2). Store in the dark (see Note 3).

4. Washing buffer: 50% Ethanol, 125 mM NaCl, 10 mM Tris–HCl, 1 mM EDTA (pH 8.0) (see Note 4)

5. Elution buffer: 10 mM Tris–HCl, 1 mM EDTA (pH 8.0).

6. 50-mL tubes.

7. 50-mL disposable serological pipettes.

8. Centrifuge capable of holding 15-mL tubes and reaching centrifugal force of 5,000 ×
g.

9. Columns (e.g. MobiCol “Classic,” MobiTec, catalog number: M1003).

10. Filter (Filter (large) 10 m m pore size, MobiTec, catalog number: M2210).

24

N. Rohland

11. Filter with 1 m m pore size (e.g., glass microfi ber binder free Grade GF/B: 1 m m, Whatman, catalog number: 1821–070).

12. Hole punch with 7 mm diameter.

13. Forceps.

14. Centrifuge capable of holding 2.0-mL tubes and reaching centrifugal force of 16,000 ×
g.

15. Vacuum manifold and vacuum pump.

16. Collection tubes without lids.

17. Disposable VacConnectors (Qiagen, catalog number: 19407).

18. 1.5-mL tubes (see Note 5).

3. Methods

All steps are to be carried out at room temperature.

3.1. Preparing

1. Weigh 4.8 g of silicon dioxide into a 50-mL tube, add water to
the Silica Suspension

bring the mixture to 40 mL, and vortex extensively.

2. Let large particles settle down for 1 h.

3. Transfer 39 mL from the top of the solution into fresh 50-mL

tube and let the solution settle for an additional 4 h.

4. Discard 35 mL from the top of the solution and add 48 m L of 30% HCl to the 4 mL pellet that remains.

5. Vortex, aliquot, and store the silica suspension at room temperature in the dark (see Note 6).

3.2. Preparing

1. Use forceps to place a large fi lter with 10 m m pore size in the
the Columns

column. Move the fi lter to the bottom of the column using the fi lter insertion tool provided with the fi lter.

2. Use a hole punch to make a smaller “fi ne fi lter” from the fi lter paper with 1 m m pore size.

3. Using the forceps, place the “fi ne fi lter” in the columns, and move it on top of the larger fi lter using the insertion tool.

3.3. Sample

1. After removing the surface of the sample with a fresh drilling
Preparation

bit at slow speed, drill into the densest part of the bone or the
and DNA Release

tooth root. Collect the powder. If a cutting tool is used instead of a drill, remove a compact part of the bone or the tooth root (again after removing the sample surface with a single-use cutting disc or blade). Grind the pieces of sample to as a fi ne powder as possible using mortar and pestle or a freezer mill.

Collect approximately 250 mg of powder per sample into separate 15-mL tubes (see Note 7).

3 DNA Extraction of Ancient Animal Hard Tissue Samples…

25

2. Add 5 mL of extraction buffer to each sample. Seal the tubes and incubate them for 16–24 h under constant agitation in the dark (see Note 8).

3.4. DNA Adsorption

1. Centrifuge the samples for 2 min at 5,000 ×
g
and transfer as
to Silica, Washing