Synthesis of RNA from cellulose-bound complementary DNA

• Published in: The Journal of biological chemistry Vol. 252; no. 16; pp. 5615 - 5618
• Main Authors: Thrash, C R, Schimke, R T
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25.08.1977
• Subjects: Cellulose; DNA, Bacterial - metabolism; DNA-Directed RNA Polymerases - metabolism; Escherichia coli - enzymology; Kinetics; Nucleic Acid Hybridization; RNA Polymerase II - metabolism; RNA, Bacterial - metabolism; Templates, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)40066-4

Radioactively labeled RNAs were synthesized from cellulose-bound cDNA templates using Escherichia coli RNA polymerase. Hybridization of this RNA to excess unlabeled cDNA approached 100%, indicating the complementarity of product and template. The average length of the RNA product, as determined by formamide gels, was approximately 40% of the template length. Hybridization of unlabeled globin RNA produced by this technique to labeled globin cDNA indicated the population of RNA sequences represented at least 80% of the template sequences. Approximately 30% of the RNA product by mass contains poly(A) tails as determined by binding to oligo(dT)-cellulose. The template can be reused for several cycles of synthesis with little loss of synthetic capability and therefore, can amplify the amount of mRNA initially used to produce the template.