Automatic synthesis of multifork-like structures for multiple labeling of oligonucleotides: application to diagnosis...

Multifork-like structures have been designed for multiple labelling of oligonucleotides. They rely on the synthesis of new non nucleosidic phosphoramidites which can be used in automatic DNA synthesis. The new compounds allowed tagging of any oligonucleotide and provided reactive primary amino groups for non isotopic labelling. Polybiotinylated oligonucleotides have been tested in hybridization assays using a corresponding complementary sequence covalently linked to polystyrene tubes. Detection limits ranged from 3.10(5) to 3.10(6) molecules, depending on the number of biotins. Multilabelled oligonucleotides have been included into a capture format to detect DNA of Borrelia burgdorferi, the causal agent of Lyme disease. In short, 5'-phosphate oligonucleotides were chemically condensed onto aminated microwells and served to capture PCR-amplified target DNA which, in turn, hybridized with polybiotinylated complementary oligonucleotides. Detection of the hybrids was achieved through streptavidin-conjugated peroxidase and a colorimetric substrate. The solid phase sandwich DNA hybridization assay proved highly specific, very sensitive, rapid and user friendly for the identification of spirochetal DNA in human biological specimens.