Electrophoretic and chromatographic separation methods used to reveal interstrand crosslinking of nucleic acids.
Several electrophoretic and chromatographic techniques, many of which have only been developed recently, provide sensitive methods for the detection and separation of DNA containing interstrand crosslinks such as those produced by many cancer chemotherapeutic drugs and photoactive psoralen derivatives. Most of the methods rely on the fact that the presence of such crosslinks prevent the complete denaturation of the two complimentary DNA strands by heat or alkali. A simple and highly sensitive neutral agarose gel electrophoresis method is particularly applicable to detailed time-course experiments of both total crosslink formation, and the "second-arm" of the crosslink reaction. This method separates denatured single-stranded from double-stranded DNA which has reannealed as a result of an interstrand crosslink. Polyacrylamide gel-based assays using denaturing gels are more suited to the separation of smaller crosslinked DNA fragments and, in particular, small oligonucleotides on high-percentage gels. In addition, they provide methods for the determination of the exact base position and sequence selectivity of crosslink formation. Sephadex chromatography and high-performance liquid chromatography can separate small crosslinked oligonucleotides from non-crosslinked duplexes, and the hydroxyapatite column chromatographic separation of single- and double-stranded cellular DNA can be used to quantitate the level of interstrand crosslinking present in the bulk of the genome. Finally, the analysis of damage by crosslinking agents, and its repair, at the level of specific genes can be achieved by hybridization with specific probes following membrane transfer from neutral agarose gels used to fractionate restricted and fully denatured genomic DNA from drug-treated cells.
Hartley
J A
JA
Department of Oncology, University College London Medical School, UK.
Souhami
R L
RL
Berardini
M D
MD
D000818:Animals; D001483:Base Sequence; D002847:Chromatography, Agarose / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000032:analysis / Q000187:drug effects / Q000235:genetics; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009696:Nucleic Acids / Q000032:analysis* / Q000378:metabolism
D003432:Cross-Linking Reagents; D009696:Nucleic Acids; D004247:DNA