The Polymerase Chain Reaction, commonly known as PCR, stands as a fundamental technique in molecular biology, widely used for amplifying specific DNA sequences. This method allows scientists to create millions of copies of a particular DNA segment from even a tiny initial sample. However, the sensitivity that makes PCR so effective also makes it vulnerable to contamination. The presence of unwanted foreign DNA or nucleic acids within a PCR reaction can compromise the accuracy and reliability of the results.
What PCR Contamination Is
PCR contamination refers to the unintended presence of extraneous nucleic acids, whether DNA or RNA, within a reaction mixture that can interfere with the intended amplification of a target sequence. This interference primarily leads to false-positive results, where a target appears to be present when it is not, or inaccurate quantification, particularly in quantitative PCR (qPCR) applications. Such issues can waste valuable time, expensive reagents, and lead to erroneous conclusions in research or diagnostic settings. Contaminants can broadly be categorized based on their origin.
Where Contamination Comes From
One of the most frequent and challenging sources of PCR contamination originates from previously amplified DNA, known as carryover contamination. PCR products from prior experiments are present in extremely high concentrations, making them highly prone to aerosolization and transfer. Even minute amounts of this amplified DNA can serve as a template in subsequent reactions, leading to false positives.
Beyond previous PCR products, exogenous DNA or RNA from various sources can enter the reaction. Human DNA, shed from skin cells, hair, or saliva of laboratory personnel, is a common contaminant. Environmental DNA can also settle into open tubes or onto work surfaces. DNA from other samples being processed in the same laboratory space can also cross-contaminate.
Reagents themselves can be a source of contamination if they are not of high purity or are handled improperly. Components like PCR-grade water, primers, DNA polymerases, and buffers can become contaminated during manufacturing, shipping, or handling.
The laboratory environment itself contributes to contamination through various pathways. Aerosols generated during pipetting can spread DNA particles throughout the workspace. Contaminated surfaces, including laboratory benches and equipment exteriors, can harbor DNA that transfers to samples. Inadequate airflow or cross-ventilation between pre-PCR and post-PCR areas can also facilitate the movement of contaminants.
Laboratory equipment, if properly maintained, can also serve as a reservoir for contaminants. Pipettes can accumulate DNA residues if not regularly cleaned and decontaminated. Reusable tube racks, rotors, and thermocycler lids can harbor amplified DNA or environmental contaminants if not routinely disinfected. Dedicated equipment for pre-PCR and post-PCR activities is therefore important to minimize cross-contamination risks.
Preventing Contamination
Implementing physical separation of laboratory activities is an effective strategy to prevent PCR contamination. This involves designating distinct, physically separated areas or rooms for different stages of the PCR workflow. Maintaining dedicated spaces ensures that high concentrations of amplified DNA from post-PCR work do not mix with pre-PCR reagents or samples.
A unidirectional workflow complements physical separation by dictating the movement of personnel and materials from clean areas to potentially contaminated areas, never in reverse. This means moving from reagent preparation to sample addition, then to amplification, and finally to post-PCR analysis, without backtracking. Establishing separate sets of equipment for pre- and post-PCR areas further reinforces this one-way flow.
Careful reagent handling is important for contamination control. Always use certified PCR-grade, nuclease-free water and reagents, as these have undergone strict quality control to ensure the absence of contaminating nucleic acids and enzymes that degrade DNA. Aliquoting reagents into small, single-use portions minimizes the number of times stock solutions are opened and exposed to the environment. This practice helps to preserve reagent integrity and reduce the risk of introducing contaminants through repeated access.
Strict equipment practices are also necessary to maintain a clean environment. Always use aerosol-barrier, or filter, pipette tips, which contain a filter barrier to prevent aerosols from entering the pipette barrel and contaminating subsequent samples. Regularly clean and decontaminate pipettes, tube racks, and other shared equipment with a 10% bleach solution, followed by a rinse with nuclease-free water, or specialized DNA-degrading solutions. Employing dedicated sets of pipettes and other small equipment exclusively for pre-PCR tasks and another set for post-PCR work further isolates potential contaminants.
Consistent use of personal protective equipment (PPE) helps to minimize the introduction of human DNA. Always wear fresh, clean laboratory coats dedicated solely to PCR work and change gloves frequently, especially when moving between different workflow areas or after touching potentially contaminated surfaces. Disposable face masks can also be considered in sensitive applications to reduce contamination from breath and saliva.
The inclusion of negative controls in every PCR run is a simple yet effective monitoring tool. A no-template control (NTC), containing all reaction components except the DNA template, should be run alongside experimental samples. A positive signal in an NTC immediately indicates contamination, alerting researchers to a problem before results are misinterpreted. This serves as a continuous check on the purity of reagents and the cleanliness of the workspace.
Routine decontamination of workspaces and equipment is a continuous preventive measure. This involves daily cleaning of laboratory benches with a 10% bleach solution, which effectively degrades DNA, or commercially available DNA-degrading reagents. Following bleach treatment, surfaces should be wiped down with ethanol to remove residues. Ultraviolet (UV) light exposure, often used in PCR workstations or laminar flow hoods, can also be employed to decontaminate surfaces by damaging nucleic acids.
Detecting and Fixing Contamination
Detecting PCR contamination often involves observing unexpected results in a typical PCR experiment. A primary indicator is the presence of unexpected DNA bands on gel electrophoresis, especially within the no-template control (NTC) lane, which should ideally show no amplification. If the NTC produces a band corresponding to the target amplicon or other non-specific products, it strongly suggests a contamination issue.
Other signs of contamination include inconsistent or uninterpretable results across multiple experimental runs, or positive signals in samples that are expected to be negative. In quantitative PCR (qPCR), contamination might manifest as an amplification curve in the NTC or a significantly lower cycle threshold (Ct) value than expected for a negative sample. These anomalies indicate that extraneous DNA is being amplified, skewing the data.
When contamination is suspected, a systematic troubleshooting approach is necessary to identify and resolve the source. The initial step involves re-running all controls with fresh reagents and consumables to confirm contamination. If the issue persists, individual reagents should be tested for purity by setting up small reactions containing only one component in question along with the necessary controls.
A thorough decontamination of the entire workspace and all equipment used in the PCR setup is often required. This involves cleaning benches, pipettes, racks, and thermocyclers with DNA-degrading solutions like a 10% bleach solution or commercial DNA removers, followed by a rinse with nuclease-free water. Preparing fresh stocks of all PCR reagents can eliminate contamination.
If the problem continues, replacing potentially contaminated equipment or consumables might be necessary. In some cases, if primer-dimer amplification is contributing to false positives, redesigning or re-synthesizing primers can mitigate the issue. Ultimately, proactive prevention through rigorous laboratory practices is far more effective and less time-consuming than reactive troubleshooting once contamination has occurred.