Analysis and purification of peptide nucleic acids by denaturing PAGE.
A flexible and convenient protocol for the analysis and purification of peptide nucleic acid (PNA) oligomers and PNA-peptide chimeras by denaturing PAGE is described. Vertical slab gel electrophoresis, 26% in polyacrylamide and 8 M urea at pH 3, was suitable for analysis of oligomers ranging in size from tetramers (4-mers) to tetradodecamers (24-mers). Single-base resolution of oligomers was achieved and separations are generally superior to those given by standard RP-HPLC techniques. The separation of a related series of PNA oligomers showed the distance migrated was linearly dependent on the logarithm of the molecular weight. The migration of oligomers through the gel is dependent on the number of basic functional groups present, such as amino groups, and the A and C content of the oligomer. PNAs are amenable to detection by UV-shadowing technique illuminated at 260 nm or Coomassie blue staining, both with similar, sub-microgram per band detection limits.
Dodd
David W
DW
Department of Chemistry, The University of Western Ontario, London, ON, Canada.
Hudson
Robert H E
RH
D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry / Q000302:isolation & purification*; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000302:isolation & purification*; D011489:Protein Denaturation; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D014508:Urea / Q000737:chemistry
D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; D014508:Urea; D004247:DNA