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Nucleic Acids Research, 1989, Vol. 17, No. 23 9957-9977
© 1989

CHEMISTRY

Immunoassays for carbodiimide modified DNA-detection of unpairing transitions in supercoiled ColE1 DNA

Altaf A. Wani, John K. Sullivan1 and Jacob Lebowitz1,*

Department of Radiology, The Ohio State University Columbus, OH 43210 1Department of Microbiology, University of Alabama at Birmingham Birmingham, AL 35294, USA

*To whom correspondence should be addressed

Received August 22, 1989. Revised October 24, 1989. Accepted October 24, 1989.

The water soluble reagent N-cyclohexyl-N'-ß-(4-methylmorpholinium) ethyl carbodiimide-p-toluene sulphonate (CMC) can be used to probe for unpaired and mismatched sites in DNA. Polyclonal antibodies for CMC modified DNA were produced in order to develop inmunological assays for the localization and qnantitation of CMC adducts. Immunoslot blot analysis of modified DNA exhibited antibody binding proportional to the extent of CMC modification with adduct detection in the femtamole range. Unmodified DNA did not cross react under the conditions of the assay. The distribution of CMC reactivity for supercoiled C0lE1 DNA modified at 100, 200 and 300mM NaCl was determined by immunoanalysia of EcoRI-Hae2-NruI restriction fragments Southern transferred to nylon membranes. Reactivity above random expectation occurred in the A2-II fragment which can be accounted for by its high A-T content of 71.3%. Reactivity below random expectation occurred in the C fragment which can be accounted for by its low AT content of 43%. CMC modification for the other restriction fragments appeared random.


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