Adaptive weighted least squares method for the estimation of DNA fragment lengths from agarose gels

The size of DNA fragments is most frequently estimated from their electrophoretic mobilities. Agarose gels are used to estimate the size of DNA fragments ranging from a few hundred nucleotides to more than 20 kbp. The common practice when estimating the unknown fragment sizes is to plot the log of the size of molecular weight standards against their mobility and read the values of unknowns from this graph. However, due to perturbations in the gel, such plots often show pronounced curvature which may introduce significant subjectivity into the interpolation process. We present a new method "adaptive weighted least squares (AWLS)" based on the significance test to choose the order of polynomial. We compare this with the method introduced by Schaffer based on the modification of the Southern method. The results obtained by AWLS are significantly better than the method introduced by Schaffer. Different lanes are tested for consistency.     

Optimization of sequencing conditions using near-infrared lifetime identification methods in capillary gel electrophoresis

We have investigated the sample preparation and electrophoresis conditions necessary to prepare DNA sequencing samples appropriate for use with near-infrared (IR) fluorescent labels with dye identification accomplished via lifetime techniques. It was found that several sample preparation protocols required attention to maximize the fluorescence yields of the labeling dyes, such as thermal cycling conditions, choice of counter ion used for the ethanol precipitation step and also, dye-primer versus dye-terminator chemistries. In addition, several different sieving matrices were investigated for their effects on both the fluorescence properties of the labeling dyes and electrophoretic resolution. Extended times used for the high temperature denaturing of duplexed DNA fragments during cycle sequencing produced cleavage products, in which the covalently attached dye to the sequencing primer was released through attack by dithiothreitol (DTT). Even under optimized thermal cycling conditions, free dye was generated that masked readable data from the sequencing traces. Ethanol precipitation was necessary to remove this free dye with the proper choice of counter ion (sodium). The results using different sieving matrices indicated that linear polyacrylamides (LPAs) were appropriate for any fluorescence measurement, since they could readily be replaced between runs minimizing deleterious memory effects associated with cross-linked polyacrylamide gels. After investigation of several different sieving LPAs, the commercially available POP6 was found to be particularly attractive, since it produced good electrophoretic resolution, single exponential behavior for the near-IR dye series investigated herein, and also, discernible lifetime differences within the dye set. Finally, dye-terminator chemistry was also found to minimize bleeding in the gel matrix produced by large amounts of unextended dye-primer within the gel lane.     

Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis.

A low cost, 0.75-mW helium neon laser, operating in the green region at 534.5 nm, is used to excite fluorescence from tetramethylrhodamine isothiocyanate-labelled DNA fragments that have been separated by capillary gel electrophoresis. The detection limit (3 sigma) for the dye is 500 ymol [1 yoctomole (1 ymol) = 10(-24) mol] or 300 analyte molecules in capillary zone electrophoresis; the detection limit for labeled primer separated by capillary gel electrophoresis is 2 zmol [1 zeptomole (1 zmol) = 10(-21) mol]. The Richardson-Tabor peak-height encoded sequencing technique is used to prepare DNA sequencing samples. In 6% T, 5% C acrylamide, 7 M urea gels, sequencing rates of 300 bases/hour are produced at an electric field strength of 200 V/cm; unfortunately, the data are plagued by compressions. These compressions are eliminated with addition of 20% formamide to the sequencing gel; the gel runs slowly and sequencing data are generated at a rate of about 70 bases/hour.     

Contaminant-induced current decline in capillary array electrophoresis

High-throughput capillary array electrophoresis (CAE) instruments for DNA sequencing suffer to varying degrees from read length degradation associated with electrophoretic current decline and inhibition or delay in the arrival of fragments at the detector. This effect is known to be associated with residual amounts of large, slow-moving fragments of template or genomic DNA carried through from sample preparation and sequencing reactions. Here, we investigate the creation and expansion of an ionic depletion region induced by overloading the capillary with low-mobility DNA fragments, and the effect of growth of this region on electrophoresis run failure. Slow-moving fragments are analytically and experimentally shown to reduce the ionic concentration of the downstream electrolyte. With injection of large fragments beyond a threshold quantity, the anode-side boundary of the nascent depletion region begins to propagate toward the anode at a rate faster than the contaminant DNA migration. Under such conditions, the depletion region expands, the capillary current declines dramatically, and the electrophoresis run yields a short read length or fails completely.     

Compensation for mobility inequalities between lanes computed from band signals in on-line fluorescence DNA sequencing.

Among on-line fluorescence DNA sequencing systems, the four-lane method exhibits the potential for reporting an erroneous sequence due to nonuniform mobility of the DNA fragments migrating among the four lanes. This error is manifest in phenomenon commonly called smiling. This paper presents a computational algorithm which compensates for the mobility inequalities between lanes using signal data obtained from the shorter DNA fragments forming the faster migrating bands. The program mainly consists of two routines: (i) calculation of calibration coefficients (mobility ratios between lanes), and (ii) examination of the coefficients by applying them to a later domain of the same signals. Both routines are connected with several feed-back branches for recalculation. Homology analysis of final sequences has shown that the accuracy rate is maximized with this algorithm and any ambiguous result can be assigned to the residual error inherent in the band identification method used.     

Quality of assurance considerations for use of the Fluorlmager SI and fragmeNT analysis software

The Fluorlmager SI (FSI) from Molecular Dynamics is one of several scanning instruments available for the detection of fluorescent emissions associated with DNA samples in a variety of matrices (agarose and polyacrylamide gels, membranes and microplates). In our laboratory, we measured the electrophoretic mobility of randomly amplified polymorphic DNA (RAPD) fragments stained with ethidium bromide in agarose using the FSI to scan gels and the associated Molecular Dynamics software (ImageQuaNT, and FragmeNT Analysis) for analysis. Initial scans and analyses resulted in inconsistent band detection across the same gel and across several scans of the same gel. To determine the best types of calibration for the instrument, several factors were considered and then evaluated. Tests of calibration acceptability were also evaluated. Band detection by FragmeNT Analysis was improved following optimization of matrices and parameters used in calibration and experimental scans. In addition, use of software templates for analysis and modifications in the staining procedure, which have resulted in decreased instrument associated variance, are discussed.     

Imaging as a tool for improving length and accuracy of sequence analysis in automated fluorescence-based DNA sequencing

A new method of signal analysis for automated fluorescence-based DNA sequencing is presented. Signal resolution is a limiting factor in obtaining accurate sequence information beyond 400-450 nucleotides per gel lane. We have developed a computer program for the imaging of DNA bands in sequencing gels. The image analysis shows that distortions in the shapes of the bands decrease resolution of peaks observed served in the standard data plots. Reconstruction of the undistorted band shape prior to signal analysis substantially improves the resolution of peaks and may improve the accuracy and length of the contiguous sequence read. Image analysis identified other factors limiting the accuracy and length of automated DNA sequence analysis and provided a tool for evaluating various remedies. Our techniques should also be applicable in other systems, for example, in gel electrophoresis of proteins and DNA restriction fragments, and in scranning densitometry.     

A general approach to the analysis of errors and failure modes in the base-calling function in automated fluorescent DNA sequencing

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.     

Electrokinetic injection of DNA from gel micropads: basis for coupling polony technology with CE separation

We propose a novel method for electrokinetic injection of DNA samples into capillaries from nanoliter gel micropads, deposited on glass slides, which are coated with electroconducting film. Theoretical and experimental proof is presented for the proposed method. The method allows efficient and highly precise injection without physical contact between the gel pad and the capillary. Read length of more than 700 bp at Q20 has been reproducibly demonstrated in fused-silica capillaries using the proposed injection technique. Based on the obtained results we discuss a novel DNA sequencing system which combines DNA amplification and cycle sequencing in arrays of subnanoliter gel micropads and high-throughput electrophoretic separation in monolith multicapillary arrays.     

Automated detection of differently expressed fragments in mRNA differential display

We present a novel method for the automated detection of fragments showing dissimilar expression in mRNA differential display. The analysis is based on aligning the numerical electrophoretic lane data in respect of a given distance function defined on a set of fragments, or signal peaks in general. We presume that significant dissimilarities between peaks result in extreme score values computed for aligned peak pairs. Whereas in sequence comparison, an overall sequence similarity score is conventionally used, the current method defines a special dissimilarity score for searching the peak pairs showing the largest relative differences between the lanes. The output of the analysis is a highly reduced list of peak pairs, along with a set of associated features extracted from the lanes. Only the peaks of this list need to be visually confirmed instead of the vast amount of peaks in the original electrophoretic results. The results obtained by the algorithm correlate well with results of visual evaluation of the same electropherograms. The current algorithm may be applied to the study of complex expression patterns in multiple lanes and, in general, to automated recognition of variously defined patterns of quantitative electrophoretic data.     

Separation and analysis of DNA sequence reaction products by capillary gel electrophoresis

This paper demonstrates the potential of capillary gel electrophoresis with laser induced fluorescence detection as a tool for DNA sequence determination. Both synthetic oligonucleotides and single-stranded phage DNA were utilized as templates in the standard chain termination procedure. Primer molecules were tagged at the 5' end with the fluorescent dye, JOE. First, baseline resolution of a dA extended primer from 18 to 81 bases long, a total of 64 fragments, was observed. A second synthetic template was designed to yield alternating stretches of dA and dT extensions of the primer. Thirdly, the sequence reaction products from a synthetic oligonucleotide template containing all four bases was analyzed in four independent runs, one for each of the four base-specific reactions. In all cases, the expected number and patterns of peaks were observed by capillary gel electrophoretic analysis. Finally, separation of sequence reaction products generated with single-strand M13mp18 phage DNA as template exhibited baseline resolution of fragments differing in length by a single nucleotide and from 18 to greater than 330 bases total length.     

Recent advances in DNA sequencing by capillary and microdevice electrophoresis

A number of significant improvements in the electrophoretic performance and design of DNA sequencing devices have culminated in the introduction of truly industrial grade production scale instruments. These instruments have been the workhorses behind the massive increase in genomic sequencing data available in public and private databases. We highlight the recent progress in aspects of capillary electrophoresis (CE) that has enabled these achievements. In addition, we summarize recent developments in the use of microfabricated devices for DNA sequencing that promise to bring the next leap in productivity.     

Analysis of electrophoretic patterns of arbitrarily primed PCR profiling

We present a mathematical algorithm for the analysis of electrophoretic patterns resulting from arbitrarily primed PCR profiling. The algorithm is based on the established mathematical procedures applied to the analysis of digital images of gel patterns. The algorithm includes (a) transformation of the image into a matrix form, (b) identification of every electrophoretic lane as a set of matrix columns that are further mathematically processed, (c) averaging of matrix columns corresponding to electrophoretic lanes that define lane representatives, (d) elimination of "smiling" bands, (e) solving the problem of a lane offset, and (f) removal of the background. Representation of individual electrophoretic lanes in the form of functions allows interlane comparisons and further mathematical analysis. Direct comparison of selected lanes was obtained by employing correlation analysis. Gel images were those obtained after arbitrarily primed PCR analysis of DNA that underwent damage induced by gamma radiation from a (60)Co source. The applied method proved to be useful for elimination of subjectivity of visual inspection. It offers the possibility to avoid overlooking important differences in case of suboptimal electrophoretic resolution. In addition, higher precision is achieved in the assessment of quantitative differences due to better insight into experimental artifacts. These simple mathematical methods offer an open-type algorithm, i.e., this algorithm enables easy implementation of different parameters that may be useful for other analytical needs.     

Alternative base-calling algorithm for DNA sequencing based on four-label multicolor detection

A simple base-calling scheme based on four-label multicolor detection is suggested for DNA sequencing. The entire spectra of the dye labels were used for identification. Specifically, the maxima of the emission spectra rather than the intensity ratios at selected wavelengths are used to provide excellent discrimination. Capillary gel electrophoresis was used for the separation of DNA fragments. Data acquisition and analysis compatible with fast and high-throughput imaging detection was accomplished. The accuracy of base calling of PGEM/U DNA from the raw data obtained with 5 nm and 7 nm spectroscopic resolution were 98.4% for 386 bases and 98.4% for 385 bases. Base calling of M13mp18 DNA showed 98.3% accuracy for 420 bases.     

Increasing voltage gradient electrophoresis of DNA

We developed a method which allows electrophoretic fractionation of DNA in an agarose matrix according to an increasing current gradient, using a previously designed [R. Barbieri, V. Izzo, M.A. Costa, G. Giudice, G. Duro, Anal. Biochem. 212 (1993) 168; M.R. Asaro, V. Izzo, R. Barbieri, J. Chromatogr. A 855 (1999) 723] voltage gradient apparatus. This method allows the separation of different DNA fragments by increasing the distances of the components fractionated in the gel, revealing small differences in the length of different DNA components.     

Activation energy of single-stranded DNA moving through cross-linked polyacrylamide gels at 300 V/cm effect of temperature on sequencing rate in high-electric-field capillary gel electrophoresis

In DNA sequencing, single-stranded DNA fragments are separated by gel electrophoresis. This separation is based on a sieving mechanism where DNA fragments are retarded as they pass through pores in the gel. In this paper, we present the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T LongRanger gels at an electric field of 300 V/cm. The temperature dependence is compared with the predictions of the biased reptation model. The model predicts that the fragment length for the onset of biased reptation with stretching increases with the square of temperature; the data show that the onset of biased reptation with stretching decreases with temperature. Biased reptation fails to model accurately the temperature dependence of mobility. We analyzed the data and extracted the activation energy for passage of sequencing fragments through the gel. For fragments containing less than ca. 200 bases, the activation energy increases linearly with the number of bases at a rate of 25 J/mol per base; for longer fragments, the activation energy increases at a rate of 6.5 J/mol per base. This transition in the activation energy presumably reflects a change in conformation of the DNA fragments; small fragments exist in a random coil configuration and larger fragments migrate in an elongated configuration.     

DNA sequencing in HydroLink matrices: extension of reading ability to greater than 600 nucleotides

All the systems for optimizing DNA sequencing published so far have introduced modifications regarding: (i) linearization of band migration via ionic strength gradients or wedge-shaped gels; (ii) automatization of band reading via introduction of fluorescent probes; (iii) direct blotting analysis; (iv) pulsed electric fields and (v) discontinuous buffer systems. In all these systems, DNA sequence reading with an accuracy of ca. 98% rarely exceeds a length of 350 bases. We have chosen, in order to increase the reading ability of a single gel, to manipulate the characteristics of the gel matrix. The Seq-HydroLink gel formation here reported allows optimal reading, from a single gel run, of at least 600 bases. In order to guarantee this reading ability in a single run, the upper and lower ends of the ladder are time-resolved, i.e. the same sample is applied to the gel matrix at three different time intervals. The present system represents an increase of at least 30% in reading ability as compared with any type of polyacrylamide gel formulation so far reported.     

Improved resolution power of electrophoretic fractionation of DNA using a voltage gradient "up and down" application

The improved resolution power of electrophoretic fractionation of DNA in a wide range of molecular masses is demonstrated using an "up and down" application of voltage gradient gel electrophoresis (VGGE). This application also allows separation of different DNA fragments which are poorly fractionated in conventional electrophoresis.     

Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.     

Monitoring of single nicks in duplex DNA by gel electrophoretic mobility-shift assay

We demonstrate that the gel electrophoretic mobility-shift assay (EMSA) can be used for site-selective and quantitative monitoring of nicks in linear double-stranded DNA (dsDNA) thus allowing to expediently follow the nicking activity of enzymes or other agents targeted to a designated dsDNA site. At elevated temperature and/or in the presence of urea, DNA fragments carrying a single nick produced by the nicking enzyme N.BstNBI exhibit a well-detectable gel retardation effect. On the basis of permutation analysis, the decreased electrophoretic mobility of nicked dsDNA fragments is attributed to a bend (or hinge) in the DNA double helix sequence-specifically generated by a nick. Since nick-induced DNA bending depends on interaction between base pairs adjacent to a nick, the change in mobility is different for nicked DNA sites with different sequences. Therefore, EMSA monitoring of differential mobility change caused by nicks within various DNA sequences could be useful for studying the differential base stacking and nearest-neighbor energetics.