Enzymatic end-to-end joining of DNA molecules

• Published in: Journal of molecular biology Vol. 78; no. 3; pp. 453,IN1,461 - 460,IN2,471
• Main Authors: Lobban, Peter E., Kaiser, A.D.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 15.08.1973
• Subjects: Centrifugation, Density Gradient; Deoxyribonucleosides; DNA; DNA Nucleotidyltransferases; DNA Repair; DNA, Circular - isolation & purification; DNA, Viral; Exonucleases; Microscopy, Electron; Phosphorus Isotopes; Salmonella Phages
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/0022-2836(73)90468-3

A way to join naturally occurring DNA molecules, independent of their base sequence, is proposed, based upon the presumed ability of the calf thymus enzyme terminal deoxynucleotidyltransferase to add homopolymer blocks to the ends of double-stranded DNA. To test the proposal, covalently closed dimer circles of the DNA of bacteriophage P22 were produced from linear monomers. It is found that P22 DNA as isolated will prime the terminal transferase reaction, but not in a satisfactory manner. Pre-treatment of the DNA with λ exonuclease, however, improves its priming ability. Terminal transferase can then be used to add oligo(dA) blocks to the ends of one population of P22 DNA molecules and oligo(dT) blocks to the ends of a second population, which enables the two DNAs to anneal to one another to form dimer circles. Subsequent treatment with a system of DNA repair enzymes converts the circles to covalently closed molecules at high efficiency. It is demonstrated that the success of the joining system does not depend upon any obvious unique property of the P22 DNA. The joining system yields several classes of by-products, among them closed circular molecules with branches. Their creation can be explained on the basis of the properties of terminal transferase and the DNA repair enzymes.