Rapid synthesis and cloning of complementary DNA from any RNA molecule into plasmid and phage M13 vectors

• Published in: Gene Vol. 68; no. 1; pp. 151 - 158
• Main Authors: Rutledge, R.G., Seligy, V.L., Côté, M.-J., Dimock, K., Lewin, L.L., Tenniswood, M.P.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 15.08.1988; Amsterdam Elsevier; New York, NY
• Subjects: Autoradiography; Biological and medical sciences; Biotechnology; cDNA; Cloning, Molecular - methods; Coliphages - genetics; DNA - biosynthesis; DNA - genetics; DNA cloning; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; gel filtration; Genetic engineering; Genetic technics; Genetic Vectors; ligation; Methods. Procedures. Technologies; Molecular cloning; oligodeoxyribonucleotide primers; phage M13; Phosphorus Radioisotopes; Plasmids; Recombinant DNA libraries; RNA; RNA - genetics; ss; DTT; oligo(dT); AMV; Recombinant DNA libraries; bp; ds; gel filtration; BSA; cfu; TCA; dNTP(s); ligation; oligodeoxyribonucleotide primers; kb; DNA cloning; cDNA; HPIV-3; RP; Enzymatic synthesis; Double stranded DNA; Complementary DNA; Primer; Gel filtration; Method; Molecular cloning
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(88)90607-5

We describe several modifications of the Gubler and Hoffman procedure [Gene 25 (1983) 263–269] for complementary DNA (cDNA) synthesis that expand the versatility of this method for the rapid synthesis and cloning of double-stranded (ds) cDNA. These modifications include: (1) The combination of first and second strand synthesis into a single two-step reaction, which reduces the time for synthesis of blunt-ended ds-cDNA to less than 4 h. (2) The use of random hexadeoxyribonucleotide primers (RP) for the synthesis of ds-cDNA, which allows the synthesis of cDNA from any RNA template. (3) The combined use of random primers and DNA ligase treatment of cDNA/RNA hybrids prior to second-strand synthesis, which promotes the production of nearly full length ds-cDNA molecules. (4) The use of gel filtration to size-fractionate ds-cDNA, which allows the selection of specific size classes of ds-cDNA for cloning. (5) The use of blunt-end ligation to insert the ds-cDNA into the vector, which reduces the total time required for the construction of cDNA libraries to less than 24 h.