Ancient DNA: Methods and Protocols (7 page)

much of the liquid as possible into new 15-mL tubes.

Steps, and Elution

2. Add 2.5 mL of binding buffer and 100 m L of well-mixed silica suspension to the extraction buffer in each tube. Incubate for 3 h in the dark under constant agitation (see Notes 9–11).

3. Place a disposable VacConnector onto the luer adapter of the vacuum manifold, then place the assembled column onto the

VacConnector (depending on the manifold used, up to 24

columns can be handled in parallel).

4. Centrifuge the sample for 2 min at 5,000 ×
g
, discard the supernatant, and resuspend the silica pellet in 400 m L of binding buffer. Transfer the suspension to the column and apply the vacuum (see Notes 12–14 ).

5. Place the column in a collection tube and centrifuge for 30 s at 16,000 ×
g
(see Note 15).

6. Place the column back onto the VacConnector of the vacuum manifold. Add 450 m L of washing buffer to the column and apply the vacuum (see Note 16).

7. Repeat the washing step at least once while the column remains on the vacuum manifold (see Note 17).

8. Insert the column into a collection tube and centrifuge for 30 s at 16,000 ×
g
(see Note 18).

9. Insert the column into a new, labeled 1.5-mL tube and allow the silica to air-dry by incubating the columns with open lids for about 3 min (see Notes 5 and 18).

10. Add 50 m L of elution buffer onto the center of the silica pellet and incubate the columns for 10 min with closed lids (see

Notes 19 and 20).

11. Centrifuge for 1 min at 16,000 ×
g
(see Note 21).

4. Notes

1. Always prepare the extraction buffer immediately before beginning the extraction, as proteinase K loses activity rapidly.

2. Recommended silicon dioxide: Sigma-Aldrich, catalog number: S5631.

3. The binding buffer is stable for at least 1 month. This buffer should be stored in the dark.

26

N. Rohland

4. The washing buffer is stable for several months.

5. Low retention or siliconized tubes are recommended, which reduce DNA loss due to tube wall effects.

6. The silica suspension is stable for at least 1 month.

7. Do not exceed 250 mg/5 mL extraction buffer. It is possible to proportionally scale the extraction up or down when more or less sample material is used; use 1 mL/50 mg. It is crucial to adjust the binding buffer volume accordingly (see Notes 10

and 11).

8. Incubation can also be performed at 37°C, where proteinase K

is more active than it is at room temperature. This may increase the DNA quantity, especially for younger samples with intact cell structures. Although increasing incubation time was not seen to have an infl uence in our test series of ancient samples
( 3 )
, incubation time can be extended in order to completely digest the material; this may also increase the quantity of extracted DNA
( 6
) .

9. Silica must be extensively vortexed before adding to the extraction and binding buffer as particles quickly settle down.

10. If more or less extraction buffer was used, adjust the volume of binding buffer accordingly so that the ratio of extraction to binding buffer is 2:1.

11. The volume of silica suspension should also be adjusted proportionally when different extraction buffer volumes are used.

The volume of silica suspension should be at least 50 m L, as too few silica particles may result in a loss of DNA molecules.

If a very large volume of extraction buffer is used, do not exceed 200 m L of silica suspension per extraction/column, as adding more per column may result in incomplete washing and elution performance; instead, concentrate the extraction buffer prior to the adsorption step using appropriate fi lter systems (e.g.,
( 2
) ), or distribute the silica over several columns when more than 200 m L of silica is used. However, the latter will result in higher elution volumes of less concentrated extract.

12. It is recommended that you keep the supernatant until the positive control gives satisfying results. If the extract of the positive control does not contain any DNA, you may repeat

the adsorption and purifi cation steps by adding freshly made silica suspension and proceed from the 3-h incubation step onwards.

13. If no columns are used, transfer the silica suspension into a 1.5-or 2.0-mL tube and perform the washing steps by resuspending the silica with washing buffer by pipetting. Then centrifuge for 30 s at 16,000 ×
g
to pelletize the silica, and discard washing buffer by pipetting it off. Dry the silica for at least 10 min and resuspend the silica in 50 m L elution buffer by pipetting.

3 DNA Extraction of Ancient Animal Hard Tissue Samples…

27

After fi nal incubation for 10 min, centrifuge for 1 min at 16,000 ×
g
and pipette off the extract into a fresh-labeled tube.

14. If you are not using a vacuum manifold, this step and all following washing steps can be performed using a microcentrifuge and collection tubes. For an even distribution of the silica particles over the fi lter and subsequently effi cient washing performance, short, slow-speed centrifugation is recommended, followed by a 180° rotation of the column and another short, slow-speed centrifugation step after the silica is applied to the columns.

15. This is a crucial step to remove remaining salts and other chemicals, as remaining GuSCN can lead to incomplete elution of the DNA from the silica and/or inhibit subsequent enzymatic reactions.

16. Fresh VacConnectors are recommended.

17. If the silica is still deeply colored after two washing steps, it is possible to wash the silica with 450 m L binding buffer, followed by centrifugation and at least two washing steps with washing buffer. Washing with binding buffer seems to reduce the amount of colored and potentially inhibiting coextracted contaminants.

18. This is a crucial step to remove any salt and ethanol remains, which may lead to incomplete elution and/or inhibition of

enzymatic reactions that follow.

19. If the silica particles are not evenly distributed on the fi lters, add the elution buffer on top of the thickest part of silica particles.

20. If more than 100 m L silica was used for adsorption, proportional increase of the elution buffer volume is recommended.

21. The elution step may be repeated. However, this increases the volume of extract, but also reduces the concentration of DNA in the extract.

Acknowledgments

I would like to thank Michael Hofreiter and Elizabeth Fels for linguistic improvements of the manuscript.

References

1. Pääbo S, Poinar H, Serre D, Jaenicke-Despres

2. Noonan JP, Hofreiter M, Smith D, Priest JR,

V, Hebler J, Rohland N, Kuch M, Krause J,

Rohland N, Rabeder G, Krause J, Detter JC,

Vigilant L, Hofreiter M (2004) Genetic analy—

Pääbo S, Rubin EM (2005) Genomic sequencing

ses from ancient DNA. Annu Rev Genet

of Pleistocene cave bears. Science 309:

38:645–679

597–599

28

N. Rohland

3. Rohland N, Hofreiter M (2007) Comparison 5. Rohland N, Hofreiter M (2007) Ancient DNA and optimization of ancient DNA extraction.

extraction from bones and teeth. Nat Protoc

Biotechniques 42:343–352

2:1756–1762

4. Rohland N, Siedel H, Hofreiter M (2010) A 6. Loreille OM, Diegoli TM, Irwin JA, Coble rapid column-based ancient DNA extraction

MD, Parson TJ (2007) High effi ciency DNA

method for increased sample throughput. Mol

extraction from bone by total demineralization.

Ecol Resour 10:677–683

Forensic Sci Int Genet 1:191–195

Case Study: Recovery of Ancient Nuclear DNA

from Toe Pads of the Extinct Passenger Pigeon *

Tara L. Fulton , Stephen M. Wagner , and Beth Shapiro Abstract

A variety of DNA extraction methods have been employed successfully to extract DNA from museum specimens. Toe pads are a common source of ancient DNA in birds, as they are generally not an informative character and can be removed without signifi cant destruction of precious specimens. However, the DNA in these tissues is often highly degraded, both by natural postmortem decay and due to treatment by preservatives. In this case study chapter, we describe the use of both a commercial DNA extraction method and a silica-based method to extract ancient DNA from desiccated toe pads from the extinct passenger pigeon,
Ectopistes migratorius
. Successful amplifi cation of nuclear DNA was achieved from both methods, representing the fi rst nuclear DNA sequence recovered from this extinct species. We describe simple modifi cations to both protocols that we employed during the DNA extraction process.

Key words:
Columbidae , Pigeons , Toe pads , DTT , Dithiothreitol , Ancient DNA , Silica extraction 1. Introduction

Specimens from museums and natural history collections are an important source of genetic information from the recent and distant past
( 2
) . Subsamples taken from museum-stored bones, skins, and other remains for ancient DNA analysis are precious, and therefore, the methods used to extract DNA from these should be effi cient both in limiting the destructiveness of the subsampling and in the amount of DNA recovered. For museum-preserved

*
Note
: In the case study presented in this chapter, we describe the extraction of DNA from toe pads of museum-preserved specimens of the passenger pigeon,
Ectopistes migratorius
, using a method similar to that presented in Chap. 3 . Other methods, such as those described in Chap. 2 , may also be appropriate to extract DNA from this type of sample. We discuss specifi c challenges associated with applying this extraction method to ancient toe pad samples, including the use of dithiothreitol (DTT) for tissue dissolution. For more information, see the original publication of the scientifi c results in
( 1 )
.

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_4, © Springer Science+Business Media, LLC 2012

29

30

T.L. Fulton
et al.

birds, a common source of DNA is toe pads. Toe pads are thick layers of tissue that can be easily removed with a sterile scalpel blade
( 3 )
. Only a single toe pad is necessary for analysis, so that while toe pads are not generally phylogenetically informative, the toe pad from the opposing foot remains intact for future analysis.

Problematically, museum specimens are often subjected to harsh chemicals as part of the process of preservation and, although toe pads may receive less focused treatment
( 3 )
, these chemicals may complicate DNA extraction and downstream molecular biology reactions.

Passenger pigeons (
Ectopistes migratorius
) were once the most abundant birds in North America, comprising an estimated 20–40%

of the total avian population and fl ock census estimates in the billions
( 4 )
. However, European settlement of the region led to the rapid decline of the species over the course of the seventeenth to nineteenth centuries, with the death of the last known individual in captivity in 1914. Overhunting and habitat loss through deforesta-tion are believed to be responsible for the extinction of the passenger pigeon, which occurred despite the enactment of some of the earliest conservation legislation in the US, which unfortunately was widely ignored
( 4
) .

Despite widespread public attention, very little is known about the evolutionary history of the passenger pigeon. Morphological analyses place it within the radiation of the New World mourning doves (
Zenaida
) ( 5
) , while mitochondrial DNA (mtDNA) suggests a relationship with the typical pigeons and doves
( 6 )
. A recent reanalysis of the available mtDNA data (1,448 base pairs (bp) of the 12S rRNA and
cytochrome b
gene) with increased taxonomic coverage and an additional 169 bp of
ATP8
(also from the mitochondrion) suggested a close but not highly supported evolutionary relationship with the New World pigeons,
Patagioenas
( 7 )
. As mtDNA and nuclear DNA phylogenies are not always congruent, we targeted a nuclear intron to further examine passenger pigeon phylogeny. Analyses of these new data confi rmed the sister relationship between the passenger pigeon and New World pigeons (
Patagioenas
) and provided moderately strong support for the Ectopistes–Patagioenas clade
( 1
) .

During the course of this study, we implemented two different extraction methods and designed a series of overlapping primers to obtain ancient nuclear intronic DNA. Here, we focus on comparison of the results of the two extraction methods, comment on technical hurdles encountered during the experiment, briefl y discuss the strategy of designing PCR primers for targeting specifi c regions of ancient nuclear DNA, and suggest further improvements to the methods used based on subsequent analyses of this and other avian taxa.

4 Case Study: Recovery of Ancient Nuclear DNA…

31

2. Materials

and Methods

We collected toe pad samples of

E. migratorius
from National

Museums Liverpool (museum ID: Canon H.B. Tristram Collection LIV T17065). We performed DNA extraction and all pre-PCR

work in a dedicated aDNA facility at the Pennsylvania State University, adhering to strict ancient DNA protocols at all stages.

We cut each toe pad tissue into small pieces and isolated DNA using two different methods. First, we used the Qiagen DNeasy Tissue Kit (Qiagen), which we modifi ed slightly to use 40 m L proteinase K and an extended initial lysis step of roughly 1 week, alternating between incubation at 50°C and at room temperature to ensure complete tissue lysis. We eluted the fi nal extract into 200 m L