High capacity and low cost detection of prion protein gene variant alleles by denaturing HPLC
| Autor: | Ricardo P. Moura, Vilma R. Martins, Danielle R. Cunha, Rosa Maria R.P.S. Castro, N. Huang, Michele Christine Landemberger, Otavia L. Caballero, Ricardo Nitrini, Roger Walz, Américo Ceiki Sakamoto, Carlos Gilberto Carlotti, Ricardo R. Brentani |
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| Rok vydání: | 2004 |
| Předmět: |
Genetics
Protein Denaturation Prions animal diseases General Neuroscience Genetic Variation Biology Molecular biology DNA sequencing nervous system diseases PRNP Denaturing high performance liquid chromatography chemistry.chemical_compound Open reading frame chemistry Humans Allele Gene Heteroduplex formation DNA Alleles Chromatography High Pressure Liquid |
| Zdroj: | Journal of neuroscience methods. 139(2) |
| ISSN: | 0165-0270 |
| Popis: | Mutations in the human prion protein gene (PRNP) are responsible for hereditary diseases called transmissible spongiform encephalopathies (TSE) and a polymorphic site at codon 129 determines sensitivity to infectious forms of these maladies. More recently, codon 129 has been related to cognition performance in the elderly, in Alzheimer disease (AD) and in Down syndrome. Furthermore, a rare polymorphism at codon 171 was described in 23% of patients with mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLE-HS), the most common form of surgically remediable epileptic syndrome. Thus, a method that permits fast and efficient screening of PRNP mutations and polymorphisms in patients, in high risk populations, and in family members is desirable. In the present study, we established the conditions for analysis of the PRNP open reading frame using denaturing high-performance liquid chromatography (DHPLC), whereby unpurified PCR products were subjected to denaturing and reannealing steps leading to heteroduplex formation. We described specific profiles for the PRNP polymorphisms at codons 129 (M/V), 117 (A/A silent), 219 (E/K), 171 (N/S), and the octarepeat deletion using amplified DNA from 562 samples. The chromatograms for TSE-associated mutations at codons 102 (P/L), 183 (T/A), and 210 (V/I) were also determined. Specificity of the DHPLC profile for each PRNP variant allele was confirmed in 100% of the samples by direct and cloned DNA sequencing in addition to endonuclease digestion when applicable. Therefore, the present study shows that DHPLC is a rapid, highly accurate and efficient technique for the detection of PRNP genetic variants. |
| Databáze: | OpenAIRE |
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