An Efficient Strategy for Sequencing-by-Synthesis

Abstract

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.

2-Color Encoding Solid Phase Minisequencing

“…15 After certain improvement, solid phase minisequencing method is also a useful sequencing-by-synthesis approach. 16 The two common strategies adopted in solid phase minisequencing are single-fluorescence method 4 and multi-fluorescence method. 12 To differentiate four varieties of nucleotides, four divided minisequencing experiments are needed by single-fluorescence method and four types of fluorescence molecules are essential by multifluorescence method.…”