RNase H-based assays utilizing modified RNA monomers

1. 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).

2. The method of claim 1 wherein the cleavage domain is a single RNA residue.

3. The method of claim 1 wherein the amplification is performed in a PCR assay that is used to discriminate between variant alleles.

4. The method of claim 3 wherein the PCR assay is used to quantitate the abundance of the target nucleic acid sequence in the sample.

5. The method of claim 1 , wherein the starting temperature is at or below 37° C.

6. The method of claim 1 wherein the RNase H2 enzyme has, at the starting temperature, less than about 15% of the activity that the RNase H2 enzyme has at the elevated temperature in step (b).

7. The method of claim 1 wherein the RNase H2 enzyme has, at the starting temperature, less than about 5% of the activity that the RNase H2 enzyme has at the elevated temperature in step (b).

8. The method of claim 1 wherein the RNase H2 enzyme has, at the starting temperature, less than about 1% of the activity that the RNase H2 enzyme has at the elevated temperature in step (b).

9. The method of claim 1 wherein the RNase H2 enzyme is Pyrococcus abyssi RNase H2.

10. The method of claim 1 , wherein the RNase H2 enzyme is encoded by SEQ ID NO: 4.

11. The method of claim 1 , wherein the RNase H2 enzyme is reversibly inactivated either by chemical modification or by a blocking antibody.