- Laboratory set-up
- aDNA clean room
- Post-PCR lab
- quantitative real-time PCR
- SABC (state agricultural biotechnology centre)
DNA is an abundant and stable molecule: given the right conditions it can persevere in the environment for thousands, possibly millions, of years. However, two decades of research in aDNA has demonstrated that characterising DNA sequences from degraded tissues requires considerable care. One of the greatest challenges is identifying authentic aDNA sequences away from contamination with exogenous (modern) DNA. Isolated pre- and post-PCR labs are strictly necessary for aDNA practices, as is wearing protective clothing (gloves, body suits, boots, facemasks) to minimise the deposition of modern DNA on ancient specimens. Another factor affecting the fidelity of aDNA sequence data is that post-mortem hydrolytic and oxidative damage to the DNA results in miscoding lesions (incorrect base incorporation). An extreme example of both contamination and damage was an aDNA extract from a single Viking tooth that yielded 23 different human mtDNA sequences (Dr Tom Gilbert, unpublished data). In 2000, a set of guidelines; “Ancient DNA do it right or not at all” (Cooper and Poinar, 2000) – was published to make researchers aware of the pitfalls of dealing with degraded DNA. Their recommendations included the use of dedicated laboratories, extraction controls, cloning and independent replication. Subsequent publications have refined and highlighted deficiencies with these guidelines (Gilbert et al. 2005), but it is clear that many molecular and procedural safeguards need to be employed to ensure the sequence data generated is in fact authentic. The information on this web page describes the set-up of the murdoch aDNA lab including the safeguards employed to minimise external contaminants.
Protective body suits used to minimise contamination
It is common place for labs who deal with low copy number DNA (usually aDNA and forensic labs) to physically isolate pre and post PCR areas. A single PCR will generate approximately 10,000,000,000,000 identical copies of the from a single starting copy of a DNA target. The majority of samples used in aDNA analyses are highly degraded and can contain little or no DNA. The potential to contaminate an ancient sample (or lab) with previously amplified DNA is very high and since the starting copy number is low it can be very problematic. Moreover, PCR carryover is very difficult to discriminate from authentic DNA as they will have a very similar genetic make-up.
The obvious solution to PCR carryover is to isolate pre and post PCR areas - and minimise/eliminate movement from a post-PCR lab into the pre-PCR environment (or clean room). Nearly all reputable ancient DNA labs comprise of a cleanroom (for DNA extractions, sample storage and PCR set-up) and a Post-PCR lab (for analysis of PCR products). The murdoch aDNA lab is no exception - a description of both the clean room and the post-PCR lab can be found below.
aDNA clean room
The ancient DNA clean room (commissioned in June 2006) is two purpose outfitted rooms dedicated to the extraction of low copy number DNA from "old" samples (bone, sediments and coprolites). The rooms have a number of safeguards (both equipment and procedural) which aim to reduce contamination of "ancient" specimens with modern DNA and/or previously amplified PCR products. However, processing a sample in a clean room will not guarantee a contamination "free" DNA extract as the sample may of been contaminated prior to arrival in the lab (for example bones washed and handled at an excavation site). In essence the goal of the clean room is to minimise further contamination.
A movie which takes a quick tour into the aDNA lab will be downloadable from this page will soon be available.
Personal wishing to enter the clean room must not of entered a post-PCR area that day - the aim of this procedural restriction is to minimise the possibility of physically carrying PCR products into the lab. DNA is everywhere - while this is is beneficial for forensic purposes it is detrimental to aDNA analyses. When you handle a sample (without gloves) or breath on it you are depositing your DNA on that sample. Entrance into the aDNA clean room is through a staging room where researchers get suited up (bodysuits, gloves, boots, face masks - pictured). The storage room also functions as a storeroom for lab supplies. Air filtering devices containing UV lights are in constant operation in this area.
PCR's that are set up in the clean room are transported to the Post-PCR lab where they are thermocycled and analysed by electrophoresis. Similar to most molecular facilities the lab is outfitted with thermocyclers, qPCR facilities, electrophoresis equipment and cameras for photographing gels. The post-PCR lab is also used for the extraction, amplification and sequencing of "modern" DNA samples. This PC2 lab is a quarantine facility and is used for the storage of customs seizure material and wildlife forensic case work. The lab is certified by the biosafety committee to use recombinant DNA techniques (cloning) to characterise PCR products generated from ancient DNA. An an avi movie that takes a quick "spin" around the aDNA and Wildlife Genetics Labs can be viewed (caution movie size approximately 4 meg).
quantitative real-time PCR
Use of quantitative PCR to optimise aDNA extractions
MyiQ real-time PCR system
Until recently, the ancient DNA field has been driven by empirical approaches to DNA recovery. Researchers have typically divided samples into two categories: those that will amplify and those that will not. Little thought has been give to the reasons for success or failure. Quantitative real-time PCR (qPCR) is a fluorescence detection system that monitors DNA amplification during the exponential phase of the reaction. In the exponential phase, the amount of fluorescence is directly proportional to the number of starting template molecules in the reaction. qPCR data are important in aDNA research and forensics for several reasons, foremost being that the results of different extraction methodologies can be compared accurately. The top panel of the adjacent figure shows a quantitative assay comparing two extraction methodologies The data show that (for this substrate) the phenol-based extraction has approximately 256 times more copies than the silica extraction. This approach is the basis for optimising extraction procedures for ancient DNA samples where methodologies can be “tailored” to suit the substrate or the environmental conditions (e.g., different pH, temperatures, moisture). The removal of inhibitors such as humic acids and tannins is also crucial to successful amplification. Many ancient DNA PCR reaction "fail" not due to the lack of DNA but due to the presence of inhibitors which inhibit polymerase activity. qPCR provideds a means by which to identify when PCR inhibition is occuring. qPCR offers another key benefit to aDNA research programs by determining the number of starting template molecules in a PCR. Reactions that start off a small number of starting templates (for example 100 copies) are more susceptible to contamination from exogenous sources and more likely to yield sequence data containing post-mortem DNA damage or contamination. Absolute quantification of targets is achieved through the use of a DNA standard shown in panel B in he adjacent figure.