Trace Research Advanced Ultra-Clean Environment Facility
Protective body suits used to minimise contamination
The ancient DNA clean room is located within the Trace Research Advanced Ultra-Clean Envrionment (TrACE) Facility at Curtin University. There are two purpose laboratories within the 400m2 facility dedicated to the extraction of low copy number DNA from ‘old’ or degraded samples (bone, sediments and coprolites). The rooms have a number of safeguards (both equipment and procedural) which aim to reduce contamination. The TrACE has extremely low ultimate particle counts that are achieved with ‘spaces within spaces’ and HEPA filtration. However, processing a sample in an ultra-clean environment will not guarantee a contamination free DNA extract as the sample may of been contaminated prior to lab arrival (for example bones that have been handled). In essence the goal of the ultra-clean environment is to minimise further contamination.
Personal wishing to conduct work in the ultra-clean environment must not have 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 ultra-clean environment DNA is everywhere - while this may be beneficial for many forensic circumatances, it is detrimental to analyses involving low copy number (LCN) DNA. When you handle a sample (without gloves) or breath on it you are depositing your DNA on that sample. Entrance into the ultra-clean environment is through a staging room where researchers get suited up (bodysuits, gloves, boots, face masks) prior to progressing through to the exclusion space and laboratories (clean 'spaces within spaces’ photograph below).
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 (see below), electrophoresis equipment and cameras for photographing DNA gels. The post-PCR lab is also used for the extraction, amplification and sequencing of "modern" DNA samples.
quantitative real-time PCR
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 forensic research 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 DNA extractions "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.