In order to facilitate genome mapping and sequencing, three approaches for targeting the chemical nuclease activity of 1,10- phenanthroline-copper(OP-Cu) to any chosen DNA sequence will be developed. In the first strategy, RNA is transcribed from the target DNA sequence using 5-allylamine-UTP in place of UTP and then chemically modified with 1,10-phenanthroline. The derivatized RNA is used to form an R-loop that provides the sequence specificity for the scission reaction which is activated by the addition of cupric ion, thiol and hydrogen peroxide. In the second method, one DNA strand of the sequence targeted for cleavage within the genome is synthesized by primer extension substituting 5-allylamine dUTP in place of TTP. .The single-strand DNA is derivatized with 1,10-phenanthroline and used as a substrate for Rec A facilitated strand invasion of the target sequence to form a D-loop. The cleavage reaction is then activated as above. Finally, the cro protein will be modified by site-directed mutagenesis to target the nuclease activity of OP-Cu. All three procedures could be used to develop sequence-specific nucleases for any preselected sequence.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG000255-03
Application #
2208670
Study Section
Biochemistry Study Section (BIO)
Project Start
1991-04-01
Project End
1995-03-31
Budget Start
1993-04-01
Budget End
1995-03-31
Support Year
3
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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