DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment

Abstract

Some prokaryotic and eukaryotic DNA polymerases are capable of adding an additional nontemplated nucleotide residue at the 3' end of a DNA fragment (Clark et al., 1987; Clark, 1988). The extra nucleotide at the 3' end of the PCR product has been shown to be a critical factor determining the efficiency of cloning PCR products into plasmids and can affect mutation analyses with a PCR-denaturing gradient gel electrophoresis (DGGE) approach (Pfeiffer and Hu, 1993). In the present work, the ability of various DNA polymerases to add an extra nontemplated nucleotide at the 3' end of DNA was studied. The results show that out of the eight studied enzymes, five can add, with varying efficiencies, an extra nucleotide residue at the 3' end of DNA. Which extra nucleotide is added depends on the terminal residue and the DNA polymerase. Among the enzymes, thermostable Pfu DNA polymerase is found to be the best choice for PCR due to its relatively high fidelity (Scott et al., 1991; Coller, unpublished), and ability to produce blunt-ended DNA fragments. The relationship between the DNA polymerases' ability to add an extra nucleotide and their 3'-->5' exonuclease activity is also discussed.