Ancient DNA: Methods and Protocols (13 page)

9. 5 M NaCl, Chloroform:Octanol (24:1).

10. PB-buffer (QIAGEN).

11. Salton wash 1 buffer (BIO 101).

12. Salton wash 2 buffer (BIO 101).

13. AW1 buffer (QIAGEN).

14. EB buffer (QIAGEN).

15. HPLC grade water.

16. Ice.

3. Methods

Sediments should be sampled in such a way so as to minimize the possibility of cross-contamination or contamination with modern DNA. Where possible, sediments should be frozen immediately after sampling.

3.1. Large Extraction

Carry out all procedures at room temperature unless otherwise stated.

1. Add 10 g wet weight of sediment to a 50-mL tube containing garnet grits.

2. Add 12 mL of Bulat extraction buffer or 15 mL of PowerMax ®

Bead Solution C2 (containing guanidine thiocyanate) (see

Note 2).

3. Add 1.2 mL of C1 solution (sodium dodecyl sulfate solution) (see Note 3).

60

J. Haile

4. Vortex for 10 min at the highest speed to ensure cell lysis and/

or release of DNA from soil particles.

5. If using Bulat buffer, incubate overnight with rotation in an oven set to 65°C. If using PowerMax ® Bead Solution, proceed to step 6.

6. Spin the 50-mL tube at 2,500 ×

g
for 3 min and transfer

the supernatant to a clean tube containing 5 mL of C3 (see Note 4).

7. Incubate at 4°C for 10 min to aid precipitation of non-DNA organic and inorganic materials, humic substances, cell debris, and proteins (see Note 5).

8. Spin the tube in the centrifuge at 2,500 ×
g
for 4 min, then transfer the supernatant to a clean tube containing 4 mL C3

solution.

9. Incubate at 4°C for 10 min.

10. Spin at 2,500 ×
g
for 4 min, then remove the supernatant to a clean 50-mL tube and add 30 mL of solution C4 (guanidine

HCl—isopropanol solution).

11. Spin the resulting solution through silicon spin fi lters (see Note 6).

12. Add 10 mL of solution C5, an ethanol-based wash solution, to clean the DNA that is bound to the silica fi lter membrane in the spin fi lter (see Note 7).

13. Add 1.5–5 mL of solution C6 (Tris buffer solution) to the spin fi lter membrane and centrifuge at 2500 ×
g
in order to elute the bound DNA (see Note 8).

14. Transfer the eluate to 1.5-mL tube(s).

15. Store the DNA extract at −20°C.

3.2. Small Extraction

1. Place up to 0.5 g (wet weight) of samples into a 2-mL

FASTPrep ® tube containing 250-mg glass beads (see Notes 9

and 10).

2. Add 600 m L of Bulat extraction buffer.

3. Place the tube in a FastPrep ® Instrument (QBIOgene) and shake for 45 s at speed 5.5. This causes samples to be pulverized and cells to be lysed.

4. Place samples on ice for 2 min.

5. Repeat steps 3 and 4 three times.

6. Place samples on a rotary mixer and incubate overnight with rotation at 65°C.

7. Adjust the mixture to 1.15 M NaCl and add 300 m L of chloroform/octanol (24:1).

8. Incubate at room temperature with rotation for 10 min.

8 Ancient DNA Extraction from Soils and Sediments

61

9. Centrifuge at 12,000 ×
g
for 2 min.

10. Remove the supernatant into a clean 1.5-mL tube.

11. Add 5× volume of PB-buffer (QIAGEN) to 1 volume of the supernatant and spin at 10,000 ×
g
for 30–60 s and discard elute.

12. Add 0.5 mL of Salton wash 1 buffer (BIO 101).

13. Spin at 10 000 ×
g
for 30–60 s and discard elute.

14. Add 0.5 mL of Salton wash 2 buffer (BIO 101).

15. Spin at 10 000 ×
g
for 30–60 s and discard elute.

16. Add 0.5 mL of AW1 (QIAGEN).

17. Spin at 10 000 ×
g
for 30–60 s and discard elute.

18. Add 0.5 mL of AW1 (QIAGEN).

19. Elute the DNA from the spin column into a clean 1.5-mL tube by spinning twice with 200 m L EB buffer (10 mM Tris–HCl, pH 8.5) (QIAGEN) 10 000 ×
g
for 30–60 s.

20. Store the DNA extract at −20°C.

4. Notes

1. Proteinase K is an endolytic serine protease that cleaves after aliphatic, aromatic, and hydrophobic amino acids to break

down protein structure
( 10
) . DTT reduces cystine crosslinks in proteins to destroy their quaternary structure and allow further degradation. Sodium dodecyl sulfate (SDS) is a detergent which acts to denature proteins (e.g., nucleases) through interfering with noncovalent subunit interactions as well as solubilizing biological membranes
( 10 )
. PTB cleaves glucose-derived protein cr
osslinks

( 11 )
and has been shown to increase the success of PCR reactions from ancient material
( 12 )
, although the exact mechanism by which it achieves this is unknown.

2. Bulat buffer often results in the fi nal extract carrying less coextracts, but can lead to clogging of proteinaceous substances on the silica fi lters. It is best used with less organic-rich samples.

3. If solution C1 contains precipitates, heat at 60°C until the precipitate has dissolved.

4. Solution C3 is a second reagent to precipitate additional non-DNA organic and inorganic material including humic acid, cell debris, and proteins.

5. It is important to remove contaminating organic and inorganic matter that may reduce DNA purity and inhibit downstream

DNA applications.

62

J. Haile

6. Solution C4 is a high concentration salt solution. Since DNA binds tightly to silica at high salt concentrations, this will adjust the DNA solution salt concentration to enable binding of

DNA to the spin fi lters, but not non-DNA organic and inorganic material that may still be present at low levels.

7. This wash solution removes residual salt, humic acid, and other contaminants while allowing the DNA to stay bound to the

silica membrane.

8. The DNA extract should be colorless. A dilution series using qPCR should be performed immediately after the extraction of DNA to assess any inhibition within the extract. If dilution does not resolve PCR inhibition, pass the extract through a 30,000

MWCO Millipore Amicon ® ultracentrifuge tube, wash twice

with ultrapure water, and elute in 200 m L of solution C6.

9. Each Lysing Matrix tube contains 1.4 mm ceramic spheres, 0.1 mm silica spheres, and one 4-mm glass bead.

10. It is important not to overload the FASTPrep ® tubes. It is possible to extract larger volumes and combine the extracts at stage 10.

Acknowledgment

This work was supported by Murdoch University, Perth, Australia.

References

1. Haile J, MacPhee R, Roberts R, Arnold L, 5. Lydolph M, Jacobsen J, Arctander P, Gilbert Brook B, Nielsen R, Gilbert M, Brock F,

M, Gilichinsky D, Hansen A, Willerslev E,

Munch K, Chivas A, Tikhonov A, Willerslev E

Lange L (2005) Beringian paleoecology

(2009) Ancient DNA reveals late survival of

inferred from permafrost-preserved fungal

mammoth and horse in interior Alaska. Proc

DNA. Appl Environ Microbiol 71:1012–1017

Natl Acad Sci U S A 106:22363–22368

6. Crecchio C, Stotzky G (1998) Binding of DNA

2. Haile J, Larson G, Owens K, Dobney K,

on humic acids: effect on transformation of

Shapiro B (2010) Ancient DNA typing of

Bacillus subtilis
and resistance to DNase. Soil

archaeological pig remains corroborates histori—

Biol Biochem 30:1061–1067

cal records. J Archaeol Sci 37:174–177

7. Khanna M, Stotzky G (1992) Transformation

3. Haile J, Holdaway R, Oliver K, Bunce M,

of
Bacillus subtilis
by DNA bound on mont—

Gilbert MTP, Nielsen R, Munch K, Ho S,

morillonite and effect of DNase on the trans—

Shapiro B, Willerslev E (2007) Ancient DNA

forming ability of bound DNA. Appl Environ

chronology within sediment deposits: are pale—

Microbiol 58:1930–1939

obiological reconstructions possible and is 8. Gilbert MTP, Bandelt HJ, Hofreiter M, Barnes DNA leaching a factor? Mol Biol Evol

I (2005) Assessing ancient DNA studies. Trends

24:982–989

Ecol Evol 20:541–544

4. Willerslev E, Hansen A, Binladen J, Brand T, 9. Bulat S, Lubeck M, Alekhina I, Jensen F, Gilbert M, Shapiro B, Bunce M, Wiuf C,

Knudsen I, Lubeck P (2000) Identifi cation of a

Gilichinsky D, Cooper A (2003) Diverse plant

universally primed-PCR-derived sequence—

and animal genetic records from Holocene and

characterized amplifi ed region marker for an

Pleistocene sediments. Science 300:791–795

antagonistic strain of

Clonostachys rosea

and

8 Ancient DNA Extraction from Soils and Sediments

63

development of a strain-specifi c PCR detection

derived protein crosslinks in vitro and in vivo.

assay. Appl Environ Microbiol 66:4758–4763

Nature 382:275–278

10. Voet D, Voet J (1995) Biochemistry. Wiley, 12. Poinar HN, Hofreiter M, Spaulding WG, New York

Martin PS, Stankiewicz BA, Bland H, Evershed

11. Vasan S, Zhang X, Zhang XN, Kapurniotu A,

RP, Possnert G, Paabo S (1998) Molecular

Bernhagen J, Teichberg S, Basgen J, Wagle D,

coproscopy: dung and diet of the extinct

Shih D, Terlecky I, Bucala R, Cerami A, Egan J,

ground sloth
Nothrotheriops shastensis
. Science

Ulrich P (1996) An agent cleaving glucose—

281:402–406

sdfsdf

Charlotte L. Oskam and Michael Bunce Abstract

Avian eggshell fragments recovered from both paleontological and archaeological deposits contain a cache of well-preserved ancient DNA. Here, we describe an extraction protocol that has been optimized to maximize the recovery of ancient DNA from fossil eggshell and minimize the copurifi cation of PCR inhibitors.

In this method, fossil eggshell fragments are powdered, then digested and heated to release DNA from the calcite matrix. The digest then undergoes a concentration step before purifi cation and washing using silica columns. The method has been used to recover aDNA from the eggshell of many avian species including moa, elephant birds, and emu, up to 19,000 years old.

Key words:
Eggshell , Silica , Ancient DNA , DNA extraction 1. Introduction

Amino acids and stable isotopes recovered from fossil eggshells have been used extensively to reconstruct palaeodiets and geo-chronology
( 1– 4 )
. Recently, we demonstrated that fossil eggshells are also a source of well-preser
ved ancient DNA ( 5 )
. As determined by confocal microscopy, DNA contained within the eggshell is protected in calcite due to its intracrystalline deposition within the eggshell matrix. This protection also provides a barrier to contaminating exogenous DNA: quantitative PCR (qPCR) results showed that moa eggshell had on average 125 times less microbial DNA than moa bone, making it an attractive substrate for high-thr
oughput sequencing applications ( 5 )
. In addition, the aDNA within eggshell has been shown to persist in a wide range of climatic conditions and has been amplifi ed from eggshell fragments many thousands of years old
( 5 )
.

In this chapter, we describe a DNA extraction protocol to isolate DNA from fossil eggshell fragments. Using qPCR to monitor DNA yields (See Chapter 16), we have optimized this protocol to 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_9, © Springer Science+Business Media, LLC 2012

65

66

C.L. Oskam and M. Bunce

maximize DNA recovery and to minimize the copurifi cation of PCR inhibitors. In this method, powdered eggshell fragments are incubated in a digestion buffer for up to 24 h, including a fi nal heat step at 95°C, which we suspect aids in solubilization of the calcite and releases the DNA from the crystalline matrix. The DNA is then concentrated on 30,000 Da MWCO columns and purifi ed

using commercial silica spin columns.

2. Materials

All reagents should be stored according to manufacturers’ requirements. Preparations should be carried out at room temperature unless indicated otherwise, using appropriate anti-contamination controls (e.g. fi lter-tipped pipettes, DNA-free consumables, etc.).

2.1. Eggshell Sampling

1. 10% bleach and 100% Ethanol (analytical grade).

2. Eggshell powdering equipment: either a Dremel tool (hand-held drill) and drill bits (part #114 or #191) (Racine, WI, USA) or fi ne grit sand paper and a mortar and pestle (see Notes 1 and 2).

3. Aluminium foil (~20 × ~30 cm 2 ).

4. 2.0-mL safelock tubes.

5. Electronic weighing scale.

2.2. Eggshell Digestion

1. Digestion buffer (700 m L per sample) containing fi nal volumes of: 0.47 M EDTA (pH 8.0), 20 mM Tris (pH 8.0), 1% Triton

X-100, 10 mM Dithiothreitol (DTT), 1 mg mL −1 proteinase K

(see Notes 3 and 4).

2. 50-mL falcon tube (one per sample).

3. Oven with a rotary mixer, wheel, or similar device to keep samples constantly in motion during incubation steps, or thermal mixer (allows temperatures up to 95°C).

4. Parafi lm.

5. Pipettes—P1000, P200, P20, and aerosol-resistant pipette tips.

2.3. Eggshell

1. 1.5-mL safelock tubes (one per sample).

Extraction

2. Vivaspin columns (30,000 MWCO).