SEQUENCE-SPECIFIC CHEMICAL NUCLEASES FOR GENOME ANALYSIS

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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.