Genotyping by Guanosine-Dependent Quenching of Single-Labeled Fluorescein Probes . ,
0.5
-,,- Wild Type
-
Heterozygous
- - - Homozygous Mutant
-0.5
" ."". No DNA
40
50
60
70
Temperature (O C)
Fig. 4. Genotyping curves for the thermolabile mutation of MTHFR (C677T). The C:T transversion resulted in a G:T mismatch in the probe-template duplex. The G:T mismatch is one of the
most stable known and destabilized the probe by only 3.9°C. Nevertheless, genotypes were clearly distinguishable. Quenching was provided by a single complementary guanosine
0.5
o
-0.5
40
(
, ,
\
, , , , , , , , , , , ,
- " - Wild Type
-
Heterozygous
, I
- - - Homozygous Mutant
"
"""" No DNA
50
60
70
Temperature (O C)
Fig. 5. Homogeneous genotyping of the factor V Leiden mutation by a single fluorescein probe.
The fluorescein probe was designed to allow quenching by two complementary guanosine
residues