Introduction
PCR-based nucleic acid detection technology is one of the most critical techniques in molecular biology. PCR allows selective amplification of specific target DNA sequences within different DNA sequences, doubling the copy number of DNA molecules in each amplification step, ultimately generating millions of copies. Initially used for pathogen detection and subtype identification, various derivative technologies of PCR have gradually become mainstream in nucleic acid detection due to their high throughput, nucleic acid quantification, and rapid characteristics. Among them, real-time quantitative PCR, nested PCR, digital PCR, and other technologies are widely applied, offering advantages such as reduced cross-contamination, rapid detection, and low cost. However, different techniques have some issues, such as poor repeatability of standard curves in RT-PCR and the tendency of nested PCR to exhibit non-specific amplification. In recent years, digital PCR has gained attention as an emerging nucleic acid amplification detection technology, praised for its excellent sensitivity, specificity, and reproducibility. Despite limitations like high cost and low sensitivity in specific applications, digital PCR still holds significant value. Overall, PCR technology and its derivative techniques play a crucial role in nucleic acid detection, meeting the demands for sensitivity, specificity, rapidity, high throughput, and cost-effectiveness.
Fig. 1 The step of polymerase chain reaction (PCR).
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Issues in Nucleic Acid Detection Technology
In the application of nucleic acid detection, PCR and its derivatives are employed for direct diagnosis requiring high levels of sensitivity and specificity. However, practical detection often falls short of requirements, leading to challenges such as low sensitivity, and poor specificity resulting in non-specific bands, dispersion, false positives, or false negatives. These issues arise due to low nucleic acid levels in early disease stages, suboptimal DNA sources for PCR, and limitations in polymerase fidelity. Addressing these challenges may involve optimizing PCR techniques, and potentially utilizing PCR enhancers to overcome limitations.
The Type of PCR Enhancers
PCR enhancers improve the sensitivity, specificity, and accuracy of PCR and related technologies, addressing issues like false positives and false negatives. When traditional optimization fails, they offer more effective strategies, crucial for rapid and accurate diagnosis of pathogens. There are various types of PCR enhancers, each providing solutions for specific nucleic acid amplification challenges.
Small-Molecule Chemical PCR Enhancers
Small-molecule chemical PCR enhancers are commonly used in PCR reactions, including dimethyl sulfoxide (DMSO) and N, N, N-trimethylglycine. DMSO is often used for standard PCR amplification optimization, while betaine, as an osmoprotectant similar to glycerol, can prevent the denaturation of DNA polymerase. Some PCR enhancer products contain betaine. Recently, various chemicals have been found to enhance PCR effects, such as formamide, glycerol, tetramethylammonium chloride, etc. Composite PCR enhancers mix multiple components in proportion, resulting in a more significant promotion effect on PCR. Commercially available, cost-effective small-molecule chemical PCR enhancers primarily consist of chemical substances. However, it is essential to note that they may affect the DNA denaturation temperature, requiring adjustments to the thermal cycling parameters. Since it is challenging to predict which reagent may be useful for a specific target, testing different additives is necessary.
Nanomaterial PCR Enhancers
Nanomaterial PCR enhancers, emerging with nanotechnology, leverage the unique physical and chemical properties of nanomaterials, such as large surface area, volume ratio, surface charge density, and excellent thermal conductivity. This approach, known as nano-polymerase chain reaction (nano-PCR), involves various nanosystems like gold nanoparticles (AuNPs), graphene oxide (GO), quantum dots (QDs), upconversion nanoparticles (UCNPs), carbon nanotubes (CNTs), and other metal nanoparticles and nanocomposites. Nanomaterials enhance PCR sensitivity and specificity, with applications in visual PCR detection, chip detection, and biosensors. Their excellent biocompatibility and thermal conductivity contribute to faster PCR diagnostics.
Protein-Based PCR Enhancers
Protein-based PCR enhancers, like single-stranded DNA binding proteins (SSBs), play a crucial role in nucleic acid amplification within organisms. SSBs nonspecifically bind to single-stranded DNA, protecting it from nucleases and preventing secondary structure formation. Research explores heat-resistant SSBs, such as TthSSB, enhancing DNA amplification efficiency. Proteins like GP32 and HU exhibit PCR and RCA enhancement. Bovine Serum Albumin (BSA) improves PCR sensitivity, while bovine thrombin (BT) effectively enhances PCR and mitigates nanomaterial-induced inhibition. These protein enhancers mimic in vivo replication, providing sensitivity and specificity in PCR-based nucleic acid detection. However, cost, stability, and potential inhibitory effects need consideration.
Other Types of PCR Enhancers
In addition to small-molecule chemicals, nanomaterials, and protein-based PCR enhancers, other types, such as element mixtures, nucleic acid compounds like 7-deaza-dGTP, and high-polymer polyethylene glycol, also exhibit PCR-enhancing effects in nucleic acid detection. Solid element mixtures of gold, titanium, nickel, bismuth, and antimony enhance PCR specificity, and yield, and reduce reaction time. 7-deaza-dGTP, a dGTP analog, significantly improves amplification in templates with low and high GC content, overcoming difficulties faced by other enhancers. These enhancers, though less commonly used, offer unique properties to address specific challenges in detection.
The public health crisis caused by the novel coronavirus outbreak in December 2019 has prompted attention to epidemic detection. In recent years, PCR enhancers have played a crucial role in enhancing the sensitivity, accuracy, and speed of nucleic acid detection in pathogen testing. This has led to the establishment of a new detection system in various fields such as infectious disease control, genetic disease diagnosis, identification of agricultural transgenic crops, and forensic analysis. PCR enhancers also optimize the synthesis of large gene segments. However, commercially available PCR enhancers are expensive, with unknown compositions and challenging concentration regulation.
References
- Karunanathie H.; et al. PCR enhancers: Types, mechanisms, and applications in long-range PCR. Biochimie. 2022, 197: 130-143.
- Yang Z.; et al. Application of nanomaterials to enhance polymerase chain reaction. Molecules. 2022, 27(24): 8854.
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