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Nucleic Acids Research, 1977, Vol. 4, No. 6 1815-1828
© 1977

Articles

A method for the isolation of cross-linked nucleosides from DNA: application to cross-links induced by nitrous acid

Samuel Dubelman and Robert Shapiro*

Department of Chemistry, New York University New York, NY 10003, USA

*Correspondence should be addressed to this author.

Received February 10, 1977. A procedure is reported for the isolation of cross-linked nucleosides from nitrous acid-treated calf thymus DNA. Cross-linked DNA was hydrolyzed enzymatically with deoxyribonuclease I and snake venom phospho-diesterase and fractionated on a DEAE-Sephadex column. After desalting, the fractions were characterized by ultraviolet spectroscopy, anion exchange high pressure liquid chromatography, gel filtration, and two dimensional thin layer chromatography. A cross-linked dinucleotide, and a series of oligonucleotides were isolated. The oligomers, which had resisted digestion by the above enzyme system, were digested to the nucleo-side level by a spleen phosphodiesterase-alkaline phosphatase combination. A second cross-linked product was isolated from this mixture. The cross-linked nucleosides were less than 0.17% of the total nucleotides of the DNA. The methods developed here are recommended for the isolation of products from DNA treated with other cross-linking agents.


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