REVERSE TRANSCRIPTION AND AMPLIFICATION OF RNA WITH SIMULTANEOUS DEGRADATION OF DNA

• US 20100015602A1
• Filed: 03/27/2006
• Published: 01/21/2010
• Est. Priority Date: 04/01/2005
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Loffert, Dirk, Peist, Ralf, Korfhage, Christian
• Granted Patent: US 8,309,303 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container

Abstract

The invention relates to a method for processing RNA, in particular, a RNA reaction method and kits for carrying out said RNA reaction method.

Claims

1. A method of analyzing RNA comprising, degradation of double-stranded DNA and an RNA reaction wherein the degradation of double-stranded DNA and the RNA reaction are carried out in the same reaction vessel at the same time and at the same temperature, wherein the degradation of double-stranded DNA is carried out with an enzyme with DNA double-strand-specific endonuclease activity.

2. The method of claim 1, wherein the RNA reaction comprises a tagging, extension or modification of the RNA.

3. The method of claim 1, wherein the RNA reaction is a specific degradation reaction, wherein the RNA is selectively degraded.

4. The method of claim 1, wherein the RNA reaction is a conversion reaction, wherein the RNA is used as a template for a polymerase reaction.

5. The method of claim 4, wherein the conversion reaction is a reverse transcription reaction.

6. The method of claim 1, wherein the RNA acts as catalyst in the RNA reaction.

7. The method of claim 1, wherein the RNA is involved in a binding reaction in the RNA reaction.

8. The method of claim 1, wherein the RNA reaction is a combined reaction, wherein at least two of the reactions selected from the group consisting of a tagging of the RNA, an extension of the RNA, a modification of the RNA, a specific degradation reaction, or a conversion reaction are involved.

9. The method of claim 1, wherein the degradation of DNA is carried out with an enzyme selected from the group consisting of DNase 1 and restriction endonucleases.

10. The method of claim 1, wherein the method is carried out at a pH value of between 6 and 10.

11. The method of claim 1, wherein the reaction comprises divalent cations.

12. The method of claim 11, wherein the divalent cations are selected from the group consisting of Mg²⁺, Mn²⁺ and Ca²⁺.

13. A kit for performing the method of claim 1, the kit comprising at least one reverse transcriptase, an enzyme with DNA double-strand-specific endonuclease activity and a reaction buffer.

14. The method of claim 3, wherein the RNA is selectively degraded by a ribozyme, RNase H or by siRNA-induced RNA degradation.

15. The method of claim 10, wherein the method is carried out at a pH value of between 7 and 9.