Protection of oligonucleotide primers against degradation by DNA polymerase I

By use of a mutational assay employing an octadecamer with a mismatch in the center, it is shown that the introduction of phosphorothioate groups near the 5'-end can protect the mismatch against degradation by the 5'-3'-exonuclease activity of Escherichia coli DNA polymerase I. An optimal level of protection is achieved when the phosphorothioate groups are incorporated in at least the second and third internucleotidic linkages from the 5'-end. However, gel electrophoretic analysis as well as the use of an octadecamer with a mismatch closer to the 5'-end in the mutational assay reveals that degradation of the oligonucleotide is not completely blocked but only slowed down.