Method for generating full-length cDNA library from single cells

• US 6,197,554 B1
• Filed: 11/20/1998
• Issued: 03/06/2001
• Est. Priority Date: 11/20/1998
• Status: Expired due to Fees

• Current Assignee: Epiclone Incorporated
• Original Assignee: Epiclone Incorporated
• Inventors: Lin, Shi-Lung, Ying, Shao-Yao, Chuong, Cheng-Ming
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): TAYLOR, JANELL E
• Application Number:
  US09/197,951
• Time in Patent Office: 837 Days
• Field of Search: 435/91.1, 435/6, 435/15, 435/91.21, 435/91.2, 435/91.5, 435/91.51, 536/23.1, 536/24.1, 536/25.3, 935/18, 935/19, 935/93, 935/16
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

The present invention provides a fast, simple and specific method for generating a complete full-length cDNA library from single cells. The first reverse transcription of intracellular mRNAs with an oligo(dT)n-promoter primer introduces a recognition site for following transcription of newly reverse-transcribed cDNAs. The poly-nucleotide tailing of above cDNAs in addition to aforementioned promoter region further forms binding templates for specific PCR amplification. After repeating the reverse transcription, transcription, reverse transcription and PCR procedure, we can multiply a single copy of mRNA to two billion folds by calculation based upon the comparison between the amount of a synthesized cDNA library and that of theoretically presumed mRNAs within a cell (0.1 pg). In conjunction with a cell fixation and permeabilization step, the complete full-length cDNA library can be directly generated from few single cells without mRNA degradation. The present invention will be very useful in preparing tissue-specific full-length cDNA libraries for future gene chip technology.

Claims

1. A method of generating a complete full-length cDNA library from single cells, comprising the steps of:

a. providing a plurality of fixed cells, wherein said fixed cells inhibit intracellular messenger RNA degradation and also increase the permeabilisation of said cells for enzyme penetration;

b. incubating said fixed cells in a reverse transcription reaction with a plurality of oligo(dT)n-promoter sequences, wherein said reverse transcription reaction is reverse transcription of a plurality of messenger RNAs by using said oligo(dT)n-promoter as primer, to form a plurality of complementary DNAs from said messenger RNAs;

c. permitting said complementary DNAs in a cDNA tailing and double-stranding reaction to form a plurality of poly(N)-tailed cDNAs, wherein said cDNA tailing and double-stranding reaction is a DNA polymerase and terminal transferase reaction capable of adding multiple copies of the same nucleotide to the tails of said complementary DNAs and then double-stranding said complementary DNAs from the tails;

d. incubating said poly(N)-tailed cDNAs in an in-vitro transcription reaction to generate a plurality of full-length aRNAs, wherein said in-vitro transcription reaction is RNA'"'"'s polymerase reaction capable of synthesizing said full-length RNA'"'"'s from said poly(N)-tailed cDNAs;

e. incubating said full-length aRNAs in said reverse transcription reaction with a plurality of oligo (anti-poly(N))-promoter sequences to form a plurality of full-length cDNAs;

wherein said oligo (anti-poly(N))-promoter sequences are complementary to the poly(N) tails of said poly(N)-tailed cDNAs and

f. amplifying said full-length cDNAs with a template-dependent extension of specific primers attached to the poly (dA)-tail and complementary promoter regions of said full-length cDNAs, and thereby providing a complete library enriched in full-length cDNAs from said fixed cells.

2. The method as defined in claim 1, further comprising the step of repeating steps of (b) through (e) on said full-length RNAs at least one time.

3. The method as defined in claim 1, further comprising the step of cloning said amplified full-length cDNA library into competent vectors after the step (f).

4. The method as defined in claim 1, further comprising the step of hybridization of said oligo(dT)n-promoter sequences to said messenger RNAs in the step (b).

5. The method as defined in claim 4, wherein said oligo(dT)n-promoter sequence is a poly(dT) primer coupled to an RNA polymerase promoter in the 5′

-end of said poly(dT) primer, of which the number of deoxythymidylate (dT) is larger than six nucleotides.

6. The method as defined in claim 5, wherein said RNA polymerase promoter is T3 RNA polymerase promoter.

7. The method as defined in claim 5, wherein said RNA polymerase promoter is T7 RNA polymerase promoter.

8. The method as defined in claim 5, wherein said RNA polymerase promoter is SP6 RNA polymerase promoter.

9. The method as defined in claim 5, wherein said RNA polymerase promoter is M13 RNA polymerase promoter.

10. The method as defined in claim 1, wherein said reverse transcription reaction is performed with AMV reverse transcriptase.

11. The method as defined in claim 1, wherein said reverse transcription reaction is performed with MMLV reverse transcriptase.

12. The method as defined in claim 1, wherein said cDNA tailing and double stranding reaction is performed with terminal transferase.

13. The method as defined in claim 1, wherein said in-vitro transcription reaction is performed with T3 RNA polymerase.

14. The method as defined in claim 1, wherein said in-vitro transcription reaction is performed with T7 RNA polymerase.

15. The method as defined in claim 1, wherein said in-vitro transcription reaction is performed with SP6 RNA polymerase.

16. The method as defined in claim 1, wherein said in-vitro transcription reaction is performed with M13 RNA polymerase.

17. The method as defined in claim 1, wherein said template-dependent extension is a polymerase chain reaction.

18. The method as defined in claim 1, wherein said poly(N)-tailed cDNAs are terminally added by multiple copies of the same nucleotide in the 3′

-end.

19. The method as defined in claim 18, wherein said same nucleotide is deoxyguanylate.

20. The method as defined in claim 18, wherein said same nucleotide is deoxycytidylate.

21. The method as defined in claim 18, wherein said same nucleotide is deoxyadenylate.

22. The method as defined in claim 18, wherein said same nucleotide is deoxythymidylate.

23. The method as defined in claim 18, wherein said same nucleotide is deoxyuridylate.

24. A method of performing improved full-length cDNA library synthesis, comprising the steps of:

a. preventing a plurality of messenger RNAs from degradation, wherein said messenger RNAs are preserved to be intact in cells;

b. generating a plurality of complementary DNAs from said messenger RNAs, wherein said complementary DNAs are reverse-transcribed from said messenger RNAs;

c. permitting said complementary DNAs to form a plurality of poly(N)-tailed cDNAs, wherein said poly(N)-tailed cDNA contains a full-length complementary DNA sequence flanked with an RNA polymerase promoter in the 5′

-end and a poly-nucleotide tail in the 3′

-end; and

d. amplifying said poly(N)-tailed cDNAs by a plurality of promoter- and tail-dependent extension systems, and thereby providing a complete library of full-length cDNAs from said messenger RNAs.

25. The method as defined in claim 24, further comprising the step of cloning said full-length cDNA library into competent vectors after the step (d).

26. The method as defined in claim 24, wherein said RNA polymerase promoter is T3 RNA polymerase promoter.

27. The method as defined in claim 24, wherein said RNA polymerase promoter is T7 RNA polymerase promoter.

28. The method as defined in claim 24, wherein said RNA polymerase promoter is SP6 RNA polymerase promoter.

29. The method as defined in claim 24, wherein said RNA polymerase promoter is M13 RNA polymerase promoter.

30. The method as defined in claim 24, wherein said messenger RNAs are protected by fixation.

31. The method as defined in claim 24, wherein said messenger RNAs are protected by RNase inhibitors.

32. The method as defined in claim 24, wherein said complementary DNAs are generated by reverse transcriptase with oligo(dT)n-promoter sequences.

33. The method as defined in claim 24, wherein said poly(N)-tailed cDNAs are formed with terminal transferase.

34. The method as defined in claim 24, wherein said poly(N)-tailed cDNAs are amplified with T3 RNA polymerase and reverse transcriptase.

35. The method as defined in claim 24, wherein said poly(N)-tailed cDNAs are amplified with T7 RNA polymerase and reverse transcriptase.

36. The method as defined in claim 24, wherein said poly(N)-tailed cDNAs are amplified with SP6 RNA polymerase and reverse transcriptase.

37. The method as defined in claim 24, wherein said poly(N)-tailed cDNAs are amplified with M13 RNA polymerase and reverse transcriptase.

38. The method as defined in claim 24, wherein said promoter- and tail-dependent extension system is a procedure of transcription, reverse transcription and polymerase chain reaction.

39. The method as defined in claim 24, wherein said poly(N)-tailed cDNAs contain multiple copies of the same nucleotide in the 3′

-end.

40. The method as defined in claim 39, wherein said same nucleotide is deoxyguanylate.

41. The method as defined in claim 39, wherein said same nucleotide is deoxycytidylate.

42. The method as defined in claim 39, wherein said same nucleotide is deoxyadenylate.

43. The method as defined in claim 39, wherein said same nucleotide is deoxythymidylate.

44. The method as defined in claim 39, wherein said same nucleotide is deoxyuridylate.