RNase H-based assays utilizing modified RNA monomers

• US 9,434,988 B2
• Filed: 03/23/2012
• Issued: 09/06/2016
• Est. Priority Date: 04/30/2008
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies (Danaher Corporation)
• Inventors: Behlke, Mark Aaron, Rose, Scott D., Dobosy, Joseph, Walder, Joseph Alan
• Primary Examiner(s): Kim, Young J
• Application Number:
  US13/429,077
• Publication Number:
  US 20120258455A1
• Time in Patent Office: 1,628 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/327 RNAse, e.g. RNAseH
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The present invention provides methods of cleaving a nucleic acid strand to initiate, assist, monitor or perform biological assays.

Claims

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.