Computer simulation of gene detection without PCR by single molecule detection

Lloyd M. Davis; John G. Williams; Don T. Lamb

doi:10.1117/12.336943

15 January 1999 Computer simulation of gene detection without PCR by single molecule detection

Lloyd M. Davis, John G. Williams, Don T. Lamb

Proceedings Volume 3570, Biomedical Sensors, Fibers, and Optical Delivery Systems; (1999) https://doi.org/10.1117/12.336943
Event: BiOS Europe '98, 1998, Stockholm, Sweden

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.

Citation Download Citation

Lloyd M. Davis, John G. Williams, and Don T. Lamb "Computer simulation of gene detection without PCR by single molecule detection", Proc. SPIE 3570, Biomedical Sensors, Fibers, and Optical Delivery Systems, (15 January 1999); https://doi.org/10.1117/12.336943

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