Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli
- 1 October 1972
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 69 (10) , 2904-2909
- https://doi.org/10.1073/pnas.69.10.2904
Abstract
We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: ( a ) converting circular SV40 DNA to a linear form, ( b ) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3′ ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase ( c ) adding complementary homodeoxypolymeric extensions to the other DNA strand, ( d ) annealing the two DNA molecules to form a circular duplex structure, and ( e ) filling the gaps and sealing nicks in this structure with E. coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule.Keywords
This publication has 14 references indexed in Scilit:
- Cell Transformation by VirusesScience, 1969
- Preparation of [α-32P]nucleoside and deoxynucleoside 5′-triphosphates from 32Pi and protected and unprotected nucleosidesBiochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1969
- Enzymatic synthesis of deoxyribonucleic acid. XXVI. Physical and chemical studies of a homogeneous deoxyribonucleic acid polymerase.1969
- Deoxyribonucleic acid nucleases. II. The effects of metals on the mechanism of action of deoxyribonuclease I.1968
- The integrated state of viral DNA in SV40-transformed cells.Proceedings of the National Academy of Sciences, 1968
- On the Mechanism of the Polynucleotide Joining ReactionCold Spring Harbor Symposia on Quantitative Biology, 1968
- A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells.Proceedings of the National Academy of Sciences, 1967
- DEOXYNUCLEOTIDE-POLYMERIZING ENZYMES OF CALF THYMUS GLAND .2. PROPERTIES OF TERMINAL DEOXYNUCLEOTIDYLTRANSFERASE1967
- AN EXONUCLEASE INDUCED BY BACTERIOPHAGE LAMBDA .I. PREPARATION OF CRYSTALLINE ENZYME1967
- AN EXONUCLEASE INDUCED BY BACTERIOPHAGE LAMBDA .2. NATURE OF ENZYMATIC REACTION1967