RNase H-based assays utilizing modified RNA monomers

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

• (a) providing a reaction mixture at a starting temperature at or below 40° C. wherein the reaction mixture comprises:
• (i) a first oligonucleotide primer having a cleavage domain positioned 5′ of a blocking group, the blocking group linked at or near the end of the 3′-end of the oligonucleotide primer, wherein the blocking group prevents primer extension and/or PCR,
• (ii) a sample nucleic acid that may or may not have the target sequence,
• (iii) a cleaving enzyme, wherein the cleaving enzyme is an RNase H2 enzyme,
• (iv) a polymerase, and
• (v) optionally, a second oligonucleotide primer in reverse orientation to support PCR;
• (b) elevating the temperature of the reaction mixture to at or above 50° C. to increase the activity of the RNase H2 enzyme;
• (c) hybridizing the blocked oligonucleotide primer to the target DNA sequence to form a double-stranded substrate;
• (d) cleaving the hybridized oligonucleotide primer with the cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the oligonucleotide primer;
• and
• (e) extending the oligonucleotide primer with the polymerase,
• wherein the RNase H2 enzyme has, at the starting temperature, less than about 16% of the activity that the RNase H2 enzyme has at the elevated temperature in step (b).