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Resolution of Linear DNA Fragments From 23 Kilobases to 6 Megabases Using Biphasic Linear Switch-Time Ramping

Contributed by Donald R. VanDevanter, Tumor Institute, Swedish Hospital Medical Center, 409 Elkind Hall, 747 Summit Avenue, Seattle, WA 98104


Introduction
Analyses of uncharacterized DNA fragments using CHEF electrophoresis often require preliminary studies to determine what size ranges DNA fragments of interest fall within. It is not uncommon to run more than one gel in order to fully characterize large DNA fragments from a single cell source1. This is because DNA fragments under 200 kilobase pairs (kbp), between 200 kbp and 2 megabase pairs (mbp), and between 2 mbp and 6 mbp are optimally separated by CHEF electrophoretic techniques using different switching frequencies, electrophoretic field strengths, angles of orientation (if using a Bio-Rad CHEF Mapper system), and/or agarose type and concentration2. Data is presented illustrating that an initial estimate of sizes can be obtained in a single gel by using a biphasic linear switch time ramping procedure.


Results
A biphasic linear switch time ramping strategy3 with a fixed electric field strength can be used with the CHEF-DR II system to resolve linear DNA fragments ranging from 23 kbp to 5.7 mbp in size on a single gel (Figure 1). The gel shown in Figure 1A was 0.5% Chromosomal Grade Agarose made in 0.5x TBE (45 mM Tris borate, 1.0 mM EDTA, pH 8.0), recirculated at 10 C. The applied electric field was held constant at 1.95 V/cm (65 V), and switch times were ramped linearly from 30 seconds to 2 minutes over 33 hours, then from 2 minutes to 50 minutes over 55 hours. Under these conditions, the chromosomes of Schizosaccharomyces pombe (closed circles, lanes 4 and 6), Saccharomyces cerevisiae (open squares, lanes 3 and 5), lambda phage concatomers (open circles, lane 2), and the larger fragments of Hind III-digested lambda DNA (closed squares, lane 1) migrate as an approximate linear function of the log of their respective molecular weights (in mbp, Figure 1B).

Lowering the concentration of agarose in gels can have the effect of decreasing run times without an apparent effect on the linearity of separation (Figure 2). The migration profiles shown in Figure 2 were derived by running the S. pombe and S. cerevisiae chromosomes on 0.5%, 0.4%, 0.3% and 0.2% Chromosomal Grade Agarose gels.


Discussion
The CHEF electrophoresis strategy outlined above is not ideal for sharp resolution of bands within a particular narrow range of DNA fragment sizes, but can be extremely useful for determining what range of fragment sizes deserve closer examination. For this reason, it is a preferable approach to running multiple pulsed-field gels under different conditions when size ranges of greatest interest remain unknown.


References
1. VanDevanter, D.R., Piaskowski, V.D., Caspar, J.T., Douglass, E.C., and Von Hoff, D.D., J. Natl. Cancer Inst. 82:1815- 1821, (1990).

2. Lai, E., Birren, B.W., Clark, S.M., Simon, M.I., and Hood, L., BioTechniques 7:34-42.

3. VanDevanter, D.R., Nucleic Acids Res. 20:1148.


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