CONTAMINATION CONTROLS AND DETECTION IN ANCIENT DNA STUDIES

With the advent of the PCR technique in molecular biology [20], DNA can now be extracted and analyzed from ancient remains [1,51]. Ancient DNA studies hold great potential for anthropologists and archaeologists to address many important issues that cannot be well dealt with in conventional ways [5,9—14,16—17]. However, the field is siill full of technical and interpretive challenges [28]. The most difflcult 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 [7]. Contamination controls and detection therefore become extremely important in ancient DNA studies. This paper 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 [28]. It is crucial to physically separate pre-PCR and post-PCR work [5]. 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 sliould 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 [5,30]. 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 [31]. UV can cause DNA to crosslink and preclude it from use in PCR amplification [7]. 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. PCR Amplification of Ancient DNA The great difficulty in the amplification of ancient DNA is due to physical and chemical degradation of DNA templates [22,51]. 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 [22]. 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 [38]. 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 [7]. For ancient human mtDNA,we have found that multiple quantified positive controls should also be

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