Genotyping by Guanosine-Dependent Quenching of Single-Labeled Fluorescein Probes 11'!.1
Results for the factor V Leiden genotyping assay are shown in Fig. 5. Quenching was provided by two consecutive guanosine residues positioned as the first
lateral base and the last hybridizing base. Figure 6 shows melting troughs for the
prothrombin G20210A genotyping assay. The probe was designed to be a perfect
match for the mutant allele. A single complementary guanosine positioned as the
first lateral base provided quenching.
Comments
Fluorescein quenching by complementary guanosine residues provides a robust
means of genotyping PCR product by melting curve analysis. The key to obtaining an adequate signal for genotyping is the design of the fluorescein probe. The
fluorescein label must be positioned such that it can readily interact with
guano sines on the complementary strand. Empirical data from synthetic probe
and template pairs suggest that the optimal position for a single complementary
guanosine is as the first base lateral to the region of hybridization. In general, single guanosines positioned as the last hybridizing base or the second, third, or
fourth lateral bases also contribute to quenching; however, their effect is weaker.
Complementary guano sines internal to the last hybridizing base in the region of
hybridization do not exhibit quenching [10].
When two or more guanosine residues are available for quenching, the interaction between the fluorophore and the complementary guanosines is greater
and the observed signal quenching is stronger. For targets with two consecutive
guanosines near the fluorescein label, the greatest quenching (approximately
32%) is demonstrated when the probe is designed to place the two residues as
the first two lateral bases outside the region of hybridization. An alternate probe
design for targets with two consecutive guanosines places the two residues as the
first lateral base and the last hybridizing base with only a slight loss of quenching from the aforementioned design (approximately 29% quenching). This alternate design was used in genotyping assays for the factor V Leiden and
hemochromatosis H63D and C282Y mutations described in this chapter. Additional complementary guanosine residues can increase the observed quenching
to about 40% (targets with four consecutive lateral guanosines); however, this
increase is not necessary for robust detection on sensitive instruments such as
the LightCycler [10].
Guanosine residues at the end of the probe near the fluorescein label can
contribute to permanent quenching of the fluorescent dye and should be avoided [15, 16].
Gain adjustments are important to collecting optimal fluorescence data for
melting curve analysis. In most systems using fluorescein quenching probes, peak
fluorescence is observed during the first few cycles of PCR. This is contrary to systems using hybridization probes in which fluorescence values increase during
amplification. Because maximum fluorescence is seen early in PCR and decreases during cycling, gain adjustments may be necessary to keep fluorescence values
from reaching low levels during amplification.
Results for the factor V Leiden genotyping assay are shown in Fig. 5. Quenching was provided by two consecutive guanosine residues positioned as the first
lateral base and the last hybridizing base. Figure 6 shows melting troughs for the
prothrombin G20210A genotyping assay. The probe was designed to be a perfect
match for the mutant allele. A single complementary guanosine positioned as the
first lateral base provided quenching.
Comments
Fluorescein quenching by complementary guanosine residues provides a robust
means of genotyping PCR product by melting curve analysis. The key to obtaining an adequate signal for genotyping is the design of the fluorescein probe. The
fluorescein label must be positioned such that it can readily interact with
guano sines on the complementary strand. Empirical data from synthetic probe
and template pairs suggest that the optimal position for a single complementary
guanosine is as the first base lateral to the region of hybridization. In general, single guanosines positioned as the last hybridizing base or the second, third, or
fourth lateral bases also contribute to quenching; however, their effect is weaker.
Complementary guano sines internal to the last hybridizing base in the region of
hybridization do not exhibit quenching [10].
When two or more guanosine residues are available for quenching, the interaction between the fluorophore and the complementary guanosines is greater
and the observed signal quenching is stronger. For targets with two consecutive
guanosines near the fluorescein label, the greatest quenching (approximately
32%) is demonstrated when the probe is designed to place the two residues as
the first two lateral bases outside the region of hybridization. An alternate probe
design for targets with two consecutive guanosines places the two residues as the
first lateral base and the last hybridizing base with only a slight loss of quenching from the aforementioned design (approximately 29% quenching). This alternate design was used in genotyping assays for the factor V Leiden and
hemochromatosis H63D and C282Y mutations described in this chapter. Additional complementary guanosine residues can increase the observed quenching
to about 40% (targets with four consecutive lateral guanosines); however, this
increase is not necessary for robust detection on sensitive instruments such as
the LightCycler [10].
Guanosine residues at the end of the probe near the fluorescein label can
contribute to permanent quenching of the fluorescent dye and should be avoided [15, 16].
Gain adjustments are important to collecting optimal fluorescence data for
melting curve analysis. In most systems using fluorescein quenching probes, peak
fluorescence is observed during the first few cycles of PCR. This is contrary to systems using hybridization probes in which fluorescence values increase during
amplification. Because maximum fluorescence is seen early in PCR and decreases during cycling, gain adjustments may be necessary to keep fluorescence values
from reaching low levels during amplification.