The Primer
The high sensitivity of the polymerase chain reaction (PCR)—theoretically with lower limits of detection as little as a single template molecule; practically, 10 to 100 copies for many assays as run—is one of its greatest strengths, but also its greatest weakness. As the method works through creating copies of its target, any positive sample can lead to large numbers of molecules which can in turn contaminate subsequent reactions and cause false positive results.
The high sensitivity of the polymerase chain reaction (PCR)—theoretically with lower limits of detection as little as a single template molecule; practically, 10 to 100 copies for many assays as run—is one of its greatest strengths, but also its greatest weakness. As the method works through creating copies of its target, any positive sample can lead to large numbers of molecules which can in turn contaminate subsequent reactions and cause false positive results.
To get a sense of the scope of this, consider a successful “average” 25μl PCR somehow getting opened and spilled in the lab. This would contain on the order of 10^12 template copies (amplicons); in other words, if a thorough cleaning reduced this by a million fold, you’d still have a million amplicon copies “floating around,” each of which could contaminate a reaction. If you’re fortunate enough to have never experienced this first-hand, you can thank the widespread acceptance of real-time PCR methods, which do away with having to open reaction tubes post amplification, and perhaps gain an appreciation of why anyone who has been through the experience treats the risk as real and ever-present.
