Ancient DNA: Methods and Protocols (9 page)

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Melanie Kuch and Hendrik Poinar

Abstract

Paleofeces are the nonmineralized remains of dung from extant and extinct fauna. They represent a surprisingly large proportion of fossil remains recovered from cave sites across the world. Paleofeces contain the DNA of the defecator as well as the DNA of ingested plant and animal remains. To successfully extract DNA from paleofeces, a balance must be achieved between the minimization of DNA loss during extraction and the removal of coeluates that would otherwise inhibit the
Taq
DNA polymerase during downstream applications. Here we present a simplifi ed version of a protocol to extract DNA from paleofecal remains.

Key words:
DNA extraction , Feces , Ancient DNA , Silica DNA-purifi cation 1. Introduction

Paleofeces, or as they are sometimes labeled, coprolites, are the nonmineralized remains of feces. As paleofeces are nonhardened fossils, they are typically assumed to be rare in the fossil record.

However, deposits of megafaunal dung, namely that of the extinct ground sloth

Nothrotheriops shastensis
found in caves of the

American southwest, rival in extent the vast deposits of mammoth bone and teeth in the permafrost
( 1
) .

Paleofeces are most often found within caves and rock shelters
( 2 )
, although some have been found at open-air sites. Paleofeces likely make up a large percentage of the sediment found within cave fl oors, and to some degree, that found within permafrost soils as well. This may explain the success in retrieving the DNA of past inhabitants of caves
( 3
) and megafauna of the high arctic
( 4
) .

The fi rst successful extraction of DNA from paleofeces involved the use of PTB, a thiazolium salt shown to be successful in the reversal of Maillard-induced crosslinks from r
educing sugars

( 5– 8 )
.

While it is unclear to what degree the PTB unlinks these nucleic 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_5, © Springer Science+Business Media, LLC 2012

37

38

M. Kuch and H. Poinar

acid/protein complexes, we have noted distinct improvements in total DNA yields when including PTB in ancient DNA extractions.

Since the fi rst publication reporting the successful extraction of DNA from paleofeces, subsequent reports have used these techniques to isolate not only the DNA of the defecator, but also DNA from
the ingested contents ( 9, 10
) ; please see r
ef. ( 11
) for a more comprehensive review.

Below, we present the most up-to-date extraction protocol for the recovery of DNA from paleofecal remains. Since the original
publication ( 12
) , we have assessed the relative success of various modifi cations using quantitative PCR assays and by measuring inhibition as gauged by the delaying of quantifi cation cycles
( 13 )
.

The protocol can be optimized for the content of the dung (plant material versus a more meat-based diet). As with many fossil and subfossil samples, feces contain potent inhibitors with similar molecular weights, sizes and charges to DNA. The extraction of DNA from feces therefore requires achieving a balance between minimizing DNA loss during extraction and removing coeluates that would otherwise inhibit downstream applications. We use a chaotropic salt/silica-based procedure that is well known for its ability to remove inhibitors
( 14
) .

2. Materials

2.1. Laboratory

Supplies (per Sample)

Forceps

1×

Small weight boat

1×

Scalpel blade

1×

15-mL tube

3×

1.5-mL tube

2×

0.5-mL tube

1×

pH paper

1+

2.2. Laboratory

1. Pipettes 1–1,000 μ L.

Equipment

2. Vortex.

3. Incubator with rotating wheel, or rotary mixer or similar device capable of being placed in an incubator.

4. Microcentrifuge as well as a large swing bucket centrifuge for 15 mL.

5. Shaking/heating block.

5 Extraction of DNA from Paleofeces

39

2.3. Solutions and

1. GuSCN extraction-buffer (14 mL per sample): 6-M guanidine
Buffers (per Sample)

thiocyanate. (GuSCN); 20 mM Tris-HCl, pH 8.0; 0.5% sodium

lauroyl sarcosinate (Sarcosyl); 8 mM Dithiothreitol (DTT); 4%

Polyvinylpyrrolidone (PVP); 10 mM

N
-phenacyl thiazolium

bromide (PTB).

2. L6-buffer (4 mL per sample): 5 M GuSCN; 0.05 M Tris-HCl, pH 8.0; 0.0225 M Natrium chloride (NaCl); 0.02 M

Ethylenediaminetetraacetic acid (EDTA), pH 8.0; 1.25%

Triton X-100; (add 200 μ L Silica, vortex, let sit).

3. Silica solution (50 μ
L per sample, see Subheading 3.1
).

4. L2-buffer (1–2 mL per sample): 5 M GuSCN; 0.05 M Tris-HCl, pH 8.0; 0.0225 M NaCl (add 200 μ L Silica, vortex, let sit).

5. New Wash substitute (1–2 mL per sample): make an 80% ethanol solution using 1× TE (10 mM Tris-HCl and 1 mM EDTA (pH 7.5)).

6. 0.1× TE pH 8.0 plus 0.05% Tween-20 (60 μ L per sample).

7. Glacial Acetic acid (~15 μ L per sample).

3. Methods

3.1. Preparing the

1. Weigh out 4.8 g silica and place it into a 50-mL gamma-

Silica Suspension

sterilized tube.

2. Add double-distilled (dd) H O to the tube containing the silica 2

to 40 mL, vortex for 2 min, then let sit for 24 h at room temperature in the dark.

3. Carefully remove 35 ml of supernatant (without distrurbing the pellet) and discard.

4. Add ddH O to 40 mL, vortex for 2 min, let sit for 6 h at room 2

temperature in the dark.

5. Carefully remove 36 ml of supernatant (without disturbing the pellet) and discard.

6. Add 48 μ L 30% hydrochloric acid (keep pH acidic <3).

7. Resuspend the pellet, and aliquot approx. 200 μ L of solution into separate tubes for later use.

8. Store in the dark at +4°C.

9. Prior to use, vortex to resuspend any pelleted material.

3.2. Paleofeces DNA

1. Add approximately 1 g of fecal material to a small weighing
Extraction

boat (see Note 1).

2. Cut the fecal remains into small pieces using scalpel blades (see Note 2).

40

M. Kuch and H. Poinar

3. Add fecal material to a fi nal volume of 14 mL of the GuSCN

extraction-buffer in a 15-mL tube and incubate, rotating

overnight at 37°C in the dark (see Notes 1, 3 and 4).

4. Centrifuge at maximum speed for 5 min and transfer supernatant to a clean 15-mL tube.

5. Centrifuge again at maximum speed for 5 min and transfer supernatant to 4 mL of room temperature

pre-incubated

L6-buffer and 50 μ L of Silica for at least 15 min (see Notes 5–8).

6. Adjust the pH to ~5 by adding glacial acetic acid (see Note 9).

7. Incubate while rotating at room temperature for 3 h in the dark (see Note 10).

8. Centrifuge for 5 min at maximum speed and discard the supernatant.

9. Add 1 mL of L2-buffer, resuspend, and transfer solution to a 1.5-mL microcentrifuge tube (see Note 11).

10. Centrifuge for 30 s and discard the supernatant.

11. If the solutions are still heavily colored, repeat steps 9–10.

12. Add 1 mL of wash buffer and resuspend the silica.

13. Centrifuge for 30 s and discard supernatant.

14. Centrifuge again briefl y and remove all remaining liquid with a pipette.

15. Dry the remaining silica pellet in a heating block for 5 min at 56°C (to remove residual ethanol).

16. Add 60 μ L of 0.1× TE pH 8.0 plus 0.05% Tween-20 and incubate with agitation for 15 min at 56°C.

17. Centrifuge for 3 min at maximum speed and transfer supernatant to a 0.5-mL tube.

18. Centrifuge again for 3 min at maximum speed and transfer supernatant to a clean 1.5-mL tube (see Note 12).

19. Freeze at −20°C.

20. Although counterintuitive, thaw the extract after completely freezing and make 10 μ L aliquots and refreeze and store at −80 or −20°C (see Note 13).

21. Prepare a 1:10 dilution from the extract for PCR.

4. Notes

1. If less than 1 g of material is available, it is possible to scale the entire procedure down to 100 or 50 mg of sample

using 1.75 mL of the GuSCN extraction-buffer. Volumes for

L6-buffer + silica and all wash buffers remain the same.

5 Extraction of DNA from Paleofeces

41

2. Cut the feces into smaller pieces to allow for more coverage of the surface area of the feces with the extraction-buffer.

3. Add the PTB directly to the extraction buffer just before using it.

4. While it may seem odd, it appears (although the evidence for this is not statistically signifi cant) to be better to add the fecal remains to the solution rather than add the solution to the fecal remains. We suspect this has something to do with the molecular availability of embedded nucleic acids and their accessibility to the salts in the buffer.

5. It appears to be benefi cial to avoid the transfer of any solid material into the binding buffer (L6).

6. We have noticed a signifi cant improvement in DNA recovery when silica and L6-buffer are incubated together prior to adding the supernatant from the GuSCN extraction-buffer/sample

mix. Pre-incubate on a rotating wheel to insure proper mixing of silica and buffer.

7. We have attempted various compositions of extraction-buffer, for example, phosphate-based buffers and other chaotropic salts such as sodium periodate; none appear to be as successful in achieving the balance between DNA recovery and inhibitor

removal as the buffer presented here.

8. Make up exactly enough fresh buffer for each use, and do not store the buffer, as guanidinium is light and temperature—

sensitive and thus loses effi cacy as it ages.

9. Keeping the buffers acidic is important
( 15, 16
) . Measure the pH of the solution after the sample has been added and adjust accordingly. The guanidinium must remain protonated to be

an effective bridge between the phosphates on the DNA and

the silica hydroxyl groups. In addition, alkaline conditions are far more conducive to DNA backbone degradation.

10. In our experience, extending the incubation time has not signifi cantly altered total DNA yield.

11. It is wise to resuspend the silica pellet by placing the tip of the pipette at the edge of the pellet in the base of the tube and pipetting up and down slowly. Be careful not to allow the solution to bleed over the edge of the tube. While 50 μ L of silica is suffi cient for more than 10 μ g of DNA, the silica clearly gets “clogged” with other polar molecules. Using less than 50 μ L

is therefore not advised. However, in our experience, increasing the amount of silica above 50 μ L has not been shown to yield quantitatively more DNA.

12. Any silica remaining in the DNA solution may inhibit downstream reactions. As a precaution, spin down the extract before taking an aliquot for subsequent PCR. See ref.
( 17
) for other helpful tips.

42

M. Kuch and H. Poinar

13.
Storage
: It is wise to freeze and thaw the extract post purifi cation and prior to PCR. It appears that many of the inhibitors are precipitated out of solution during this step.

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