RNase H-based assays utilizing modified RNA monomers

• US 9,644,198 B2
• Filed: 08/05/2013
• Issued: 05/09/2017
• Est. Priority Date: 04/30/2008
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies (Danaher Corporation)
• Inventors: Walder, Joseph Alan, Behlke, Mark Aaron, Rose, Scott, Dobosy, Joesph
• Primary Examiner(s): HOLLAND, PAUL J
• Application Number:
  US13/959,637
• Publication Number:
  US 20140106433A1
• Time in Patent Office: 1,373 Days
• Field of Search: None
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2549/101 Hot start

Abstract

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3′ end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

Claims

1. A hot start enzyme composition comprising an RNase H enzyme and a chemical modification to said RNase H enzyme, wherein said chemical modification reversibly inactivates said RNase H enzyme, and wherein said chemical modification is formed by reacting said RNase H enzyme with a maleic acid anhydride analog.

2. The hot start enzyme composition of claim 1, wherein said RNase H enzyme is Pyrococcus abyssi RNase H enzyme.

3. The hot start enzyme composition of claim 2, wherein said Pyrococcus abyssi RNase H enzyme comprises the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:

4.

4. The hot start enzyme composition of claim 1, wherein said maleic anhydride analog is citraconic anhydride.

5. A hot start enzyme composition comprising an RNase H enzyme and an antibody bound to said RNase H enzyme, wherein said antibody reversibly inactivates said RNase H enzyme when said antibody is bound to said RNase H enzyme.

6. The hot start enzyme composition of claim 5, wherein said RNase H enzyme is Pyrococcus abyssi RNase H enzyme.

7. The hot start enzyme composition of claim 6, wherein said Pyrococcus abyssi RNaseH enzyme comprises the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:

4.

8. The hot start enzyme composition of claim 6, wherein said antibody is an antibody that specifically binds said Pyrococcus abyssi RNase H enzyme.