Laboratory Facilities | Murdoch University ancient DNA research laboratory | Mike

quantitative real-time PCR

Fri, 16 Jun 2006 10:15:54 +0800

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.

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.

This lab is equipped with a Bio-Rad MyIQ real-time detection system (pictured). The lab is grateful to the School of Biological Sciences and Biotechnology equipment fund (2006) for financing this system.

Post-PCR lab

Thu, 15 Jun 2006 21:33:00 +0800

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).

SABC (state agricultural biotechnology centre)

Thu, 15 Jun 2006 17:09:00 +0800

The Murdoch aDNA lab is lucky enough to have access to the equipment and expertise (Dr David Berryman, Frances Briggs) located in the state agricultural biotechnology centre (SABC). This equipment in this facility include:

- 3730 capillary sequencer

- Pyrosequencing

- PCR facilities

- Centrifugation facilities

- Spectrophotometers

- Proteomics/2D gel facilities

- Mass spec

- Robotic platforms

aDNA clean room

Tue, 06 Jun 2006 23:06:55 +0800

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.

This clean room is under positive air pressure. The air handelling unit mounted on the wall (pictured) is HEPA filtered, ensuring clean air circulates through the clean room and into the staging room. The airflow then exits the lab and ensures a constant pressure gradient always exists. The clean room is equipped with two glove boxes (pictured) which are regularly treated with UV lights. One glove box is used for DNA extractions and aliquoting DNA , the other for reagent handling (no DNA). Other equipment include UV lighting, centrifuges with aerosol control and a ductless cabinet for dealing with toxic chemicals. Work surfaces, reagents and equipment is also routinely irradiated or cleaned with bleach to remove any DNA.

The clean room environment plays a key role in minimising contamination - so do do procedures used in DNA extraction and PCR set-up. These procedures include:

- Multiple DNA extraction and PCR controls

- Using reagents/labwear suitable for use in a DNA clean room (e.g. Filter tips)

- Changing gloves often

- Processing only single samples or small batches

- common sense approaches to minimise handling and potential of cross contamination

Laboratory set-up

Tue, 06 Jun 2006 13:42:55 +0800

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.