The Polymerase Chain Reaction (PCR) is a molecular biology technique that creates millions to billions of copies of a specific DNA segment rapidly. This high amplification ability makes PCR sensitive, capable of detecting minute quantities of target genetic material. However, this sensitivity also makes the reaction highly susceptible to contamination by unwanted foreign DNA or RNA, which interferes with the accuracy of results. Understanding the nature and origin of these contaminating materials is crucial for maintaining the reliability of any PCR experiment.
Understanding the Types of Contaminants
Contamination in a PCR setting falls into two distinct categories based on the source of the foreign genetic material. The most frequent contaminant is amplicon carryover, which consists of previously amplified product from other PCR reactions performed in the same laboratory. This material is problematic because it is present in extremely high concentrations, sometimes reaching $10^{12}$ copies per milliliter, and can easily outcompete the new, intended target DNA.
The second category is template contamination, which involves non-target genetic material that has not been previously amplified. This material can include human DNA shed by laboratory personnel, environmental microbial DNA, or DNA from a different sample run concurrently. Although template contamination is usually present at a lower concentration than amplicon carryover, it provides a false template for the polymerase enzyme to copy. This can lead to spurious amplification and complicate the interpretation of results.
Identifying Contamination Sources
Contaminants enter the PCR workflow through several physical pathways originating from routine laboratory practices and reaction components. One common vector is reagent contamination, where foreign DNA is introduced through components like water, primers, buffers, or the enzyme mix used to assemble the reaction. While manufacturers take precautions, trace amounts of DNA can occasionally be present and subsequently amplified.
Another pervasive source is aerosol and environmental contamination, particularly from fine droplets generated during pipetting, vortexing, or opening tubes containing highly concentrated amplicon DNA. These microscopic aerosols drift through the air and settle on laboratory surfaces, equipment, or open reaction tubes. Improper airflow or the lack of designated workspaces exacerbates this spread.
Contamination is also introduced via personnel and shared equipment, highlighting the need for meticulous laboratory hygiene. Surfaces such as benchtops, thermal cyclers, and shared pipettes can harbor DNA residues transferred by improperly gloved hands or accidental splashing. Any piece of equipment that moves between the post-amplification area and the pre-amplification setup area acts as a bridge for transferring millions of copies of amplified product back into new reactions.
Impact on Experimental Results
The introduction of foreign genetic material has direct consequences for the reliability of experimental results. The most common outcome is the generation of false positive results, especially with amplicon carryover. A sample negative for the target sequence will incorrectly register a positive signal because the contaminating DNA is amplified. False positives are particularly damaging in diagnostic or forensic applications, as they undermine the accuracy of the entire testing process.
Contamination can also lead to false negative results if the contaminating DNA sequence shares primer binding sites with the actual target. In this scenario, the overwhelming presence of the contaminant competitively inhibits the amplification of the true target DNA, preventing its detection.
Prevention and Mitigation Strategies
A robust defense against contamination relies on a combination of strict physical segregation, routine decontamination procedures, and disciplined procedural habits.
Unidirectional Workflow and Segregation
The most effective strategy is implementing a unidirectional workflow, which physically separates the pre-PCR setup area (where reagents and samples are prepared) from the post-PCR analysis area (where amplified products are handled). Designated equipment, including dedicated sets of pipettes, tube racks, and vortexers, must remain isolated within each zone to prevent the transfer of amplified DNA.
Decontamination Methods
Routine decontamination of the work environment is necessary for eliminating residual DNA from surfaces and equipment. Chemical cleaning with a 10% bleach solution (sodium hypochlorite) is effective at destroying DNA on benchtops and non-electronic equipment. For sensitive equipment, shortwave ultraviolet (UV) light can be used to damage the DNA structure, making it unsuitable for amplification.
Laboratories can also employ specialized enzymatic strategies, such as the Uracil-DNA Glycosylase (UNG) system, for decontamination of the master mix. This involves incorporating dUTP instead of dTTP into the reaction mix, causing newly amplified product to contain uracil. Before the next reaction, UNG is added to excise any uracil-containing contaminant DNA, neutralizing it without affecting the original sample DNA.
Procedural Habits
Meticulous procedural habits must be maintained by all personnel to minimize the risk of introducing template contamination. This includes the consistent use of barrier-filter pipette tips, which physically block aerosols from entering the pipette barrel. Frequent changing of gloves, especially when moving between work zones or handling highly concentrated products, prevents the transfer of DNA from hands to equipment and reagents.