SENSITIVITY AND CONTAMINATION
Figure 5.4 (a) The target sequence on the template DNA molecule has been amplified using two different primer pairs: using primers a + c generates a 460 bp product and the a + b primer pair leads to a 260 bp product. (b) Following amplification of template DNA in two separate reactions the products were separated on a 2% agarose gel and stained with ethidium bromide. The amplified products can be seen in lane 2 (460 bp) and lane 4 (260 bp). Lane 1 contains a 100 bp ladder and lanes 3 and 5 contain negative controls from bone and will inhibit PCR unless removed as it binds ions such as magnesium ions that are essential for PCR . In a forensic science context, the blue dye in clothing such as denim, called indigo, has an inhibitor effect on PCR . Extraction methods have been developed to remove commonly encountered PCR inhibitors and, for example, the silica binding methods that are commonly used in forensic analysis are effective at removing most inhibitors whereas the methods that produce a cruder extract such as the Chelex® resin are more prone to inhibition. When it is not possible to remove all the potential inhibitors from a DNA extract, the addition of theproteinbovineserumalbumin(BSA)tothePCRcaninmanycasespreventorreduce the inhibition of the Taq polymerase. The BSA acts as a binding site for some inhibitors and can competitively remove or reduce the concentration of the inhibitor [30, 38]. The action of inhibitors can be detected, for example by spiking a PCR with a known amount of DNA, this alerts the analyst that further purification steps are required .
Sensitivity and contamination
The great advantage of PCR is that it will amplify DNA from a template of only a few cells. This high level of sensitivity can also be a potential disadvantage, as DNA from incidental sources can be present and contamination can be introduced. Throughout the handling and analysis of DNA samples extreme care needs to be taken to minimize the chance of introducing this extraneous DNA. When samples are collected from the scene of an incident, there may be cellular material from persons who had been present at the scene prior to the incident and hence DNA profiles will be generated from people unconnected with the incident. This type of DNA can be termed as incidental as it is not a contamination of the samples. At the time of the incident there is an opportunity for transfer from the perpetrator and
it is this cellular material that is pertinent to the investigation. Consider an event such as theft from a house. Prior to the incident there will be cellular material from the owners and from any recent visitors. At the time of the break in there may be transfer from the thief. If the incident is discovered by a neighbour then they will introduce their cellular material after the incident and prior to the scene being secured. When the police are called they have the potential to introduce their cellular material. Once the scene is secured then those entering should be wearing full protection to minimize the opportunity for transfer of their cellular material [41, 42]. If there is any introduction of DNA from those at the crime scene, during collection and transportation, or from laboratory staff, then this is considered as contamination.
The PCR laboratory
Once evidential samples have reached the forensic laboratory there is further potential to introduce contamination. A fundamental feature of PCR laboratories, to reduce the possibility of introducing contamination, is that they are clearly divided into pre- and post-PCR areas (Figure 5.5).
Once the samples reach the laboratory, potential contamination comes from the reagents, equipment and the forensic scientists undertaking the analysis. To prevent contamination being introduced from the scientist, protective clothing is worn, includ- ing a lab coat, gloves, a face mask, safety glasses/visor and a head cover. Even with these precautions it is still possible to get the scientist’s DNA profile showing up – a database of all the people who enter/work in the laboratory can be used to detect when contamination could have been introduced within the laboratory. When laboratories are engaged in analysing both samples from suspects and from crime scenes it is common to have dedicated areas for the two classes of sample; this prevents any potential cross-contamination of crime scene and suspect DNA. Special dedicated facilities may also be used when dealing with samples that contain very small amounts of DNA, such as hair shafts. DNA extraction and PCR set-up are commonly carried out in specialized clean hoods that provide a very controlled environment. The hoods have stainless steel surfaces and are easy to keep clean; they have filtered air to prevent any dust or other contaminant getting into the reaction, and they are fitted with a UV-light source that is used to remove any contaminating DNA effectively. The pipetting of any liquids involved in the extraction and PCR set-up is performed using pipette tips with barriers to prevent any DNA carry over. During the DNA extraction process negative control extractions must always be car- ried out to monitor for contamination; positive controls that involve extracting material similar to the casework samples, for example buccal swabs or blood stains, can be carried out to monitor that the extraction and amplification procedures are working