Method for linear mRNA amplification

• US 6,916,633 B1
• Filed: 10/16/2000
• Issued: 07/12/2005
• Est. Priority Date: 05/28/1999
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Shannon, Karen W.
• Primary Examiner(s): LeGuyader, John L.
• Assistant Examiner(s): BOWMAN, AMY HUDSON
• Application Number:
  US09/690,173
• Time in Patent Office: 1,730 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.21, 435/91.51, 536/24.1, 536/24.3, 536/23.1, 536/24.5
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• 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 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 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

Methods for linearly amplifying mRNA to produce antisense RNA are provided. In the subject methods, mRNA is converted to double-stranded cDNA using a promoter-primer having a poly-dT primer site linked to a promoter sequence so that the resulting double-stranded cDNA is recognized by an RNA polymerase. The resultant double-stranded cDNA is then transcribed into antisense RNA in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods find use a variety of different applications in which the preparation of linearly amplified amounts of antisense RNA is desired. Also provided are kits for practicing the subject methods.

Claims

1. A kit for use in linearly amplifying mRNA, said kit comprising:

(a) an oligonucleotide promoter-primer comprising an RNA polymerase promoter sequence;

(b) an RNaseh-polymerase;

(c) an RNaseH+polymerase; and

(d) a reverse transcriptase inhibitor.

2. The kit according to claim 1, wherein said kit further comprises:

instructions to convert the mRNA to cDNA, and to then transcribe the cDNA into RNA in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step.

3. The kit according to claim 1, wherein said kit further comprises MMLV-RT.

4. The kit according to claim 3, wherein said kit further comprises an RNA polymerase.

5. The kit according to claim 4, wherein said RNA polymerase is T7 RNA polymerase.

6. The kit according to claim 1, wherein said reverse transcriptase inhibitor is at least one ddNTP.

7. The kit according to claim 6, wherein said reverse transcriptase inhibitor comprises ddNTPs.

8. The kit according to claim 7, wherein said ddNTPs are selected from ddATP, ddTTP, ddCTP and ddGTP.

9. The kit according to claim 8, wherein said ddNTPs are selected from ddATP and ddGTP.

10. A kit for use in linearly amplifying mRNA, said kit comprising:

(a) an oligonucleotide promoter-primer comprising an RNA polymerase promoter sequence;

(b) an RNaseH−

polymerase;

(c) an RNaseH+ polymerase;

(d) ddNTPs; and

(e) an RNA polymerase.

11. The kit according to claim 10, wherein said RNA polymerase is T7 RNA polymerase.

12. The kit according to claim 10, wherein said kit further comprises:

instructions to convert the mRNA to cDNA, and to then transcribe the cDNA into RNA in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step.

13. The kit according to claim 10, wherein said kit further comprises MMLV-RT.

14. A kit for use in linearly amplifying mRNA, said kit comprising:

(a) an oligonucleotide promoter-primer comprising an RNA polymerase promoter sequence; and

(b) a reverse transcriptase inhibitor.

15. The kit according to claim 14, wherein said reverse transcriptase inhibitor is at least one ddNTP.

16. The kit according to claim 14, wherein said reverse transcriptase inhibitor comprises ddNTPs.

17. The kit according to claim 16, wherein said ddNTPs are selected from ddATP, ddTTP, ddCTP and ddGTP.

18. The kit according to claim 14, wherein said kit further comprises:

instructions to convert the mRNA to cDNA, and to then transcribe the cDNA into RNA in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step.