PCR with deoxyinosine-containing primers using DNA polymerases with proofreading activity.

1Department of Dermatology, Wayne State University, Detroit, Michigan 48201; 2Department of Dermatology, Niigata University, Niigata, Japan As the application of PCR has expanded, more amplified products have been used for cloning and mutagenesis. Although Taq polymerase (from eubacterium Thermus aquaticus) is the most convenient and intensively studied enzyme to date, one of its drawbacks is a relatively high misincorporation (error) rate ( 1 x 10s), which may be problematic in some applications. Recently several DNA polymerases with 3'--~ 5' exonuclease (proofreading) activity have become commercially available, namely Deep Vent (from archaebacterium Pyrococcus sp.), Pfu (from archaebacterium Pyrococcus furiosus), and U1Tma DNA polymerases (from eubacterium Thermotoga maritima). They could yield lower misincorporation rates ( -2• -6) and greater stability at high temperature, according to the manufacturers' specification. Degenerate primers with mixed bases have been used in many applications, such as identification of members of gene families, (1> and simultaneous detection of viruses with conserved genomic sequences. (2~ Substitution of a mixed-base position with deoxyinosine (dI) was reported to make the amplification more effective. (3> Although we tried to identify paramyxovirus RNA in lupus erythematosus tissues with degenerate primers, we were able to amplify the sequence with Taq polymerase but not with Pfu or Deep Vent polymerases. In this report we present a successful amplification of a viral sequence with primers containing dI in various positions, using U1Tma DNA polymerase.