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Nucleic Acids Research, 2001, Vol. 29, No. 21 e105
© 2001 Oxford University Press

A versatile in vivo footprinting technique using 1,10-phenanthroline–copper complex to study important cellular processes

Shashwati Basak1 and V. Nagaraja1,2,*

1Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India and 2Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 012, India

A number of reagents have been used to define the sequence-specific protein–DNA contacts by footprinting analysis. We report a new in vivo technique using the complex of 1,10-phenanthroline and copper [(OP2)Cu] as a probe to study various intracellular DNA–protein interactions in whole cells. The versatility of the protocol is demonstrated by applying the technique to address various processes. The protocol is applied to (i) detect structural alterations in DNA as a result of single base substitution, (ii) footprint site-specific DNA-binding proteins, (iii) analyze promoter occupancy by RNA polymerase and (iv) analyze molecular interactions during transcription initiation. The results demonstrate that in vivo (OP)2Cu probing is a useful tool in studying important cellular processes involving DNA–protein interactions and has potential applications in post-genomic research.

* To whom correspondence should be addressed at: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India. Tel: +91 80 360 0668; Fax: +91 80 360 2697; Email: vraj{at}mcbl.iisc.ernet.in


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