Dynamic molecular combing: a 'low tech' method for genetic mapping and diagnosis

Abstract

So much of molecular genetics involves invisible samples at the bottom of minuscule tubes, that the attraction of something you can actually see is virtually irresistible. Recent developments in high-resolution fluorescent in situ hybridization (FISH) are both visually appealing and a valuable addition to the repertoire of gene-mapping and diagnostic techniques. The latest of these, dubbed dynamic molecular combing allows precise, high-resolution mapping of markers on cloned or total genomic DNA. In this technique, a coverslip coated with silane is dipped into a DNA solution. After a few minutes' incubation, during which the ends of the DNA molecules bind to the coverslip, a small motorized device is used to withdraw the coverslip vertically, by its edge, at a constant rate of 300 microseconds −1. The force exerted on the DNA molecules by the meniscus stretches all the molecules uniformly in the same direction, and they dry instantly onto the silanized surface as it is exposed to air. The result is a surface covered with a high density of parallel DNA molecules, which can then be probed with fluorescent-labelled markers and visualized under a miscroscope. Because of the constant stretching factor of 2 kb per micrometer of fibre length, no internal control is needed for calibration, unlike other recent fibre-FISH methods. In pilot experiments, the technique has been used successfully to measure microdeletions involving the tuberous sclerosis 2 gene, for fine-mapping and orienting of sets of contiguous cosmid clones (contigs) on a yeast artificial chromosome within its yeast genome, and to measure gaps in a cosmid contig by hybridizing pairs of cosmid probes to combed total human genomic DNA. Dynamic molecular combing will also be useful for a variety of applications in genetic diagnosis, such as mapping the breakpoints where segments of different chromosomes have been interchanged, and analysing sequence rearrangements within genes. Probes as small as 3 kb can be used when mapping cloned DNA; when total genomic DNA is the template, however, the probe needs to be at least 5 kb to enable the signal to be distinguished from background. The length of DNA that can be visualized in any one field of view, approximately 400-600 kb, is determined by the characteristics of the camera used to record the image and the objective on the microscope. The new equipment needed is simple and the protocol very straightforward, which should put it within the reach of most genetics laboratories. The motorized device used to withdraw the coverslip from the DNA solution at the required speed of 300 micrometer s −1 will be available commercially from early 1998 (contact the authors of the Science paper for details). Alternatively, such a device could be readily constructed by most departmental workshops. The silane-coated coverslips are best prepared in the gaseous phase, especially for combing total genomic DNA, when a homogeneous surface is essential. The researchers in Paris and London who developed the technique are, therefore, also considering making the coverslips commercially available.