Method for linear mRNA amplification

• US 6,132,997 A
• Filed: 05/28/1999
• Issued: 10/17/2000
• Est. Priority Date: 05/28/1999
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies Europe BV (Agilent Technologies, Inc.)
• Inventors: Shannon, Karen W.
• Primary Examiner(s): Elliott, George C.
• Assistant Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/322,692
• Time in Patent Office: 508 Days
• Field of Search: 435/91.21, 435/91.2, 435/6, 435/91.51, 536/23.1, 536/24.1, 536/25.3
• US Class Current: 435/91.21
• 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 method for producing linearly amplified amounts of antisense RNA from mRNA, said method comprising:

(a) converting mRNA to double-stranded cDNA, wherein one terminus of said double-stranded cDNA comprises an RNA polymerase promoter region; and

(b) transcribing said double-stranded cDNA with an RNA polymerase and ribonucleotides into antisense RNA in the presence of a reverse transcriptase that is incapable of RNA-dependent DNA polymerase activity during said transcribing step.

2. The method according to claim 1, wherein said method further comprises inactivating said reverse transcriptase prior to said transcribing step.

3. The method according to claim 2, wherein said inactivation is accomplished by heating the reaction mixture.

4. The method according to claim 1, wherein said method further comprises inhibiting said reverse transcriptase with an inhibitor during said transcribing step.

5. The method according to claim 4, wherein said inhibitor is at least one ddNTP.

6. The method according to claim 1, wherein said converting step comprises a single cDNA synthesis step, wherein the same polymerase is employed for the synthesis of first and second cDNA strands.

7. The method according to claim 1, wherein said converting step comprises a first strand cDNA synthesis step and a second strand cDNA synthesis step.

8. The method according to claim 7, wherein a first polymerase is employed for synthesis of said first strand cDNA and a second polymerase is employed for synthesis of said second strand cDNA, wherein said first polymerase is lacking RNaseH activity.

9. The method according to claim 1, wherein said converting step employs a promoter-primer comprising an mRNA binding site linked to a promoter sequence.

10. A method for producing linearly amplified amounts of antisense RNA from mRNA, said method comprising:

(a) converting mRNA to cDNA with a promoter-primer comprising an mRNA binding site linked to a promoter sequence, wherein one terminus of said cDNA comprises an RNA polymerase promoter region; and

(b) transcribing said cDNA with an RNA polymerase and ribonucleotides into antisense RNA in the presence of a reverse transcriptase that has been rendered ineffective for RNA-dependent DNA polymerase activity prior to said transcribing step.

11. The method according to claim 10, wherein said method further comprises inactivating said reverse transcriptase prior to said transcribing step.

12. The method according to claim 11, wherein said inactivation is accomplished by heating the reaction mixture.

13. The method according to claim 10, wherein said method further comprises inhibiting said reverse transcriptase with an inhibitor during said transcribing step.

14. The method according to claim 13, wherein said inhibitor is at least one ddNTP.

15. The method according to claim 10, wherein said RNA-dependent DNA polymerase activity is inhibited with ddNTPs.

16. A method for producing linearly amplified amounts of antisense RNA from mRNA, said method comprising:

(a) converting mRNA to double-stranded cDNA, wherein one terminus of said double-stranded cDNA comprises an RNA polymerase promoter region by;

(i) contacting mRNA with a promoter-primer under conditions wherein said mRNA forms a complex with said promoter-primer, wherein said promoter-primer comprises an mRNA binding site linked to a promoter sequence; and

(ii) converting said complex to double-stranded cDNA using a combination of RNA-dependent DNA polymerase activity, RNaseH activity and DNA-dependent DNA polymerase activity; and

(b) transcribing said double-stranded cDNA with an RNA polymerase and ribonucleotides into antisense RNA in the presence of a reverse transcriptase that is incapable of RNA-dependent DNA polymerase activity during said transcribing step;

whereby said mRNA is linearly amplified into antisense RNA.

17. The method according to claim 16, wherein said RNA-dependent DNA polymerase activity, RNaseH activity and DNA-dependent DNA polymerase activity are contributed by a single polymerase.

18. The method according to claim 17, wherein said polymerase is the reverse transcriptase of Moloney Murine leukemia virus (MMLV-RT).

19. The method according to claim 17, wherein said polymerase is the reverse transcriptase of avian myeloblastosis virus (AMV-RT).

20. A method for producing linearly amplified amounts of antisense RNA from mRNA, said method comprising:

(a) contacting mRNA with a promoter-primer in the presence of a first polymerase having RNA-dependent DNA polymerase activity and lacking RNaseH activity under conditions sufficient for first strand cDNA synthesis to occur to produce a hybrid of said mRNA and a first strand cDNA, wherein said promoter-primer comprises an mRNA binding site linked to a RNA polymerase promoter sequence;

(b) contacting said hybrid with an enzyme catalyzing RNaseH activity under conditions sufficient to convert said complex to a double-stranded cDNA molecule; and

(c) transcribing said double-stranded cDNA with an RNA polymerase and ribonucleotides into antisense RNA in the presence of ddNTPs;

whereby said mRNA is linearly amplified into antisense RNA.

21. The method according to claim 20, wherein said enzyme catalyzing RNaseH activity is the reverse transcriptase of Moloney Murine leukemia virus (MMLV-RT).

22. The method according to claim 20, wherein said enzyme catalyzing RNaseH activity is the RNaseH of Escherichia coli (E. coli RNaseH).

23. The method according to claim 20, wherein said RNA polymerase promoter is the T7 promoter and said RNA polymerase is T7 RNA polymerase.

24. The method according to claim 20, wherein said RNA polymerase promoter is the T3 promoter and said RNA polymerase is T3 RNA polymerase.

25. The method according to claim 20, wherein said ddNTPs are selected from the group consisting of:

ddATP and ddGTP.

26. A kit for use in linearly amplifying mRNA into antisense RNA, said kit comprising:

an oligonucleotide promoter-primer comprising an RNA polymerase promoter sequence; and

ddNTPs.

27. The kit according to claim 26, wherein said kit further comprises at least one polymerase.

28. The kit according to claim 26, wherein said polymerase is MMLV-RT.

29. The kit according to claim 26, wherein said kit comprises a first RNaseH-polymerase and a second RNAseH+ polymerase.

30. The kit according to claim 26, wherein said kit further comprises an RNA polymerase.

31. The kit according to claim 26, wherein said RNA polymerase is T7 RNA polymerase.