Sunday, 19 June 2011

Understanding PCR Contamination

Early in the history of PCR, its pioneers recommended certain techniques and practices for preventing and recognizing contamination.  A parallel with sterile technique in medical clinics is often drawn.  By definition, sterile means the absence of all living organisms, including bacteria and viruses.  For example, sterile technique is used when working in the vicinity of an open wound.  PCR technique is similar to sterile technique and even borrows many basics concepts from it.  This includes the use of sterile instruments and pipettes that may contact the samples under analysis.  Similar sterile techniques are used by scientists who grow cells in culture dishes which are easily contaminated. 
PCR technique differs from sterile technique in that a clinically sterile solution or instrument may still harbor DNA.  DNA usually survives heat sterilization used to make clinical solutions and instruments sterile.  Presence of a single bacterium or virus would violate sterility.  Doctors and nurses think in terms of a sterile "field", an area where everything present is sterile and meticulous effort expended to maintain that condition of sterility.  Once a non-sterile object, or even one whose sterility can be questioned, enters the area, the field is no longer considered sterile.  Sterile technique training involves the development of a mental image of the sterile field and how to protect it.  Finally, one does not assume success, just because the mental picture seems un-breached.  Post-sterile technique practices include monitoring patients for fever and other sign of infection and giving antibiotics in advance, actions that basically assume that technique may well have failed.  As rigorous as sterile technique concepts are, PCR technique involves the same concepts and more since a properly sterilized item of equipment, or a sterilized solution, may contain DNA that would potentially influence a PCR.  For example, large pressure cookers called autoclaves are effectively used to sterilize instruments and some solutions by heating to temperatures (slightly higher than the temperature of boiling water) that most infectious organisms can't survive.  However, such temperatures are insufficient to destroy contaminating DNA.  Thus, autoclaves, while they achieve the condition of clinical sterility by getting rid of all bacteria, are not infallible for PCR.  In short, PCR technique needs to go beyond sterile technique.  Disposable instruments and pipettes and proper design of PCR laboratories are helpful considerations in this regard. 
Good PCR technique is no guarantee that contamination didn't influence the results.  Steps must be taken to try and detect contamination.  Negative controls are blank PCRs that have all the components of the evidentiary PCRs but have no other DNA added intentionally.  Fortunately, there are often two negative controls used, one when the DNA is extracted, and another when the PCR is set up.  Any PCR signal in the negative control would warn that contamination has occurred.  Unfortunately, the negative controls are virtually the only warning of PCR contamination.  Negative controls may alert the analyst to general contamination occurring within the lab or the lab reagents.  These controls don't offer protection against contamination occurring before the samples arrived at the PCR lab.  Negative controls also can't rule out contamination of individual samples.  The individual samples lack individual signs of contamination if it occurs.  Unlike a human patient, a PCR is incapable of showing signs of infection (contamination) such as fever or undue pain.  PCRs also have no immune system to ward off contaminants.  
It is often said that the most critical source of PCR contamination is DNA from previous PCRs.  Again, a PCR produces many DNA copies of the target DNA sequences.  Due to shear number, these copies (called amplicons) are a hazard for future PCRs.  In terms of DNA typing, stray amplicons could contribute single or multiple alleles to a genetic profile.  This would manifest itself in the form of producing, for example, an extra dot on a DQA1 or PM typing strip or an extra band in an STR profile.  The fact that the contaminating dot or band is in fact extra may or may not reveal itself.  Thus, amplicons can lead to mistyping.  
However, a more dangerous source of contamination is what is called genomic DNA.  This is DNA that hasn't yet been amplified.  Genomic DNA doesn't have the high concentration of the target DNA copies but is a hazard because genomic DNA could produce an entirely false DNA profile.  Full profile contaminants have been documented on multiple occasions and in multiple laboratories.  Partial profile contaminants are more common and sometimes constitute a poorly recognized risk in using partial profiles in evidentiary samples as evidence.  When contamination occurs there is rarely any way to confirm how it happened.  
For example, suppose evidence item #1 has little to no DNA or has DNA degraded beyond the ability to function in a PCR.  Suppose further that item #2 is a defendants reference blood stain that would typically have a high concentration of undegraded genomic DNA from the defendant.  If item #2 comes in close proximity with item #1, or comes in contact with item #1, the genomic DNA from item #2 may contaminate item #1.  Subsequent DNA typing of contaminated item #1 will give the false impression that the defendant contributed DNA to item #1 during a crime.  Similarly, when there are multiple items of evidence with some having larger amounts of DNA and some much lower, cross-contamination is an important consideration.  
This is not to say that all PCR-based results are due to cross-contamination.  However, the ease of cross-contamination and its potentially misleading effects may sometimes be under-appreciated, especially in the context of match probabilities reported to be extremely rare.

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