On the Mechanism of Oligonucleotide‐Primed DNA Synthesis

Oligodeoxyribonucleotides from various sources and of defined chain length were examined for their capacity to prime DNA synthesis on base complementary and non‐complementary single‐stranded DNA templates. Using Escherichia coli DNA polymerase I as enzyme, primer activity could be detected at chain lengths as few as five to six nucleotides. The activity was higher on base complementary templates and increased progressively with chain length from 0.07% priming efficiency to about 20% at chain length 16. On non‐complementary templates primer activity was low with maximal efficiencies around 0.1% at chain lengths of 7–10. Polypyrimidine nucleotides and homooligonucleotides of dT, dA and dC showed very low primer efficiencies (0.0001%–0.03%). The high priming activity of the complementary oligonucleotides strongly depended on the preincubation with the template and was paralleled by the stable annealing of primer molecules to the template DNA. It is concluded that the annealing reaction of template and primer is the rate‐limiting step in chain initiation and that the stability of the base‐paired primer template duplex determines the primer efficiency of an oligonucleotide.