Polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) assays form a foundation for molecular biology research and applications. For decades, they have enabled the detection and quantification of nucleic acids as well as the production of large volumes of DNA for cloning or sequencing purposes. Today, they play a critical role in a massive number of applications including a wide swath of genomic analysis, gene expression studies, pathogen detection, and genotyping techniques. With so much work reliant on PCR-based methodologies, ensuring reaction efficiency and accuracy is an absolute requirement for obtaining reproducible results in molecular biology labs. Increasing productivity and throughput with automation and high-throughput instrumentation is a major concern for many labs for the same reason.
Achieving success in PCR methodologies depends on three key factors: the technology used, the optimization of reaction mix and conditions, and the quality of both samples and reagents. Thermal cyclers play a crucial role, where precise temperature control and speed of transitions directly impact reaction efficiency and ultimately determine the quality of results. With multiple technologies to choose from, understanding their differences, which features to look for, and the options for most effectively increasing throughput is important. Optimizing reaction conditions and components, including primer design and template preparation, helps ensure efficiency and reproducibility. Additionally, selecting the best reagents and maintaining proper storage and handling practices are vital to quality control and preventing cross-contamination, leading to more consistent and reliable data.
The ongoing miniaturization of materials and solutions that accompany increasingly high-throughput formats presents challenges for maintaining data quality. Microfluidics and digital PCR (dPCR) techniques can minimize amplification bias and background interference by partitioning the sample into nanovolume droplets.
Download the full resource guide to learn:
- Key tips and insights for successful PCR experiments
- An overview of thermal cycler technology, including high-throughput options
- Expert guidance on selecting the best reagents for your application
- A best practices checklist to ensure reliable and reproducible results
- A comparison of PCR, qPCR, and dPCR techniques, plus how dPCR enhances genetic diagnostics by analyzing single DNA molecules
