Polymerase Chain Reaction (PCR) is a powerful molecular biology technique used to amplify specific DNA sequences, creating millions of copies from a small initial sample. To ensure the reliability of experimental outcomes, controls are routinely incorporated. These controls act as benchmarks, verifying that the experimental setup works as expected and that observed results are genuinely due to the variables being studied.
What is Negative Control PCR
A negative control in PCR, also known as a “no template control” (NTC), detects contamination within the experimental setup. This control contains all reagents for standard PCR amplification—primers, DNA polymerase, nucleotides (dNTPs), and reaction buffer—but nuclease-free water is added instead of sample DNA. The expectation is no DNA amplification, resulting in no detectable product. If amplification occurs, it indicates contaminating DNA, which could originate from the laboratory environment or reagents.
Why Negative Controls are Crucial
A negative control in every PCR experiment directly checks for contamination. PCR is an exceptionally sensitive technique, capable of amplifying even minute quantities of DNA. This high sensitivity means stray DNA from previous experiments, dust, or skin cells can lead to amplification and false positive results. Without a negative control, it is impossible to determine if a positive signal from a sample is truly from the intended target DNA or due to unwanted contaminating DNA.
Unidentified contamination can lead to misleading conclusions, wasting time and resources. The negative control validates the absence of extraneous DNA, ensuring the integrity of experimental results.
Interpreting Negative Control Outcomes
A successful negative control in PCR shows no DNA amplification. When analyzing PCR products, through gel electrophoresis, a valid negative control lane appears blank, with no DNA bands. This confirms reagents are free from contaminating DNA and no unintended amplification occurred, increasing confidence in positive results from experimental samples.
Conversely, a failed negative control shows DNA amplification, appearing as one or more bands on the gel. If a band is observed, especially one similar in size to the expected product, it indicates contamination of reagents, equipment, or the workspace with target DNA. Amplification in the negative control compromises the entire PCR run, making it impossible to distinguish true positive results from contamination. In such cases, experimental sample results are unreliable, and the experiment should be repeated after addressing the contamination. Sometimes, smaller, diffuse bands, known as primer dimers, may appear, which are typically not the target amplification and are less concerning than a full-sized product band.
Preventing Contamination in PCR
Implementing strict practices to prevent contamination is important due to PCR’s high sensitivity. A common strategy involves establishing separate workstations for pre-PCR activities (reagent preparation and reaction setup) and post-PCR analysis (gel electrophoresis and product handling). This physical separation helps minimize the transfer of amplified DNA products, a major source of contamination.
Using dedicated equipment, such as pipettes and centrifuges, for each area is also advised. Employing sterile, filtered pipette tips prevents aerosol formation and cross-contamination between samples. Regular decontamination of work surfaces with cleaning agents like a 10-15% bleach solution, followed by a 70% alcohol wipe, before and after each PCR session helps eliminate residual DNA. Changing gloves frequently and using fresh reagents are additional measures to maintain a clean and reliable PCR environment.