Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis

DC Schwartz, CR Cantor - cell, 1984 - cell.com
DC Schwartz, CR Cantor
cell, 1984cell.com
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 fig 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 set
to 90 set to achieve optimal separations for DNAs with sizes from 30 to 2000 kb. This pulsed …
Summary
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 fig 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 set to 90 set 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.
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