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Generation of cDNA expression libraries enriched for in-frame sequences

Davis, Claytus A. and Benzer, Seymour (1997) Generation of cDNA expression libraries enriched for in-frame sequences. Proceedings of the National Academy of Sciences of the United States of America, 94 (6). pp. 2128-2132. ISSN 0027-8424. PMCID PMC20052. https://resolver.caltech.edu/CaltechAUTHORS:20141203-112209793

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1073/pnas.94.6.2128 DOIArticle
http://www.pnas.org/content/94/6/2128PublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC20052/PubMed CentralArticle
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.
Funders:
Funding AgencyGrant Number
Human Frontier Science ProgramUNSPECIFIED
Medical Research Council of CanadaUNSPECIFIED
NSFMCB-9408718
NIHAG12289
NIHEY09278
McKnight FoundationUNSPECIFIED
James G. Boswell FoundationUNSPECIFIED
Subject Keywords:cloning vector; ORF; ORF vector; in-frame vector
Issue or Number:6
PubMed Central ID:PMC20052
Record Number:CaltechAUTHORS:20141203-112209793
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20141203-112209793
Official Citation:Claytus A. Davis and Seymour Benzer Generation of cDNA expression libraries enriched for in-frame sequences PNAS 1997 94 (6) 2128-2132
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:52332
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:03 Dec 2014 20:42
Last Modified:03 Oct 2019 07:41

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