Computer simulation of gene detection without PCR by single molecule detection

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. Our findings demonstrate the feasibility of the approach for selected fluorophores, and identify suitable 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 occuring 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.

Dealing with reduced data acquisition times in Fluorescence Correlation Spectroscopy (FCS) for High-Throughput Screening (HTS) applications

“…Figure 11 illustrates a simple algorithm by which the ACF may be accumulated in real time as a histogram of cumulative delay times [11], obviating the need for a hardware correlator. Reference [12] includes an algorithm for accumulation of cross-correlation functions (CCFs). Figure 11 also illustrates the accumulation of a family of histograms of delay times between successive numbers of photons (SPIDA) [10].…”

Simulations for guiding the delivery and trapping of single biomolecules in a nanofluidic device

“…The use of Monte Carlo simulations for validating experiments and determining feasibility limits was described in the first reports on the detection of single-chromophore molecules in solution [2]. Later simulations have been used to study efficiency of detection [20], two-color coincidence detection limits [21], and the efficiency of neural network analysis for distinguishing single molecules with different spectroscopic properties [22]. Section 2 presents an overview of the experiments and results.…”

<title>Analysis of ultrasensitive fluorescence experiments</title>

“…Details and key sections of the computer code for these evaluations can be found in Ref. 1. The WSS is evaluated using the Gaussian weighting function that corresponds to the mean fluorescence signal expected from a fluorophore that passes in a straight line through the center of the probe region, without photodegradation or intersystem crossing.…”

“…1 For example, for 'conventional' DNA sequencing by the Sanger method, the electrophoresis within a capillary or slab gel may be multiplexed by the use of spectroscopically identifiable fluorescent labels. Lieberwirth et al 2 have recently demonstrated such multiplex dye DNA sequencing in capillary gel electrophoresis by the use of four dyes with disparate fluorescence lifetimes.…”