3'-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures

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2000
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Abstract
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 (T(m)) 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 T(m)(65 degrees 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 T(m)and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.
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openalex_W2170081920 Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Igor V. Kutyavin, Irina Afonina, Andrew Mills, Vladimir V. Gorn, E A Lukhtanov, Evgeniy Belousov, M. J. Singer, David K. Walburger, S. G. Lokhov, Alexander Gall, Robert O. Dempcy, Malcolm Reed, Rich B. Meyer, Joe Hedgpeth
Journal Nucleic Acids Research
Year 2000
DOI
10.1093/nar/28.2.655
URL
https://doi.org/10.1093/nar/28.2.655
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