DNA Extraction from Keratin and Chitin - Springer

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Dec 8, 2011 - Natural History Museum of Denmark, University of Copenhagen, Østervoldgade 5-7, DK, 1350, Copenhagen, Denmark ...
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 purification 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 biogenesis. 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 purified from the solution using either a silica-based purification or by extraction with organic solvents followed by isopropanol purification.

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

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2. Materials Prepare the digestion buffer using molecular biology reagents at room temperature, using appropriate anti-contamination controls (e.g., filter-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 Requirements

1. Bleach solution, diluted in H2O to a final NaClO concentration of approximately 0.5%. 2. “Molecular Biology Grade” H2O (ddH2O). 3. Stable digestion buffer: 10 mM Tris buffer (pH 8.0), 10 mM NaCl, 5 mM CaCl2, 2.5 mM EDTA (pH 8.0), 2% SDS. Store 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 purifications 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 Purification (3.3)

1. Qiaquick DNA purification kit (Qiagen, Valencia, CA) including “Qiaquick” silica columns, and buffers “PB”, “PE”, and “EB.”

2.3. For Organic Purification (3.4)

1. Tris-buffered phenol, pH 8.0. 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).

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3. Methods Carry out all procedures at room temperature unless otherwise specified. 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 Pre-Preparation

1. For most materials, proceed directly to step 2. For large pieces 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 briefly centrifuge the mixture to pellet the powder. Pour off the bleach. 3. Rinse material several times in ddH2O to remove all traces of 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 ddH2O then repeat.

3.2. Tissue Digestion

1. Add 40 mL 1 M DTT solution and 100 mL proteinase K solution per 860 mL 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 briefly 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 mL 1 M DTT solution and 100 mL proteinase K solution to the mixture, vortex briefly, 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 purification using either the silica (see Subheading 3.3) or organic (see Subheading 3.4) method (see Note 4).

3.3. DNA Purification: Silica Method (see Note 5)

1. Centrifuge the digestion mixture for 3–5 min at high speed (>10,000 × g) to pellet any solid remains. Carefully pipette the liquid fraction of the digestion into a new tube. Avoid transferring any solids that may block the spin filter.

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2. Add 5 volumes Qiaquick buffer PB to the solution. 3. Mix thoroughly. 4. Add 700 mL 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 filter without binding. However, if the liquid does not pass through the filter 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 mL Qiagen wash buffer PE to the filter. 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 filter. Any residual ethanol from the PE buffer will inhibit downstream applications. 10. Place the filter in a new 1.5-mL tube. Add 50–100 mL Qiagen elution buffer EB directly to the centre of the filter, and leave at room temperature for 5 min (see Note 7). EB can be replaced with molecular biology grade ddH2O (pH 7–8). 11. Centrifuge for 1 min at maximum speed to collect the EB and DNA. 3.4. DNA Purification: Organic Extraction (see Note 8)

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

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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 mL 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 briefly incubating the dry pellet at a relatively high temperature (e.g., 55–75°C). 14. Resuspend the pellet in elution TE buffer or ddH2O. If the 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 sufficient to cover the surface of the material. 4. DNA can be purified from the digestion mixture in a number of different ways. Selecting a method depends ultimately on convenience and user preference. For small volumes, silica spin-columns are convenient, but for larger volumes these rapidly become very labour-intensive. For larger volumes of digestion mix (e.g., >1 mL), organic extractions are often preferable, in particular if large amounts of undigested melanin, dirt or

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other material are present in solution, as these tend to block silica filters. For a silica protocol, refer to Subheading 3.3. For organic purification, refer to Subheading 3.4. 5. As recommended by Yang et al. (6), Qiagen’s “Qiaquick” PCR clean-up kits are an excellent and quick tool for purifying DNA. The instructions in the kit manual can be followed almost directly if one replaces the phrase “PCR product” with “DNA extract”. The only change we recommend is the modification of the centrifugal speeds. 6. Qiagen buffers contain guanidinium salts, and relevant local disposal regulations should be consulted. 7. The volume of EB to use in this step depends on final concentration of DNA required and can be modified. 8. Organic extractions use phenol and chloroform to help purify the DNA. Both phenol and chloroform are toxic, and phenol in particular is extremely dangerous. Neither should be used without appropriate training. Always handle both liquids and their containers with extreme care, using appropriate face, hand, and body protection. Do not handle using latex gloves, as these are permeable to phenol and chloroform; use only nitryl gloves. The fumes of both chemicals are dangerous; therefore, these steps should always be performed in a vented fume hood. Disposal of both requires conformation to specific regulations, thus relevant local disposal regulations should be consulted. 9. Isopropanol precipitation is most effective at relatively high centrifugal forces and in small tubes (the DNA pellet is easiest to see and resuspend if 1.5-mL tubes or smaller are used). If large volumes are to be precipitated, we recommend first concentrating the liquid, for example with a centrifugal concentrator such as an Amicon centricon (Millipore, Billerica, MA) with 30 kD or less molecular weight cut-off. 10. Melanin pigments often copurify with the DNA and coprecipitate with the DNA during isopropanol precipitation. This results in a brown concentrated DNA pellet and a brown extract after resuspension. As melanin can inhibit enzymatic reactions (e.g., PCR), an additional purification step may be followed, for example using a silica procedure (e.g., see Subheading 3.3).

Acknowledgments MTPG was supported by the Danish National Science Foundation’s “Skou” grant program.

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References 1. Bonnichsen R, Hodges L, Ream W et al (2001) Methods for the study of ancient hair: radiocarbon dates and gene sequences from individual hairs. J Archaeol Sci 28:775–785 2. Gilbert M, Wilson A, Bunce M et al (2004) Ancient mitochondrial DNA from hair. Curr Biol 14:463 3. Rawlence N, Wood J, Armstrong K et al (2009) DNA content and distribution in ancient feathers and potential to reconstruct the plumage of extinct avian taxa. Proc Biol Sci 276:3395 4. Willerslev E, Gilbert MT, Binladen J et al (2009) Analysis of complete mitochondrial

genomes from extinct and extant rhinoceroses reveals lack of phylogenetic resolution. BMC Evol Biol 9:95 5. King G, Gilbert M, Willerslev E et al (2009) Recovery of DNA from archaeological insect remains: first results, problems and potential. J Archaeol Sci 36:1179–1183 6. Yang DY, Eng B, Waye JS et al (1998) Technical note: improved DNA extraction from ancient bones using silica-based spin columns. Am J Phys Anthropol 105:539–543