An improved DNA sequencing-by-synthesis method

This paper reported an improved sequencing-by-synthesis method. The read length was 19 bases for the sequence with a hairpin structure immobilized onto the surface of aldehyde glass slides. The Cy5-labeled deoxyribonucleoside triphosphate (dNTP) species were incorporated in the extension reactions and the fluorescein label was destroyed by hydrogen peroxide(H2O2) after the readout. The optimal conditions for the fluorescence quenching and the mechanism of reaction were explored. The extension efficiency in the single step was about 80% after quenching of the fluorescence. The improved sequencing-by-synthesis method could sequence more than 80 bases in principle. The images of fluorescence collected from the array during the extension process were reanalyzed with the prepared program of Matlab software.