Quantifying DNA damage by gel electrophoresis, electronic imaging and number-average length analysis.

DNA damages that can be converted to single- or double strand breaks can be quantified by separating DNA by gel electrophoresis and obtaining a quantitative image of the resulting distribution of DNA in the gel. We review the theory of this method and discuss its implementation, including the charge-coupled device (CCD) camera systems we developed to acquire images of fluorophore labeled DNA.

The integrating ion imager: a device for determining heavy ion doses during irradiations.

We have designed and built an integrating ion imaging system (I3) that records the spatial distribution of the dose of heavy ions incident on samples irradiated at the radiobiology beamline of the Alternating Gradient Synchrotron at Brookhaven National Laboratory. The images of dose are integrated over the duration of the exposure. Unlike the images formed on X-ray film, these images are linear with the incident dose. Heavy ions are incident on a phosphor that is located just behind the sample position. Visible light emitted from the phosphor is collected by a lens and focused onto a scientific grade charge coupled device (CCD) cooled to about -45 degrees C. The phosphor and CCD camera are integral parts of a modular sample holder designed for irradiating molecular samples, which is easily mounted on the sample platform of the beamline. The imager can be adapted to other types of samples. The present CCD image is digitized to 14 bits (16,384 intensity levels), but the dynamic range is extended by adjusting the aperture of the CCD camera lens. Digital images from the CCD are routinely transferred over the BNL local area network for archival storage on a UNIX server, from which they can be opened from any authorized computer with access to the Internet. Images obtained with no sample in place record the dose at all points on the target field. When a sample is in place, an image of the sample appears providing its exact location with respect to fiducial marks recorded for all images. Areas surrounding the image of the sample are used in comparison with companion no-sample images to get exact doses over the sample. The contrast mechanism responsible for image formation is the shift along the Bragg curve resulting from loss of energy of the ions as they pass through the sample--not from a change in ion flux reaching the phosphor. The sharpness of the images formed with the DNA samples we have recorded indicates that neither scattering of the incident heavy ions or the generation of secondary ions contribute significantly.

Clustered DNA damages induced by high and low LET radiation, including heavy ions.

Clustered DNA damages--here defined as two or more lesions (strand breaks, oxidized purines, oxidized pyrimidines or abasic sites) within a few helical turns--have been postulated as difficult to repair accurately, and thus highly significant biological lesions. Further, attempted repair of clusters may produce double strand breaks (DSBs). However, until recently, there was no way to measure ionizing radiation-induced clustered damages, except DSB. We recently described an approach for measuring classes of clustered damages (oxidized purine clusters, oxidized pyrimidine clusters, abasic clusters, along with DSB). We showed that ionizing radiation (gamma rays and Fe ions, 1 GeV/amu) does induce such clusters in genomic DNA in solution and in human cells. These studies also showed that each damage cluster results from one radiation hit (and its track), thus indicating that they can be induced by very low doses of radiation, i.e. two independent hits are not required for cluster induction. Further, among all complex damages, double strand breaks comprise--at most-- ~20%, with the other clustered damages being at least 80%.

A robust, inexpensive filter for blocking UVC radiation in broad-spectrum 'UVB' lamps.

Accurate studies of the biological effects of UBV radiation require suitable laboratory sources. Lamps labeled as UVB sources often emit UVC radiation that contributes significantly to the levels of DNA damage. The UVC from an unfiltered UVB source produced more pyrimidine dimers in soybean DNA than a lamp filtered by a Pyrex dish that removes wavelengths of < 280 nm. Calculations based on action spectra and on the emission spectra of unfiltered lamps indicate that UVC contributes approximately 13%, 4% and approximately 1% of the total dimers induced in unshielded cells or DNA, alfalfa cotyledons, and human skin, respectively. Further, relative to a Pyrex dish-filtered lamp, an unfiltered lamp would produce approximately 7-, 2.4- or 2.8-fold more dimers in these three biological systems. We report here that a Pyrex dish provides an effective, stable, robust and inexpensive filter for reducing or excluding the contribution of UVC to damage induced by broad-spectrum 'UVB' lamps.

A comparison of electrophoretic resolution for snapshot and finish-line imaging.

Finish-line imaging, in which DNA or other macromolecules are detected after electrophoresis for a constant distance, usually improves resolution compared to snapshot imaging, in which molecules are electrophoresed for a constant time in an apparatus of comparable dimensions. Resolving power, which is an objective measure of the ability of different separatory methods to detect closely spaced molecular species, can be used to compare directly the performance of systems employing both snapshot and finish-line imaging [E. A. Ribeiro and J. C. Sutherland, Anal. Biochem. 210, 378-388 (1993)]. Experimentally determined values of resolving power are influenced both by the method of imaging (snapshot vs finish-line) and by instrument-specific factors that affect resolution. Previous comparisons of the resolving power obtained with finish-line and snapshot imaging involved data sets acquired by different instruments with different instrumental resolutions. To reduce the influence of instrumental effects, we constructed a scanning laser fluorometer that can measure both snapshot and finish-line images of fluorochrome-labeled DNA. Snapshot and finish-line images of a DNA sample containing HaeII restriction fragments of the DNA from bacteriophage T7, which range in length from 474 to 6514 base pairs, were obtained under otherwise identical electrophoretic conditions. Snapshot and finish-line imaging give similar resolving powers for DNA molecules up to about 1.5 kbp long. For both imaging modes, maximum resolving power was achieved for DNA molecules between 2 and 3 kbp in length. For larger DNA molecules, finish-line imaging provided higher resolving power. The ratio of the resolving power of finish-line images to that of snapshot images increased monotonically as a function of DNA length. For the longest restriction fragments studied (6514 bp), the resolving power for finish-line images exceeded that of snapshot images by about 50%.

Fluorescence of matrix isolated guanine and 7-methylguanine.

We have prepared argon and nitrogen matrices containing guanine and 7-methylguanine, and measured their absorption, fluorescence excitation and fluorescence emission spectra. The fluorescence excitation spectrum of guanine shows four well-resolved bands in the range from 170 to 290 nm; excitation at the wavelengths of each of these bands results in a fluorescence emission with maximum intensity near 350 nm and a single-exponential decay with a lifetime of about 10 ns. There are significant differences between the fluorescent excitation and emission spectra of guanine and of 7-methylguanine, suggesting that the fluorescence observed from the guanine sample does not arise from a minority tautomer.

Circular dichroism user facility at the National Synchrotron Light Source: estimation of protein secondary structure.

The ultraviolet circular dichroism of a protein can be used to estimate the net fraction of its amino acids in different classes of secondary structure. Recent advances in the accuracy of such calculations have resulted from improved computational techniques, as well as extension of the spectral region analyzed to wavelengths less than 180 nm, a wavelength range beyond the limit of most laboratory-based circular dichroism spectrometers. We describe a spectrometer that uses UV radiation from the National Synchrotron Light Source at the Brookhaven National Laboratory to record circular dichroism spectra of proteins (and other biologically important molecules) in aqueous solution over the optimum wavelength range required for calculation of secondary structures. This instrument is available for use by scientists from academic, commercial and research institutions.

Separation of chromosomal length DNA molecules: pneumatic apparatus for rotating gels during electrophoresis.

The maximum length of DNA molecules that can be separated by gel electrophoresis can be increased greatly by periodically altering the direction of the electric field with respect to the gel by an angle that exceeds 90 degrees. One method involves rotating the gel by the desired angle in alternate directions periodically during electrophoresis. We describe a modification of the rotating gel electrophoresis apparatus developed by Serwer (Electrophoresis 1987, 8, 301-304) that uses a pneumatic rotary actuator instead of a stepping motor, hence reducing the cost by about 50%. Other advantages of our design are a lower center of gravity that makes the apparatus more stable and the removal of all electrical power from beneath the fluid-filled electrophoresis chamber. We present data demonstrating the separation of chromosomal length DNA molecules from Saccharomyces cerevisiae strain 334 into 14 resolved bands in parallel lanes.

Electronic imaging system for direct and rapid quantitation of fluorescence from electrophoretic gels: application to ethidium bromide-stained DNA.

We have built an electronic imaging system based on a modified charge-coupled-device television camera that directly quantitates the distribution of fluorescence from electrophoretic gels, chromatograms, and other stationary sources. Exposure times can exceed 1 min. Unlike the photographic system that it replaces, the response of the camera is directly proportional to the intensity of incident fluorescence, and image data are digitized and stored in computer memory ready for analysis immediately upon completion of an exposure. We describe procedures for the display, normalization, and archival storage of image data and programs that use images of ethidium bromide-stained DNA in alkaline agarose gels to quantitate single-strand breaks in DNA.

Unidirectional pulsed-field electrophoresis of single- and double-stranded DNA in agarose gels: analytical expressions relating mobility and molecular length and their application in the measurement of strand breaks.

Unidirectional pulsed-field electrophoresis improves the separation of single-stranded DNA molecules longer than 20 kilobases (kb) in alkaline agarose gels compared to static-field electrophoresis. The greatest improvement in separation is for molecules longer than 100 kb. The improved resolution of long molecules with unidirectional pulsed-field electrophoresis makes possible the measurement of lower frequencies of single-strand breaks. The analytical function that relates the length and mobility of single-stranded DNA electrophoresed with a static field also applies to unidirectional pulsed field separations. Thus, the computer programs used to measure single-strand breaks are applicable to both undirectional pulsed- and static-field separations. Unidirectional pulsed-field electrophoresis also improves the separation of double-stranded DNA in neutral agarose gels. The function relating molecular length and mobility for double-stranded DNA separated by unidirectional pulsed-field electrophoresis is a superset of the function for single-stranded DNA. The coefficients of this function can be determined by iterative procedures.

Two-dimensional, computer-controlled film scanner: quantitation of fluorescence from ethidium bromide-stained DNA gels.

A two-dimensional scanner based on a digital plotter is described. The device is used to analyze photographic negatives of ethidium bromide-stained DNA-agarose gels. Scanning is controlled by and photometric data transferred to a computer for processing, storage, display, and analysis such as integration of the areas under bands and determination of the mean distances of migration of polydisperse samples. An integral light source and detector module designed for reading optical "bar-codes" is mounted in place of the pen of the plotter. Spatial resolution and reproducibility are about 0.2 and 0.005 mm, respectively. Photometric precision as good as one part per thousand is achieved by sinusoidal modulation of the intensity of the light source and synchronous, phase-sensitive detection of the signal from the detector by a lock-in amplifier. No part of the sensor assembly touches the surface of the negative. In contrast to a densitometer, the computer transforms photometric data to values directly proportional to the amount of DNA at given points on the original gel. The ability to move the sensor in two dimensions over the negative allows for the integration across the width of a lane correctly allowing for the nonuniform distribution of the DNA.