Method for unbiased mRNA amplification

• US 5,932,451 A
• Filed: 11/19/1997
• Issued: 08/03/1999
• Est. Priority Date: 11/19/1997
• Status: Expired due to Fees

• Current Assignee: Incyte Pharmaceuticals, Inc.
• Original Assignee: Incyte Pharmaceuticals, Inc.
• Inventors: Wang, Bruce, Chung, Alicia
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US08/974,273
• Time in Patent Office: 622 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.21, 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 1/6844 Nucleic acid amplification reactions

Abstract

Methods of enzymatically producing unbiased amounts of nucleic acid from mRNA are provided. In the subject methods, a sample of mRNA is converted to ds cDNA using a primer containing an RNA polymerase site and at least one priming site. The resultant ds cDNA is then asymmetrically amplified with captureable primer to produce captureable cDNA which is then converted to captured ds cDNA. The resultant ds cDNA may then be used in a number of different applications, such as in the preparation of amplified amounts of aRNA, in the preparation of cDNA probes, and the like.

Claims

1. A method for producing amplified amounts of nucleic acids from mRNA, said method comprising:

(a) converting mRNA to ds cDNA with a first primer comprising an RNA polymerase promoter region and at least one priming site;

(b) asymmetrically amplifying said ds cDNA with a second captureable primer to produce an amplified amount of captureable antisense cDNA;

(c) capturing said captureable antisense cDNA; and

(d) converting said captured antisense cDNA to captured ds DNA.

2. The method according to claim 1, wherein said asymmetric amplification step (b) comprises a plurality of cycles.

3. The method according to claim 1, wherein said first primer comprises:

(i) an oligo dT region;

(ii) an RNA polymerase promoter region 5'"'"' of the oligo dT region; and

(iii) at least one priming site 5'"'"' of the RNA polymerase promoter region.

4. The method according to claim 1, wherein said converting of said captured antisense cDNA to ds cDNA of step (d) comprises:

homopolymeric tailing of the 3'"'"' terminus of said captured antisense cDNA; and

hybridization of a complementary homopolymeric primer to said homopolymeric tail; and

enzymatic primer extension of said hybridized primer.

5. The method according to claim 1, wherein said converting of said captured antisense cDNA to ds cDNA of step (d) comprises:

hybridizing random oligomer primers to said captured antisense cDNA; and

enzymatically extending said hybridized primers.

6. The method according to claim 5, wherein said method further comprises transcribing said captured ds cDNA to aRNA.

19. The method according to claim 1, wherein said method further comprises denaturing said captured ds DNA and recovering the sense cDNA.

20. The method according to claim 19, wherein said method further comprises amplifying assymetrically said recovered sense cDNA.

21. The method according to claim 5, wherein said method further comprises denaturing said captured ds DNA and recovering the sense cDNA.

22. The method according to claim 21, wherein said method further comprises amplifying assymetrically said recovered sense cDNA.

7. A method for producing amplified amounts of nucleic acids from mRNA, said method comprising:

(a) converting mRNA to ds cDNA with a first primer comprising an RNA polymerase promoter region and at least one priming site;

(b) asymmetrically amplifying said ds cDNA with a second captureable primer to produce an amplified amount of captureable antisense cDNA;

(c) capturing said captureable antisense cDNA; and

(d) converting said captureable antisense cDNA to captured ds DNA by hybridizing random oligomer primers to said captured antisense cDNA and enzymatically extending said primers.

8. The method according to claim 7, wherein said oligo dT primer comprises a T7 promoter region.

9. The method according to claim 7, wherein priming site is from 14 to 60 nt in length.

10. The method according to claim 7, wherein said captureable primer is biotinylated.

11. The method according to claim 10, wherein said method further comprises transcribing said captured ds cDNA to aRNA.

12. A kit for use in the preparation of amplified amounts of nucleic acids from mRNA, said kit comprising:

a first primer comprising in the 3'"'"' to 5'"'"' direction an oligo dT region, an RNA polymerase promoter site and at least one priming site; and

a second captureable primer.

13. The kit according to claim 12, wherein said first primer comprises at least two priming sites.

14. The kit according to claim 12, wherein said first primer comprises an A, G or C at its 3'"'"' terminus.

15. The kit according to claim 12, wherein said captureable primer comprises a member of a specific binding pair bonded to its 5'"'"' terminus.

16. The kit according to claim 15, wherein said member of a specific binding pair is biotin.

17. The kit according to claim 12, wherein said kit further comprises a thermostable polymerase.

18. The kit according to claim 12, wherein said kit further comprises an RNA polymerase.

23. The kit according to claim 12, wherein said kit further comprises random oligomer primers.