Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

• US 5,846,721 A
• Filed: 09/19/1996
• Issued: 12/08/1998
• Est. Priority Date: 09/19/1996
• Status: Expired due to Term

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Bonaldo, Maria de Fatima, Soares, Marcelo Bento
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/715,941
• Time in Patent Office: 810 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/25.4
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection

Abstract

This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

Claims

1. A method to normalize a cDNA library comprising:

(a) providing a cloned library containing cDNA inserts in circular double-stranded form wherein the inserts are capable of being amplified by polymerase chain reaction using appropriate primers;

(b) converting the double-stranded cDNA library into single-stranded DNA circles;

(c) generating single-stranded nucleic acid molecules complementary to the inserts of the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers and melting double-stranded cDNA inserts produced by the polymerase chain reaction;

(d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes at an appropriate Cot; and

(e) separating unhybridized single-stranded DNA circles from hybridized DNA circles, thereby generating a normalized cDNA library.

2. A method of claim 1, wherein the cDNA library is constructed in the pT7T3-Pac vector.

3. A method of claim 1, wherein the cDNA library is a directional cDNA library.

4. A method of claim 1, wherein the cDNA library is a randomly-primed cDNA library.

5. A method of claim 3, wherein the directional cDNA library is generated by using a primer of oligodT stretch.

6. A method of claim 5, wherein the directional cDNA library is generated by using a primer having a rare restriction enzyme recognition site for the first strand cDNA synthesis, upstream of the oligodT stretch.

7. A method of claim 6, wherein the rare restriction enzyme recognition site is Not I or Pac I site.

8. A method of claim 6, wherein the primer contains a specific sequence between the sequence of the rare restriction site and the oligodT stretch.

9. A method of claim 1 wherein in step (b) the double-stranded cDNA library constructed in a vector capable of being converted into single-stranded DNA circles in vivo is converted into the single-stranded DNA circles in vivo.

10. A method of claim 9 wherein the double-stranded cDNA library is converted into the single-stranded DNA circles by transforming the cDNA library into bacteria and infecting the transformed bacteria with helper phage capable of converting the double-stranded cDNA library into the single-stranded DNA circles.

11. A method of claim 10 wherein the bacteria used is Escherichia coli DH5α

F'"'"' and the helper phage used is M13K07.

12. A method of claim 1 wherein in step (b) the double-stranded cDNA library constructed in a vector capable of being converted into single-stranded DNA circles in vitro is converted into the single-stranded DNA circles in vitro.

13. A method of claim 12 wherein the double-stranded cDNA library is converted into the single-stranded DNA circles by digestion with a site-specific endonuclease and an exonuclease.

14. A method of claim 13 wherein the site-specific endonuclease used is the replication initiator protein of bacteriophage f1, Gene II, and the exonuclease used is Exonuclease III.

19. A method of claim 1 or 15 wherein the single-stranded DNA circles are separated from the unconverted double-stranded cDNA library.

20. A method of claim 19 wherein the single-stranded DNA circles are separated from the unconverted double-stranded cDNA library by hydroxyapatite column chromatography.

22. A method of claim 1 wherein the single-stranded nucleic acid molecules generated in step (c) span the entire length of the cDNA inserts.

23. A method of claim 1 wherein the single-stranded nucleic acid molecules are generated by PCR amplification of all cDNA inserts of the cDNA library.

24. A method of claim 1 or 15, wherein in step (d) the complementary nucleic acid molecules and the single-stranded DNA circles are hybridized under conditions used for hydroxyapatite column chromatography.

25. A method of claim 24, wherein the complementary nucleic acid molecules and the single-stranded DNA circles are hybridized in a solution comprising 0.12M NaCl-50% formamide-1% SDS-5 mM EDTA pH 8.0 at 30°

C. for an appropriate amount of time which corresponds to a Cot of about 5.

15. A method to normalize a cDNA library comprising:

(a) providing a directionally cloned library containing cDNA inserts in circular double-stranded form;

(b) converting the double-stranded cDNA library into single-stranded DNA circles;

(c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by

excising cDNA inserts from the double-stranded cDNA library;

purifying the cDNA inserts from cloning vectors; and

digesting the cDNA inserts with an exonuclease;

(d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes at an appropriate Cot; and

(e) separating unhybridized single-stranded DNA circles from hybridized DNA circles, thereby generating a normalized cDNA library.

16. A method of claim 15 wherein in step (b) the double-stranded cDNA library is converted into the single-stranded DNA circles in vitro.

17. A method of claim 15 wherein in step (b) the double-stranded cDNA library is converted into the single-stranded DNA circles in vivo.

18. A method of claim 15 wherein the exonuclease used is Exonuclease III.

21. A method of claim 15 wherein the single-stranded nucleic acid molecules generated in step (c) comprise the 5'"'"' halves of all cDNA inserts of the double-stranded cDNA library used in step (a).

26. A method to construct a subtractive cDNA library comprising:

(a) converting a double-stranded circular cDNA library into single-stranded DNA circles;

(b) treating a pool of double-stranded circular DNAs which are to be eliminated from the subtractive cDNA library with a site-specific endonuclease and an exonuclease to generate single-stranded circular DNA molecules;

(c) separating the single-stranded circular DNA molecules from step (a) from double-stranded DNAs;

(d) amplifying inserts of the separated single-stranded DNA molecules derived from step (b) by PCR using appropriate primers to form amplification products;

(e) hybridizing the single-stranded DNA circles derived from step (a) with the amplification products generated in step (d) to produce partial duplexes at an appropriate Cot; and

(f) separating unhybridized single-stranded DNA circles from hybridized DNA circles, thereby generating a subtractive cDNA library.

27. A method of claim 26 wherein in step (a) the double-stranded circular cDNA library is converted into the single-stranded DNA circles by treating the double-stranded cDNA circular library with a site-specific endonuclease and an exonuclease.

28. A method of claim 27 wherein the site-specific endonuclease used is the replication initiator protein of bacteriophage f1 (Gene II) and the exonuclease used is Exonuclease III.

29. A method of claim 26 wherein the single-stranded DNA circles are separated from an unconverted double-stranded circular cDNA library by hydroxyapatite column chromatography.

30. A method of claim 26 wherein in step (b) the site-specific endonuclease used is the replication initiator protein of bacteriophage f1 (Gene II) and the exonuclease used is Exonuclease III.