Expression vectors permitting cDNA cloning and enrichment for specific sequences by hybridization/selection.
A set of vectors is described which allow the efficient cloning of full-length cDNAs, using a modification of the method of Okayama and Berg [Mol. Cell Biol. 2 (1982) 161-170], and enrichment of specific sequences directly from cDNA libraries by hybridization/selection. The vectors pcDpolyB+ and pcDpolyB- are derived from an expression vector described previously [Okayama and Berg, Mol. Cell Biol. 3 (1983) 280-289] and allow expression of cloned cDNAs in eukaryotic cells from the simian virus 40 early region promoter. The vectors BSB+ and BSB- contain convenient priming sites for sequence analysis and the T3 and T7 RNA polymerase promoters, allowing synthesis of transcripts homologous to either strand of the cDNA. Each of these vectors also contains the intergenic region from the bacteriophage f1 permitting synthesis of single-stranded (ss) copies of the cDNA libraries. Enrichment for cDNAs containing sequences homologous to the hypoxanthine phosphoribosyl transferase gene from an ss copy of a cDNA library by hybridization/selection is demonstrated. Levels of enrichment sufficient for the direct cloning of specific sequences without requiring colony or plaque hybridizations were obtained. Libraries constructed from different cell types can be screened against each other to create sublibraries highly enriched in sequences specific to a single cell type. The availability of cDNA expression libraries enriched for cell-type-specific cDNAs should greatly enhance the efficiency with which cDNAs can be identified on the basis of functional assays.
Pruitt
S C
SC
Department of Molecular and Cellular Biology, Roswell Park Memorial Institute, Buffalo, NY 14263.
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D004262:DNA Restriction Enzymes; D004270:DNA, Circular / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific; D004526:Efficiency; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010957:Plasmids; D014170:Transformation, Genetic
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D004262:DNA Restriction Enzymes; C047564:CCANNNNNNTGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific