HARTLEY, JA; SOUHAMI, RL; BERARDINI, MD; (1993) ELECTROPHORETIC AND CHROMATOGRAPHIC-SEPARATION METHODS USED TO REVEAL INTERSTRAND CROSS-LINKING OF NUCLEIC-ACIDS. J CHROMATOGR-BIOMED , 618 (1-2) 277 - 288.
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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.
|Title:||ELECTROPHORETIC AND CHROMATOGRAPHIC-SEPARATION METHODS USED TO REVEAL INTERSTRAND CROSS-LINKING OF NUCLEIC-ACIDS|
|Keywords:||CROSS-LINKING AGENTS, SINGLE-NUCLEOTIDE RESOLUTION, DNA-SEQUENCE SPECIFICITY, MITOMYCIN-C, DEOXYRIBONUCLEIC-ACID, DEOXYGUANOSINE RESIDUES, DUPLEX, FRAGMENTS, SITES, MECHLORETHAMINE|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Wolfson Institute and Cancer Institute Administration > Cancer Institute > Research Department of Oncology|
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