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Abstract
A new type of gel electrophoresis separates DNA molecules up to 2000 kb with resolutions
exceeding the logarithmic molecular weight dependence of conventional electrophoresis.
The technique uses 1.5% agarose, 10 to 20 micrograms of DNA per well, and low ionic
strength buffers. It employs alternately pulsed, perpendicularly oriented electrical
fields, at least one of which is inhomogeneous. The duration of the applied electrical
pulses is varied from 1 sec to 90 sec to achieve optimal separations for DNAs with
sizes from 30 to 2000 kb. This pulsed field gradient gel electrophoresis fractionates
intact S. cerevisiae chromosomal DNA, producing a molecular karyotype that greatly
facilitates the assignment of genes to yeast chromosomes. Each yeast chromosome consists
of a single piece of DNA; the chromosome sizes are consistent with the genetic linkage
map. We also describe a general method for preparing spheroplasts, and cell lysates,
without significant chromosomal DNA breakage.