An Efficient Strategy for Sequencing-by-Synthesis

Currently, the products of polymerase chain reaction (PCR) provide the templates for most of sequencing methods. RCA is a method for amplifying DNA. We report an approach for preparing sequencing templates based on rolling-circle amplification (RCA) to reduce the cost of sequencing when compared to the cost of conducting PCR. We describe a method using modified slides: acrylamide-modified slides, streptavidin-modified slides and aldehyde-modified slides. The products of RCA can be immobilized onto an acryl-modified slide surface. The Cy5-labeled deoxyribonucleoside triphosphate (dNTP) species are incorporated in extension reactions which can be read by determining the level of fluorescence. Then the fluorescence is deactivated using hydrogen peroxide (H₂O₂) as one of the oxidants. With the fluorescence deactivated, further extension and reading along the DNA chain is possible. Using this method, we successfully read a sequence of 27 bases. This strategy shows potential as a next-generation sequencing method.