Ancient DNA: Methods and Protocols (18 page)

3.3. Challenges of a

Warm, wet environments are expected to dramatically increase the
Warm Environment

thermal age of ancient DNA, resulting in extensive damage and fragmentation
( 2 )
. Our results suggest that Pleistoceneage DNA remains in some specimens were preserved in subtropical regions.

However, it should be noted that the sample from which DNA was recovered had been preserved in a cave microenvironment. Cave interiors provide highly stable environments with little annual fl uctuation in temperature or humidity and are known to promote the preservation of DNA. Mefford Cave may be exceptional among caves for long-term preservation of DNA: the current entrance is recent, and the small opening through which animal remains entered (presumably by washing in) during the Pleistocene has since been closed
( 11 )
. Therefore, it is conceivable that Mefford Cave was sealed to the outside environment for an extended period of time following the deposition of this specimen.

References

1. Ramakrishnan U, Hadly EA (2009) Using 6. Voorhies MR (1987) Fossil Armadillos in phylochronology to reveal cryptic population

Nebraska: the Northernmost Record.

histories: review and synthesis of 29 ancient

Southwestern Nat 32:237–243

DNA studies. Mol Ecol 18:1310–1330

7. Huchon D, Delsuc F, Catzefl is FM, Douzery

2. Smith CI, Chamberlain AT, Riley MS, Stringer

EJP (1999) Armadillos exhibit less genetic

C, Collins MJ (2003) The thermal history of

polymorphism in North America than in South

human fossils and the likelihood of success—

America: nuclear and mitochondrial data con—

ful DNA amplifi cation. J Hum Evol 45:

fi rm a founder effect in
Dasypus novemcinctus

203–217

(Xenarthra). Mol Ecol 8:1743–1748

3. Mitchell D, Willerslev E, Hansen A (2005) 8. Hill RV (2006) Comparative anatomy and his-Damage and repair of ancient DNA. Mutat Res tology of xenarthran osteoderms. J Morphol

571:265–276

267:1441–1460

4. Klippel W, Parmalee P (1984) Armadillos in 9. Eckert KA, Kunkel TA (1991) DNA poly-North American late Pleistocene contexts. Spec merase fi delity and the polymerase chain reac—

Publ Carnegie Mus Nat Hist 8:149–160

tion. PCR Methods Appl 1:17–24

5. Gillette DD, Ray CE (1981) Glyptodonts of 10. Stiller M, Green RE, Ronan M, Simons JF, Du North America. Smithsonian Contrib Paleobiol

L, He W, Egholm M, Rothberg JM, Keats SG,

40:1–262

Ovodov ND, Antipina EE, Baryshnikov GF,

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Kuzmin YV, Vasilevski AA, Wuenschell GE,

DNA. Proc Natl Acad Sci USA 103:

Termini J, Hofreiter M, Jaenicke-Despres V,

13578–13584

Paabo S (2006) Patterns of nucleotide misin—

11. Auffenberg W (1957) A note on an unusually

corporations during enzymatic amplifi cation

complete specimen of
Dasypus bellus
(Simpson)

and direct large-scale sequencing of ancient

from Florida. Q J Fla Acad Sci 20:233–237

Nondestructive DNA Extraction from Museum Specimens

Michael Hofreiter

Abstract

Natural history museums around the world hold millions of animal and plant specimens that are potentially amenable to genetic analyses. With more and more populations and species becoming extinct, the importance of these specimens for phylogenetic and phylogeographic analyses is rapidly increasing.

However, as most DNA extraction methods damage the specimens, nondestructive extraction methods are useful to balance the demands of molecular biologists, morphologists, and museum curators. Here, I describe a method for nondestructive DNA extraction from bony specimens (i.e., bones and teeth). In this method, the specimens are soaked in extraction buffer, and DNA is then purifi ed from the soaking solution using adsorption to silica. The method reliably yields mitochondrial and often also nuclear DNA.

The method has been adapted to DNA extraction from other types of specimens such as arthropods.

Key words:
Ancient DNA , Arthropods , Bones , Teeth , Museum specimens , Silica 1. Introduction

The research fi eld of ancient DNA is generally accepted to have started in 1984, with the publication of short mitochondrial (mt) DNA fragments from the extinct quagga
( 1
) . However, it is important to note that the samples investigated in this study were a mere 140 years old, a typical age for many museum specimens of extant species. Since then, the number of studies using museum specimens for genetic investigations has risen sharply, be it for phyloge-

netic (e.g. ( 2– 8 ) ), phylogeographic, (e.g. ( 9– 14
) ) or population genetic analyses (e.g.
( 15– 20 )
). Sometimes, even studies on human
genetic diversity ( 21 )
or paternity analyses of animal populations
( 22 )
rely on museum specimens.

This rising demand of molecular biologists to sample museum specimens is putting an increasing pressure on the collections of 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_13, © Springer Science+Business Media, LLC 2012

93

94

M. Hofreiter

natural history museums. Although a variety of tissue types can be
used for genetic analyses (see ( 23
) for a review), including hair (e.g.
( 24 )
), skin (e.g.
( 11
) ), or bird toe pads (e.g.
( 25
) ), the most common tissues used are bony specimens. However, while many studies yield exciting r
esults, “consumptive sampling” ( 26 )
, i.e., the removal and destruction of parts of the specimen, often irre-versibly damages specimens and is in the long run unsustainable.

As many specimens housed in natural history museums are from
now-extinct populations or species

( 23 )
, their preservation for future morphological as well as genetic studies is vital. Therefore, less destructive methods for DNA sampling have been developed, such as sampling from maxilloturbinal bone material (i.e. “the thin bones attached anteriorly to ridges inside the nasal cavity,”
( 26
) ), a part of the skeleton that is not used for morphological studies.

However, the ideal sampling method does not require any consumption of material, but rather preserves the morphological characters for future studies.

The method described below has been developed with exactly this aim, i.e., obtaining suffi cient DNA for genetic analyses from bones and teeth without affecting the morphology of the specimens studied
( 27 )
. Although it was initially developed for mtDNA analyses, subsequent studies
( 6, 8
) have shown that many samples yield suffi cient DNA to also allow analysis of nuclear DNA, at least up to a length of ar
ound 250 base-pairs (bp) ( 6
) . The method involves incubation of whole bone or teeth specimens in the extraction buffer for one to several days, followed by DNA recovery from the incubation solution using adsorption to silica in the presence of a chaotropic salt (generally guanidinium isothiocyanate, GuSCN). After extraction, samples are washed in double-distilled water to remove any traces of the extraction buffer and air-dried.

This treatment has no visible effect on the morphology of solid bone specimens (apart from them looking cleaner after the extraction; see Figs. 2 in
( 6, 27
)
), but very fragile specimens such as jaws or rostra from small mammals such as golden moles
( 8
) may show signs of bone dissolution on the surface of the specimens.

The protocol described below is based on the initial publication of the method on bones and teeth
( 27
) . However, the method has been used for the extraction of DNA from arthropod specimens using both the original buffer conditions
( 28 )
and modifi ed
conditions

( 29, 30 ) . In one of these studies

( 30 )
, DNA was

extracted from beetles up to 26,000 years old. Similarly, while the protocol below describes a silica-batch method for DNA purifi cation, depending on the extraction buffer used, other DNA purifi -

cation methods may be considered
( 31
) .

13 Nondestructive DNA Extraction from Museum Specimens

95

2. Materials

Prepare all solutions using HPLC grade water or water with a similar purity grade. Both the extraction and the binding buffer as well as the silica suspension are stable for at least 1 month. The washing buffer and the TE for elution are stable for several months.

1. Extraction/binding buffer: 5 M guanidinium isothiocyanate, 50 mM Tris–HCl, pH 8.0, 25 mM NaCl, 1.3% Triton-X100,

20 mM EDTA, 50 mM DTT (see also Notes 1–3).

2. Silica suspension: Weigh 4.8 g of silicon dioxide (recommended: Sigma-Aldrich, catalog number: S5631), add ddH O to 40 mL, 2

and vortex until the silica is completely in suspension. Allow to settle for 1 h, transfer upper 39 mL into fresh tube, and allow to settle for another 4 h. Discard the upper 35 mL, leaving 4 ml of suspension/pellet, and add 48 m L 30% HCl. Vortex, aliquot, and store at room temperature in the dark.

3. Washing buffer 1: 5 M Guanidinium thiocyanate, 0.3 M sodium acetate (pH 5.2); store at RT in the dark.

4. Washing buffer 2: 50% Ethanol, 125 mM NaCl, 10 mM Tris–HCl, 1 mM EDTA (pH 8.0); store at RT.

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

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

7. Table top centrifuge for 1.5/2-mL tubes going up to

12,000 rpm.

3. Methods

All steps are performed at room temperature.

3.1. Incubation

1. Obtain an appropriate sample for extraction. For small species such as rodents, tenrecs, or insectivores, complete bones such as jaws or rostra can be used. For larger species, teeth are a good source, although when using incubation dishes of appropriate size, larger samples such as complete ape skulls can be extracted. In such cases, the extraction buffer volume needs to be adjusted accordingly, and DNA purifi cation usually has to be done in multiple aliquots (see Notes 3 and 4).

2. When working in 15–50-mL tubes, add between 5 and 20 mL

of extraction buffer to each sample. Seal tube with parafi lm and incubate for 5 days under constant agitation in the dark (see also Notes 3–6).

96

M. Hofreiter

3.2. DNA Purifi cation

1. Either remove bone specimen from tube or transfer supernatant to a new tube.

2. Centrifuge the supernatant for 2 min at 12,000 ×
g
to pellet any particles that have come off the sample. This is particularly important for samples that contain large amounts of dried soft tissue. Transfer as much of the liquid as possible into a new tube.

3. Add 100 m L of resuspended silica suspension and incubate for 3 h under constant movement in the dark (see Notes 6

and 7).

4. Centrifuge for 2 min at 5,000 ×
g
, remove supernatant (see Note 8), and resuspend the silica pellet in 1 mL washing buffer 1 (see Note 9).

At this step, you can also resuspend the silica pellet in 0.4 mL

washing buffer 1 and proceed from step 4 of Subheading 3.2

of Chap. 3 .

5. Centrifuge for 2 min at 5,000 ×
g
, discard supernatant, and resuspend the silica pellet in 1 mL washing buffer 2.

6. Repeat step 4.

7. Centrifuge for 2 min at 16,000 ×
g
and discard supernatant (see Note 10).

8. To completely remove any remaining supernatant, centrifuge again for 30 s at 16,000 ×
g,
and remove any remaining supernatant (see Note 11).

9. Air-dry the silica by leaving the tubes with open lids at RT for about 15 min.

10. Add 50 m L elution buffer to the silica pellet, resuspend by carefully pipetting up and down and stirring with the pipette tip until you have a homogenous suspension (see Note 12).

11. Incubate for 10 min with closed lid.

12. Centrifuge for 2 min at 16,000 ×
g
, transfer supernatant to a new, labeled tube, preferably a 0.5-mL tube; aliquot extract if required (see Note 13).

13. You may want to repeat steps 10 and 11, but the DNA yields of the second elution are generally much lower, so pooling of both elutions will result in a lower DNA concentration of the fi nal extract.

3.3. Sample Curation

To avoid any salts of the extraction buffer infi ltrating the samples, after removal from the extraction buffer, transfer them to a tube with double-distilled water. Incubate them overnight at RT, transfer them to a new tube with double-distilled water, and incubate for another few hours. Remove them from tube and let them air-dry slowly at room temperature.

13 Nondestructive DNA Extraction from Museum Specimens

97

4. Notes

1. When using fragile specimens such as bones from small mammals, it is advisable to adjust the extraction buffer, either by reducing the volume or the concentration of EDTA, which has a dissolving effect on bone. It is also possible to use a completely different buffer for extraction, as has been done as an adaptation of this method for DNA extraction from beetle

specimens
( 29
) .

2. Although the initial study gave the best results using the GuSCN buffer, two other buffers (one Tris–NaCl-based, the

other one sodium-phosphate-based) also yielded results with teeth
( 27 )
.

3. Recent studies have shown that the optimal GuSCN concentration for maximizing DNA yields is around 1.4–1.7 M, rather than 5 M
( 31
) . However, it is unknown how a reduced GuSCN