Method for construction of normalized cDNA libraries

• US 5,830,662 A
• Filed: 06/21/1996
• Issued: 11/03/1998
• Est. Priority Date: 09/24/1993
• 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: Efstratiadis, Argiris, Soares, Marcelo B.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/619,542
• Time in Patent Office: 865 Days
• Field of Search: 435/91.1, 435/6, 536/23.1, 536/25.4
• US Class Current: 506/9
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3'"'"' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

Claims

1. A method of producing a tagged normalized directional cDNA library containing cDNA clones constructed in a vector that allows propagation in single-stranded circle form comprising:

(a) propagating a directional cDNA library in single-stranded circles;

(b) annealing the single-stranded circles to an appropriate primer, wherein the primer contains a specific sequence tag and performing controlled extension reaction with an appropriate polymerase in the presence of an appropriate ratio between dideoxynucleotide triphosphates and deoxynucleotide triphosphates to generate fragments complementary to the 3'"'"' noncoding sequence of the single-stranded circles in the library to produce partial duplexes;

(c) purifying the partial duplexes;

(d) melting and reassociating the purified partial duplexes to appropriate Cot; and

(e) purifying unassociated single-stranded circles, thereby generating a tagged normalized directional cDNA library.

2. A normalized cDNA library generated by the method of claim 1.

3. A library of claim 2, wherein the cDNA library is derived from liver and spleen.

4. A human cDNA catalogue comprising at least two tagged normalized directional cDNA libraries generated by the method of claim 1.

5. A method to normalize the cDNA catalogue of claim 4 comprising:

(a) propagating the directional cDNA libraries in single-stranded circles;

(b) annealing the single-stranded circles to an appropriate primer and performing controlled extension reactions with an appropriate polymerase in the presence of an appropriate ratio between dideoxynucleotide triphosphates and deoxynucleotide triphosphates to generate fragments complementary to the 3'"'"' noncoding sequence of the single-stranded circles in the libraries to produce partial duplexes;

(c) purifying the partial duplexes;

(d) melting and reassociating the purified partial duplexes to appropriate Cot; and

(e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA catalogue.

6. A normalized cDNA catalogue generated by the method of claim 5.

7. A method of isolating cDNA clones specific to a tissue comprising:

(a) hybridizing single-stranded DNA circles from a normalized library of claim 2 with excess RNAs derived from other tissue; and

(b) separating the unhybridized DNA circles from the hybridized DNA circles, thereby isolating cDNA clones specific to the tissue.

8. A method of claim 7, wherein the RNAs are synthesized in vitro from at least one normalized cDNA library.

9. A method of claim 7 or 8, wherein the RNAs are at least one hundred fold excess than the single-stranded DNA circles.

10. A method of claim 8, wherein the normalized libraries used are identified by being tagged with a different specific sequence.

11. A method of claim 10, further comprising inputting single-stranded DNA circles of normalized library from other tissue in step (a).

12. A method of isolating cDNA clones specific to a tissue comprising:

(a) hybridizing approximately an equal amount of single-stranded DNA circles from a tagged normalized library produced by the method of claim 1 and single-stranded DNA circles from at least one normalized tagged library of other tissue said library produced by the method of claim 1 with excess in vitro synthesized RNAs from the tagged normalized library of the other tissue;

(b) separating the hybridized DNA circles from unhybridized DNA circles;

(c) determining the specific sequence tag on the unhybridized DNA circles, an absence of the specific sequence tag of the normalized library of the other tissue indicating the completeness of the hybridization in step (a), thereby isolating cDNA clones specific to the tissue.

13. A method of identifying cDNA clones capable of hybridizing a genomic clone comprising:

(a) hybridizing the genomic clone with the single-stranded circles of a normalized cDNA library of claim 2; and

(b) separating the hybridized cDNA circles from the unhybridized circles, thereby identifying cDNA clones capable of hybridizing the genomic clone.

14. A method of identifying cDNA clones capable of hybridizing a genomic clone comprising:

(a) immobilizing the genomic clone on a solid matrix;

(b) hybridizing the genomic clone with the single-stranded circles of a normalized cDNA library of claim 2;

(c) separating the hybridized cDNA circles from the unhybridized circles; and

(d) eluting the hybridized cDNA circles from the solid matrix, thereby identifying cDNA clones capable of hybridizing the genomic clone.

15. A method of claim 14, where in step (c), the unhybridized circles are separated from the hybridized circles by washing the matrix with an appropriate buffer.

16. A method of identifying cDNA clones capable of hybridizing a genomic clone comprising:

(a) growing the genomic clones from a genomic library on a master plate;

(b) duplicating the genomic clones on a solid matrix such that the positions of the clones on the master plate and the matrix can be correlated;

(c) hybridizing the genomic clones on the solid matrix with the single-stranded circles of a normalized cDNA library of claim 2;

(d) washing the matrix to separate the hybridized cDNA circles from the unhybridized circles;

(e) labelling the hybridized cDNA circles of step

(d) with a probe such that the position of the genomic clone on the master plate could be determined; and

(f) eluting the hybridized cDNA circles from the solid matrix, thereby identifying cDNA clones capable of hybridizing the genomic clone which is determined on the master plate.

17. A method of claim 16, wherein the solid matrix is a filter.

18. A method of claim 17, wherein the probe is nucleic acid molecule capable of hybridizing to the single-strand circle and is labelled.

19. A method of claim 7, further comprising converting the isolated unhybridized DNA circles to partial duplexes by primed extension.

20. A method of claim 1, wherein the specific sequence tag is between a sequence of a rare restriction site and an oligodT stretch.

21. A method of claim 20, wherein the number of the oligodT stretch ranges from 12 to 18.

22. A method of claim 5, wherein the primer is a primer comprising an oligo(dt) stretch.

23. A method of either of claims 14 or 16, further comprising converting the eluted single-stranded DNA circles to partial duplexes by primed extension.

24. A method of any of claims 19, 20, or 21, further comprising introduction of the partial duplexes into competent host cells.

25. A method of claim 24, wherein the duplexes are introduced into the host cells by electrophoration.