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3'-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures
Open accessAbstract
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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Metadata
- Authors
- Kutyavin, I. V.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2000-01-15
- Volume
- 28
- Issue
- 2
- Pages
- 655-661
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- OpenAlex
- W2170081920
- Cited by
- 797
- References
- 0
- FWCI
- 7.77
- Type
- journal article
- Language
- en
- Open access
- green
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