The four dye fluorescence detection strategy is a widely used approach to automated DNA sequence analysis. An important aspect of data processing in this approach is the multicomponent analysis to deduce the concentrations of four fluorophores from fluorescence emission intensities at four different wavelengths. This requires knowledge of the correct transformation matrix M. The matrix M is a function both of the fluorophores employed and the fluorescence detection system. M is typically determined either by a calibration process with individual dyes, or by choosing four well-separated individual peaks corresponding to the four different dyes. Both are time-consuming and complicated procedures for routine use. An automatic scheme for finding M directly from raw sequence data is presented here. This facilitates data analysis and the underlying algorithm may also find utility in other multispectral applications.
In a previous paper (Yin et al., Electrophoresis 1996, 17, 1143-1150), an automated method for matrix determination in four-dye fluorescence-based DNA sequencing was presented. As a continuation of that work, we have developed an alternative method to estimate the matrix from raw sequence data. The method uses an iterative clustering technique to associate each 4 x 1 data vector with one column of the desired filter matrix, using Kullback's I-divergence as a distance measure. The method requires less preprocessing of the data and less computation than the approach described by Yin et al. (Electrophoresis 1996, 17, 1143-1150). An example demonstrating applicability of the proposed method to Applied Biosystems sequencer data is given.
We present a sensitive and rapid screening method for the determination of β‐lactamase activity of antibiotic‐resistant bacteria, by designing a pH‐sensitive fluorescent dye‐doped mesoporous silica nanoparticle encapsulated with penicillin G as a substrate. When penicillin G was hydrolysed by β‐lactamase and converted into penicilloic acid, the acidic environment resulted in fluorescence quenching of the dye. The dye‐doped mesoporous nanoparticles not only enhanced the β‐lactamase‐catalyzed reaction rate but also stablized the substrate, penicillin G, which degrades into penicilloic acid in a water solution without β‐lactamase. Twentyfive clinical bacterial samples were tested and the antibiotic resistant and susceptible strains were identified. The proposed method may detect the presence of β ‐lactamases of clinically relevant samples in less than 1 hour. Moreover, the detection limit of β‐lactamase activity was as low as 7.8×10−4 U/mL, which was determined within two hours.
In four-color fluourescence-based automated DNA sequencing, a 4 x 4 filter matrix parameterizes the relationship between the dye-intensity signals of interest and the data collected by an optical imaging system. The filter matrix is important because the estimated DNA sequence is based on the dye intensities that can only be recovered via inversion of the matrix. In this paper, we present a calibration method for the estimation of the columns of this matrix, using data generated through a special experiment in which DNA samples are labeled with only one fluorescent dye at a time. Simulations and applications of the method to real data are provided, with promising results.
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