Methods and compositions for full-length cDNA Cloning using a template-switching oligonucleotide

• US 5,962,272 A
• Filed: 01/03/1997
• Issued: 10/05/1999
• Est. Priority Date: 01/03/1996
• Status: Expired due to Term

• Current Assignee: Clontech Laboratories Incorporated
• Original Assignee: Clontech Laboratories Incorporated
• Inventors: Diatchenko, Luda, Zhu, York, Chenchik, Alex, Siebert, Paul
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/778,494
• Time in Patent Office: 1,005 Days
• Field of Search: 435/91.1, 435/91.2, 435/810, 536/23.1, 536/24.2, 536/25.3
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6855 Ligating adaptors

Abstract

The present invention pertains to methods for the synthesis and cloning of full-length cDNA, or cDNA fragments, that correspond to the complete sequence of 5'"'"'-ends of mRNA molecules. The method of the present invention comprises contacting RNA with a cDNA synthesis primer which can anneal to RNA, a suitable enzyme which possesses reverse transcriptase activity, and a template switching oligonucleotide under conditions sufficient to permit the template-dependent extension of the primer to generate an mRNA-cDNA hybrid. The template switching oligonucleotide hybridizes to the CAP site at the 5'"'"'-end of the RNA molecule and serves as a short, extended template for CAP-dependent extension of the 3'"'"'-end of the ss cDNA that is complementary to the template switching oligonucleotide. The resulting full-length ss cDNA includes the complete 5'"'"'-end of the RNA molecule as well as the sequence complementary to the template switching oligonucleotide, which can then serve as a universal priming site in subsequent amplification of the cDNA.

The subject invention also pertains to the template switching oligonucleotides that can be used according to the subject method. Kits containing the template switching oligonucleotide are also included within the scope of the present invention.

Claims

1. A method for preparing DNA that is complementary to an RNA molecule and a template switching oligonucleotide, said method comprising the steps of:

annealing a cDNA synthesis primer to said RNA molecule and synthesizing a first cDNA strand to form an RNA-cDNA intermediate;

contacting said RNA-cDNA intermediate with a template switching oligonucleotide, wherein said template switching oligonucleotide has at it'"'"'s 3'"'"' end at least one nucleotide which forms a basepair(s) with a nucleotide(s) at a 3'"'"' end of said first cDNA strand of said RNA-cDNA intermediate, said template switching oligonucleotide serves as a template for extension of said 3'"'"' end of said first cDNA strand, and said template switching oligonucleotide has a pre-selected arbitrary nucleotide sequence at its 5'"'"' end and at least one ribonucleotide residue at its 3'"'"' end; and

extending said 3'"'"' end of said first cDNA strand to produce a DNA that is complementary to said RNA molecule and said template switching oligonucleotide.

2. The method according to claim 1, wherein said at least one ribonucleotide residue of said template switching oligonucleotide is riboguanine.

3. The method according to claim 1, wherein said template switching oligonucleotide comprises at least three ribonucleotide residues.

4. The method according to claim 3, wherein at least two of said at least three ribonucleotide residues are riboguanine.

5. The method according to claim 1, further comprising amplifying said DNA strand that is complementary to said RNA molecule and said template switching oligonucleotide using an oligonucleotide primer that comprises a nucleotide sequence comprising at least a portion of said pre-selected nucleotide sequence of said template switching oligonucleotide.

6. The method according to claim 1, wherein said RNA molecule has a 7-methylguanosine CAP structure attached at the 5'"'"'-end of said RNA.

7. The method according to claim 1, wherein said template switching oligonucleotide has at least three riboguanine residues at its 3'"'"' end.

8. The method according to claim 1, wherein a 3'"'"'-OH group of said at least one ribonucleotide comprises a chemical group selected from the group consisting of amino, biotin, phosphate, glycerol and fluoro.

9. The method according to claim 1, wherein said template switching oligonucleotide comprises a protein that binds to a 5'"'"' CAP structure of said RNA.

10. The method according to claim 9, wherein said protein is selected from the group consisting of eukaryotic initiation factor 4E and anti-CAP structure antibody.

11. The method according to claim 1, wherein said template switching oligonucleotide is represented by a formula of 5'"'"'-dN₁ -dN₂ - . . . dN_m -rN₁ -rN₂. . . rN_n -3'"'"', wherein dN represents a deoxyribonucleotide;

m represents an integer;

rN represents an ribonucleotide at least one of which is GMP and n represents an integer.

12. The method according to claim 1, wherein said template switching oligonucleotide is selected from the group consisting of SEQ ID Nos. 1-7, 9, 13, 18-66, 68, and 84-108.

13. The method according to claim 1, wherein said cDNA synthesis primer comprises a sequence complementary to at least one target RNA.

14. The method according to claim 1, wherein said cDNA synthesis primer comprises a sequence complementary to a poly(A) portion of said RNA.

15. The method according to claim 1, wherein said cDNA synthesis primer is selected from the group consisting of SEQ ID No.8, 16, 67, 109, 110 and 111.

16. The method according to claim 1, wherein said pre-selected arbitrary nucleotide sequence of said template switching oligonucleotide comprises at least one nucleotide with a hapten group, and further comprises the steps of incubating said RNA-cDNA-hapten intermediate with a binding ligand of said hapten group, where said binding ligand is conjugated to a support, under conditions sufficient to permit said hapten to become bound to said binding ligand to produce an intermediate-hapten-ligand complex;

washing said intermediate-hapten-ligand complex; and

separating said intermediate-hapten from said binding ligand.

17. The method according to claim 1, further comprising the step of incubating said DNA strand that is complementary to said RNA molecule and said template switching oligonucleotide under conditions wherein a second DNA strand complementary to said DNA strand that is complementary to said RNA molecule and said template switching oligonucleotide is synthesized.

18. The method according to claim 1, further comprising the steps of incubating said DNA strand that is complementary to said RNA molecule and said template switching oligonucleotide with at least one primer, wherein said primer comprises a nucleotide sequence comprising at least a portion of said pre-selected arbitrary sequence of said template switching oligonucleotide, and an effective amount of reagents necessary to perform PCR to produce a PCR cocktail, and incubating said PCR cocktail under conditions suitable for generating a full-length double-stranded cDNA.

19. The method according to claim 18, wherein said primer is selected from the group consisting of SEQ ID No. 10, 11, 12, 17, 69, and 70-83.

20. A template switching oligonucleotide having at it'"'"'s 3'"'"' end at least one nucleotide which forms a basepair(s) with a nucleotide(s) at a 3'"'"' end of a first cDNA strand of an RNA-cDNA intermediate, wherein said template switching oligonucleotide is capable of serving as a template for extension of said 3'"'"' end of said first cDNA strand, and said template switching oligonucleotide has a pre-selected arbitrary nucleotide sequence at its 5'"'"' end and at least three ribonucleotide residues at its 3'"'"' end.

21. The template switching oligonucleotide of claim 20, wherein at least one of said three ribonucleotide residues is a riboguanine residue.

22. The template switching oligonucleotide of claim 20, wherein said template switching oligonucleotide has a general formula of 5'"'"'-dN₁ -dN₂ - . . . dN_m -rN₁ -rN₂. . . rN_n -3'"'"', wherein dN represents a deoxyribonucleotide;

m represents an integer;

rN represents an ribonucleotide at least one of which is GMP and n represents an integer.

23. The template switching oligonucleotide of claim 22, wherein m represents an integer between 10 and 50.

24. The template switching oligonucleotide of claim 22, wherein n represents an integer between 3 and 7.

25. The template switching oligonucleotide of claim 22, wherein an 3'"'"'-OH group of said GMP comprises a chemical group selected from the group consisting of amino, biotin, phosphate, glycerol and fluoro.

26. The template switching oligonucleotide of claim 22, wherein said template switching oligonucleotide further comprises a protein capable of binding to a 5'"'"' CAP structure of said mRNA.

27. The template switching oligonucleotide of claim 26, wherein said protein is selected from the group consisting of eukaryotic initiation factor 4E and anti-CAP structure antibody.

29. A kit comprising the template switching oligonucleotide of claim 20.

28. A template switching oligonucleotide selected from the group consisting of SEQ ID Nos. 1-7, 9, 13, 18-66, 68, and 84-108.