Worm Breeder's Gazette 9(2): 108
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Schwartz and Cantor (Cell 37, 67-75 (1984) and Carle and Olson (NAR 12, 5647-5664, 1984) have devised an orthogonal field agarose gel system that allows the separation of large DNA molecules (>2000 kb). A recent modification of the method by Carle et al. (Science 232, 65- 68, 1986) allows these separations to be obtained using a conventional gel box by periodically reversing the field. Control of the switch times through a microcomputer is convenient and allows the switch time to be varied at will during the run. This is important as the switch time controls the range of sizes that can be separated in a given run. Chris Bond of the MRC electronics laboratory designed and built an extremely convenient switch box employing Mosfett power bridges, which have an excellent response time and a long life time. Details of the set up I'm using are available to anyone interested (see also Carle and Olson's Science paper). To make high molecular weight (chromosomal?) DNA from C. elegans, I am currently using pure populations of L1's as starting material. These are embedded in 0.5% agarose in 0.125 M EDTA 0.125 M Tris pH9 and then lysed by overlayering with 1% sarcosyl, 1 mg/ml proteinase K and 7% -ME as described by Carle and Olson (1985) for yeast. Without restriction of the lysed worms very little ethidium stained material enters the gel under a variety of conditions with the exception of some low molecular weight material, assumed to be degraded RNA, and 2 or 3 ethidium stained bands with a mobility corresponding to about 100 kb but of unknown origin. After digestion with any of a series of restriction enzymes large amounts of DNA enter the gel. Two enzymes, NotI and SfiI which both have 8 bp recognition sequences composed entirely of G:C pairs yield a smear extending from about 100 kb to more than 1000 kb. A few distinct bands are visible against the background smear but it is impossible to directly estimate the number of fragments present. From parallel experiments using digests of random cosmid clones, NotI sites are estimated to occur every 600-700 kb on average, predicting be less than 150 bands. SfiI sites are slightly more frequent. After BglI digest, a 6 bp recognition site enzyme that does not cut within the ribosomal repeat sequence (Ellis et al. NAR 14, 2345-2364, 1986), most DNA appears to be about 50-150 kb although bands are visible at 300 and 350-400 kb and a single distinct band is present with a mobility between that of yeast chromosomes XI (~700 kb) and X. This band hybridizes strongly and specifically with an rDNA probe provided by R. Fishpool, and places an upper limit of 100 rDNA of 7.2 kb repeats. Current efforts are being directed toward using this methodology to characterize genome rearrangements in the sma-1 region of LGV. I am also trying to identify the free duplication fragments mnDp30 and eDp6 in unrestricted DNA preps. The methodology may provide a level of resolution intermediate between that of cytogenetics and the cosmid mapping.