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Proceedings Paper

Computer simulation of gene detection without PCR by single molecule detection
Author(s): Lloyd M. Davis; John G. Williams; Don T. Lamb
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Paper Abstract

Pioneer Hi-Bred is developing a low-cost method for rapid screening of DNA, for use in research on elite crop seed genetics. Unamplified genomic DNA with the requisite base sequence is simultaneously labeled by two different colored fluorescent probes, which hybridize near the selected gene. Dual-channel single molecule detection (SMD) within a flow cell, then provides a sensitive and specific assay for the gene. The technique has been demonstrated using frequency- doubled Nd:YAG laser excitation of two visible-wavelength dyes. A prototype instrument employing infrared fluorophores and laser diodes for excitation has been developed. Here, we report results from a Monte Carlo simulation of the new instrument, in which experimentally determined photophysical parameters for candidate infrared dyes are used for parametric studies of experimental operating conditions. Fluorophore photostability is found to be a key factor in determining the instrument sensitivity. Most infrared dyes have poor photostability, resulting in inefficient SMD. However, the normalized cross-correlation function of the photon signals from each of the two channels can still yield a discernable peak, provided that the concentration of dual- labeled molecules is sufficiently high. Further, for low concentrations, processing of the two photon streams with Gaussian -weighted sliding sum digital filters and selection of simultaneously occurring peaks can also provide a sensitive indicator of the presence of dual-labeled molecules, although accidental coincidences must be considered in the interpretation of results.

Paper Details

Date Published: 15 January 1999
PDF: 12 pages
Proc. SPIE 3570, Biomedical Sensors, Fibers, and Optical Delivery Systems, (15 January 1999); doi: 10.1117/12.336943
Show Author Affiliations
Lloyd M. Davis, Univ. of Tennessee Space Institute (United States)
John G. Williams, Pioneer Hi-Bred International, Inc. and LI-COR, Inc. (United States)
Don T. Lamb, Pioneer Hi-Bred International, Inc. (United States)

Published in SPIE Proceedings Vol. 3570:
Biomedical Sensors, Fibers, and Optical Delivery Systems
Francesco Baldini; Nathan I. Croitoru; Martin Frenz; Ingemar Lundstroem; Mitsunobu Miyagi; Riccardo Pratesi; Otto S. Wolfbeis, Editor(s)

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