Ancient DNA: Methods and Protocols (10 page)

BOOK: Ancient DNA: Methods and Protocols
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ses from ancient DNA. Annu Rev Genet

Gilbert MT, Shapiro B, Bunce M, Wiuf C,

38:645–679

Gilichinsky DA, Cooper A (2003) Diverse plant

12. Poinar HN, Hofreiter M, Spaulding WG,

and animal genetic records from Holocene and

Martin PS, Stankiewicz BA, Bland H, Evershed

Pleistocene sediments. Science 300:791–795

RP, Possnert G, Pääbo S (1998) Molecular

5. Cerami A, Vlassara H, Brownlee M (1987)

coproscopy: dung and diet of the extinct

Glucose and aging. Sci Am 256:90–96

ground sloth
Nothrotheriops shastensis
. Science

281:402–406

6. Vasan S, Zhang X, Zhang X, Kapurniotu A,

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

13. King C, Debruyne R, Kuch M, Schwarz C,

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

Poinar H (2009) A quantitative approach to

Ulrich P (1996) An agent cleaving glucose—

detect and overcome PCR inhibition in ancient

derived protein crosslinks in vitro and in

DNA extracts. Biotechniques 47:941–949

vivo. Nature 382:275–278

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7. Wolffenbuttel BH, Boulanger CM, Crijns FR,

Wertheim-van Dillen PM, van der Noordaa J

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(1990) Rapid and simple method for purifi caS, Egan JJ, Ulrich P, Cerami A, Levy BI (1998)

tion of nucleic acids. J Clin Microbiol

Breakers of advanced glycation end products

28:495–503

restore large artery properties in experimental

15. Fujiwara M, Yamamoto F, Okamoto K,

diabetes. Proc Natl Acad Sci U S A

Shiokawa K, Nomura R (2005) Adsorption of

95:4630–4634

duplex DNA on mesoporous silicas: possibility

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of inclusion of DNA into their mesopores. Anal

new dimension for the ichnologist. Ichnos

Chem 77:8138–8145

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(1996) Driving forces for DNA adsorption to

K, Martin PS, Possnert G, Pääbo S (2000) A

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Chapter 6

DNA Extraction from Keratin and Chitin

Paula F. Campos and Thomas M. P. Gilbert Abstract

DNA extracted from keratinous and chitinous materials can be a useful source of genetic information. To effectively liberate the DNA from these materials, buffers containing relatively high levels of DTT, proteinase K, and detergent are recommended, followed by purifi cation using either silica-column or organic methods.

Key words:
Chitinous tissue , Keratinous tissue , Silica , DNA extraction, , Ancient DNA , Hair , Feathers , Nail , Cuticle , Exoskeleton

1. Introduction

 

The DNA preserved in ancient or historic keratinous and chitinous materials is generally of lower quality than that preserved in other tissues, due to extensive DNA degradation during tissue biogene-sis. Despite this, keratinous (e.g., hair, nails, feather, hoof, and horn sheath) and chitinous (e.g., insect cuticles) tissues are becoming increasingly popular as sources of ancient DNA (aDNA) (e.g.

( 1– 5 )
). Although not always available, these tissues offer advantages over bone or soft tissue in that their surfaces are relatively simple to decontaminate, and that they can often be sampled unobtrusively.

Key to the extraction of DNA from keratinous tissues is to break down the keratin in order to liberate the DNA. This is achieved by using digestion buffers that contain large amounts of detergents and reducing agents (e.g., SDS, DTT, or Cleland’s reagent) and proteinase K. DNA is then purifi ed from the solution using either a silica-based purifi cation or by extraction with organic solvents followed by isopropanol purifi cation.

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_6, © Springer Science+Business Media, LLC 2012

43

44

P.F. Campos and T.M.P. Gilbert

 

2. Materials

Prepare the digestion buffer using molecular biology reagents at room temperature, using appropriate anti-contamination controls (e.g., fi lter-tipped pipettes, DNA free consumables, etc.). To minimise the risk of contamination, we recommend purchasing ready-made stock solutions as opposed to making stock solutions in the lab.

2.1. General

1. Bleach solution, diluted in H O to a fi nal NaClO concentra-2

Requirements

tion of approximately 0.5%.

2. “Molecular Biology Grade” H O (ddH O).

2

2

3. Stable digestion buffer: 10 mM Tris buffer (pH 8.0), 10 mM

NaCl, 5 mM CaCl , 2.5 mM EDTA (pH 8.0), 2% SDS. Store

2

at 4°C.

4. 1 M Dithiothreitol (DTT) solution. Make up fresh for each digestion and discard unused solution (see Note 1).

5. Proteinase K solution (see Note 1).

6. Centrifuge(s) for 1.5/2 mL (>10,000 ×
g
) and 15-mL tubes (>3,000 ×
g
), dependent on size of digestions and purifi cations to be performed.

7. Oven prepared at 55°C, within which a rotary device (below) can be placed.

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

9. Tabletop vortex.

10. Sterile 1.5-mL and 15-mL tubes, depending on the size of the extraction being performed.

2.2. For Silica-Column

1. Qiaquick DNA purifi cation kit (Qiagen, Valencia, CA) includ-

Purifi cation
( 3.3
)

ing “Qiaquick” silica columns, and buffers “PB”, “PE”, and “EB.”

2.3. For Organic

1. Tris-buffered phenol, pH 8.0.

Purifi cation
( 3.4
)

2. Chloroform.

3. Isopropanol.

4. 3 M sodium acetate, approximate pH 5.

5. (
Optional
) DNA precipitation “carrier,” e.g., Glycoblue (Ambion, Inc., Austin, TX).

6. “Molecular Biology Grade” ethanol, 85%.

7. TE elution buffer: 10 mM Tris–HCl, 1 mM EDTA (pH 8.0).

6 DNA Extraction from Keratin and Chitin

45

 

3. Methods

Carry out all procedures at room temperature unless otherwise specifi ed. Always incorporate extraction blanks in the analysis. This protocol assumes the use of pure keratin or chitin,
e.g.
hair, horn, nail, feather, arthropod exoskeleton/wing carapaces.

3.1. Tissue

1. For most materials, proceed directly to step 2. For large pieces
Pre-Preparation

of nail or horn, drill a suitable amount of powder (e.g., 100 mg) directly from the specimen and collect the powder in an appropriate container.

2.
For non-powdered material,
clean the tissue via a brief wash in dilute bleach solution, taking care to remove all obvious sources of contaminant matter.
For powdered material
, clean the powder by immersing it in the bleach solution for 10–20 s, then briefl y centrifuge the mixture to pellet the powder. Pour off the bleach.

3. Rinse material several times in ddH O to remove all traces of 2

bleach (see Note 2).
For powdered material
, use a vortex to ensure the pellet from step 2 is homogenised after adding the water. After 10–20 s of incubation, re-pellet the powder. Pour off the ddH O then repeat.

2

3.2. Tissue Digestion

1. Add 40 m L 1 M DTT solution and 100 m L proteinase K solution per 860 m L stable digestion buffer to make the active digestion buffer. Mix well (see Note 3).

2. Add a suitable amount of digestion buffer to the sample (see Note 3). Vortex briefl y to ensure that any pelleted powder is homogenised in the solution. Incubate the sample plus buffer overnight at 55°C with gentle agitation.

3. Keratinous samples may not fully digest after this incubation. If full digestion is required, add an additional 40 m L 1 M DTT

solution and 100 m L proteinase K solution to the mixture, vortex briefl y, and return to incubation with agitation for at least 1

more hour. Chitinous samples rarely fully digest; however, in both tissues, DNA is usually liberated into solution even if digestion does not appear to be complete upon visual inspection.

4. Proceed to DNA purifi cation using either the silica (see
Subheading 3.3 ) or or
ganic (see Subheading 3.4 ) method (see

Note 4).

3.3. DNA Purifi cation:

1. Centrifuge the digestion mixture for 3–5 min at high speed
Silica Method

(>10,000 ×
g
) to pellet any solid remains. Carefully pipette the
( see Note 5 )

liquid fraction of the digestion into a new tube. Avoid transferring any solids that may block the spin fi lter.

46

P.F. Campos and T.M.P. Gilbert

2. Add 5 volumes Qiaquick buffer PB to the solution.

3. Mix thoroughly.

4. Add 700 m L of this mixture to the Qiaquick spin column.

5. Centrifuge for 1 min at 6,000 ×
g
. This speed is useful to limit how much target DNA passes through the fi lter without binding. However, if the liquid does not pass through the fi lter in 1 min, the speed can be increased.

6. Empty the liquid waste from the spin column (see Note 6).

Repeat steps 5–6 with the remaining PB buffer-digestion mix until all the liquid passes through the spin column.

7. Add 500 m L Qiagen wash buffer PE to the fi lter.

8. Centrifuge for 1 min at 10,000 ×
g
. Empty the waste and repeat if extra purity is required.

9. Centrifuge for 3 min at maximum speed to dry the fi lter. Any residual ethanol from the PE buffer will inhibit downstream applications.

10. Place the fi lter in a new 1.5-mL tube. Add 50–100 m L Qiagen elution buffer EB directly to the centre of the fi lter, and leave at room temperature for 5 min (see Note 7). EB can be replaced with molecular biology grade ddH O (pH 7–8).

2

11. Centrifuge for 1 min at maximum speed to collect the EB and DNA.

3.4. DNA Purifi cation:

1. Add phenol (pH 8) to the digestion mix at a ratio of 1:1 with
Organic Extraction

the total digestion volume.

( see Note 8 )

2. Agitate gently at room temperature for 5 min.

3. Centrifuge for 5 min to separate the layers. The speed of centrifugation will depend on the volume of the digestion mix, the centrifuge capacity, and the maximum speed designation of the tubes being used. It is generally advisable to use the maximum speed possible. If after 5 min the layers have not fully separated, extend the centrifugation time.

4. Carefully remove the upper aqueous layer. Be careful not to remove the protein-containing interface. Discard the lower, phenol layer (see Note 8).

5. Add to 1 volume of new phenol. Repeat steps 2–

4 in

Subheading 3.4
. After the second centrifugation, add the aqueous layer to 1 volume chloroform.

6. Agitate gently at room temperature for 5 min.

7. Centrifuge for 5 min to separate the layers. Remove the upper aqueous layer. Discard the lower, chloroform layer (see Note 8).

8. Add 0.6–1 volume isopropanol and 0.1 volume 3 M sodium acetate (approx. pH 5). A small amount of commercial carrier 6 DNA Extraction from Keratin and Chitin

47

solutions can also be added if required to facilitate pellet visualisation, such as Glycoblue (Ambion, Inc., Austin, TX), following the manufacturers’ guidelines. Mix well (see Note 9).

9. Immediately centrifuge at high speed (>10,000 ×
g
) for 30 min at room temperature.

10. Immediately following centrifugation, decant the liquid from the tube carefully. The DNA will have precipitated into a pellet at the bottom of the tube and may not be visible.

11. To rinse the pellet, gently add 500–1,000 m L 85% ethanol, slowly invert the tube once, then centrifuge for 5 min at high speed.

12. Gently decant the ethanol. Repeat if necessary.

13. All ethanol must be removed from the pellet as any residual ethanol will inhibit downstream applications. This can be

achieved by using a small bore pipette and by briefl y incubating the dry pellet at a relatively high temperature (e.g., 55–75°C).

14. Resuspend the pellet in elution TE buffer or ddH O. If the 2

pellet has become very dry, this may require leaving the pellet at room temperature in the liquid for 5–10 min, followed by gentle pipetting (see Note 10).

4. Notes

 

1. Neither DTT nor proteinase K are stable once added to the active digestion solution, thus the active buffer needs to be freshly made for each digestion. At 4°C, the SDS will precipitate out of solution. Prior to the addition of DTT and proteinase K, the buffer should be warmed up until the SDS is fully dissolved.

2. Any bleach carryover will degrade the DNA and reagents in subsequent steps of the DNA extraction, thus it is extremely important that bleach is removed completely.

3. The volume of digestion buffer needed is sample dependent, but generally should be at least suffi cient to cover the surface of the material.

BOOK: Ancient DNA: Methods and Protocols
13.56Mb size Format: txt, pdf, ePub
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