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US 2003/0211462 A1Published application
Directionally cloned random cDNA expression vector libraries, compositions and methods of use
AssigneeRigel Pharmaceuticals, Inc.
InventorsMary Shen, Simon Yu, Xian Wu, Donald Payan
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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.
Claims26
A method for producing an expression vector library, comprising:
- a) providing a plurality of mRNAs;
- b) synthesizing a plurality of cDNAs from said mRNAs, comprising:
- i) synthesizing a first cDNA strand using a primer, which primer comprises:
- a) a random polynucleotide from about 6 to about 9 nucleotides in length;
- b) a 5′ terminal nucleotide comprising the base cytosine;
- and
- c) a nucleotide immediately 3′ to the 5′ terminal nucleotide, comprising the base cytosine;
- and
- ii) synthesizing a second cDNA strand complementary in sequence to the first cDNA strand, whereby the second cDNA strand comprises a 3′ terminal nucleotide comprising the base guanine, and a nucleotide immediately 5′ to the 3′ terminal nucleotide, comprising the base guanine, to produce a double stranded cDNA;
- c) ligating a first adaptor to the 5′ end and a second adaptor to the 3′ end of said double stranded cDNA, to produce an adaptor-modified cDNA, wherein said first and said second adaptors are each double stranded nucleic acids comprising the sequence
- 6 5′-p-C C N1 N2 N3N4N5 G G C C Nx G G C C N6N7 N8 N9-3′ 3′-G G N′1N′2N′3N′4N′5 C C G G N′x C C G G N′6-p -5′;
- d) cleaving the adaptor-modified cDNA with the restriction endonuclease Sfil, to produce a first nucleic acid which is a cleaved adaptor-modified cDNA;
- and
- e) cloning said first nucleic acid into an expression vector, which vector comprises:
- i) a transcription regulatory sequence;
- and
- ii) a cloning site comprising a 5′ Sfil-a recognition sequence
- 5′-G G C C N N′9N′8N′7N G G C C-3′,
- and a 3′ Sfil-b recognition sequence
- 5′-G G C C N N2N3N4N G G C C-3′, as read on the same strand;
- wherein N and N1 through N9 are each a nucleotide selected from the group consisting of dAMP, dTMP, dGMP and dCMP, wherein nucleotides of the vector site and the adaptor having the same numerical denotation are the same nucleotide type, wherein Nx represents “x” number of nucleotides wherein x is between 1 and 9, which nucleotides are each selected from the group consisting of dAMP, dTMP, dGMP and dCMP, wherein N′ denotes a nucleotide which is complementary to a nucleotide denoted N, and wherein the transcription regulatory sequence and the cloning site are positioned relative to each other such that cloning of the first nucleic acid into the cloning site of the expression vector operably links the cDNA of the first nucleic acid to said transcription regulatory sequence.
18 more elements
A method for producing an expression vector library, comprising:
- a) providing a plurality of mRNAs;
- b) synthesizing a plurality of cDNAs from said mRNAs, comprising:
- i) synthesizing a first cDNA strand using a primer, which primer comprises:
- a) a random polynucleotide from about 6 to about 9 nucleotides in length;
- b) a 5′ terminal nucleotide comprising the base cytosine;
- and
- c) a nucleotide immediately 3′ to the 5′ terminal nucleotide, comprising the base cytosine;
- and
- ii) synthesizing a second cDNA strand complementary in sequence to the first cDNA strand, whereby the second cDNA strand comprises a 3′ terminal nucleotide comprising the base guanine, and a nucleotide immediately 5′ to the 3′ terminal nucleotide, comprising the base guanine, to produce a double stranded cDNA;
- c) ligating a first adaptor to the 5′ end and a second adaptor to the 3′ end of said double stranded cDNA, to produce an adaptor-modified cDNA, wherein said first and said second adaptors are each double stranded nucleic acids comprising the sequence
- 8 5′-p-C C N1 N2 N3N4N5 G G C C Nx G G C C N6N7 N8 N9-3′ 3′-G G N′1N′2N′3N′4N′5 C C G G N′x C C G G N′6-p -5′;
- d) cleaving the adaptor-modified cDNA with the restriction endonuclease Sfil, to produce a first nucleic acid which is a cleaved adaptor-modified cDNA;
- and
- e) cloning said first nucleic acid into an expression vector, which vector comprises:
- i) a transcription regulatory sequence;
- and
- ii) a cloning site comprising a 5′ Sfil-b recognition sequence
- 5′-G G CC N N′4N′3N′2N G G C C-3′,
- and a 3′ Sfil-a recognition sequence
- 5′-G G C C N N7N8N9N G G C C-3′, as read on the same strand;
- wherein N and N1 through N9 are each a nucleotide selected from the group consisting of dAMP, dTMP, dGMP and dCMP, wherein nucleotides of the vector site and the adaptor having the same numerical denotation are the same nucleotide type, wherein Nx represents “x” number of nucleotides wherein x is between 1 and 9, which nucleotides are each selected from the group consisting of dAMP, dTMP, dGMP and dCMP, wherein N′ denotes a nucleotide which is complementary to a nucleotide denoted N, and wherein the transcription regulatory sequence and the cloning site are positioned relative to each other such that cloning of the first nucleic acid into the cloning site of the expression vector operably links the cDNA of the first nucleic acid to said transcription regulatory sequence.
18 more elements
Dependencies are inferred from the claim text.