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


Gill P, Ivanov Pl, Kimpton C, Piercy R, 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 origin of modern humans. Cell 90:19-30.

MacHugh DE, Edwards CJ, Bailey JF, Bancroft DR, and Bradley DG. 2000. The extraction and analysis of ancient DNA from bone and teeth: a survey of current methodologies. Ancient Biomolecules 3:81-103.

Mullis KB and Faloona FA. 1987. Specific synthesis of DNA in Vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology 155:335-350.

O'Rourke DH, Hayes MG, and Carlyle SW. 2000. Ancient DNA studies in physical anthropology. Annual Review of Anthropology 29:217-242.

Pääbo  S, Higuchi RG, and Wilson AC. 1989. Ancient DNA and the 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- based spin columns. American Journal of Physical Anthropology 105:539-543.

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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