The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 ؋ 10 ؊6 ) than Taq DNA polymerase (11.98 ؋ 10 ؊6 ). Uniquely, Neq DNA polymerase also amplified DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two-to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.DNA polymerase (EC 2.7.7.7) plays essential roles in cellular DNA replication and repair. In recent years, thermostable archaeal DNA polymerases have been identified in many species (6,7,11,19,20,34,35). On the basis of amino acid sequences, DNA polymerases can be classified into at least six distinct families (A, B, C, D, X, and Y) (27), and most archaeal DNA polymerases have been identified as members of family B, along with eukaryotic replicative DNA polymerases and Escherichia coli DNA polymerase II (6). Previously reported archaeal family B DNA polymerases from hyperthermophiles possess a 3Ј35Ј proofreading exonuclease activity and so are capable of carrying out PCR with higher fidelity than Thermus aquaticus (Taq) DNA polymerase. Another notable feature of archaeal family B DNA polymerases is the recognition of uracil in DNA templates, which causes DNA synthesis to stall (14,22). The archaeal family B DNA polymerases have a readahead function concerning uracil detection, based on an Nterminal pocket specific for uracil (12). In addition, archaeal family B DNA polymerases interact with another deaminated base, hypoxanthine (13).Archaea have been recognized as a third domain of living organisms, distinct from the Bacteria and Eukarya (38). From a phylogenetic perspective on the basis of rRNA sequences, Archaea have been classified into four phyla: Crenarchaeota, Euryarchaeota, Korarchaeota, and Nanoarchaeota (3, 17). Nanoarchaeum equitans, which belongs to Nanoarchaeota, is a nano-sized and hyperthermophilic anaerobe and is the firstknown obligate archaeal symbiont, which grows on the surface of a specific crenarchaeal host, Ignicoccus sp. strain KIN4/I (17, 18). The 490,885-bp N. equitans genome is one of the smallest microbial genomes and has been completely sequenced (37).N. equitans family B DNA polymerase (Neq DNA polymerase) is encoded by two open reading fra...