Generation of cDNA expression libraries enriched for in-frame sequences
- Creators
- Davis, Claytus A.
- Benzer, Seymour
Abstract
Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60–80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.
Additional Information
© 1997 National Academy of Sciences. Contributed by Seymour Benzer, December 24, 1996. We thank Lynette Dowling and Viveca Sapin for expert technical assistance. This work was supported by fellowships to C.A.D. from the Human Frontiers in Science Program and the Canadian Medical Research Council and by grants to S.B. from the National Science Foundation (MCB-9408718), the National Institutes of Health (AG12289 and EY09278), the McKnight Foundation, and the James G. Boswell Foundation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.Attached Files
Published - PNAS-1997-Davis-2128-32.pdf
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Additional details
- PMCID
- PMC20052
- Eprint ID
- 52332
- Resolver ID
- CaltechAUTHORS:20141203-112209793
- Human Frontier Science Program
- Medical Research Council of Canada
- NSF
- MCB-9408718
- NIH
- AG12289
- NIH
- EY09278
- McKnight Foundation
- James G. Boswell Foundation
- Created
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2014-12-03Created from EPrint's datestamp field
- Updated
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2021-11-10Created from EPrint's last_modified field