Directional cloning of cDNA using a selectable SfiI cassette.
To increase the efficiency of directionally cloning cDNA, we have constructed a pair of vectors and devised a cDNA cloning strategy that improves upon previously published methods. The vectors, pLIB: AZ and pLIB: ZA, have two unique (distinct religation specificities; GGCCN/NNNNGGCC) SfiI sites (SfiI.A and SfiI.B) flanking a stuffer fragment which contains the tetracycline-resistance element. These vectors permit the directional cloning of cDNA in both sense (pLIB: AZ) and antisense (pLIB: ZA) orientations relative to the promoter for phage T3 RNA polymerase. cDNA that was synthesized using a primer with a 5' sequence of a SfiI.B site followed by an oligo(dT)16 3' tail was then ligated to an adaptor with the sequence of a SfiI.A site produced directional molecules that could be cloned into the pLIB vectors. Complex libraries with 10(7) members were produced from as few as 6 x 10(5) cells. The SfiI sites and stuffer can be subcloned as a cassette to permit directional cloning in other vectors, as there are several restriction enzyme sites flanking this region to the 5' and 3'.
Zelenetz
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AD
Stanford Medical School, Department of Medicine, CA 94305-5306.
Levy
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R
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D015723:Gene Library; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D015183:Restriction Mapping; D013379:Substrate Specificity; D013753:Tetracycline Resistance / Q000235:genetics
D015345:Oligonucleotide Probes; D004262:DNA Restriction Enzymes; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific