Nucleic Acids Research, 2000, Vol. 28, No. 2 655-661
© 2000 Oxford University Press
3'-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures
Epoch Pharmaceuticals, Inc., 12277 134th Court NE #110, Redmond, WA 98052, USA
DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3'-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (Tm) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5'-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same Tm (65°C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe Tm and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.
* To whom correspondence should be addressed. Tel: +1 425 821 7535; Fax: +1 425 825 0306; Email: mreed@epochpharm.com Present address: Rich B. Meyer, Genelabs Technologies, Inc., Redwood City, CA, USA
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I. V. Kutyavin, S. G. Lokhov, I. A. Afonina, R. Dempcy, A. A. Gall, V. V. Gorn, E. Lukhtanov, M. Metcalf, A. Mills, M. W. Reed, et al. Reduced aggregation and improved specificity of G-rich oligodeoxyribonucleotides containing pyrazolo[3,4-d]pyrimidine guanine bases Nucleic Acids Res., November 15, 2002; 30(22): 4952 - 4959. [Abstract] [Full Text] [PDF]
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M. Gotoh, T. Yada, T. Sato, T. Akashima, H. Iwasaki, H. Mochizuki, N. Inaba, A. Togayachi, T. Kudo, H. Watanabe, et al. Molecular Cloning and Characterization of a Novel Chondroitin Sulfate Glucuronyltransferase That Transfers Glucuronic Acid to N-Acetylgalactosamine J. Biol. Chem., October 4, 2002; 277(41): 38179 - 38188. [Abstract] [Full Text] [PDF]
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M. Gotoh, T. Sato, T. Akashima, H. Iwasaki, A. Kameyama, H. Mochizuki, T. Yada, N. Inaba, Y. Zhang, N. Kikuchi, et al. Enzymatic Synthesis of Chondroitin with a Novel Chondroitin Sulfate N-Acetylgalactosaminyltransferase That Transfers N-Acetylgalactosamine to Glucuronic Acid in Initiation and Elongation of Chondroitin Sulfate Synthesis J. Biol. Chem., October 4, 2002; 277(41): 38189 - 38196. [Abstract] [Full Text] [PDF]
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C. Schmitt, A. Humeny, C.-M. Becker, K. Brune, and A. Pahl Polymorphisms of TLR4: Rapid Genotyping and Reduced Response to Lipopolysaccharide of TLR4 Mutant Alleles Clin. Chem., October 1, 2002; 48(10): 1661 - 1667. [Abstract] [Full Text] [PDF]
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I. M. Mackay, K. E. Arden, and A. Nitsche Real-time PCR in virology Nucleic Acids Res., March 15, 2002; 30(6): 1292 - 1305. [Abstract] [Full Text] [PDF]
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S. N. Agoff, T. A. Brentnall, D. A. Crispin, S. L. Taylor, S. Raaka, R. C. Haggitt, M. W. Reed, I. A. Afonina, P. S. Rabinovitch, A. C. Stevens, et al. The Role of Cyclooxygenase 2 in Ulcerative Colitis-Associated Neoplasia Am. J. Pathol., September 1, 2000; 157(3): 737 - 745. [Abstract] [Full Text] [PDF]
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