Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions.
Temperature-gradient gel electrophoresis (TGGE) is applied to analyze conformational transitions and sequence variations of nucleic acids and protein-nucleic acid interactions. A linear and highly reproducible temperature-gradient is established perpendicular or parallel to the direction of the electrophoresis. The instrument consists of an electrically insulated metal plate, which is heated at one edge and cooled at the other edge by two thermostating baths and is used as an ancillary device for commercial horizontal gel electrophoresis instruments. Biopolymers are separated in TGGE according to size, shape and thermal stability of their conformational transitions. If the temperature-gradient is established perpendicular to the electrophoresis, monomolecular conformational transitions of nucleic acids show up as continuous transition curves; strand-separation leads to discontinuous transitions. In the studies on viroid RNA it was shown that natural circular viroid RNA undergoes one highly cooperative transition detected by TGGE as a drastic retardation in mobility. Oligomeric replication intermediates of viroids exhibit coexisting structures which could not be detected by any other technique. Double-stranded satellite RNA from cucumber mosaic virus is a mixture of sequence variants, all of which have the identical length of 335 nucleotides. In TGGE six different strains were resolved. Sequence variants of viroids were analyzed by hybridizing viroid RNA to (-)strand viroid RNA transcripts from viroid cDNA clones. Sequence variations lead to mismatches in the double strands and thereby to a shift of the transition curve to lower temperature. Mutations in plasmids, particularly in cloned inserts, were detected by mixing plasmids of two different clones, linearizing, denaturing, renaturing, and searching for shifts in the transition curves, which are generated by mismatch-formation during the renaturation of (+)- and (-)strands from different clones. Examples are given for different viroid clones and HIV-clones from one and the same patient. In another example, clones with point mutations from site-directed mutagenesis are analyzed and selected by TGGE. TGGE is also applied to study the effect of amino acid exchanges in the Tet repressor from E. coli on the thermal stability of the repressor and on the mode of binding of the repressor to the operator DNA. The results are discussed under the aspect that TGGE may be applied as routine analytical laboratory procedure.
Riesner
D
D
Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Federal Republic of Germany.
Steger
G
G
Zimmat
R
R
Owens
R A
RA
Wagenhöfer
M
M
Hillen
W
W
Vollbach
S
S
Henco
K
K
D001483:Base Sequence; D004247:DNA / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009029:Mosaic Viruses / Q000032:analysis; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009695:Nucleic Acid Renaturation; D009696:Nucleic Acids / Q000032:analysis*; D010957:Plasmids; D012313:RNA / Q000032:analysis; D013194:Staining and Labeling; D013696:Temperature; D014772:Viroids / Q000032:analysis
D004268:DNA-Binding Proteins; D009696:Nucleic Acids; D012313:RNA; D004247:DNA