Subtractive amplification

• US 5,712,127 A
• Filed: 04/29/1996
• Issued: 01/27/1998
• Est. Priority Date: 04/29/1996
• Status: Expired due to Term

• Current Assignee: Alethia BioTherapeutics, Inc.
• Original Assignee: Genscape, Inc. (Verisk Analytics Incorporated)
• Inventors: Sooknanan, Roy R., Malek, Lawrence T.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/639,763
• Time in Patent Office: 638 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.21, 435/91.3, 435/91.5, 435/91.51
• US Class Current: 435/91.21
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• 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 2525/121 incorporating both deoxyribonucleotides and ribonucleotides

Abstract

A method is provided for preferentially amplifying target RNA relative to non-target RNA in a sample of tester RNA. According to the method, a sample of tester RNA is contacted with driver sequences which are complementary to the non-target RNA under conditions where the driver sequences hybridize to the non-target RNA. A nucleic acid primer is then extended using the target RNA as a template, forming a DNA template complementary to some or all of the target RNA. The DNA template formed using the target RNA as template is then rendered single-stranded to enable the hybridization of the DNA template to a promoter template. The DNA template is then extended using the promoter template to form an extended DNA template comprising a functional double-stranded promoter. Multiple copies of the target RNA sequence can now be transcribed from the extended DNA template through recognition of the contained functional double-stranded promoter by RNA polymerase.

Claims

1. A subtractive amplification method for preferentially amplifying target RNA relative to non-target RNA in a sample of tester RNA, the method comprising;

a) contacting a sample of tester RNA with driver nucleic acids under hybridizing conditions, wherein tester RNA which hybridizes to driver nucleic acids is non-target RNA and is inhibited by the hybridized driver nucleic acids from functioning as a template for reverse transcription, and wherein tester RNA which does not hybridize to driver nucleic acids comprises target RNA;

b) reverse transcribing the target RNA using a first primer to form a DNA template complementary to at least a portion of the target RNA;

c) forming an extended DNA template comprising an RNA polymerase promoter sequence by extending the 3'"'"' end of the DNA template using a hybridized promoter template; and

d) transcribing the extended DNA template formed in step c) to form synthetic target RNA.

2. The method according to claim 1, wherein step a) further comprises inactivating the non-target RNA by rendering the non-target RNA incapable of functioning as a template for reverse transcription.

3. The method according to claim 1, wherein the DNA template is rendered single stranded prior to step c) by digesting target RNA hybridized to the DNA template.

4. The method according to claim 1, wherein the DNA template is rendered single stranded prior to step c) by performing strand separation.

5. The method according to claim 1, further comprising the steps of:

e) contacting the synthetic target RNA formed in step d) with a second set of driver nucleic acids under hybridizing conditions;

f) reverse transcribing synthetic target RNA which does not hybridize to the second set of driver nucleic acids in step e) to form DNA complementary to at least a portion of the synthetic target RNA; and

g) copying the DNA formed in step f) to form double stranded DNA.

6. The method according to claim 1 wherein the driver sequences nucleic acids are DNA.

7. The method according to claim 2, wherein step a) further comprises digesting non-target RNA hybridized to a driver nucleic acid.

8. The method according to claim 7, wherein digesting non-target RNA hybridized to a driver nucleic acid is performed using a ribonuclease.

9. The method according to claim 8, wherein the ribonuclease is ribonuclease H.

10. The method according to claim 1, wherein the first primer is DNA.

11. The method according to claim 1, wherein reverse transcription is performed using reverse transcriptase.

12. The method according to claim 3 wherein digesting target RNA hybridized to the DNA template is performed using a ribonuclease.

13. The method according to claim 4, wherein strand separation is performed by heat denaturation.

14. The method according to claim 1, wherein the promoter template is DNA.

15. The method according to claim 14, wherein the promoter template includes sequences encoding a promoter and a transcription initiation site from a bacteriophage.

16. The method according to claim 15, wherein the bacteriophage is T7.

17. The method according to claim 1, wherein extending the DNA template in step c) is performed using a DNA polymerase.

18. The method according to claim 1, further including the step of amplifying the DNA template formed in step b).

19. The method according to claim 18, wherein amplification is performed by a polymerase chain reaction.

20. The method according to claim 1, further including the step of amplifying the extended DNA formed in step c) template.

21. The method according to claim 20, wherein amplification is performed by a polymerase chain reaction.

22. The method according to claim 1 wherein the synthetic target RNA is synthesized by a RNA amplification reaction.

23. The method according to claim 22, wherein amplification is performed by NASBA.

24. The method according to claim 1, wherein transcription is performed using a bacteriophage RNA polymerase.

25. The method according to claim 1, wherein the sample of tester RNA is synthetic target RNA obtained by a method of substractive amplification.

26. A subtractive amplification method for preferentially amplifying target RNA relative to non-target RNA in a sample of tester RNA, the method comprising:

a) contacting a sample of tester RNA with driver nucleic acids under hybridizing conditions, wherein tester RNA which hybridizes to driver nucleic acids is non-target RNA and is inhibited by the hybridized driver nucleic acids from functioning as a template for reverse transcription, and wherein tester RNA which does not hybridize to driver nucleic acids comprises target RNA;

b) contacting the target RNA with nucleic acid primers under hybridizing conditions;

c) reverse transcribing the target RNA using the hybridized primers to form a DNA template complementary to at least a portion of the target RNA;

d) rendering the DNA template single stranded;

e) contacting the single stranded DNA template with a promoter template under hybridizing conditions;

f) forming an extended DNA template comprising an RNA polymerase promoter sequence by extending the 3'"'"' end of the DNA template using the hybridized promoter template; and

g) transcribing the extended DNA template of step f) to form synthetic target RNA.

27. A subtractive amplification method for preferentially amplifying target RNA relative to non-target RNA in a sample of tester RNA, both the target and non-target RNA containing terminal priming sequences for amplification, the method comprising:

a) contacting a sample of tester RNA with driver nucleic acids under hybridizing conditions, wherein tester RNA which hybridizes to driver nucleic acids is non-target RNA and is inhibited by the hybridized driver nucleic acids from functioning as a template for reverse transcription, and wherein tester RNA which does not hybridize to driver nucleic acids comprises target RNA;

b) reverse transcribing the target RNA using nucleic acid primers that hybridize to the target RNA to form a DNA template complementary to at least a portion of the target RNA;

c) forming an extended DNA template comprising a functional double stranded RNA polymerase promoter by rendering the DNA template formed in step b) single stranded and extending the 3'"'"' end of the DNA template using a hybridized promoter template; and

d) transcribing the extended DNA template formed in step c) with an RNA polymerase to form one or more copies of synthetic target RNA.