Real-Time PCR Measurement by Fluorescence Anisotropy

Abstract

Real-time polymerase chain reaction (PCR) is the gold-standard for quantitation in both mutation and gene expression analyses. Already this technique has found valuable clinical application in disease diagnosis and progression evaluation. As the number of known gene-disease correlations continues to rise, there will be increased demand for higher throughput and decreased cost for these analyses. Present real-time PCR measurement is based upon the fluorescent intensity of either intercalating dyes or oligonucleotide probes. Intercalating dye methods suffer from a lack of binding specificity, while probe methods are expensive and require increased assay optimization.In this thesis, a new method is presented for monitoring real-time PCR that utilizes the fluorescent anisotropy (FA) of labeled primers. FA, when measured at constant temperature, is indicative of the molecular mass to which the fluorophore is attached. Specificity is improved with the FA method over the use of intercalating dyes since the selective binding of primers is required for signal change. Assay complexity and cost are reduced compared to fluorogenic probe methods since the probes are eliminated.The design of a prototype instrument, which successfully implements this new method, is presented. Instrument and assay performance are compared to intercalating dye assays run in commercially available instrumentation. Theoretical limits on performance are also presented and compared to experimental results. Excellent repeatability and linearity are observed with respect to these benchmarks. This new method, having both high specificity and low optimization complexity, is expected to be particularly applicable to the demanding robustness requirements of nano-scale PCR.

Taylor dispersion in polymerase chain reaction in a microchannel

“…To validate this choice of k av , we compare the predicted amplification in a batch PCR with the experimental data of Crane et al 36 ͑Fig. 2͒.…”

A Novel Poisson Distribution-Based Approach for Testing Boundaries of Real-Time PCR Assays for Food Pathogen Quantification

“…Application of Poisson distribution as a statistical tool for this purpose was suggested by Wang and Spadoro (47) for conventional PCR. In general, the analysis of positive and negative results in a cohort of samples was used by incidence of rare events such as frequency of alleles, mutations, levels of viruses, and clone frequencies in complex populations (1,6,11,22,34,35,41), and by several studies presenting new nucleic acid-based methods (5,9,12,14,19). Nevertheless, Poisson analysis was applied qualitatively, and the quantitative information was neglected.…”

Self-Catalytic Growth of Unmodified Gold Nanoparticles as Conductive Bridges Mediated Gap-Electrical Signal Transduction for DNA Hybridization Detection

“…Ever-growing interests in genomics and clinical diagnostics have promoted the development of efficient tools for sensitive assay of low-abundant DNA biomarkers. , Recently, polymerase chain reaction (PCR) coupled with fluorescence spectroscopy has been viewed as the most widely used technique for sensitive detection of DNA. , Although with high sensitivity, the PCR-based techniques for DNA detection encounter the problems of complicated procedures, easy contamination, and high cost. Gap-based electrical biosensors devices can provide a viable alternative route to PCR for the rapid quantification of DNA, attributing that such devices can directly transduce nucleic acid hybridization events into useful electrical signals through pairs of microgapped or nanogapped electrodes .…”