Characterization of interfacial gene transcription with on-line acoustic wave sensor network and surface analysis techniques.
Contributors: University of Toronto.  Dept. of Chemistry.
Published: 2005
Language: eng
Number of pages: 216
Description: Variations in the binding interaction between strong, weak, open and closed DNA promoter sequences and the T7 RNA polymerase during the critical step of transcriptional activation were identified using the TSM sensor. Furthermore, the use of the TSM sensor to monitor the transcription reaction in real time was successfully demonstrated with control experiments that clearly distinguish features in the data unique to transcription initiation and elongation, along with supporting evidence from the other surface characterization methods. Acoustic physics in biosensor applications thus presents an optimal solution for the need to investigate gene transcription in real time on the micro- to nano-scale.The thickness shear mode (TSM) acoustic wave sensor is introduced as a viable, label-free technique for the signaling of interfacial gene transcription in real-time.The T7 RNA Polymerise model system was used to evaluate and understand the sensor response to important steps in gene transcription. The sensing device is a gold-coated piezoelectric quartz crystal onto which the template DNA is immobilized. RNA polymerise binding to promoter DNA sequence in transcriptional activation, as well as initiation and elongation in the presence of nucleotide tri phosphates were detected from the change in resonant frequency of the crystal. The real-time impedance data collected from the sensor is fit to an equivalent circuit model using a network analysis method, from which multidimensional data is retrieved. Of this data set, the series resonant frequency and motional resistance were monitored simultaneously to identify the contribution of different experimental factors. For instance, motional resistance is an energy dissipation parameter, while the resonant frequency is influenced by mass accumulation on the sensor surface.Accordingly, experimental factors contributing to the sensor data were independently examined with Time of Flight Secondary Ion Mass Spectrometry, Atomic Force Microscopy, and Cyclic-Voltammetry. The application of principal components analysis to ToF-SIMS spectra supported the notion that conformational alteration of the T7 RNA polymerase in transcription initiation could contribute to the observed change in motional resistance.The sensitivity to multiple factors that change during an experiment allows for extracting potent information concerning the complex transcriptional machinery.
Notes: Thesis (Ph.D.)--University of Toronto, 2005.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 66-06, Section: B, page: 3107.