Recovery of DNA fragments inserted by the "tailing" method: regeneration of PstI restriction sites.
A general method has been developed for the recovery of any DNA fragment inserted into a cloning vehicle containing a single endonuclease PstI site. Endonuclease PstI sites are regenerated by the addition of one or more deoxyguanosine residues to the 3' termini of the PstI-cleaved vehicle by terminal deoxynucleotidyl transferase. Chain elongation by terminal deoxynucleotidyl transferase is then continued with dITP, dATP or dGTP. A plasmid vehicle, pAO1, containing a single PstI site has been constructed. Insertional (foreign) DNA fragments that were "tailed" with dCTP have been annealed to PstI-cleaved pAO1 that was "tailed" with dGTP. When the annealed fragments were used to transform competent Escherichia coli cells, the single-stranded DNA gaps in the recombinant plasmids were repaired. Plasmids recovered from transformed bacteria could be cleaved by PstI into the insertional DNA with dG:dC tracts and linear pAO1 molecules.
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D005822:Genetic Vectors; D010957:Plasmids
D004247:DNA; D004262:DNA Restriction Enzymes