g Applications in Genetics
acid change at position 158. APOE4 alleles differ by the point mutation C3932T,
causing an amino acid change at position 112 [9,10]. The homozygous APOE2
genotype leads to familial dysbetalipoproteinemia with increased plasma levels
of cholesterol and triglycerides [11]. The APOE4 genotype has been associated
with decreased longevity, hypercholesterolemia and an increased prevalence of
Alzheimer's disease [12].
To evaluate patients for the presence of these point mutations, we established a
LightCyder-assisted polymerase chain reaction (PCR), which greatly simplifies
the identification of all mutations in a parallel analysis within 60 min.
Materials
Equipment MagNa Pure LC, version 2.0 (Roche Diagnostics, Mannheim, Germany)
LightCyder instrument (Roche Diagnostics)
LightCyder software, verso 3.5 (Roche Diagnostics)
LightCyder Carousel centrifuge (Roche Diagnostics)
Reagents
MagNa Pure LC DNA Isolation Kit I (Roche Diagnostics)
LightCyder Factor V Leiden Mutation Detection Kit (Roche Diagnostics)
LightCyder Apo B Mutation Detection Kit (codon 3500) (Roche Diagnostics)
LightCyder Apo EMutation Detection Kit (codons 112 and 158) (Roche Diagnostics)
AAT PCR primers and hybridization probes (TIB MOLBIOL, Berlin, Germany)
HFE (H63D and C282Y mutations) PCR primers (MWG-BIOTECH, Ebersberg,
Germany)
HFE (H63D and C282Y mutations) hybridization probes (TIB MOLBIOL)
Procedure
DNA Extraction Genomic DNA was isolated from 200 III peripheral blood anticoagulated with
EDTA on the MagNa Pure LC instrument using the MagNa Pure LC DNA Isolation
Kit I, according to the manufacturer's instructions. The isolated DNA was eluted
in 200 !-t1 elution buffer and a 5-lll aliquot was transferred to PCR.
Primers and For the detection of the HFE H63D mutation, PCR primers amplifying a 169-bp
Hybridization Probes fragment of the HFE gene and two hybridization probes labeled with fluorescein
and LightCyder Red 640 (LCRed640) were constructed as described in Table 1.
The LCRed640-labeled probe hybridizes to the HFE sequence, which harbors the
C187G mutation. For analysis of the HFE C282Y mutation, different PCR primers
and two hybridization probes also labeled with fluorescein and LCRed640 were
used as described in Table 1. The primer pair amplifies a 347-bp fragment of the
HFE gene. The fluorescein-labeled hybridization probe hybridizes to the HFE
sequence, which contains the G845A mutation. To identify the PiZ mutation, we
used a primer pair to amplify a 182-bp fragment of exon V of the AAT gene
(Table l). The two hybridization probes were labeled with fluorescein and
acid change at position 158. APOE4 alleles differ by the point mutation C3932T,
causing an amino acid change at position 112 [9,10]. The homozygous APOE2
genotype leads to familial dysbetalipoproteinemia with increased plasma levels
of cholesterol and triglycerides [11]. The APOE4 genotype has been associated
with decreased longevity, hypercholesterolemia and an increased prevalence of
Alzheimer's disease [12].
To evaluate patients for the presence of these point mutations, we established a
LightCyder-assisted polymerase chain reaction (PCR), which greatly simplifies
the identification of all mutations in a parallel analysis within 60 min.
Materials
Equipment MagNa Pure LC, version 2.0 (Roche Diagnostics, Mannheim, Germany)
LightCyder instrument (Roche Diagnostics)
LightCyder software, verso 3.5 (Roche Diagnostics)
LightCyder Carousel centrifuge (Roche Diagnostics)
Reagents
MagNa Pure LC DNA Isolation Kit I (Roche Diagnostics)
LightCyder Factor V Leiden Mutation Detection Kit (Roche Diagnostics)
LightCyder Apo B Mutation Detection Kit (codon 3500) (Roche Diagnostics)
LightCyder Apo EMutation Detection Kit (codons 112 and 158) (Roche Diagnostics)
AAT PCR primers and hybridization probes (TIB MOLBIOL, Berlin, Germany)
HFE (H63D and C282Y mutations) PCR primers (MWG-BIOTECH, Ebersberg,
Germany)
HFE (H63D and C282Y mutations) hybridization probes (TIB MOLBIOL)
Procedure
DNA Extraction Genomic DNA was isolated from 200 III peripheral blood anticoagulated with
EDTA on the MagNa Pure LC instrument using the MagNa Pure LC DNA Isolation
Kit I, according to the manufacturer's instructions. The isolated DNA was eluted
in 200 !-t1 elution buffer and a 5-lll aliquot was transferred to PCR.
Primers and For the detection of the HFE H63D mutation, PCR primers amplifying a 169-bp
Hybridization Probes fragment of the HFE gene and two hybridization probes labeled with fluorescein
and LightCyder Red 640 (LCRed640) were constructed as described in Table 1.
The LCRed640-labeled probe hybridizes to the HFE sequence, which harbors the
C187G mutation. For analysis of the HFE C282Y mutation, different PCR primers
and two hybridization probes also labeled with fluorescein and LCRed640 were
used as described in Table 1. The primer pair amplifies a 347-bp fragment of the
HFE gene. The fluorescein-labeled hybridization probe hybridizes to the HFE
sequence, which contains the G845A mutation. To identify the PiZ mutation, we
used a primer pair to amplify a 182-bp fragment of exon V of the AAT gene
(Table l). The two hybridization probes were labeled with fluorescein and