Contamination
Controls and Detection
in Ancient DNA
Studies*
Dongya
Yang, Ph.D.
With the advent of the
PCR technique in molecular biology(Mullis
and Faloona, 1987), DNA can now be extracted and analyzed
from ancient remains (Pääbo
et al., 1989;Hofreiter et al., 2001b). Ancient DNA
studies hold great potential for anthropologists and archaeologists to address
many important issues that cannot be well dealt with in conventional way.
However, the field is still full of technical and interpretive challenges (Wayne
et al., 1999).
The most difficult is proving that amplified DNA is authentic ancient DNA. The
high risk of contamination is due to the fact that ancient DNA is highly
degraded and only minute amounts are preserved, while the PCR technique is
extremely sensitive and can easily pick up tiny amounts of contaminant DNA(O'Rourke
et al., 2000).
Contamination controls and detection therefore become extremely important in
ancient DNA studies. This web page will discuss some practical guidelines that
can be used to carry out effective contamination controls and detection in
order to obtain authentic ancient DNA.
Dedicated
Laboratory for Ancient DNA Studies
A dedicated laboratory
is required for ancient DNA extraction and other pre-PCR work(Wayne et al., 1999). It is crucial
to physically separate pre-PCR and post-PCR work (Herrmann
and Hummel, 1994). The
laboratory and all equipment should be dedicated to ancient DNA work. No modern
DNA work should ever be carried out in the dedicated ancient DNA laboratory.
Ideally, the pre-PCR laboratory should have UV-filtered ventilation system and
positive pressure airflow. Sterile disposables and filtered tips should be
used. Gloves, masks, boots and lab coats should be worn. 10% bleach and UV
light should be used to clean and irradiate the surfaces of benches and
equipment to destroy contaminant DNA.
Selection
and Decontamination of Ancient Remain for DNA Studies
When selecting
specimens for ancient DNA extraction, besides other criteria, the ease of
decontaminating remains must be considered carefully since most remains
excavated in the past had been contaminated by subsequent handling and
analysis. There are several methods currently available for specimen
decontamination(Herrmann
and Hummel, 1994;MacHugh et al., 2000). Physical
methods remove the contaminated surface using sandpaper or electronic drills.
Chemical decontamination uses chemicals such as 10% bleach to damage and
destroy surface contaminant DNA. Ultraviolet (UV) irradiation is another
effective method for decontaminating specimens, reagents and other supplies(Tofanelli
and Nencioni, 1999). UV can cause DNA to crosslink and
preclude it from use in PCR amplification(O'Rourke
et al., 2000).
DNA Extraction from
Ancient Remains
Selection of optimal
DNA extraction methods and setup of blank extractions should be carried out in
this step. Blank extraction should be used to monitor possible contamination of
extraction reagents, commercial kits and the entire extraction process.
Experiments that involve less steps or less human involvement should be
considered advantageous (Yang
et al., 1998).
PCR Amplification of
Ancient DNA
The great difficulty
in the amplification of ancient DNA is due to physical and chemical degradation
of DNA templates(Hofreiter et al.,
2001b;Handt et al., 1994). Ancient DNA
can only be extracted in minute amounts, with small fragments and is often
associated with PCR inhibitors, therefore, protocols for ancient DNA
amplification must be optimized accordingly. Shorter target DNA fragments
should be sought since extracted DNA is usually less than 300 bp. The shorter
the target fragment, the more templates will be potentially available for
amplification. Obviously, with older remains, the difficulty to amplify longer
fragments increases (Handt
et al., 1994).
This fact can be used in the authentication of ancient DNA. Both negative and
positive controls should be setup along with ancient DNA samples for PCR
amplification(Yang
et al., 2003).
Positive controls can be used to indicate whether PCR conditions are set up
correctly and negative controls including blank extracts will show
amplification products if contamination occurs. Multiple negative controls
should be setup in order to more effectively monitor contamination(O'Rourke
et al., 2000).
For ancient human mtDNA, we have found that multiple quantified positive
controls should also be used to indicate the sensitivity of individual PCR
amplification and the level of contamination if it occurs (Yang
et al., 2003).
Quantification
of Ancient DNA Templates
Accurate estimation of
the number of ancient DNA templates is of great assistance in determining whether
amplified DNA is from authentic ancient DNA since greater numbers of ancient
DNA templates result in a diminished likelihood of contamination(Handt
et al., 1996;Hofreiter et al., 2001a). Competitive
PCR can be used for the quantification of ancient DNA (Krings
et al., 1997),
but the estimated amount of templates may also include contaminant DNA.
Another method has
been proposed to examine the preservation state of ancient DNA through amino
acid racemization analysis (Poinar
et al., 1996). Although the analysis cannot produce
accurate estimation of ancient DNA templates, the preservation state can
clearly indicate the possibility of extracting DNA from very ancient remains
such as fossils.
Sequencing
and Cloning PCR Products and Sequence Analysis
Once ancient DNA is
amplified, it can be treated as any modern DNA sample would be and no special
laboratory or equipment requirements are needed. Electrophoresis of multiple
positive and negative controls should be used to quickly examine whether
contamination occurs and the level of contamination if it occurs. Sequencing results can be of assistance
in detecting contamination. For example, a DNA sample from one individual
usually only contains one mtDNA sequence. A good indication of possible
contamination is the presence of more than one type of mtDNA sequence or if the
same type of mtDNA sequence is detected from many unrelated individuals.
PCR
products can be cloned to determine the number and percentage of different
types of sequences present in PCR products. For more ancient remains such as
fossils, cloning should be carried out since it is not only good for detecting
contamination but also very useful in reconstructing an authentic ancient DNA
sequence. When the number of DNA templates is extremely low and DNA itself is
highly degraded, incorrect nucleotides may be incorporated into the synthesis
of new DNA molecules and generate incorrect DNA sequences(Hofreiter
et al., 2001a),
or prematurely terminated DNA fragments may jump from one template to another
and produce chimeric DNA sequences (jumping PCR) (Pääbo
et al., 1990).
These amplification errors are generally random and can be detected though
cloning and repeat experiments.
Obtained ancient DNA
sequences must make a phylogenetic sense(Handt
et al., 1996)
and/or at very least should not contradict genetic rules and patterns. Otherwise,
contamination should be suspected. For example, dinosaur DNA should be more
similar to reptilian DNA than to mammalian DNA. In humans, one individual
should only have two copies of nuclear DNA fragments. Special attention should
also be given to the presence and detection of nuclear mtDNA amplification(van
der Kuyl et al., 1995) and mtDNA’s heteroplasmy (Gill
et al., 1994).
When these occur, they may complicate the detection of contaminant sequences.
Reproducibility
Test and Ancient DNA Authentication
The reproducibility
test is an integral part of ancient DNA research(Hofreiter
et al., 2001a). Replication of the entire ancient DNA
process should be undertaken to examine whether the same results can be
obtained. Authentic ancient DNA and contaminant modern DNA have different
“behavioral patterns” in the test. If DNA is authentic, the same DNA should be
extracted, amplified and sequenced from different bones of the same
individuals, in different laboratories and by different groups of researchers.
Thus, it should be expected that different repeats generate the same DNA
sequence. However, due to its random nature, contaminant DNA generally fails in
reproducibility tests.
Ancient DNA
authentication is to examine all contamination controls, laboratory procedures
and amplified DNA sequences to demonstrate that extracted and amplified DNA is
authentic ancient DNA and not contaminant modern DNA. Strict contamination
controls are required but they cannot guarantee contamination-free results.
There are also no absolute physical, chemical and biological criteria one can
use to simply determine DNA’s antiquity. Thus, there is no way to directly
authenticate ancient DNA based only on the DNA itself.
Though we cannot
directly determine whether amplified DNA is authentic or contaminant,
logically, we can exclude one source and indirectly prove the other. Compared
to the scarcity of ancient DNA templates, contaminant DNA is much more
plentiful, making contamination of modern DNA an inevitable reality in ancient
DNA studies. Therefore, one must analyze the possibility of contaminant DNA
first before one can accept the result as authentic ancient DNA. Negative,
positive controls and DNA sequence analyses are all capable of indicating
contamination. Each individual control itself may not have a strong power in
excluding contamination, however, when all controls and analyses do not
indicate contamination, statistically, there is likely no contamination.
Literature
Cited
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Benson N, Tully G, Evett I, Hagelberg E, and Sullivan K. 1994. Identification
of the remains of the Romanov family by DNA analysis. Nature Genetics
6:130-135.
Handt O, Hoss M, Krings M, and
Pääbo S. 1994. Ancient DNA:
methodological challenges. Experientia 50:524-529.
Handt O, Krings M, Ward RH, and
Pääbo S. 1996. The retrieval of
ancient human DNA sequences. American Journal of Human Genetics
59:368-376.
Herrmann B and Hummel S. 1994. Ancient
DNA: Recovery and Analysis of Genetic Material from Paleontological,
Archaeological, Museum, Medical and Forensic Specimens. New York: Springer
Verlag.
Hofreiter M, Jaenicke V, Serre D,
Haeseler AA, and Pääbo S. 2001a.
DNA sequences from multiple amplifications reveal artifacts induced by cytosine
deamination in ancient DNA. Nucleic Acids Research 29:4793-4799.
Hofreiter M, Serre D, Poinar HN, Kuch
M, and Pääbo S. 2001b. Ancient
DNA. Nature Reviews Genetics 2:353-359.
Krings M, Stone A, Schmitz RW,
Krainitzki H, Stoneking M, and Pääbo S. 1997. Neandertal DNA sequences and the
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MacHugh DE, Edwards CJ, Bailey JF,
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Mullis KB and Faloona FA. 1987.
Specific synthesis of DNA in Vitro via a polymerase-catalyzed chain
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O'Rourke DH, Hayes MG, and Carlyle SW.
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polymerase chain reaction. The emerging field of molecular archaeology. Journal
of Biological Chemistry 264:9709-9712.
Poinar HN, Hoss M, Bada JL, and
Pääbo S. 1996. Amino acid
racemization and the preservation of ancient DNA. Science 272:864-866.
Pääbo S, Irwin DM, and Wilson AC. 1990.
DNA damage promotes jumping between templates during enzymatic smplification. Journal
of Biological Chemistry 265:4718-4721.
Tofanelli S and Nencioni L. 1999.
Recovering sncient DNA by dtreptavidin-coated magnetic beads and biotinylated
oligonucleotides. Ancient Biomolecules 2:307-320.
van der Kuyl AC, Kuiken CL, Dekker JT,
Perizonius WR, and Goudsmit J. 1995. Nuclear counterparts of the cytoplasmic
mitochondrial 12S rRNA gene: a problem of ancient DNA and molecular
phylogenies. Journal of Molecular Evolution 40:652-657.
Wayne RK, Leonard JA, and Cooper A.
1999. Full of sound and fury: The recent history of ancient DNA. Annual
Review of Ecology and Systematics 30:457-477.
Yang DY, Eng B, Waye JS, Dudar JC, and
Saunders SR. 1998. Improved DNA extraction from ancient bones using silica-
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Yang DY, Eng.B. and Saunders SR. 2003.
Hypersensitive PCR, ancient human mtDNA and contamination. Human Biology, 75:355-364.
* ---- Summarized from an article (Yang, D.Y. 2003. Contamination
Controls and Detection in Ancient DNA Studies. Acta Anthropologica Sinica 22:163-173.)
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