title: Effect of transition metal ions on the fluorescence and Taq-catalyzed polymerase chain reaction of tricyclic cytidine analogs.
abstract: The cytosine analogs 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) stand out among fluorescent bases due to their unquenched fluorescence emission in double-stranded DNA. Recently, we reported a method for the generation of densely tCo-labeled DNA by polymerase chain reaction (PCR) that relied on the use of the extremely thermostable Deep Vent polymerase. We have now developed a protocol that employs the more commonly used Taq polymerase. Supplementing the PCR with Mn(2+) or Co(2+) ions dramatically increased the amount of tCo triphosphate (dtCoTP) incorporated and, thus, enhanced the brightness of the PCR products. The resulting PCR products could be easily detected in gels based on their intrinsic fluorescence. The Mn(2+) ions modulate the PCR by improving the bypass of template tCo and the overall catalytic efficiency. In contrast to the lower fidelity during tCo bypass, Mn(2+) improved the ability of Taq polymerase to distinguish between dtCoTP and dTTP when copying a template dA. Interestingly, Mn(2+) ions hardly affect the fluorescence emission of tC(o), whereas the coordination of Co(2+) ions with the phosphate groups of DNA and nucleotides statically quenches tC(o) fluorescence with small reciprocal Stern-Vollmer constants of 10-300μM.
authors: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
authors: Gudrun
authors: G
authors: Stengel
authors: Byron W
authors: BW
authors: Purse
authors: Robert D
authors: RD
authors: Kuchta
references: Biochemistry. 2009 Aug 11;48(31):7547-55
references: 19580325
references: J Fluoresc. 2010 Mar;20(2):557-61
references: 20020318
references: Biophys Chem. 1993 Dec;48(2):193-204
references: 8298057
references: Nucleic Acids Res. 2004 Sep 27;32(17):5087-95
references: 15452275
references: EMBO J. 1998 Dec 15;17(24):7514-25
references: 9857206
references: Nucleic Acids Res. 2000 Oct 15;28(20):3962-71
references: 11024176
references: J Biol Chem. 1979 Jul 10;254(13):5718-25
references: 376517
references: Biochemistry. 2003 Jun 24;42(24):7467-76
references: 12809503
references: Biotechniques. 1996 Dec;21(6):1102-8
references: 8969839
references: J Am Chem Soc. 2006 Jan 25;128(3):780-90
references: 16417367
references: PCR Methods Appl. 1994 Jun;3(6):S136-40
references: 7920233
references: Nat Struct Mol Biol. 2009 Feb;16(2):212-8
references: 19151724
references: Acta Biochim Biophys Acad Sci Hung. 1981;16(3-4):131-3
references: 7347985
references: Anal Biochem. 1999 Mar 15;268(2):278-88
references: 10075818
references: Mol Biosyst. 2006 Feb;2(2):122-7
references: 16880929
references: J Am Chem Soc. 2003 Apr 30;125(17):4972-3
references: 12708835
references: DNA Repair (Amst). 2008 Oct 1;7(10):1647-58
references: 18638572
references: Methods Mol Biol. 2009;521:381-96
references: 19563118
references: Chem Rev. 2010 May 12;110(5):2579-619
references: 20205430
references: Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4267-72
references: 17360513
references: Nucleic Acids Res. 2002 Aug 1;30(15):3323-32
references: 12140316
references: Carcinogenesis. 1995 Aug;16(8):1775-82
references: 7634403
references: Gene. 1994 Mar 11;140(1):1-5
references: 8125324
references: J Biomol Struct Dyn. 2001 Oct;19(2):285-91
references: 11697733
references: Anal Chem. 2009 Nov 1;81(21):9079-85
references: 19810708
references: J Am Chem Soc. 2007 May 16;129(19):6240-8
references: 17455934
references: Biochemistry. 2002 Apr 16;41(15):4771-8
references: 11939771
references: J Am Chem Soc. 2009 Apr 1;131(12):4288-93
references: 19317504
references: J Biol Chem. 2009 Jan 16;284(3):1732-40
references: 18984581
references: DNA Repair (Amst). 2008 Nov 1;7(11):1824-34
references: 18692600
references: Biochim Biophys Acta. 1985 Sep 10;818(3):365-72
references: 4041444
references: Nucleic Acids Res. 2008 Jan;36(1):157-67
references: 18003656
references: Biochemistry. 2001 Feb 27;40(8):2580-7
references: 11327881
references: J Mol Biol. 2003 Jul 18;330(4):687-703
references: 12850140
references: J Phys Chem A. 2010 Sep 30;114(38):10388-94
references: 20828117
references: J Biol Chem. 1987 Oct 25;262(30):14648-54
references: 3667596
references: Biochemistry. 1996 Dec 10;35(49):15715-25
references: 8961934
references: Nucleic Acids Res. 1990 Dec 25;18(24):7465
references: 2259646
references: Biochemistry. 1985 Oct 8;24(21):5810-7
references: 3910084
references: Biotechnology (N Y). 1993 Sep;11(9):1026-30
references: 7764001
references: Proc Natl Acad Sci U S A. 1989 Jun;86(11):4076-80
references: 2657738
references: DNA Repair (Amst). 2007 Sep 1;6(9):1333-40
references: 17475573
references: Biochemistry. 2007 Oct 30;46(43):12289-97
references: 17915941
references: J Biol Chem. 2007 Aug 24;282(34):24689-96
references: 17609217
references: Nature. 1998 Jan 15;391(6664):231-2
references: 9440683
references: Anal Chem. 2010 Feb 1;82(3):1082-9
references: 20067253
references: Biochemistry. 1977 Aug 9;16(16):3647-54
references: 889813
references: Biochemistry. 2006 Jun 20;45(24):7563-73
references: 16768452
references: Genome Res. 1996 Jul;6(7):633-8
references: 8796351
references: Biochemistry. 2002 Oct 8;41(40):12051-61
references: 12356305