3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.
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.
Kutyavin
I V
IV
Epoch Pharmaceuticals, Inc., 12277 134th Court NE #110, Redmond, WA 98052, USA.
Afonina
I A
IA
Mills
A
A
Gorn
V V
VV
Lukhtanov
E A
EA
Belousov
E S
ES
Singer
M J
MJ
Walburger
D K
DK
Lokhov
S G
SG
Gall
A A
AA
Dempcy
R
R
Reed
M W
MW
Meyer
R B
RB
Hedgpeth
J
J
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
D017931:DNA Primers; D015342:DNA Probes; D009838:Oligodeoxyribonucleotides