Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase

• Published in: Applied and Environmental Microbiology 2008-11, Vol.74 (21), p.6563-6569
• Main Authors: Choi, Jeong Jin, Song, Jae-Geun, Nam, Ki Hoon, Lee, Jong Il, Bae, Heejin, Kim, Gun A, Sun, Younguk, Kwon, Suk-Tae
• Format: Article
• Language: English
• Subjects: Bacteriophage lambda - genetics; Biochemistry; Biological and medical sciences; Deoxyribonucleic acid; DNA; DNA polymerase; DNA, Viral - genetics; DNA-Directed DNA Polymerase - isolation & purification; DNA-Directed DNA Polymerase - metabolism; Enzymology and Protein Engineering; Fundamental and applied biological sciences. Psychology; Glycosylation; Hypoxanthine - metabolism; Microbiology; Microorganisms; Molecular Weight; Nanoarchaeota - enzymology; Nanoarchaeum equitans; Polymerase Chain Reaction - methods; Substrate Specificity; Uracil - metabolism
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.00624-08

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 λ DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 x 10⁻⁶) than Taq DNA polymerase (11.98 x 10⁻⁶). Uniquely, Neq DNA polymerase also amplified λ 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.