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

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Hoskins RA,Stapleton M,George RA,Yu C,Wan KH,Carlson JW,Celniker SE

doi

10.1093/nar/gni184

keywords:

subject

Has Abstract

pub_date

2005-12-02 00:00:00

pages

e185

issue

21

eissn

0305-1048

issn

1362-4962

pii

33/21/e185

journal_volume

33

pub_type

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