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.

Reversed Field Gel Electrophoresis of C. elegans DNA

R.H. Waterston

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 
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