DNA sequencing in high-throughput

The completion of the Human Genome Project has set the stage for screening genetic mutations to identify disease genes on a genome-wide scale. To overcome the limitations of the current sequencing technology based on electrophoresis and to develop more rapid and accurate DNA sequencing in high-throughput manner, a variety of new methods have been explored. We wish to develop a state-of-the-art sequencing technology based on step-by-step nucleotide extension. In this method, unknown single DNA strands bearing complementary sequence against primer are hybridised onto the solid (chip)-immobilised primer and then chain extension by DNA polymerase is performed with four specially modified nucleotide analogues (A, T, C, G). Each nucleotide analogue has a fluorescent label with a unique colour and the fluorescent label is connected through the peptide substrate. At this stage, only one matched nucleotide analogue out of four nucleotide analogues is incorporated to the growing DNA strand and chain extension is temporarily terminated due to huge moiety of the unnatural nucleotide analogue. Therefore, unknown sequence next to the primer can be identified by the colour of fluorophore. Afterward, fluorescent reporter is cleaved by protease and second polymerase reaction is following. As the same procedure is repeated, unknown sequences can be monitored step-by-step; chain extension by polymerase and reporter release by protease (Figure1).

Figure1. Schematic view for DNA sequencing by chain extension of polymerase and tag release of protease