ErlI Methods Useful in Genetics and Oncology
While developing a mutation detection assay on the LightCycler using
hybridization probes, we noticed that one fluorescein-labeled probe produced a
fluorescent signal strong enough to allow genotyping without addition of a second probe.
This genotyping probe produced a significant decrease in fluorescein emissions measured in F 1 of the LightCycler upon annealing to complementary strand
DNA. As the probe-template hybrid melted during temperature ramping, the fluorescein residue was released from quenching and the observed fluorescence
emissions increased. Analysis of the fluorescence data throughout the melting
transition produced melting troughs analogous to the melting peaks produced by
hybridization probes upon melting (see Figs. 1-6). The observed fluorescein
quenching effect was experimentally attributed to the interaction of the dye with
complementary, native guanosine residues [10].
Fluorescein quenching by guanosine residues is an attractive technique for
monitoring real-time PCR. Fluorescein quenching probes are inexpensive and
simple to design, synthesize, and purify and are capable of fine sequence detection by melting curve analysis. In order to demonstrate the practicality of fluorescein quenching probes for genotyping, we developed assays to genotype:
1. The hemochromatosis mutation C282Y (G845A)
2. The hemochromatosis mutation H63D (C187G)
3. The cystic fibrosis-associated deletion F508del
4. The thermolabile mutation of methylenetetrahydrofolate reductase (MTHFR;
C677T)
5. The factor V Leiden mutation (G1691A) responsible for heritable activated
protein C resistance
6. The G20210A transition in the 3'-untranslated region of prothrombin
Materials
Equipment LightCycler Instrument (Roche Diagnostics, Mannheim, Germany)
LightCycler Capillary Tubes (Roche Diagnostics)
Reagents
LightCycler-DNA Master Hybridization Probes (Roche Molecular Biochemicals,
Mannheim, Germany)
Amplification primers (Operon Technologies, Alameda, CA, USA)
Fluorescein-labeled probes (Operon Technologies)
Sample Preparation Procedure
Human genomic DNA was isolated from EDTA anticoagulated whole blood by
phenol-chloroform extraction and ethanol precipitation [11]. After precipitation,
the extracted DNA was resuspended in TE', denatured by heating at 95°C for
5 min, and diluted to 1 OD (50 ng/~). Extracted samples were frozen at -20°C or
stored at 4°C until genotyping was performed.
While developing a mutation detection assay on the LightCycler using
hybridization probes, we noticed that one fluorescein-labeled probe produced a
fluorescent signal strong enough to allow genotyping without addition of a second probe.
This genotyping probe produced a significant decrease in fluorescein emissions measured in F 1 of the LightCycler upon annealing to complementary strand
DNA. As the probe-template hybrid melted during temperature ramping, the fluorescein residue was released from quenching and the observed fluorescence
emissions increased. Analysis of the fluorescence data throughout the melting
transition produced melting troughs analogous to the melting peaks produced by
hybridization probes upon melting (see Figs. 1-6). The observed fluorescein
quenching effect was experimentally attributed to the interaction of the dye with
complementary, native guanosine residues [10].
Fluorescein quenching by guanosine residues is an attractive technique for
monitoring real-time PCR. Fluorescein quenching probes are inexpensive and
simple to design, synthesize, and purify and are capable of fine sequence detection by melting curve analysis. In order to demonstrate the practicality of fluorescein quenching probes for genotyping, we developed assays to genotype:
1. The hemochromatosis mutation C282Y (G845A)
2. The hemochromatosis mutation H63D (C187G)
3. The cystic fibrosis-associated deletion F508del
4. The thermolabile mutation of methylenetetrahydrofolate reductase (MTHFR;
C677T)
5. The factor V Leiden mutation (G1691A) responsible for heritable activated
protein C resistance
6. The G20210A transition in the 3'-untranslated region of prothrombin
Materials
Equipment LightCycler Instrument (Roche Diagnostics, Mannheim, Germany)
LightCycler Capillary Tubes (Roche Diagnostics)
Reagents
LightCycler-DNA Master Hybridization Probes (Roche Molecular Biochemicals,
Mannheim, Germany)
Amplification primers (Operon Technologies, Alameda, CA, USA)
Fluorescein-labeled probes (Operon Technologies)
Sample Preparation Procedure
Human genomic DNA was isolated from EDTA anticoagulated whole blood by
phenol-chloroform extraction and ethanol precipitation [11]. After precipitation,
the extracted DNA was resuspended in TE', denatured by heating at 95°C for
5 min, and diluted to 1 OD (50 ng/~). Extracted samples were frozen at -20°C or
stored at 4°C until genotyping was performed.