m ApplicatioD~ in GeDetic~
Sample Preparation
Primer and Probe
Design
Artificial
Complements for
Evaluation of Probe
Quality
Restriction
Fragment Length
Polymorphism
Analysis
Procedure
Genomic DNA was extracted from either 200-fJl samples of blood containing anticoagulant (EDTA blood) or cotton swab samples of mouth mucosa with the
QIAamp DNA Blood Mini Kit, according to the manufacturer's instructions. Cotton
swabs were eluted in 1 ml 0.1 M Tris/HCl, pH 8.3; 1% Tween 20. We used 200 fJl of
the eluant for DNA extraction. DNA was eluted from the columns with 50 fJl H20.
The concentration of the DNA was not determined. For PCR, DNA from EDTA
blood was diluted 1:5 with H 2 0; swab DNA was used without further dilution.
Primers and probes were designed using a beta version of the LightCycler probe
design software (version 0.99.11; Roche Molecular Biochemicals, Mannheim, Germany). This novel software tool screens the target sequence for suitable primers
and probes and calculates their melting temperatures (T m). For the probe complementary to the region with the variable nucleotide, the software provides the T m
for match and mismatch. Two different pairs of primers and hybridization probes
were designed. The sequences of all oligonucleotides and their binding sites on the
human prion protein gene (GenBank accession #HSU29185) are given in Table 1.
Figure 1 shows the binding sites of the two different sets of hybridization probes.
Both probe systems bind to the target with a gap of two bases between anchor and
detection probes. All oligonucleotides were purchased from TIB MOLBIOL.
Two different oligonucleotides (complement-methionine and complement-valine;
Table 1) were designed to be complementary to hybridization probe system 1 (anchor
1 and detection probe 1; Table 1; Fig. 1). Each complement contained seven additional bases at the 5' -end and six additional bases at the 3' -end of the probe binding
region. The complements were synthesized and HPLC-purified by TIB MOLBIOL.
The genotype of different samples was verified by restriction fragment length
polymorphism (RFLP) analysis of products from a conventional PCR. It was not
possible to analyze PCR products from the LightCycler reaction, since the LightCycler reaction mix contains dU instead of dT and dU is not recognized by the
restriction enzyme used for RFLP.
DNA for RFLP analysis was amplified by conventional PCR in a Perkin Elmer
model PE9600 thermo cycler. The 50-fll PCR reaction mix contained 20 pmol each
of forward primer and reverse primer (primer set 1), 0.2 mM of each dNTP, 3 mM
MgC1 2 ,5 fll PCR buffer (lOx, Qiagen), 0.25 fJl HotStarTaq DNA polymerase (Qiagen) and 5 fll genomic DNA. The PCR thermocycling profile included 15 min at
95°C, followed by 35 cycles of 94°C for 30 s, 55°C for 30 sand noc for 30 s. We
added 1 fJl of restriction buffer M and 0.5 fJl of restriction endonuclease NspI
(both Roche Molecular Biochemicals) to 10 fll PCR product and incubated at 37°C
for 1 h. Fragments were separated on 3% agarose gels and visualized under UV
light after staining with ethidium bromide. The following fragment pattern was
obtained for the different genotypes: 169 bp and 16 bp for valine homozygous
samples; 94 bp, 75 bp and 16 bp for methionine homozygous samples; and 169 bp,
94 bp, 75 bp and 24 bp for methionine/valine heterozygous samples.
Sample Preparation
Primer and Probe
Design
Artificial
Complements for
Evaluation of Probe
Quality
Restriction
Fragment Length
Polymorphism
Analysis
Procedure
Genomic DNA was extracted from either 200-fJl samples of blood containing anticoagulant (EDTA blood) or cotton swab samples of mouth mucosa with the
QIAamp DNA Blood Mini Kit, according to the manufacturer's instructions. Cotton
swabs were eluted in 1 ml 0.1 M Tris/HCl, pH 8.3; 1% Tween 20. We used 200 fJl of
the eluant for DNA extraction. DNA was eluted from the columns with 50 fJl H20.
The concentration of the DNA was not determined. For PCR, DNA from EDTA
blood was diluted 1:5 with H 2 0; swab DNA was used without further dilution.
Primers and probes were designed using a beta version of the LightCycler probe
design software (version 0.99.11; Roche Molecular Biochemicals, Mannheim, Germany). This novel software tool screens the target sequence for suitable primers
and probes and calculates their melting temperatures (T m). For the probe complementary to the region with the variable nucleotide, the software provides the T m
for match and mismatch. Two different pairs of primers and hybridization probes
were designed. The sequences of all oligonucleotides and their binding sites on the
human prion protein gene (GenBank accession #HSU29185) are given in Table 1.
Figure 1 shows the binding sites of the two different sets of hybridization probes.
Both probe systems bind to the target with a gap of two bases between anchor and
detection probes. All oligonucleotides were purchased from TIB MOLBIOL.
Two different oligonucleotides (complement-methionine and complement-valine;
Table 1) were designed to be complementary to hybridization probe system 1 (anchor
1 and detection probe 1; Table 1; Fig. 1). Each complement contained seven additional bases at the 5' -end and six additional bases at the 3' -end of the probe binding
region. The complements were synthesized and HPLC-purified by TIB MOLBIOL.
The genotype of different samples was verified by restriction fragment length
polymorphism (RFLP) analysis of products from a conventional PCR. It was not
possible to analyze PCR products from the LightCycler reaction, since the LightCycler reaction mix contains dU instead of dT and dU is not recognized by the
restriction enzyme used for RFLP.
DNA for RFLP analysis was amplified by conventional PCR in a Perkin Elmer
model PE9600 thermo cycler. The 50-fll PCR reaction mix contained 20 pmol each
of forward primer and reverse primer (primer set 1), 0.2 mM of each dNTP, 3 mM
MgC1 2 ,5 fll PCR buffer (lOx, Qiagen), 0.25 fJl HotStarTaq DNA polymerase (Qiagen) and 5 fll genomic DNA. The PCR thermocycling profile included 15 min at
95°C, followed by 35 cycles of 94°C for 30 s, 55°C for 30 sand noc for 30 s. We
added 1 fJl of restriction buffer M and 0.5 fJl of restriction endonuclease NspI
(both Roche Molecular Biochemicals) to 10 fll PCR product and incubated at 37°C
for 1 h. Fragments were separated on 3% agarose gels and visualized under UV
light after staining with ethidium bromide. The following fragment pattern was
obtained for the different genotypes: 169 bp and 16 bp for valine homozygous
samples; 94 bp, 75 bp and 16 bp for methionine homozygous samples; and 169 bp,
94 bp, 75 bp and 24 bp for methionine/valine heterozygous samples.