RNase H-Based Assays Utilizing Modified RNA Monomers

A method of amplifying a target DNA sequence, said method comprising the steps of:

• a) providing a reaction mixture comprising (i) an upstream oligonucleotide primer and a downstream oligonucleotide primer each having a cleavage domain positioned 5′ of a blocking group, said blocking group linked at or near the end of the 3′-end of each oligonucleotide primer wherein said blocking group prevents primer extension, (ii) a sample nucleic acid that may or may not have the target sequence, (iii) a cleaving enzyme and (iv) a polymerase wherein said cleaving enzyme is a hot start cleaving enzyme which is thermostable and has reduced activity at lower temperatures;
• b) hybridizing either the upstream or downstream primer to the target DNA sequence to form a double-stranded substrate;
• c) cleaving the hybridized primer with said cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the primer;
• and
• d) extending the primer with the polymerase.