Polymerase Chain Reaction (PCR), a fundamental technique in molecular biology and genetics, was invented in 1983 by biochemist Kary Mullis. This technique allows scientists to amplify and detect DNA and RNA sequences rapidly, facilitating nucleic acid cloning within hours using minimal template material. Often described as molecular photocopying, PCR’s sensitivity enables the generation of substantial DNA quantities from small nucleic acid samples.
Quantitative PCR (qPCR) enhances standard PCR by offering greater sensitivity and specificity for DNA sequence detection and quantification. Unlike conventional PCR which provides endpoint analysis, qPCR monitors DNA amplification in real-time, quantifying products after each cycle by measuring fluorescence from dyes or probes. This fluorescence, directly proportional to DNA quantity, allows precise calculation of initial template DNA copies with high accuracy across a broad range. While traditional PCR is considered qualitative or semi-quantitative, qPCR can deliver both qualitative and fully quantitative results.
qPCR instrumentation relies on thermal cyclers which precisely control temperature for PCR stages: initialization, denaturation, annealing, and elongation. Standard qPCR machines, beyond temperature control, incorporate fluorescence sources and detectors to track fluorescence during amplification. These systems utilize diverse optics to simultaneously detect multiple targets in a single reaction using multiplexing, a common feature in modern PCR systems. Light sources include LEDs, halogen lamps, and lasers. Lamp-based systems can have light path variations across wells, leading to fluorescence measurement differences. However, internal reference dyes or ROX dyes can normalize these variations through qPCR software, though sometimes at the cost of a detection channel.
Bio-Rad’s CFX Opus Real-Time PCR Systems employ a unique optical shuttle design using solid-state LEDs for excitation and photodiodes for detection. Available in CFX Opus 96, 384, and Deepwell configurations, these instruments feature an optics shuttle that scans across the sample plate, individually illuminating and detecting fluorescence from each well to prevent crosstalk. This design ensures identical light paths and intensities for all wells, eliminating the need for passive reference normalization and simplifying data processing to baseline subtraction.
CFX Opus Systems are presented as powerful tools for translational research and biopharmaceutical testing, featuring rapid heating and cooling for improved thermal uniformity and minimized edge effects, crucial for protein thermal shift assays. The systems enable temperature gradient programming up to 24°C across the block, optimizing primer annealing in a single experiment, saving time and resources. This technology aims to accelerate the preclinical to clinical trial transition in therapeutics development by improving model system development and therapeutic/biomarker testing. The innovative shuttle optical design offers channel multiplexing without reference dyes; the CFX Opus 96 System supports true five-target multiplexing for detailed analysis of stem cells, gene expression, and biomarker analysis, facilitating real-time monitoring of biological reactions. FRET experiments are also possible with a dedicated channel on the CFX Opus 96.
CFX Maestro Software with PrimePCR Assays simplifies experimental setup, data acquisition, analysis, and visualization. The software facilitates multi-plate gene study analysis and visualization tools like hierarchical clustering enhance data interpretation for target and cluster identification. Applications extend to viral load studies, mutation detection, residual DNA testing, biodistribution/persistence, and copy number variation. Bio-Rad qPCR systems are promoted for user-friendliness, adaptability, sensitivity, dynamic range, and high-throughput automation integration, advancing translational research.
qPCR assays are highlighted as widely used for rapid nucleic acid determination in diverse research fields. Bio-Rad CFX Opus Systems, utilizing the optical shuttle, aim to provide enhanced accuracy, reliability, and consistency in qPCR analyses, supported by CFX Maestro Software’s statistical tools. The system is presented as a comprehensive solution for advancing translational research and biopharmaceutical product testing.
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