111 Methods Useful in Genetics and Oncology
Results
During the melting transition, fluorescein quenching probes produce a detectable
increase in raw fluorescence as the probe melts from the template. This increase
in probe fluorescence is converted into readily interpretable melting curves by the
standard LightCycler Data Analysis software (LCDA). Because LCDA plots the
negative first derivative of fluorescence vs temperature, the increase in raw fluorescence results in negative melting troughs, not positive peaks (see Figs. 1-6).
Fluorescein quenching by guanosine residues was used to genotype the C282Y
hemochromatosis mutation as shown in Fig. 1. Fluorescence data collected in Fl
of the LightCcycler during the melting protocol are plotted in Fig. la as fluorescence vs temperature. The plot shows an increase in fluorescence corresponding
to the probe melting transition. A plot of the negative derivative of fluorescence
vs temperature (-d{Fl}/dt) for the melting curve is shown in Fig. lb. The resulting melting troughs were readily interpretable for genotyping. Homozygous samples that were complementary to the probe (wild type) melted in a single transition at a relatively high temperature. Homozygotes that were mismatched to the
probe sequence (homozygous mutant) melted in a single transition at a relatively low temperature. Heterozygotes (heterozygous) melted in two transitions.
Quenching was provided by two consecutive complementary guano sines with
one positioned as the last hybridizing base and the other as the first lateral base.
Other assays that use fluorescein quenching for genotyping are shown in
Figs. 2-6. Melting troughs for genotyping the hemochromatosis mutation H63D
are shown in Fig. 2. The fluorescein-labeled probe used to genotype the H63D
locus was designed to be perfectly complementary to the mutant sequence
(C187G) and made use of quenching by two complementary guano sines positioned as the first lateral base and the last hybridizing base.
Figure 3 shows melting troughs for genotyping the cystic fibrosis-associated
mutation F508del. The three base pair deletions resulted in a mismatched 3-bp
loop in the probe that destabilized the probe-template hybrid by approximately
lOoC. Quenching was provided by a single complementary guanosine positioned
as the first lateral base to the region of hybridization. While genotyping samples
for the F508del mutation, two other variant alleles were identified whose mutations resulted in melting troughs distinguishable from F508del. One variation was
the F508C mutation caused by a T:G transversion within codon 508. This mutation resulted in an A:G mismatch that destabilized the probe-template hybrid by
approximately 3°C. The second allele identified was a pathologic I507del variant.
The deletion of codon 507 resulted in a 9°C shift from wild type and was differentiable from the F508del mutation by a 1°C difference in T m' Both variant alleles
were identified in heterozygotes.
Melting troughs from the genotyping assay for the thermolabile mutation of
MTHFR (C677T) are shown in Fig. 4. The C:T transversion resulted in a G:T
mismatch in the probe-template hybrid. The G:T mismatch is one of the most
thermostable known and resulted in a Tm shift of only 3.9°C. A single complementary guanosine positioned as the first lateral base provided quenching for
genotyping.
Results
During the melting transition, fluorescein quenching probes produce a detectable
increase in raw fluorescence as the probe melts from the template. This increase
in probe fluorescence is converted into readily interpretable melting curves by the
standard LightCycler Data Analysis software (LCDA). Because LCDA plots the
negative first derivative of fluorescence vs temperature, the increase in raw fluorescence results in negative melting troughs, not positive peaks (see Figs. 1-6).
Fluorescein quenching by guanosine residues was used to genotype the C282Y
hemochromatosis mutation as shown in Fig. 1. Fluorescence data collected in Fl
of the LightCcycler during the melting protocol are plotted in Fig. la as fluorescence vs temperature. The plot shows an increase in fluorescence corresponding
to the probe melting transition. A plot of the negative derivative of fluorescence
vs temperature (-d{Fl}/dt) for the melting curve is shown in Fig. lb. The resulting melting troughs were readily interpretable for genotyping. Homozygous samples that were complementary to the probe (wild type) melted in a single transition at a relatively high temperature. Homozygotes that were mismatched to the
probe sequence (homozygous mutant) melted in a single transition at a relatively low temperature. Heterozygotes (heterozygous) melted in two transitions.
Quenching was provided by two consecutive complementary guano sines with
one positioned as the last hybridizing base and the other as the first lateral base.
Other assays that use fluorescein quenching for genotyping are shown in
Figs. 2-6. Melting troughs for genotyping the hemochromatosis mutation H63D
are shown in Fig. 2. The fluorescein-labeled probe used to genotype the H63D
locus was designed to be perfectly complementary to the mutant sequence
(C187G) and made use of quenching by two complementary guano sines positioned as the first lateral base and the last hybridizing base.
Figure 3 shows melting troughs for genotyping the cystic fibrosis-associated
mutation F508del. The three base pair deletions resulted in a mismatched 3-bp
loop in the probe that destabilized the probe-template hybrid by approximately
lOoC. Quenching was provided by a single complementary guanosine positioned
as the first lateral base to the region of hybridization. While genotyping samples
for the F508del mutation, two other variant alleles were identified whose mutations resulted in melting troughs distinguishable from F508del. One variation was
the F508C mutation caused by a T:G transversion within codon 508. This mutation resulted in an A:G mismatch that destabilized the probe-template hybrid by
approximately 3°C. The second allele identified was a pathologic I507del variant.
The deletion of codon 507 resulted in a 9°C shift from wild type and was differentiable from the F508del mutation by a 1°C difference in T m' Both variant alleles
were identified in heterozygotes.
Melting troughs from the genotyping assay for the thermolabile mutation of
MTHFR (C677T) are shown in Fig. 4. The C:T transversion resulted in a G:T
mismatch in the probe-template hybrid. The G:T mismatch is one of the most
thermostable known and resulted in a Tm shift of only 3.9°C. A single complementary guanosine positioned as the first lateral base provided quenching for
genotyping.