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

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.

Development of real-time PCR using Minor Groove Binding probe to monitor the biological control agent Candida oleophila (strain O)

“…The presence of a MGB stabilises the duplex between the probe and its target by folding into the minor groove formed by the terminal 5-6 bp of the duplex. The addition of a MGB conjugate can increase the specificity of a probe, especially when a mismatch is situated in the MGB region, but also its melting temperature (Kutyavin et al, 2000). This second property allows the design of shorter probes, which maintain an optimal melting temperature.…”

“…This second property allows the design of shorter probes, which maintain an optimal melting temperature. Due to their small size, these probes are more specific for single base mismatches and fluorescence quenching is more efficient, resulting in an increased sensitivity (Kutyavin et al, 2000, Salmon, 2002. For these reasons, the covalent conjugation of the MGB moiety to the Taqman probe was preferred over the classical Taqman probe.…”

“…The MGB probe was designed using the software Primer Express 5.1 (PE Applied Biosystems, Foster City, USA) and following criteria of Livak, K., Marmaro, J. and Flood, S. (Perkin-Elmer Research News 57: 1-5) and Kutyavin et al (2000). The MGB probe was supplied by Applied Biosystem with a 5V covalently attached 6-carboxyfluorescine (FAM) reporter dye, a nonfluorescent quencher and MGB moiety at the 3Vend.…”

“…Real-time PCR using the Taqman fluorescent chemistry has already been used to quantify microorganisms in their environment, like strains of Candida species in water samples (Brinkman et al, 2003), Stachybotrys chartarum in water and dust (Haugland et al, 2002) or plant pathogens in soil (Filion et al, 2003b). A more sophisticated Taqman probe, called 3V-Minor Groove Binder-DNA (MGB) probes, can be used to modify the detection specificity (Kutyavin et al, 2000;Marbot et al, 2003;Salmon, 2002).…”

Detection of apple chlorotic leaf spot virus using a 5′ nuclease assay with a fluorescent 3′ minor groove binder-DNA probe

“…The MGB binds the minor groove of double strand DNA and stabilizes the probe/ target duplex through Van der Waals contacts, hydrophobic and electrostatic interactions. Therefore the melting temperature (T m ) of the probe increases dramatically so that a T m in a range of 65-70°C can be reached with a probe of 12-18 nucleotides (Kutyavin et al, 1997(Kutyavin et al, , 2000Walburger et al, 2001). The hybridized probe is then cleaved by the enzyme during strand elongation, resulting in the separation of the fluorescent dye and the quencher, and a subsequent increase in fluorescence.…”