DNA probes with conjugated minor groove binder (MGB) groups form extremely
stable duplexes with single-stranded DNA targets, allowing shorter probes t
o be used for hybridization based assays. In this paper, sequence specifici
ty of 3'-MGB probes was explored. In comparison with unmodified DNA, MGB pr
obes had higher melting temperature (T-m) and increased specificity, especi
ally 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 (realtime PCR assay, TaqMan assay). A 12mer MGB probe h
ad 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 quen
ching was more efficient, giving increased sensitivity. ATT rich duplexes w
ere 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 hyb
ridization temperatures.