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.

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%.