Method for linear mRNA amplification

• US 7,927,838 B2
• Filed: 07/11/2005
• Issued: 04/19/2011
• Est. Priority Date: 05/28/1999
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Shannon, Karen W.
• Primary Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US11/179,813
• Publication Number:
  US 20060024799A1
• Time in Patent Office: 2,108 Days
• Field of Search: None
• 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 method for producing RNA, comprising:

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

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

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

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

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

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

6. The method according to claim 1, wherein the 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 the 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 the first strand cDNA, which is lacking RNaseH activity.

9. The method according to claim 1, wherein the converting step employs a promoter-primer comprising an RNA binding site for the RNA molecule linked to a promoter sequence.

26. The method according to claim 1, wherein no more than one thermal cycling reaction occurs.

27. The method according to claim 1, wherein the double-stranded cDNA is not physically separated from the reverse transcriptase following double-stranded cDNA preparation.

28. The method of claim 1 wherein the reverse transcriptase is irreversibly incapable of RNA-dependent DNA polymerase activity during the transcribing step.

10. A method for producing RNA comprising:

(a) converting an RNA molecule to cDNA with a promoter-primer comprising a binding site for the RNA molecule linked to an RNA polymerase promoter sequence; and

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

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

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

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

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

15. A method for producing RNA comprising:

(a) converting an RNA molecule to double-stranded cDNA by;

(i) contacting the RNA molecule with a promoter-primer under conditions wherein the RNA molecule forms a complex with the promoter-primer, wherein the promoter-primer comprises a binding site for the RNA molecule linked to a RNA promoter sequence; and

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

16. The method according to claim 15, wherein the RNA-dependent DNA polymerase activity, RNaseH activity and DNA-dependent DNA polymerase activity are contributed by a single polymerase.

17. The method according to claim 16, wherein the polymerase is the reverse transcriptase of Moloney Murine leukemia virus (MMLV-RT).

18. The method according to claim 16, wherein the polymerase is the reverse transcriptase of avian myeloblastosis virus (AMV-RT).

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

20. A method for producing RNA comprising:

(a) contacting an RNA molecule with a promoter-primer in the presence of a first reverse transcriptase under conditions sufficient for first strand cDNA synthesis to occur to produce a hybrid of the RNA molecule and a first strand cDNA, wherein the promoter-primer comprises an binding site for the RNA molecule linked to a RNA polymerase promoter sequence;

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

(c) transcribing the double-stranded cDNA with an RNA polymerase and ribonucleotides in the presence of ddNTPs and said reverse transcriptase, wherein said reverse transcriptase is incapable of RNA-dependent DNA polymerase activity.

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

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

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

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

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

ddATP and ddGTP.