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LNA hybridisation iFRET OFP fluorescent
Abstract:
Oligonucleotide fingerprinting (OFP) is a high-throughput alternative to tag sequencing methods to determine the spectrum and abundance of genes in genomic DNA or cDNA libraries. This method currently relies on the sequential hybridisations of at least 200 short (8-12 mer), radioactively labeled DNA oligonucleotides to arrayed dsDNAs. After image analysis, a sequence fingerprint is generated for each clone based on the hybridisation signals. Then according to the fingerprint similarity, the clones from the same gene can be grouped together using clustering algorithms. The main problems of the classic OFP method include high oligoprobe number and the use of radioactivity. To resolve these problems, here we have exploited the high affinity and good mismatch discrimination of oligoprobes modified with locked nucleic acid (LNA). In our method, short (hexamer or heptamer) LNA-modified oligoprobes are labelled with fluorescent dyes (e.g. Cy5). Hybridisation results are recorded with a CCD-camera developed for this purpose. The sensitivity of the fluorescence detection permits the convenient use of homogenous liquid assay as well as nylon membrane support. Thus hybridisation data quality is improved, and the process accelerated, simplified, and less expensive.
