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.

The development of good quantitative assays allows systematic assessment of DNA preservation. qPCR approaches will not only maximise our ability to extract and amplify older and more degraded templates, but will also contribute to the prediction of other sites favourable to the preservation of aDNA. 

Our labs are equipped with a single colour Bio-Rad MyIQ and an 3 colour ABI StepOne real-time detection system. Our labs are grateful to the Murdoch University School of Biological Sciences and Biotechnology equipment fund for assistance in purchasing these systems. 

Content and images in this website cannot be reproduced without permission and may be under copyright restrictions. University Disclaimer: This Content is the responsibility of the institution, organisation, or individual that has provided it. No endorsement, approval or authorisation is attributable to Murdoch University for information, ideas, views, or expressions of opinion within this Content. In no event shall Murdoch University be liable for any damages whatsoever resulting from any action arising in connection with the use of information within this Content, or its publication by means of a Server run on a computer on the Murdoch University Network, including any action for infringement of copyright or defamation.