Ancient DNA: Methods and Protocols (4 page)

BOOK: Ancient DNA: Methods and Protocols
3.06Mb size Format: txt, pdf, ePub

3.2. Reagents

One should assume that all reagents and tools are contaminated
and Materials

with human DNA, even those labeled as sterile. Although few products are certifi ed to be DNA-free, many are certifi ed as nuclease-free; using these is key so that what little aDNA remains in an extract is not degraded upon contact with nuclease-contaminated materials. For the greatest assurance that potential contaminants 8

T.L. Fulton

are removed, especially for human studies, all solutions should be fi lter sterilized.

All equipment must be decontaminated before use, for example by UV irradiation (45 W, 72 h), baking at 180°C for 12 h, acid-treatment with 2.5 M HCl for 48 h, or 3–5% bleach (up to 50%

dilution of store bleach; sodium hypochlorite) for 48 h
( 26, 35
) . It has been recommended that autoclaving is not used for steriliza-tion, as it breaks DNA into short (~100 bp) fragments and may increase the potential for bacterial contamination
( 35
) . However, many of the methods listed above may result in problems if they are not performed effectively. As all function to break down DNA, if the reaction is incomplete (i.e., not high enough chemical concentration; insuffi cient time of exposure to UV), the resulting fragmented, damaged, modern DNA appears and behaves as

aDNA. Though widely used, UV destruction of DNA mimics that of natural environmental processes, causing photoproducts in
DNA that lead primarily to C–T transitions ( 36
) . Bleach causes oxidative damage to DNA, producing chlorinated base products and cleaving DNA into progressively smaller fragments
( 37 )
. If this process is incomplete, modern contaminant DNA will be fragmented and appear ancient. Thus, it is important to adhere fully to decontamination procedures, so as not to disguise modern contamination as the targeted aDNA.

Studies of domestic animals should also consider that PCR

reagents are potentially contaminated not only by human DNA, but also by animal DNA
( 27 )
. Thus, negative controls including carrier DNA are important in both human and animal studies, as is considering the source of the reagents. For example, if ancient cattle or bison DNA is the target, rabbit serum albumin (RSA) may be a better choice than bovine serum albumin (BSA) in PCR.

3.3. Maintaining

To maintain a sterile working environment, all personnel should
a Sterile Space

wear a full body suit including a hood, mask, shoe covers, and gloves at all times. Personnel should not enter the lab unless they have showered and changed into clothing that has not been in the PCR lab (see Note 1). Every surface should be cleaned before and after any work is performed; an additional thorough weekly cleaning is also important to maintain a sterile work environment.

Surfaces can be cleaned in a number of ways to destroy any contaminant DNA present: 3–10% bleach followed by 70% ethanol (to clean away bleach and avoid corrosion), acid, and/or nightly UV

irradiation. Keep in mind that the same problems of insuffi cient decontamination apply as discussed in the previous section.

3.4. Sample

The most effective way to avoid contamination of samples is to

adhere to stringent aDNA sterility protocols from the moment
and Storage

that the sample is excavated onward. Contamination at the point of collection is a serious problem, particularly for bones and teeth
( 38, 39 )
. However, it is not always possible to be certain of the 1 Setting Up an Ancient DNA Laboratory


collection and storage history of every specimen. Even those samples collected in the most sterile manner may still have surface contaminants from the depositional environment. Many protocols have been proposed that attempt to remove surface contaminants from ancient bones and teeth. These include the physical removal of the outer surface, washing or prolonged exposure of the outer surface to chemicals including water, EDTA, bleach, ethanol, acid, or hydrogen peroxide, UV irradiation of the sample, and/or extrac-

tion of the inner material ( 37 )
. If the sample is well preserved, bleaching the bone powder can be effective to degrade contaminant DNA preferentially over endogenous DNA
( 40
) .

Ancient DNA has been obtained from many different kinds of substrates. Hair has recently shown excellent promise for the preservation of aDNA and exclusion of environmental contaminants
( 41
) , as was highlighted recently with the publication of the fi rst ancient human genome sequenced from permafrost-preser
ved hair

( 42
) .

However, because of their comparative abundance, bones and teeth remain the most commonly used substrates for aDNA work. When obtaining powder from bones or teeth for extraction (see Note 3), it is important to protect the area where PCR is set up, either by separating or enclosing the bone powdering area or by installing an air vent that will gently draw the bone powder out of the air and separately enclosing the PCR setup area in a fl ow hood or glove box.

The most appropriate protocol for long-term storage of ancient specimens varies depending on how the species were collected. If a sample was frozen upon collection, it is ideal to maintain that temperature. If a sample was collected at room temperature, it should be stored in a cool, dry environment but may not benefi t from being frozen, in particular if several freeze/thaw cycles are anticipated. In general, simply avoiding environmental conditions that are known to promote DNA damage is key to sample preservation.

A cool, dry, temperature-stable environment is ideal. Avoid heat, freeze/thaw cycles, and moisture. Although the depositional and preservation conditions are most important in the survival of DNA through time, poor treatment after sample collection can quickly degrade DNA that has persisted over thousands of years.

Work with aDNA is time-consuming and expensive. However,

when care and appropriate precautions are taken from the outset, it can be a powerful tool for investigating evolutionary processes that cannot be addressed using modern data alone.

4. Notes


1. This also applies to notebooks, carrier bags, jackets,
It is practical to have a storage area outside the clean lab where personnel can leave their “PCR-contaminated” belongings.


T.L. Fulton

2. Carrier DNA should be selected to be a type of DNA that will not be amplifi ed in the PCR reaction. For example, lambda or vector DNA is easily accessible and makes good carrier DNA for mammalian studies.

3. A Dremel rotary tool with a cutting blade is useful for scraping off the surface of a bone, as well as for cutting out sections that can later be powdered using a bone mill. Other bits, such as a Dremel engraving cutter or a regular drill bit, are useful for hollowing out sections of bone underneath the surface, reducing the amount of damage that is visible as well as accessing the inner material that is less likely be contaminated or exposed to damaging agents.


1. Higuchi R, Bowman B, Freiberger M et al 10. Shapiro B, Drummond AJ, Rambaut A et al (1984) DNA-sequences from the Quagga, an

(2004) Rise and fall of the Beringian steppe

extinct member of the horse family. Nature

bison. Science 306:1561–1565


11. Campos PF, Willerslev E, Sher A et al (2010)

2. Saiki RK, Scharf S, Faloona F et al (1985)

Ancient DNA analyses exclude humans as the

Enzymatic amplifi

cation of beta-globin

driving force behind late Pleistocene musk ox

genomic sequences and restriction site analysis

Ovibos moschatus
) population dynamics. Proc

for diagnosis of sickle-cell anemia. Science

Natl Acad Sci U S A 107:5675–5680


12. Leonard JA, Wayne RK, Cooper A (2000)

3. Pääbo S, Higuchi RG, Wilson AC (1989)

Population genetics of ice age brown bears.

Ancient DNA and the polymerase chain-reac—

Proc Natl Acad Sci U S A 97:1651–1654

tion—the emerging fi eld of molecular archae—

13. Pinsky ML, Newsome SD, Dickerson BR et al

ology. J Biol Chem 264:9709–9712

(2010) Dispersal provided resilience to range

4. Green RE, Krause J, Briggs AW et al (2010) A

collapse in a marine mammal: insights from the

draft sequence of the Neandertal genome.

past to inform conservation biology. Mol Ecol

Science 328:710–722


5. Edwards CJ, Bollongino R, Scheu A et al (2007)

14. Shapiro B, Sibthorpe D, Rambaut A et al

Mitochondrial DNA analysis shows a Near

(2002) Flight of the dodo. Science 295:1683

Eastern Neolithic origin for domestic cattle and

15. Orlando L, Metcalf JL, Alberdi MT et al (2009)

no indication of domestication of European

Revising the recent evolutionary history of

aurochs. Proc Biol Sci 274:1377–1385

equids using ancient DNA. Proc Natl Acad Sci

6. Larson G, Liu RR, Zhao XB et al (2010)

U S A 106:21754–21759

Patterns of East Asian pig domestication, 16. Krause J, Unger T, Nocon A et al (2008) migration, and turnover revealed by modern

Mitochondrial genomes reveal an explosive

and ancient DNA. Proc Natl Acad Sci U S A

radiation of extinct and extant bears near the


Miocene-Pliocene boundary. BMC Evol Biol

7. Leonard JA, Wayne RK, Wheeler J et al (2002)


Ancient DNA evidence for Old World origin of

17. Lindahl T (1993) Instability and decay of the

New World dogs. Science 298:1613–1616

primary structure of DNA. Nature 362:

8. Goloubinoff P, Pääbo S, Wilson AC (1993)


Evolution of Maize inferred from sequence 18. Pääbo S (1989) Ancient DNA—extraction, diversity of an Adh2 gene segment from

characterization, molecular-cloning, and enzy—

archaeological specimens. Proc Natl Acad Sci

matic amplifi cation. Proc Natl Acad Sci U S A

U S A 90:1997–2001


9. Stiller M, Baryshnikov G, Bocherens H et al 19. Poinar HN, Schwarz C, Qi J et al (2006) (2010) Withering away-25,000 years of genetic

Metagenomics to paleogenomics: large-scale

decline preceded cave bear extinction. Mol Biol

sequencing of mammoth DNA. Science 311:

Evol 27:975–978


1 Setting Up an Ancient DNA Laboratory


20. Hoss M, Jaruga P, Zastawny TH et al (1996)

34. Hebsgaard MB, Phillips MJ, Willerslev E

DNA damage and DNA sequence retrieval

(2005) Geologically ancient DNA: fact or arte—

from ancient tissues. Nucleic Acids Res

fact? Trends Microbiol 13:212–220


35. Willerslev E, Hansen AJ, Poinar HN (2004)

21. Rohland N, Pollack JL, Nagel D et al (2005)

Isolation of nucleic acids and cultures from fos—

The population history of extant and extinct

sil ice and permafrost. Trends Ecol Evol

hyenas. Mol Biol Evol 22:2435–2443


22. Hofreiter M (2008) Long DNA sequences and

36. Griffi ths AJF (2005) Introduction to genetic

large data sets: investigating the Quaternary via

analysis, 8th edn. W.H. Freeman, New York

ancient DNA. Quat Sci Rev 27:2586–2592

37. Kemp BM, Smith DG (2005) Use of bleach to

23. Lindahl T (1993) Recovery of antediluvian

eliminate contaminating DNA from the surface

DNA. Nature 365:700

of bones and teeth. Forensic Sci Int 154:

24. Hofreiter M, Serre D, Poinar HN et al (2001)


Ancient DNA. Nat Rev Genet 2:353–359

38. Gilbert MTP, Hansen AJ, Willerslev E et al

25. Pääbo S, Poinar H, Serre D et al (2004) Genetic

(2006) Insights into the processes behind the

analyses from ancient DNA. Annu Rev Genet

contamination of degraded human teeth and


bone samples with exogenous sources of DNA.

Int J Osteoarchaeol 16:156–164

26. Willerslev E, Cooper A (2005) Ancient DNA.

Proc Biol Sci 272:3–16

39. Sampietro ML, Gilbert MTP, Lao O et al

(2006) Tracking down human contamination

27. Leonard JA, Shanks O, Hofreiter M et al

in ancient human teeth. Mol Biol Evol 23:

(2007) Animal DNA in PCR reagents plagues


ancient DNA research. J Archaeol Sci


40. Salamon M, Tuross N, Arensburg B et al

(2005) Relatively well preserved DNA is pres—

28. Gilbert MTP, Bandelt HJ, Hofreiter M et al

ent in the crystal aggregates of fossil bones.

(2005) Assessing ancient DNA studies. Trends

Proc Natl Acad Sci U S A 102:13783–13788

Ecol Evol 20:541–544

41. Gilbert MTP, Menez L, Janaway RC et al

29. Handt O, Hoss M, Krings M et al (1994)

(2006) Resistance of degraded hair shafts to

Ancient DNA—methodological challenges.

contaminant DNA. Forensic Sci Int 156:

Experientia 50:524–529


30. Cooper A, Poinar HN (2000) Ancient DNA: 42. Rasmussen M, Li YR, Lindgreen S et al (2010) do it right or not at all. Science 289:1139

Ancient human genome sequence of an extinct

31. Poinar HN, Hoss M, Bada JL et al (1996)

Palaeo-Eskimo. Nature 463:757–762

Amino acid racemization and the preservation

43. Hofreiter M, Jaenicke V, Serre D et al (2001)

of ancient DNA. Science 272:864–866

DNA sequences from multiple amplifi cations

BOOK: Ancient DNA: Methods and Protocols
3.06Mb size Format: txt, pdf, ePub

Other books

Alligator Playground by Alan Sillitoe
Signs and Wonders by Alix Ohlin
Falling for Fate by Caisey Quinn
The Case of the Curious Bride by Erle Stanley Gardner
Frigid by J. Lynn
Invasion: Colorado by Vaughn Heppner