PCR is a powerful tool in molecular biology, widely employed in diagnostics to detect and amplify specific DNA sequences. However, the success of PCR can be hindered by various inhibitors present in complex sample matrices, potentially leading to false-negative results.
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Understanding PCR Inhibition Mechanisms
PCR inhibition can arise from various sources, impacting DNA polymerization or fluorescence detection. Inhibitory molecules may directly affect polymerase activity, bind to nucleic acids, or alter ion content, hindering primer extension.
Fig. 1 PCR inhibitors possess the potential to impact any of the essential subreactions in PCR (Sidstedt M., et al. 2020).
DNA Polymerase Inhibitors
Certain substances, such as hemoglobin and haematin, have been identified as DNA polymerase inhibitors, causing reduced polymerase activity and amplification efficiency. In soil samples, humic substances like humic acid and fulvic acid have long been recognized as PCR inhibitors, possibly due to their binding to DNA or direct impact on polymerase activity.
Fluorescence Inhibition
Fluorescence inhibition can lead to false-negative results. Various substances, including humic acid and hemoglobin, have been shown to quench fluorescence, affecting the accuracy of detection. This is particularly critical in quantitative PCR (qPCR) and digital PCR (dPCR) applications where fluorescence signals directly correlate with amplicon generation.
Understanding the Importance of Monitoring PCR Inhibition
To achieve accurate and dependable results in routine diagnostics, it is imperative to monitor and control PCR inhibition. Proper quality assurance and control measures, including method validation, are fundamental prerequisites before implementing PCR for specific sample types. Validation studies should encompass a thorough investigation of general performance characteristics and, importantly, address the issue of PCR inhibition.
One crucial aspect of method validation is the inclusion of relevant sample types and matrices expected in the analysis. Reference materials, mimicking commonly encountered matrices, can be prepared and applied to assess PCR inhibition accurately. The outcome of such experiments, where DNA is analyzed with quantitative PCR (qPCR) in the presence of increasing amounts of inhibitors, provides insights into whether the inhibitors affect DNA polymerization or fluorescence signals.
Two main approaches are commonly employed to assess the impact of PCR inhibition during routine analysis: internal amplification controls (IAC) and kinetic outlier detection (KOD). IAC, involving the addition of a non-target DNA fragment co-amplified with the target, is well-established and highly recommended in diagnostics. KOD, although not yet broadly introduced, may serve as a complement to IAC.
Strategies for Overcoming PCR Inhibition
Overcoming Polymerization Inhibition
To overcome polymerization inhibition, selecting appropriate DNA polymerases and buffer systems is crucial. Various commercial DNA polymerases tailored for specific applications are available. Pre-PCR processing, optimizing PCR composition, proves effective in overcoming inhibition for challenging samples, and improving detection limits.
Studies show considerable differences in DNA polymerase tolerance to inhibitors. Combining complementary or synergistic DNA polymerases enhances tolerance. Novel polymerases with improved inhibitor resistance have been developed through mutagenesis, fusion strategies, or compartmentalized self-replication.
Buffer composition adjustments, such as elevated pH, altering ion content, and the addition of molecules like bovine serum albumin (BSA) or trehalose, enhance polymerization in the presence of inhibitors. Quality control measures like CE-based fragment analysis or qPCR before sequencing monitor PCR inhibition effects.
Overcoming Fluorescence Inhibition
Fluorescence inhibition, common in challenging samples like those containing soil or blood, can be addressed by choosing appropriate detection methods. Hydrolysis probes are preferable over dsDNA-binding dyes to avoid inhibition. Blending qPCR dyes optimizes detection in the presence of quenching molecules, ensuring reliable results.
In summary, addressing PCR inhibition challenges involves comprehensive method validation, selection of suitable DNA polymerases and buffer systems, and consideration of fluorescence inhibition. These strategies ensure the reliability and accuracy of PCR analysis in routine diagnostics.
Reference
- Sidstedt M., et al. PCR inhibition in qPCR, dPCR and MPS-mechanisms and solutions. Analytical and Bioanalytical Chemistry. 2020, 412(9): 2009-2023.
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