Abstract

A new technique has been developed for analyzing DNA for modifications induced by carcinogens and mutagens. The assay advocates a novel approach combining the basic idea of postlabeling the enzymatically digested DNA with a fluorophore and fluorescence detection. Development of He-Cd laser induced fluorescence detection in combination with microbore high performance liquid chromatography has offered a sub-femtomol limit of detection for real-time analysis of dansylated nucleotides. Further improvement in the performance of the assay system as well as application to emerging biological problems requiring highly sensitive detection of DNA modifications are the general objective of the proposed application.
The specific aims are: 1) to introduce capillary electrophoresis with laser- induced fluorescence detection as an instrumental method for analysis of the postlabeled nucleotides, 2) to extend the current in vitro DNA damage analysis to in vivo study using well-suited carcinoma cell lines and white blood cells from cancer patients exposed to fluoropyrimidines and platinated drugs.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA046896-06
Application #
2092361
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1989-04-01
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
6
Fiscal Year
1994
Total Cost
Indirect Cost

  Institution

Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
City
Buffalo
State
NY
Country
United States
Zip Code
14263

  Publications

Sharma, M; Jain, R (1998) Isolation and analysis of dityrosine from enzyme-catalyzed oxidation of tyrosine and X-irradiated peptide and proteins. Chem Biol Interact 108:171-85
Jain, R; Freund, H G; Budzinsky, E et al. (1997) Radiation-induced formation of 3,4-dihydroxyphenylalanine in tyrosine-containing peptides and proteins as a function of X-irradiation dose. Bioconjug Chem 8:173-8
Sharma, M; Jain, R; Ionescu, E et al. (1995) Quantitative analysis of 3'-azido-3'-deoxythymidine incorporation into DNA in human colon tumor cells. Bioconjug Chem 6:536-40
Sharma, M; Jain, R; Ionescu, E et al. (1995) Capillary electrophoretic separation and laser-induced fluorescence detection of the major DNA adducts of cisplatin and carboplatin. Anal Biochem 228:307-11
Azadnia, A; Campbell, R; Sharma, M (1994) The scope of dansyl vs fluorescein label in fluorescence postlabeling assay for DNA damage. Anal Biochem 218:444-8
Jain, R; Isac, T V; Sharma, M (1994) Fluorescence postlabeling assay of RNA modification. Biochem Biophys Res Commun 200:1239-44
Jain, R; Sharma, M (1993) Fluorescence postlabeling assay of DNA damage induced by N-methyl-N-nitrosourea. Cancer Res 53:2771-4
Lee, T; Yeung, E S; Sharma, M (1991) Micellar electrokinetic capillary chromatographic separation and laser-induced fluorescence detection of 2'-deoxynucleoside 5'-monophosphates of normal and modified bases. J Chromatogr 565:197-206
Sharma, M; Jain, R (1991) Nuclease P1-mediated fluorescence postlabeling assay of AAF modified DNA model d(TACGTA) and calf-thymus DNA. Biochem Biophys Res Commun 177:151-8
Sharma, M; Jain, R; Isac, T V (1991) A novel technique to assay adducts of DNA induced by anticancer agent cis-diamminedichloroplatinum(II). Bioconjug Chem 2:403-6

Showing the most recent 10 out of 12 publications