Unique substrate spectrum and PCR application of Nanoarchaeum equitans family B DNA polymerase.
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 lambda 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 lambda 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.
Choi
Jeong Jin
JJ
Department of Genetic Engineering, Sungkyunkwan University, Jangan-Gu, Suwon 440-746, Republic of Korea.
Song
Jae-Geun
JG
Nam
Ki Hoon
KH
Lee
Jong Il
JI
Bae
Heejin
H
Kim
Gun A
GA
Sun
Younguk
Y
Kwon
Suk-Tae
ST
D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D019271:Hypoxanthine / Q000378:metabolism; D008970:Molecular Weight; D046588:Nanoarchaeota / Q000201:enzymology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013379:Substrate Specificity; D014498:Uracil / Q000378:metabolism
D004279:DNA, Viral; D019271:Hypoxanthine; D014498:Uracil; D004259:DNA-Directed DNA Polymerase
J Clin Microbiol. 1993 Sep;31(9):2356-60
8408555
J Bacteriol. 2000 Feb;182(3):655-63
10633098
Nat Struct Biol. 2002 Dec;9(12):922-7
12415291
Extremophiles. 2000 Aug;4(4):215-25
10972190
FEBS Lett. 1996 Jun 10;388(1):1-4
8654578
Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):596-601
11782527
J Bacteriol. 1999 Oct;181(19):5984-92
10498710
Res Microbiol. 2003 Apr;154(3):165-71
12706504
Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9045-50
10430892
Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):12984-8
14566062
Nature. 2002 May 2;417(6884):63-7
11986665
Nature. 1990 Jun 28;345(6278):773-4
2359454
Appl Environ Microbiol. 1997 Nov;63(11):4504-10
9361436
Anal Biochem. 1993 May 15;211(1):164-9
8323030
Virol J. 2005 Apr 11;2:29
15823209
Gene. 1991 Dec 1;108(1):1-6
1761218
Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):9188-93
8799176
J Biol Chem. 1996 Jul 26;271(30):17692-6
8663453
J Biol Chem. 2000 Jun 23;275(25):19146-9
10777501
Gene. 1990 Sep 1;93(1):125-8
2227421
Nat Protoc. 2007;2(3):603-14
17406621
J Bacteriol. 1998 Apr;180(8):2232-6
9555910
Proc Natl Acad Sci U S A. 1990 Jun;87(12):4576-9
2112744
Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216-20
8134376
Nucleic Acids Res. 2008 Feb;36(3):705-11
18032433
Anal Biochem. 2003 Oct 15;321(2):226-35
14511688
Mol Cell. 2001 Jul;8(1):7-8
11515498
Nucleic Acids Res. 1993 Sep 11;21(18):4427-8
8415021
J Mol Biol. 2006 Mar 10;356(5):1093-106
16412462
J Mol Biol. 2007 Sep 28;372(4):855-863
17692870
Biotechniques. 2000 Aug;29(2):268-70
10948427
Nucleic Acids Res. 1996 Sep 15;24(18):3546-51
8836181
Mol Cell Probes. 2002 Aug;16(4):261-7
12270267
J Mol Biol. 1982 Dec 25;162(4):729-73
6221115
FEMS Microbiol Lett. 2002 Nov 19;217(1):89-94
12445650