Worm Breeder's Gazette 9(2): 109

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.


M. Finney and B. Horvitz

We have been using Maynard Olson's one-dimensional field-reversal 
gels (an improvement on Schwartz-Cantor orthogonal gels; see below) to 
look at large pieces of DNA.  This technique is potentially useful for 
worm breeders in several ways.  First, it may be possible to separate 
small free duplications from worm chromosomes, making quick clone 
mapping or region-specific libraries possible.  Second, restriction 
enzymes that cut worm DNA infrequently can be used to determine the 
structure of DNA in a large region; specifically, they can be used to 
find breakpoints of chromosomal aberrations.  Third, resolution can be 
improved over normal gels in the 10-50 kb range.
Intact (>2Mb) DNA was prepared (see below) from SP957, a strain 
carrying mnDp30 balanced by a deficiency.  On gels that resolve a 1.6 
Mb yeast chromosome, mnDp30 is excluded from the gel (using a probe 
kindly supplied by Barbara Meyer).  We were not particularly surprised,
as 1.6 MB is about one-eighth of a chromosome, about the genetic size 
of mnDp30.  (Contrary to Olson, we find that DNA larger than a certain 
cutoff size remains at the origin; he probably did not see this 
phenomenon because he used nothing larger than yeast chromosomes.)
The eight-base recognition sequence enzymes NotI and SfiI both cut 
worm DNA into a smear of fragments ranging in size from about 50 kb to 
about 300 kb (and a small amount of DNA of very high molecular weight).
This range is somewhat smaller than was predicted based on the AT 
content of worm DNA.  Getting a complete digest can be difficult if 
there is very much DNA; nevertheless, we do see bands on Southerns, 
and we are looking for the eT1 breakpoint using probes linked to unc-
Intact worm DNA: Prepare nuclei suspended in agarose as follows.  
Freeze worms at -80 C (or in liquid nitrogen) in a buffered solution 
containing 1mM spermidine, 5mM EDTA, and 1% NP40.  Grind the frozen 
worms in a mortar until powdered.  Scrape the powder into a microfuge 
tube and let thaw on ice.  Spin in a microfuge for 1 second to pellet 
debris and transfer the sup to a new tube.  Spin for 10 minutes at 5 C 
to pellet nuclei and discard sup.  Resuspend pellet in the same buffer 
without detergent and add and mix in quickly an equal volume of 50mM 
EDTA 1% agarose, melted and cooled to 55 C.  Let the agarose harden 
and slice the plug into convenient sized pieces.  Add a ml of your 
favorite SDS-proteinase K solution and incubate at 65 C for an hour.  
Soak the slices in several changes of TE.  Store them at 5 C in TE.  
To digest, soak a slice in the appropriate enzyme buffer containing 
1mM of the protease inhibitor PMSF, and then change the buffer and add 
enzyme.  Before trying to load the slice into a gel well, it helps to 
soak it in TE containing dye, so you can find it if you drop it.
Field reversal electrophoresis: First read Carle, Frank, and Olson, 
Science 232, 65, 1986.  Initially we reversed the field using a 
computer-controlled apparatus built by Leon Avery and MF.  Plans are 
available on request.  The disadvantage of this arrangement is that 
the computer is tied up for the entire run of the gel.  Now we are 
using a switching apparatus with an integral microprocessor built by 
MF.  We are still experimenting with conditions--for the latest, give 
us a call.