Sequence analysis of heteropolymeric DNA synthesized in vitro by the enzyme terminal deoxynucleotidyl transferase and cloned in Escherichia coli.

We have synthesized in vitro single strands of DNA in reaction mixtures containing the terminal deoxynucleotidyl transferase (Bollum's enzyme), an oligo-dG as primer, the four common deoxynucleoside triphosphates and both Mg and Co ions. The resulting heteropolymers have been converted into double strands, tailed with oligo-dC sequences, annealed with an oligo-dG tailed plasmid vector and cloned in E. coli. Six recombinant plasmids have been isolated and characterized. Two of them have been sequenced. The heteropolymeric chains produced by the terminal transferase, ranging in size between 200 and 400 nucleotides, are richer in purines than in pyrimidines, except in the last portions. Open reading frames for 7-20 amino acids with repeated, in phase translational stop codons are present in these sequences and in their complements.