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An Efficient Strategy for Sequencing-by-Synthesis

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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.

Keywords: HYDROGEN PEROXIDE (H2O2); ROLLING-CIRCLE AMPLIFICATION (RCA); SEQUENCING-BY-SYNTHESIS (SBS)

Document Type: Research Article

DOI: http://dx.doi.org/10.1166/jnn.2010.2183

Publication date: May 1, 2010

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  • Journal for Nanoscience and Nanotechnology (JNN) is an international and multidisciplinary peer-reviewed journal with a wide-ranging coverage, consolidating research activities in all areas of nanoscience and nanotechnology into a single and unique reference source. JNN is the first cross-disciplinary journal to publish original full research articles, rapid communications of important new scientific and technological findings, timely state-of-the-art reviews with author's photo and short biography, and current research news encompassing the fundamental and applied research in all disciplines of science, engineering and medicine.
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