Blog Against Cancer

PULSED-FIELD GEL ELECTROPHORESIS

One application for Southern blotting is the direct demonstration of physical linkage between two genes. If two different gene probes were to hybridize to the same restriction fragment in a Southern blot, this would prove that the loci of the two genes were closely linked. Unfortunately, eukaryotic genetic linkages ordinarily extend over millions of bases (megabases) of DNA, and the largest DNA fragments that can be resolved by conventional agarose gel electrophoresis are less than 100 thousand bases (100 kilobases). The reason for this limitation is the tendency for all DNA molecules above a certain size to become oriented with their
long axes parallel to the electric field.

This prevents any appreciable interaction between the DNA molecules and the agarose gel. In the absence of such interaction, the DNA molecules are not retarded during
electrophoresis, and will migrate at the same rate regardless of size. If the long axes of these large molecules are periodically reoriented perpendicular to the direction of migration, they once again
interact with the agarose. Such interactions force the DNA molecules to migrate at rates inversely proportional to their lengths, and resolution by size can then be achieved.

A number of ingenious techniques have been designed to accomplish this purpose: pulsed-field gel electrophoresis, in which two electrical fields are oriented perpendicularly and are alternately pulsed;20
field-inversion gel electrophoresis in which a single field is periodically inverted;21 and contour-clamped homogeneous gel electrophoresis (CHEF) in which multiple fields of various orientations can be alternately applied.22 These techniques now allow the separation of DNA fragments from 2 to 5 megabases in length.
Pulsed-field gel electrophoresis has been used in the analysis of gene linkage on the long arm of chromosome 5. Genetic losses and alterations involving 5q have been associated with a variety of hematologic malignancies. The mapping of many of the genes encoding growth factors and growth factor receptors for hematologic cells on 5q has led to the suggestion that alterations in these genes are etiologic in these diseases. By digesting DNA with restriction endonucleases that have rarely occurring recognition sites, and performing Southern blotting experiments after pulsed-field gel electrophoresis, the genes
for IL-3 and granulocyte macrophage (GM)-CSF could be shown to lie on the same 436 kb fragment of DNA.24 Ultimately, it was demonstrated that these genes are separated by only 9 kb of DNA.