Computer-based image analysis of one-dimensional electrophoretic gels used for the separation of DNA restriction fragments

Abstract

A stand-alone, interactive computer system has been developed that automates the analysis of ethidium bromide-stained agarose and acrylamide gels on which DNA restriction fragments have been separated by size. High-resolution digital images of the gels are obtained using a camera that contains a one-dimensional, 2048-pixel photodiode array that is mechanically translated through 2048 discrete steps in a direction perpendicular to the gel lanes. An automatic band-detection algorithm is used to establish the positions of the gel bands. A color-video graphics system, on which both the gel image and a variety of operator-controlled overlays are displayed, allows the operator to visualize and interact with critical stages of the analysis. The principal interactive steps involve defining the regions of the image that are to be analyzed and editing the results of the band-detection process. The system produces a machine-readable output file that contains the positions, intensities, and descriptive classifications of all the bands, as well as documentary information about the experiment. This file is normally further processed on a larger computer to obtain fragment-size assignments.

Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis

“…We achieve this sensitivity using a protocol that involves staining the gels with ethidium bromide for 10-30 min at a concentration of 0.1 pg/ml in 0.5xTBE, de-staining for 20 min in 0.5xTBE, and employing an ultraviolet transilluminator with a wavelength maximum at 300nm (Spectronics, Model TR-302). The combination of camera, film, and filters that we employ has been described previously (7). High-speed instant film (Polaroid Type 667) gives adequate results for day-to-day use with exposures of 30 sec or less, while the photographs for this paper were prepared using a wet-process film (Eastman Kodak No.…”

A New Method for Purifying Lambda DNA From Phage Lysates

“…As X cloning is used to analyze the sequences present in increasingly complex DNA sources -such as entire mammalian chromosomes-the requirement for information on the restriction sites present in essentially random sets of clones is likely to become the limiting factor in numerous studies. The effort expended on earlier steps in the X-cloning cycle is largely independent of the complexity of the source DNA, whereas the steps following the acquisition of a high-quality gel photograph are readily automated (Gray et al, 1984). In contrast, the effort devoted to the experimental steps addressed in the present paper increases in direct proportion to the complexity of the system.…”

“…The gel photographs in this paper were obtained by staining the gels in 0.1 /ig/ml ethidium bromide in water for 30 min, stimulating the fluorescence with 300-nm light (using a Spectronics TR-302 transilluminator), and carrying out the photography with a wet-process film (Eastman Kodak No. 4155) as previously described (Gray et al, 1984). For dayto-day use, a high-speed instant film (Polaroid Type 667) is employed.…”

A comprehensive package for DNA sequence analysis in FORTRAN IV for the PDP-11

“…promoter sequences, ribosome binding sites, etc.) Computers in molecular biology are now being adapted to a broader class of applications (17,20): (i) experimental design (3,4,16,57); (ii) data collection (2,19,22,25,27,50); (iii) data cleaning (33); (iv) data base management (54, see also 44,45,46); (v) exploratory analysis (44,46,57); (vi) modeling (30,31,52,53,57). Three areas deserve particular attention.…”