Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP).
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.
Hoskins
Roger A
RA
Berkeley Drosophila Genome Project, Department of Genome Biology, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Stapleton
Mark
M
George
Reed A
RA
Yu
Charles
C
Wan
Kenneth H
KH
Carlson
Joseph W
JW
Celniker
Susan E
SE
D000818:Animals; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D004331:Drosophila melanogaster / Q000235:genetics; D020224:Expressed Sequence Tags; D015723:Gene Library; D017344:Genes, Insect; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics
D018076:DNA, Complementary; D014157:Transcription Factors