Directional cDNA library construction assisted by the in vitro recombination reaction.
We report here a new directional cDNA library construction method using an in vitro site-specific recombination reaction, based on the integrase-excisionase system of bacteriophage lambda. Preliminary experiments revealed that in vitro recombinational cloning (RC) provided important advantages over conventional ligation-assisted cloning: it eliminated restriction digestion for directional cloning, generated low levels of chimeric clones, reduced size bias and, in our hands, gave a higher cloning efficiency than conventional ligation reactions. In a cDNA cloning experiment using an in vitro synthesized long poly(A)(+) RNA (7.8 kb), the RC gave a higher full-length cDNA clone content and about 10 times more transformants than conventional ligation-assisted cloning. Furthermore, characterization of rat brain cDNA clones yielded by the RC method showed that the frequency of cDNA clones >2 kb having internal NotI sites was approximately 6%, whereas these cDNAs could not be cloned at all or could be isolated only in a truncated form by conventional methods. Taken together, these results indicate that the RC method makes it possible to prepare cDNA libraries better representing the entire population of cDNAs, without sacrificing the simplicity of current conventional ligation-assisted methods.
Ohara
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Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan. ohara@kazusa.or.jp
Temple
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D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D010582:Bacteriophage lambda / Q000201:enzymology; D001921:Brain / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015723:Gene Library; D019426:Integrases / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011995:Recombination, Genetic / Q000235:genetics*; D013049:Spectrin / Q000235:genetics
D018076:DNA, Complementary; D013049:Spectrin; D019426:Integrases; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases