Genotyping of the Methionine-Valine Polymorphjsm at Codon 129 of the Human Prion Protein IIBJ
• Melting Curve Analysis
Parameter
Value
Cycles
Type
Target temperature [0C]
Incubation tjrne [s]
Temperature transition rate [OCls]
Acquisition mode
Results
Melting curve
Segment 1
95
10
20
None
Segment 2
Segment 3
45
75
60
0
20
0.1
None
ConI.
Using a LightCycler high-speed PCR protocol for amplification of different DNA
samples with equimolar concentrations of primer set 1 and probe set 1, we detected
a strong fluorescence signal at the end of the PCR amplification. Agarose gel electrophoresis of the products showed unique bands with the expected size of 185 bp
(data not shown). However, the results of the melting point analysis were unexpected. Both methionine/valine heterozygous samples and a valine homozygous sample
lacked a valine peak, which was supposed to occur at approximately 65°C (i.e., the
calculated melting temperature for the detection probe without mismatch).
To evaluate the quality of the hybridization probes, we used single-stranded
oligonucleotides complementary to probe set 1 in a melting point experiment
without prior PCR amplification. Both so-called complements, one corresponding
to the valine genotype (no mismatch) and the other corresponding to the methionine genotype (one mismatch), showed the calculated melting peaks of approximately 65°C and 58°C, respectively (Fig. 2).
Since the probes had now been shown to work well on a single-stranded template,
an asymmetric PCR system was devised to increase the amount of single-stranded PCR product available as target for the probes. In this system, the target DNA
was amplified in the presence of a fixed amount of forward primer and various
dilutions of reverse primer. A heterozygous (methionine/valine) sample could be
successfully amplified with all concentrations of the reverse primer, from 5 pmol
(equal to forward primer) to only 0.5 pmol (Fig. 3a, c).
Melting point analysis of the PCR reaction containing equal amounts of
primers revealed only one peak around 58°C (Fig. 3b, reaction 2). The correct
result for the heterozygous sample, with the two expected peaks (approximately at
58°C and 68°C), was obtained only with the lower concentration of the reverse
primer (Fig. 3b, reactions 3-5).
Similar PCR experiments were performed with the methionine homozygous
(Fig. 4) and the valine homozygous sample (Fig. 5). Melting point analysis revealed
the expected peak around 58°C in all reactions with the methionine homozygous
Use of Artifical
Complements for
Evaluation of Probe
Quality
Asymmetric PCR
to Produce SingleStranded Template
for Melting Point
Analysis
• Melting Curve Analysis
Parameter
Value
Cycles
Type
Target temperature [0C]
Incubation tjrne [s]
Temperature transition rate [OCls]
Acquisition mode
Results
Melting curve
Segment 1
95
10
20
None
Segment 2
Segment 3
45
75
60
0
20
0.1
None
ConI.
Using a LightCycler high-speed PCR protocol for amplification of different DNA
samples with equimolar concentrations of primer set 1 and probe set 1, we detected
a strong fluorescence signal at the end of the PCR amplification. Agarose gel electrophoresis of the products showed unique bands with the expected size of 185 bp
(data not shown). However, the results of the melting point analysis were unexpected. Both methionine/valine heterozygous samples and a valine homozygous sample
lacked a valine peak, which was supposed to occur at approximately 65°C (i.e., the
calculated melting temperature for the detection probe without mismatch).
To evaluate the quality of the hybridization probes, we used single-stranded
oligonucleotides complementary to probe set 1 in a melting point experiment
without prior PCR amplification. Both so-called complements, one corresponding
to the valine genotype (no mismatch) and the other corresponding to the methionine genotype (one mismatch), showed the calculated melting peaks of approximately 65°C and 58°C, respectively (Fig. 2).
Since the probes had now been shown to work well on a single-stranded template,
an asymmetric PCR system was devised to increase the amount of single-stranded PCR product available as target for the probes. In this system, the target DNA
was amplified in the presence of a fixed amount of forward primer and various
dilutions of reverse primer. A heterozygous (methionine/valine) sample could be
successfully amplified with all concentrations of the reverse primer, from 5 pmol
(equal to forward primer) to only 0.5 pmol (Fig. 3a, c).
Melting point analysis of the PCR reaction containing equal amounts of
primers revealed only one peak around 58°C (Fig. 3b, reaction 2). The correct
result for the heterozygous sample, with the two expected peaks (approximately at
58°C and 68°C), was obtained only with the lower concentration of the reverse
primer (Fig. 3b, reactions 3-5).
Similar PCR experiments were performed with the methionine homozygous
(Fig. 4) and the valine homozygous sample (Fig. 5). Melting point analysis revealed
the expected peak around 58°C in all reactions with the methionine homozygous
Use of Artifical
Complements for
Evaluation of Probe
Quality
Asymmetric PCR
to Produce SingleStranded Template
for Melting Point
Analysis