Multi-allele genotyping platform for the simultaneous detection of mutations in the Wilson disease related ATP7B gene

•Multi-allele genotyping platform for the detection of mutations in the Wilson disease related ATP7B gene.•Few cycles of primer extension reaction is used to discriminate allele specific sequences.•Spatial resolution of alleles on a solid support is achieved by (in flow) hybridization.•Low-cost assa...

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Bibliographic Details
Published in:Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2015-12, Vol.1006, p.201-208
Main Authors: Amvrosiadou, Maria, Petropoulou, Margarita, Poulou, Myrto, Tzetis, Maria, Kanavakis, Emmanuel, Christopoulos, Theodore K., Ioannou, Penelope C.
Format: Article
Language:English
Subjects:
Adenosine Triphosphatases - genetics
Alleles
ATP7B gene mutations
Biosensing Techniques
Cation Transport Proteins - genetics
Copper-transporting ATPases
DNA Mutational Analysis - methods
Genotyping
Genotyping Techniques - methods
Hepatolenticular Degeneration - genetics
Humans
Magnesium
Multi-allele DNA biosensor
Mutation - genetics
Primer extension reaction
Reproducibility of Results
Sensitivity and Specificity
Temperature
Visual detection
Wilson disease
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Summary:•Multi-allele genotyping platform for the detection of mutations in the Wilson disease related ATP7B gene.•Few cycles of primer extension reaction is used to discriminate allele specific sequences.•Spatial resolution of alleles on a solid support is achieved by (in flow) hybridization.•Low-cost assay in a lateral flow ‘dipstick’ format precluding the need for specialized instrumentation. Wilson’s disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel.
ISSN:1570-0232
1873-376X
DOI:10.1016/j.jchromb.2015.10.036