Locked nucleic acid (LNA) probes in high-throughput genetic analysis: application to an assay for type 1 diabetes-related HLA-DQB1 alleles.

ABSTRACT

OBJECTIVES: In large-scale genetic screening, an assay that is reliable, fast and easy to perform, and straightforwardly adapted to new analytes is a necessity. We describe a one-step assay for analyzing HLA-DQB1 alleles which are associated with susceptibility to type 1 diabetes. DESIGN AND METHODS: The assay is based on asymmetric PCR amplification and a homogeneous hybridization method. The specificity of the probes was improved by substituting LNA (locked nucleic acid) for DNA at the critical bases. RESULTS: The functionality of the LNA containing probes was found to be superior compared to probes consisting of DNA only. The homogeneous assay gave a correct genotyping result in 100% of the cases, which included both extracted DNA samples and blood samples dried on sample collection cards. CONCLUSION: This homogeneous approach provides a simple method to define disease risk associated with HLA alleles for large-scale screening projects.