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Nucleic Acids Research 2005 33(21):e185; doi:10.1093/nar/gni184
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Published online 2 December 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP)

Roger A. Hoskins, Mark Stapleton, Reed A. George, Charles Yu, Kenneth H. Wan, Joseph W. Carlson and Susan E. Celniker*

Berkeley Drosophila Genome Project, Department of Genome Biology, Life Sciences Division, Lawrence Berkeley National Laboratory Berkeley, CA 94720, USA

*To whom correspondence should be addressed at Berkeley Drosophila Genome Project, Lawrence Berkeley National Laboratory, One Cyclotron Road MS 64-121, Berkeley, CA 94720, USA. Tel: 510 486 6258; Fax: 510 486 6798; Email: celniker{at}fruitfly.org

Received September 7, 2005. Revised November 11, 2005. Accepted November 11, 2005.

cDNA cloning is a central technology in molecular biology. cDNA sequences are used to determine mRNA transcript structures, including splice junctions, open reading frames (ORFs) and 5'- and 3'-untranslated regions (UTRs). cDNA clones are valuable reagents for functional studies of genes and proteins. Expressed Sequence Tag (EST) sequencing is the method of choice for recovering cDNAs representing many of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a cDNA library at random, and it recovers transcripts with low expression levels inefficiently. We describe a PCR-based method for directed screening of plasmid cDNA libraries. We demonstrate its utility in a screen of libraries used in our Drosophila EST projects for 153 transcription factor genes that were not represented by full-length cDNA clones in our Drosophila Gene Collection. We recovered high-quality, full-length cDNAs for 72 genes and variously compromised clones for an additional 32 genes. The method can be used at any scale, from the isolation of cDNA clones for a particular gene of interest, to the improvement of large gene collections in model organisms and the human. Finally, we discuss the relative merits of directed cDNA library screening and RT–PCR approaches.


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