Genotyping of the Methionine-Valine Polymorphism
at Codon 129 of the Human Prion Protein by Melting Point
Analysis of Fluorescently Labeled Hybridization Probes
SIEGFRIED BURGGRAF"", SIEGFRIED KOSEL, SABINE LOHMANN,
REINHARD BECK, BERNHARD OLGEMOLLER
Introduction
Human prion diseases include Creutzfeldt-Jakob disease (CJD), the GerstmannStraussler-Scheinker syndrome, fatal familial insomnia, and kuru. Since 1996 a
new variant of CJD (vCJD) has been known, which is associated with the bovine
spongiform encephalopathy (BSE). Prion diseases are characterized by the accumulation of abnormal prion protein deposits within the brain. The inherited
forms are caused by mutations in the human prion protein gene on chromosome
20. In addition, a common methionine/valine polymorphism at codon 129 of the
prion protein gene modulates disease susceptibility and phenotypic variability of
human prion diseases [1-4]. Several studies have shown that methionine homozygo city at codon 129 is significantly more frequent in sporadic and iatrogenic CJD
compared with the genotype distribution of the Caucasian population and confers
an increased risk of developing CJD [1,5-7]. Interestingly, all vCJD patients tested
were methionine homozygotes at codon 129 [8,9].
Here we present an assay for fast determination of the human prion protein
genotype at codon position 129 by the use of rapid cycle PCR and melting point
analysis of fluorogenic hybridization probes. During evaluation of the assay we
encountered an artifact that can lead to incorrect results. We describe troubleshooting experiments and asymmetric PCR as a way to obtain reliable genotyping results.
Materials
LightCycler instrument
Equipment
QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany)
Reagents
Amplification Primer (TIB MOLBIOL, Berlin, Germany)
Hybridization Probes (TIB MOLBIOL)
LightCycler-FastStart DNA Master Hybridization Probes (Roche Diagnostics,
Mannheim, Germany)
,. Siegfried Burggraf (~) (e-mail: burggraf@labor-bo.de)
Labor Becker, Olgemoller und Kollegen, Fiihrichstr. 70,81671 MUnchen, Germany
at Codon 129 of the Human Prion Protein by Melting Point
Analysis of Fluorescently Labeled Hybridization Probes
SIEGFRIED BURGGRAF"", SIEGFRIED KOSEL, SABINE LOHMANN,
REINHARD BECK, BERNHARD OLGEMOLLER
Introduction
Human prion diseases include Creutzfeldt-Jakob disease (CJD), the GerstmannStraussler-Scheinker syndrome, fatal familial insomnia, and kuru. Since 1996 a
new variant of CJD (vCJD) has been known, which is associated with the bovine
spongiform encephalopathy (BSE). Prion diseases are characterized by the accumulation of abnormal prion protein deposits within the brain. The inherited
forms are caused by mutations in the human prion protein gene on chromosome
20. In addition, a common methionine/valine polymorphism at codon 129 of the
prion protein gene modulates disease susceptibility and phenotypic variability of
human prion diseases [1-4]. Several studies have shown that methionine homozygo city at codon 129 is significantly more frequent in sporadic and iatrogenic CJD
compared with the genotype distribution of the Caucasian population and confers
an increased risk of developing CJD [1,5-7]. Interestingly, all vCJD patients tested
were methionine homozygotes at codon 129 [8,9].
Here we present an assay for fast determination of the human prion protein
genotype at codon position 129 by the use of rapid cycle PCR and melting point
analysis of fluorogenic hybridization probes. During evaluation of the assay we
encountered an artifact that can lead to incorrect results. We describe troubleshooting experiments and asymmetric PCR as a way to obtain reliable genotyping results.
Materials
LightCycler instrument
Equipment
QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany)
Reagents
Amplification Primer (TIB MOLBIOL, Berlin, Germany)
Hybridization Probes (TIB MOLBIOL)
LightCycler-FastStart DNA Master Hybridization Probes (Roche Diagnostics,
Mannheim, Germany)
,. Siegfried Burggraf (~) (e-mail: burggraf@labor-bo.de)
Labor Becker, Olgemoller und Kollegen, Fiihrichstr. 70,81671 MUnchen, Germany