Methods And Compositions For Transcription-Based Nucleic Acid Amplification

• US 20080182300A1
• Filed: 10/31/2007
• Published: 07/31/2008
• Est. Priority Date: 06/26/2000
• Status: Abandoned Application

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith
• Application Number:
  US11/933,332
• Publication Number:
  US 20080182300A1
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

Methods for isothermal exponential amplification of a target polynucleotide are disclosed. The methods employ two transcription modules, the first module providing linear amplification resulting in RNA transcripts, and a second module providing for further (generally cyclical) amplification resulting in more RNA transcripts. In one aspect, the amplification of the first module is composite primer based. In a second aspect, the amplification of the first module is based on target switching to generate a primer extension product comprising a promoter sequence. In all aspects, the RNA transcripts of the first transcription module are subjected to further amplification by creating an intermediate product comprising a double stranded promoter region from which transcription can occur. The invention further provides compositions and kits for practicing said methods, as well as methods which use the amplification results.

Claims

1. A kit comprising (a) a propromoter template switch oligonucleotide (TSO), (b) a first primer, (c) a DNA dependent DNA polymerase, (d) an RNA dependent DNA polymerase, (e) an enzyme that cleaves RNA from an RNA/DNA hybrid, and (f) an RNA polymerase.

2. The kit of claim 1 further comprising a second primer.

3. The kit of claim 1 wherein the enzyme which cleaves RNA from an RNA/DNA hybrid has RNAse H activity.

5. The kit of claims 1 or 4 further comprising deoxynucleoside triphosphates or ribonucleoside triphosphates.

6. The kit of claim 5 wherein a deoxynucleoside triphosphate or a ribonucleoside triphosphate is labeled.

7. The kit of claim 1 or 4 further comprising a nucleotide analog that upon incorporation into a primer extension product effects termination of nucleotide polymerization.

8. The kit of claim 7 wherein the nucleotide analog which effects termination is labeled.

9. The kit of claims 1 or 4 wherein one or more components is provided as a dry powder.

4. A kit comprising one or more containers comprising a first primer comprising a propromoter TSO, a first primer, an enzyme which cleaves RNA from an RNA/DNA hybrid and any two of:

(a) a second primer, (b) an enzyme with DNA dependent DNA polymerase activity, (c) an enzyme with RNA dependent DNA polymerase activity, and (d) an RNA polymerase.

10. A system for amplifying a sequence of interest, comprising:

(a) a propromoter TSO;

(b) a first primer;

(c) an RNA-dependent DNA polymerase;

(d) an enzyme that cleaves RNA from an RNA/DNA hybrid; and

(e) an RNA polymerase.

11. The system of claim 10 further comprising a DNA-dependent DNA polymerase.

12. The system of claim 10, wherein the RNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.

13. The system of claim 11, wherein the DNA-dependent DNA polymerase and the RNA-dependent DNA polymerase are the same enzyme.

14. The system of claim 11, wherein the DNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.

15. The system of claim 11, wherein the RNA-dependent DNA polymerase, DNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same.

16. The system of claim 10 further comprising a second primer.

17. The system of claim 10 wherein the first primer comprised DNA.