Quantitative Multiplex Analysis Using a Novel Universal Real-time Transcription-Mediated Amplification–based Format
Norman C. Nelson, Director of Biochemistry, Gen-Probe
Date Posted: Wednesday, November 18, 2009
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Launch presentation
About the speaker
Dr. Nelson received his BS in Chemistry from The California Institute of Technology in 1976 and his PhD in Chemistry from the University of California at San Diego in 1982. He joined Gen-Probe (San Diego, CA) in 1985, where he remains today as the Senior Director of Biochemistry. Dr Nelson is the sole or co-inventor on 20 issued patents and numerous pending applications. His primary focus is new technology discover and development for use in molecular diagnostic assays.
Abstract
A real-time Transcription-Mediated Amplification (TMA)–based multiplex assay has been developed that reduces or eliminates the adverse primer reactions observed in other multiplex formats. In this assay, a pair of universal tags is incorporated into the amplicon of each analyte of interest during the initiation phase using a unique, target capture–mediated approach. The exponential phase of amplification for each analyte being tested is then driven by the same universal primer/provider set. Amplification is detected in real time using a different-colored fluorescent self-reporting probe for each analyte. In a proof of principle study, a quantitative triplex assay for the detection of PCA3 (a prostate cancer marker), PSA (prostate-specific antigen), and internal control was developed. The assay was sensitive, and had excellent accuracy and precision characteristics over a wide dynamic range. Interference between PCA3 and PSA was very low, even when the analytes were present at 1000-fold different levels. In conclusion, a sensitive, accurate, and precise universal real-time TMA format that displays very low primer/provider interaction has been developed. This assay holds great promise for the simultaneous detection of multiple analytes in a variety of molecular diagnostic applications.
Launch presentation