Fractionation, phosphorylation and ligation on oligonucleotide microchips to enhance sequencing by hybridization

Abstract

Oligonucleotide microchips are manufactured by immobilizing presynthesized oligonucleotides within 0.1 x 0.1 x 0.02 mm or 1 x 1 x 0.02 mm polyacrylamide gel pads arranged on the surface of a microscope slide. The gel pads are separated from each other by hydrophobic glass spacers and serve as a kind of 'microtest tube' of 200 pl or 20 nl volume, respectively. Fractionation of single-stranded DNAs is carried out by their hybridization with chip pads containing immobilized 10mers. DNA extracted separately from each pad is transferred onto a sequencing chip and analyzed thereon. The chip, containing a set of 10mers, was enzymatically phosphorylated, then hybridized with DNA and ligated in a site-directed manner with a contiguously stacked 5mer. Several cycles of successive hybridization-ligation of the chip-bound 10mers with different contiguously stacked 5mers and hybridized with DNA were carried out to sequence DNA containing tetranucleotide repeats. Combined use of these techniques show significant promise for sequence comparison of homologous regions in different genomes and for Sequence analysis of comparatively long DNA fragments or DNA containing internal repeats.