Directionally cloned random cDNA expression vector libraries, compositions and methods of use

• US 7,034,145 B2
• Filed: 09/03/2004
• Issued: 04/25/2006
• Est. Priority Date: 05/08/2002
• Status: Active Grant

• Current Assignee: Rigel Pharmaceuticals, Inc.
• Original Assignee: Rigel Pharmaceuticals, Inc.
• Inventors: Yu, Simon, Shen, Mary, Payan, Donald, Wu, Xian
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US10/933,855
• Publication Number:
  US 20050032110A1
• Time in Patent Office: 599 Days
• Field of Search: 536/24.3, 536/24.33
• US Class Current: 536/24.3
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2740/13043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/70 Vectors containing special elements for cloning, e.g. topoisomerase, adaptor sites
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important

Abstract

The present invention provides random cDNA expression vector libraries, comprising expression vectors which comprise random cDNAs positioned in sense orientation. Also provided are random cDNA expression vector libraries, comprising expression vectors which comprise random cDNAs positioned in antisense orientation. Methods for producing these libraries through directional cloning of random cDNAs are also provided. Also provided herein are methods of using these libraries to screen for agents capable of modulating cell phenotype in desirable ways.

Claims

1. A composition comprising combinable reagents for making a directionally cloned cDNA library, comprising:

a primer having at its 5′

terminus a first nucleotide sequence of a 3′

portion of a restriction site for an infrequently cutting restriction endonuclease; and

a double stranded adaptor having a second nucleotide sequence of a 5′

end of said restriction site for said infrequently cutting restriction endonuclease.

2. The composition of claim 1, wherein said first and second nucleotide sequences, when ligated together, form a complete sequence of a restriction site for said infrequently cutting restriction endonuclease.

3. The composition of claim 1, wherein said infrequently cutting restriction endonuclease is Sfi1, BstAP1, PfiM1, Mwo1 or AlwN1.

4. The composition of claim 3, wherein said infrequently cutting restriction endonuclease is Sfi1.

5. The composition of claim 1, wherein said primer contains a CC at its 5′

terminus.

6. The composition of claim 1, wherein said primer is a random primer.

7. The composition of claim 6, wherein said random primer contains 4 to 10 random nucleotides at its 3′

end.

8. The composition of claim 1, wherein said primer comprises oligodT.