A method for two-dimensional DNA electrophoresis (2D-DE): application to the immunoglobulin heavy chain variable region.
The scarcity of single-copy probes creates difficulty in the generation of large-scale physical maps of mammalian gene families. A simple method of two-dimensional DNA electrophoresis (2D-DE) has been developed to overcome this problem. DNA (2 micrograms) is digested with a rare-cutting restriction endonuclease and size separated by pulsed-field gel electrophoresis (PFGE). The DNA, still contained within the lane of the PFGE gel, is digested with a second frequent-cutting restriction enzyme and is subjected to an electrical field perpendicular to that of the PFGE. 2D-DE allows the simultaneous mapping, to large restriction fragments, of all the genes detected by a particular probe. The human immunoglobulin variable region was used as an example for this procedure. Two VH5 genes, on 8- and 9-kb EcoRI fragments, were mapped to 200- and 65-kb SfiI fragments, respectively, by 2D-DE. This technique will be particularly useful in the generation of physical maps of complex human gene families and of repeat families.
Walter
M A
MA
Research Institute, Hospital for Sick Children, Ontario, Canada.
Cox
D W
DW
D002874:Chromosome Mapping; D004247:DNA / Q000032:analysis / Q000235:genetics; D004262:DNA Restriction Enzymes; D004587:Electrophoresis, Agar Gel / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D005810:Multigene Family
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D004247:DNA; D004262:DNA Restriction Enzymes