In many settings, molecular testing is needed but unavailable due to complexity and cost. Simple, rapid, and specific DNA detection technologies would provide important alternatives to existing detection methods. Here we report a novel, rapid nucleic acid detection method based on the accelerated photobleaching of the light-sensitive cyanine dye, 3,3′-diethylthiacarbocyanine iodide (DiSC 2 (3) I − ), in the presence of a target genomic DNA and a complementary peptide nucleic acid (PNA) probe. On the basis of the UV-vis, circular dichroism, and fluorescence spectra of DiSC 2 (3) with PNA-DNA oligomer duplexes and on characterization of a product of photolysis of DiSC 2 (3) I − , a possible reaction mechanism is proposed. We propose that (1) a novel complex forms between dye, PNA, and DNA, (2) this complex functions as a photosensitizer producing 1 O 2 , and (3) the 1 O 2 produced promotes photobleaching of dye molecules in the mixture. Similar cyanine dyes (DiSC 3 (3), DiSC 4 (3), DiSC 5 (3), and DiSC py (3)) interact with preformed PNA-DNA oligomer duplexes but do not demonstrate an equivalent accelerated photobleaching effect in the presence of PNA and target genomic DNA. The feasibility of developing molecular diagnostic assays based on the accelerated photobleaching (the smartDNA assay) that results from the novel complex formed between DiSC 2 (3) and PNA-DNA is under way.Photobleaching of cyanine dyes is usually seen as an undesirable characteristic which hampers their use in nucleic acid detection. However, we have discovered that in the presence of a target specific peptide nucleic acid (PNA) oligomer probe, the rate of 3,3′-diethylthiacarbocyanine iodide (DiSC 2 (3)) photobleaching is directly related to the amount of target DNA present. We call this type of reaction "smartDNA" and this rapid color loss can be used as a sensitive indicator for the presence of a specific DNA sequence.PNAs, which are used in the current assay as hybridization probes, are oligonucleotide analogues where the negatively charged phosphoribose backbone has been replaced with a neutral N-(2-aminoethyl) glycine group. 1,2 The absence of the negatively charged backbone gives PNAs unique physiochemical properties for binding to nucleic acid targets. PNAs rapidly *Corresponding

We present a new method for the linearization and alignment of data traces generated by multilane automated DNA sequencing instruments. Application of this method to data generated with the Visible Genetics Open Gene DNA sequencing system (using MicroCel 700 gel cassettes, with a 25 cm separation distance) allows read lengths of > 1,000 nucleotides to be routinely obtained with high confidence and > 97% accuracy. This represents an increase of 10-15% in average read length, relative to data from this system that have not been processed in the fashion described herein. Most importantly, the linearization and alignment method allows usable sequence to be obtained from a fraction of 10-15% of data sets which, because of original trace misalignment problems, would otherwise have to be discarded. Our method involves adding electrophoretic calibration standards to the DNA sequencing fragments. The calibration standards are labeled with a dye that differs spectrally from the dye attached to the sequencing fragments. The calibration standards are identical in all the lanes. Analysis of the mobilities of the calibration standards allows correction for both systematic and random variation of electrophoretic properties between gel lanes. We have successfully used this method with two-dye and three-dye DNA sequencing instruments.

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We describe the analysis of errors and failure modes in the base-calling function in automated DNA sequencing, on instruments in which fluorescently-labeled Sanger dideoxy-sequencing ladders are detected via their times of migration past a fixed detector. A general approach entails the joint use of: (i) well-defined control samples such as M13mp18, and (ii) mathematical simulation of sequencing electropherograms, with the deliberate introduction of different types of distortion and noise. An algorithm, the electrophoretic trace simulator (ETS), is used to calculate electrophoresis traces corresponding to the output data stream of an automated fluorescent DNA sequencer. The ETS accepts a user-defined sequence of nucleotide bases (A, C, G, T) as input, and employs user-adjustable functions to compute the following critical parameters of an electropherogram: peak intensity, peak spacing, peak shape as a function of base number; background, noise, and spectral cross-talk correction (for a sequencer using multiple dyes). We use a combination of M13mp18 controls and simulated electropherograms to analyze two problems of considerable practical importance: (i) variation in electrophoretic migration rates between different lanes of a gel, and (ii) variation in signal intensity due to user-dependent loading artifacts. The issue of base-calling errors and failure modes, for electropherograms that contain noise and distortion, is addressed.

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