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.
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- 86.Technical Section 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.