title: 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.
authors.affiliation: Epoch Pharmaceuticals, Inc., 12277 134th Court NE #110, Redmond, WA 98052, USA.
authors.forename: I V
authors.initials: IV
authors.lastname: Kutyavin
authors.forename: I A
authors.initials: IA
authors.lastname: Afonina
authors.forename: A
authors.initials: A
authors.lastname: Mills
authors.forename: V V
authors.initials: VV
authors.lastname: Gorn
authors.forename: E A
authors.initials: EA
authors.lastname: Lukhtanov
authors.forename: E S
authors.initials: ES
authors.lastname: Belousov
authors.forename: M J
authors.initials: MJ
authors.lastname: Singer
authors.forename: D K
authors.initials: DK
authors.lastname: Walburger
authors.forename: S G
authors.initials: SG
authors.lastname: Lokhov
authors.forename: A A
authors.initials: AA
authors.lastname: Gall
authors.forename: R
authors.initials: R
authors.lastname: Dempcy
authors.forename: M W
authors.initials: MW
authors.lastname: Reed
authors.forename: R B
authors.initials: RB
authors.lastname: Meyer
authors.forename: J
authors.initials: J
authors.lastname: Hedgpeth
mesh_terms: D020137:Base Pair Mismatch; D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes / Q000378:metabolism*; D006358:Hot Temperature; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D009838:Oligodeoxyribonucleotides