METHODS FOR CLEAVING DNA WITH NUCLEOTIDE INTEGRASES

摘要

The present invention provides methods, using a nucleotide integrase, for cleaving nucleic acids substrates at specific sites and inserting a nucleic acid molecule into the cleaved substrate. The method of cleaving one strand of a double-stranded DNA substrate comprises providing a nucleotide integrase comprising a group II-intron RNA having two hybridizing sequences capable of hybridizing with two intron RNA binding sequences on the one strand of the substrate and a group II-intron encoded protein which binds to a first sequence element of the substrate. The method of cleaving both strands of a double-stranded DNA substrate comprises providing a nucleotide integrase comprising a group II-intron RNA having two hybridizing sequences capable of hybridizing with two intron RNA binding sequences on one strand of the substrate and a group II-intron encoded protein capable of binding to first and second sequence elements in the recognition site of the substrate. The method of cleaving a single-stranded nucleic acid substrate comprises providing an integrase having two hybridizing sequences capable of hybridizing with two intron RNA-binding sequences of the substrate and a group II-intron encoded protein.