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Nucleic Acids Res. 2000 Jan 15;28(2):655-61.

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

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  • 1Epoch Pharmaceuticals, Inc., 12277 134th Court NE #110, Redmond, WA 98052, USA.

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.

PMID:
10606668
[PubMed - indexed for MEDLINE]
PMCID:
PMC102528
Free PMC Article
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