Rapid typing of the codon 129 polymorphism of the human prion protein gene by combined real-time PCR and melting curve analysis.
Clin Lab
; 48(1-2): 25-30, 2002.
Article
en En
| MEDLINE
| ID: mdl-11833672
ABSTRACT
Homozygosity of methionine (m/m) at amino acid residue 129 (codon 129) of the human prion protein (PrP) has been reported for all so far analyzed cases of the new variant Creutzfeldt-Jakob disease (vCJD). This contrasts with its general distribution in the healthy Caucasian population of only about 43%. For this reason a predisposition for carriers of the corresponding genotype to develop vCJD after infection with the bovine spongiform encephalopathy (BSE) agent is assumed, and PCR based methods such as allele-specific oligonucleotide hybridization or restriction analysis and sequencing have been developed for codon 129 genotyping. These methods are cumbersome and time-consuming and the need for extensive post-amplification manipulations increases the risk of carry-over contamination with amplified products. To overcome these shortcomings, the authors developed a real-time PCR assay on the LightCycler (LC) instrument combining PCR and temperature melting curve analysis (Tm) in a closed vessel format. Forty-six swabs and blood samples from healthy donors were tested. Of these 23 (50%) were heterozygous at codon 129, 4 (8.7%) homozygous for valine and 19 (41.3%) homozygous for methionine. Accuracy of LC-genotyping was confirmed by automated sequencing of the amplified products. Taken together, genotyping of the codon 129 polymorphism by combined LC-PCR and melting-curve analysis with the LC-instrument is a reliable and easy to perform method even in a screening context with numerous samples. Results can be obtained within 2 hours, including sample preparation.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Priones
/
Codón
/
Pruebas Genéticas
/
Polimorfismo de Nucleótido Simple
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Clin Lab
Asunto de la revista:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Año:
2002
Tipo del documento:
Article
País de afiliación:
Alemania