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A homogeneous high-throughput genotyping method based on competitive hybridization.

Minna Kiviniemi·Jussi Nurmi·Hannu Turpeinen·Timo Lövgren·Jorma Ilonen
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Abstract

OBJECTIVES: A reliable high-throughput assay system is necessary for the analysis of the ever-increasing numbers of single-nucleotide polymorphisms (SNP) relevant to genetic screening studies. We describe an assay suitable also for large-scale screening programs. DESIGN AND METHODS: The one-step assay is based on asymmetric PCR amplification of the target sequence and subsequent time-resolved fluorescence measurement. Asymmetric amplification results in a single-stranded PCR product that is detected in the amplification vessel with a highly sensitive, homogeneous hybridization method. RESULTS: A dual label, homogeneous high-throughput platform for nucleic acid sequence analysis was developed and validated using a C/T single-nucleotide polymorphism in the insulin gene as a model analyte and applied also to two other SNP-assays (poliovirus receptor A/G-polymorphism and CD86-gene exon 2 A/G-polymorphism). CONCLUSIONS: The described high-throughput genotyping technology is very competitive in price, simple in design and easily applied to any analyte sequence.

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Genetic TestingGenotypeHumansInsulinNucleic Acid HybridizationPolymerase Chain ReactionPolymorphism, Single NucleotideJournal Article

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Minna Kiviniemi, Jussi Nurmi, Hannu Turpeinen, Timo Lövgren, Jorma Ilonen. 2003. A homogeneous high-throughput genotyping method based on competitive hybridization.. https://doi.org/10.1016/s0009-9120(03)00106-1

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Exome sequencing and large-scale analysis of electronic medical record-linked biobank data identify candidate deafness genes.

INTRODUCTION: Rapid advances in whole-exome sequencing (WES) have enabled large-scale detection of pathogenic variants. Although hundreds of genes are implicated in hearing loss, up to half of inherited cases remain unsolved, limiting eligibility for gene therapy trials that require genetic diagnosis. Biobanks and electronic medical records (EMRs) offer opportunities to integrate genomic and clinical data at scale and expand the spectrum of hearing loss genes. Despite clinical value, EMRs often lack key information such as inheritance patterns, posing challenges for accurate interpretation. METHODS: WES was performed on DNA samples from 1038 hearing-impaired patients enrolled in the Maccabi Research and Innovation Center Tipa Biobank. Clinical data were extracted from EMRs. Audiograms were available for all cases, although data on age of onset, family history and mode of inheritance were mostly unavailable. We applied a scalable bioinformatics analysis strategy for high-throughput annotation, filtering and prioritisation of WES variants across more than 1000 patients, designed to accommodate incomplete and heterogeneous clinical records. RESULTS: Using this approach, 15% of cases were solved or potentially solved through known or novel variants in established deafness genes. Homozygous variants in novel candidate genes were identified in 3% of cases. Functional characterisation was performed for promising candidate genes to validate their role in the ear. CONCLUSION: These findings demonstrate that WES can determine disease aetiology in large, genetically heterogeneous populations, even in the context of incomplete clinical data. This approach supports large-scale genetic screening and provides a framework for identifying patients who may benefit from emerging gene-based therapies.

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