Saturday, September 25, 2010

PCR Contamination

            It is worth considering contamination early in this discussion since this is a well-recognized limitation.  Unfortunately, the importance contamination in PCR is often underestimated.  PCR copies DNA efficiently if the initial DNA is in good condition.  A single DNA entity (molecule) can become millions or billions of DNA molecules in about three hours.  The PCR process is sometimes compared to a Xerox machine since many copies are made.  While initially, this is a useful comparison, it doesn't communicate the true, chain-reaction nature of PCR.  In PCR, the original DNA is copied, then the copies are copied, those copies are copied and so on.  This results in dramatic increases in the amount of DNA that couldn't be easily accomplished in the Xeroxing analogy.  The PCR process deserves its classification as a "chain-reaction" because it has much in common with other chain reactions such as avalanches. 
            PCR is also very similar to what happens when a clinical infection occurs.  Clinicians have known for many years that a single germ (bacterial cell or virus) contaminating a wound can produce a massive infection.  Similarly, a DNA molecule can contaminate (infect) a PCR and become a significant problem.  The ability of small amounts of DNA to produce false and misleading results is well-known and well-documented within the research community, where the technology originated.  Anyone who has caught a cold from an unknown source, or who has a pollen allergy should have some sense of how easily PCRs are contaminated.  Actually, it is probably easier to contaminate a PCR than to catch a cold since unlike our bodies, PCRs lack immune systems.  The only protection PCRs have is the technique of the analyst, use of control samples to monitor contaminants and careful interpretation. 
            Prevention of false results involves the use of carefully applied controls and techniques.  As described later, such controls and techniques can rarely guarantee that contamination hasn't influenced the results.  In forensic DNA testing, some of the scientifically worst-case scenarios can be prevented by keeping DNA samples from known individuals well out of range of other items of evidence at all stages.  Most forensic DNA laboratories perform negative controls, blank samples that will often detect contaminants in the laboratory.  The blanks detect contaminants by showing partial or full DNA profiles representing the contaminants.  Alternatively, the blank may show no profile, consistent with, but not proving that contamination didn't occur.  Unfortunately, a few forensic DNA laboratories omit their controls.  A few favor the controls by using special equipment on them, or by not carrying them through the entire procedure.  Such practices are hazardous, especially when an important evidentiary sample has a low amount of DNA, degraded DNA, or otherwise presents as a minimal or partial (see below) sample.  In short, while PCR is a useful research tool, all applications require extreme care and vigilance.  

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