Excision of DNA segments introduced into cloning vectors by the poly(dA-dT) joining method.
A method is described for excising cloned DNA segments that have been inserted into their vectors by poly(dA-dT) joins. The recombinant DNA is cleaved within the vector DNA portion by one or more restriction endonucleases to generate a linear DNA molecule with the insert DNA sequence flanked by the poly(dA-dT) joins. After denaturation, the single strands "snap back" because of the intrastrand poly(dA) and poly(dT) sequences to form circular structures with "tails" of vector DNA. The vector portion of the DNA is then digested by Escherichia coli exonuclease VII, while the insert portion remains resistant to attack. The resistant strands are annealed and purified by electrophoresis in agarose. The insert DNA segment free of contaminating vector sequences can be used as a hybridization probe and for insertion into a new vector since suitable cohesive termini are generated from the retained poly(dA) and poly(dT) tails by an appropriate exonuclease.
D004269:DNA, Bacterial; D004270:DNA, Circular; D004274:DNA, Recombinant; D004926:Escherichia coli; D005092:Exonucleases / Q000378:metabolism; D010957:Plasmids; D011067:Poly dA-dT; D011089:Polydeoxyribonucleotides
D004269:DNA, Bacterial; D004270:DNA, Circular; D004274:DNA, Recombinant; D011089:Polydeoxyribonucleotides; D011067:Poly dA-dT; D005092:Exonucleases