Methods and compositions for producing full length cDNA libraries
AssigneeIncyte Genomics, Inc.
InventorsKarl Guegler, Ruoying Tan, Michael J. Rose
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Abstract
Methods and compositions are provided for producing full-length CDNA libraries. In the subject methods, full length first strand cDNAs are isolated using a fusion protein of an eIF-4E domain and an eIF-4G domain separated by a flexible linker. Also provided is the novel fusion protein employed in the subject methods, as well as nucleic acids encoding, and host cells capable of expressing, the same. Finally, kits for use in practicing the subject methods are provided. The subject invention finds use in a variety of applications in which full-length cDNA libraries are employed.
Claims20
A method of isolating a nucleic acid having a 5′ end cap structure, said method comprising:
- contacting said nucleic acid with a Fusion Protein comprising an eIF-4E domain and an eIF-4G domain joined by a linker domain, whereby a complex of said Fusion Protein and nucleic acid is produced;
- and
- isolating said complex.
A method for generating a cDNA library, said method comprising:
- (a) generating a population of blunt-ended mRNA/cDNA duplexes;
- (b) contacting said population of blunt-ended mRNA/cDNA duplexes with a fusion protein comprising an eIF-4E domain and an eIF-4G domain joined by a linker domain, to produce a reaction mixture including complexes of said Fusion Protein and blunt-ended mRNA/cDNA duplexes;
- and
- (c) separating said complexes from the remainder of said reaction mixture.
A method for generating a cDNA library, said method comprising:
- (a) contacting a population of mRNAs with a reverse transcriptase under conditions sufficient to produce a population of mRNA/cDNA duplexes;
- (b) contacting said population of mRNA/cDNA duplexes with an RNAse under conditions sufficient to degrade single stranded RNA to produce a population of blunt-ended mRNA/cDNA duplexes;
- (c) contacting said population of blunt-ended mRNA/cDNA duplexes with a fusion protein comprising an eIF-4E domain and an eIF-4G domain joined by a linker domain, to produce complexes of said Fusion Protein and blunt-ended mRNA/cDNA duplexes in an aqueous mixture;
- (d) separating said complexes from the remainder of said aqueous mixture;
- and
- (e) disrupting said complexes to produce free blunt-ended mRNA/cDNA duplexes.
A Fusion Protein comprising an eIF-4E domain and a eIF-4G domain separated by a flexible linking domain.
A kit for use in the preparation of a cDNA library, said kit comprising:
- a Fusion Protein comprising an eIF-4E domain and a eIF-4G domain separated by a flexible linking domain.
Dependencies are inferred from the claim text.