Abstract
Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration.
Highlights
Quantitative Polymerase Chain Reaction is one of central techniques in molecular biology and important tool in medical diagnostics
While being a golden standard Quantitative Polymerase Chain Reaction (qPCR) techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision
Digital PCR technologies should alleviate the need for calibration by providing absolute quantitation using binary signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met
Summary
Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. The assays that we describe in this report can be used to analyze difficult and exotic samples on standard qPCR (real-time) devices and to design new digital-analogue assays that maximize information gain from the partitions and reduce to minimum their number required to reach a particular level of precision in a given dynamic range of concentrations. The fact that the estimate relies on comparison to a reference compromises the accuracy and precision of the measurement by variation in multiple experimental factors, such as quality of the reference samples, the type and quality of the buffer, the activity of the polymerase, the amount and specificity of primers, and the reproducibility of thermal cycling and/or of detection in the apparatus In spite of these limitations, qPCR remains the ‘golden standard’. The advantages include simple liquid handling protocols that do not require extensive partitioning of the sample, and straightforward mathematical procedures for obtaining the final result from the measurements on the sample and from the calibration curve
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