Use of the T4 Polynucleotide Ligase in The Joining of Flush‐Ended DNA Segments Generated by Restriction Endonucleases

Open Access
Publisher Website
Google Scholar
Abstract
Double-stranded DNA segments with completely base-paired ends were obtained by the action of various restriction endonucleases on phage and plasmid DNA. These segments were joined covalently by the [phage] T4 polynucleotide ligase [EC 6.5.1.1]. The joining was monitored by the EM count of intramolecularly circularized segments. The highest extent of joining, close to 75%, was observed at 15-25.degree. C with the segments resulting from the action of the Bacillus subtilis (strain R) restriction endonuclease Bsu on the DNA of bacteriophage SPP1 or of the plasmid pSC 101. The joining of double-stranded termini required about 10 times more enzyme than the short single-stranded termini produced by the Escherichia coli restriction endonuclease EcoRI. A shortened purification of the T4 ligase gave an enzyme devoid of interfering nucleases.