3′-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures
AUTOR(ES)
Kutyavin, Igor V.
FONTE
Oxford University Press
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=102528Documentos Relacionados
- Detection of a Point Mutation Associated with High-Level Isoniazid Resistance in Mycobacterium tuberculosis by Using Real-Time PCR Technology with 3′-Minor Groove Binder-DNA Probes
- Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate.
- Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder.
- Smallpox and pan-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms
- DNA sequence preferences of several AT-selective minor groove binding ligands.