A peptide nucleic acid (PNA) oligomer, an analogue of DNA, was examined for its ability to function as a primer or a template to support DNA synthesis catalyzed by DNA polymerases. The analogue, (PNA)19-TpG-OH, comprised of 19 bases in the form of PNA followed by a dinucleotide (TpG-OH) with a single phosphate and a free 3'OH terminus, was recognized as a bona fide primer by 2 reverse transcriptases and also by the Klenow fragment of E. coli DNA polymerase I. The 21-mer PNA chimera is extended on both RNA and DNA templates by all three polymerases. The specificity of binding of the PNA chimeric primer/DNA template at the template-primer binding site of the enzyme was shown by its photo-cross-linking ability to the enzyme which could be effectively competed out by another TP but not by template or primer alone. Furthermore, the chimeric TP-enzyme covalent complex was found to be catalytically active as judged by its ability to incorporate one nucleotide onto the 3'OH terminus of the immobilized primer. PNA sequences were also recognized as template when annealed with a DNA primer. These observations are in variance with the conventional suggestion that the phosphate backbone in the duplex region is essential for recognition and binding by DNA polymerases. The efficient extension of (PNA)19-TpG-OH suggests that the diameter of the duplex region of template primer rather than the phosphate backbone may be sufficient for recognition by DNA polymerases.