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High-resolution capillary electrophoresis sizing of preformed complexes of bis-intercalating fluorescent dyes with double-stranded DNA has been demonstrated using hydroxyethylcellulose and 3-[tris-(hydroxymethyl) methylamino]-1-propanesulfonic acid-tetrapentylammonium (Taps-NPe+4) buffers (S. M. Clark and R. A. Mathies, Anal. Chem. 69, 1355-1363, 1997). Such capillary electrophoresis separations were unattainable in conventional buffers containing other cations such as Tris+, Na+, and NH+4. We report here the behavior of preformed double-stranded DNA-dye complexes on agarose slab gel electrophoresis in 40 mM Taps-NPe+4, 1 mM H2EDTA, pH 8.2. Upon electrophoresis in this buffer (a) complexes formed at DNA base pairs:dye ratios ranging from 100:1 to 5:1 show the same mobility; (b) the half-lives of DNA-dye complexes with monointercalators are two- to threefold longer than those in commonly used Tris buffers; (c) there is little dye transfer between labeled and unlabeled DNA molecules; and (d) precise two-color sizing of preformed restriction fragment-dye complexes with fluorescent bisintercalators is achieved.

Fluorescence-detected capillary electrophoresis separations of phi X174/HaeIII DNA restriction fragments have been performed using monomeric and dimeric intercalating dyes. Replaceable hydroxyethyl cellulose solutions were used as the separation medium. Confocal fluorescence detection was performed following 488-nm laser excitation. The limits of DNA detection for on-column staining with monomeric dyes (ethidium bromide, two propidium dye derivatives, oxazole yellow, thiazole orange, and a polycationic thiazole orange derivative) were determined. The thiazole orange dyes provide the most sensitive detection with limiting sensitivities of 2-4 amol of DNA base pairs per band, and detection of the 603-bp fragment was successful, injecting from phi X174/HaeIII samples containing only 1-2 fg of this fragment per microliter. Separations of preformed DNA-dimeric dye complexes were also performed. The breadth of the bands observed in separations of preformed DNA-dimeric dye complexes is due to the presence of DNA fragments with different numbers of bound dye molecules that can be resolved as closely spaced subbands in many of our separations. The quality of these DNA-dye complex separations can be dramatically improved by performing the electrophoresis with 9-aminoacridine (9AA) in the column and running buffers. The optimum concentrations of 9AA for the separation of complexes preformed with the dimeric dyes TOTO, EthD, TOTAB, and YOYO were determined to be 100, 1, 1, and 0.5 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

We report here a system for automation of the dideoxynucleotide DNA sequencing method. The system consists of a Beckman Biomek 1000 robotic workstation which has been modified by the addition of a thin heater/cooler block directly on the instrument table. The heater/cooler block, which is regulated by a user-specified program integrated into the Biomek software, facilitates the use of both single- and double-stranded DNA sequencing procedures. Using this system, we are able to perform 24 sets of dideoxynucleotide DNA sequencing reactions in approximately 45 min. The reactions are performed in 96-well microtiter plates, using a prepared reagent pack which includes primer, enzyme, nucleotide mixes and radioactive label. Using standard gel electrophoresis technology, we are able to resolve over 500 nucleotides per sequencing reaction in about 5 h. Currently, we are able to perform three runs of 24 samples each, with subsequent gel analysis per 8-h period. Excluding autoradiography, this represents a daily data output of 36,000 base pairs.

A new instrument has been developed for the electrophoretic separation of large DNA molecules that can independently regulate the voltage of each of 24 electrodes and allow the magnitude, orientation, homogeneity, and duration of the electric field to be precisely controlled. Each parameter can be varied at any time during the electrophoretic process. Thus distinct sets of conditions can be combined to optimize the separation of various fragment sizes in a single run. Independent control of electrode voltage allows all of the fields to be generated with electrodes arranged in a closed contour, independent of a particular geometry. This device increases both the resolution in any size range and the speed of separation, especially for DNA molecules larger than 3 megabases.

Quantitative measurement of DNA migration in gel electrophoresis requires precisely controlled homogeneous electric fields. A new electrophoresis system has allowed us to explore several parameters governing DNA migration during homogeneous field pulsed field gel (PFG) electrophoresis. Migration was measured at different switch times, temperatures, agarose concentrations, and voltage gradients. Conditions which increase DNA velocities permit separation over a wider size range, but reduce resolution. We have also varied the angle between the alternating electric fields. Reorientation angles between 105 degrees and 165 degrees give equivalent resolution, despite significant differences in DNA velocity. Separation of DNA fragments from 50 to greater than 7000 kilobases (Kb) can easily be optimized for speed and resolution based on conditions we describe.