A New Strategy for Next Generation Sequencing: Merging the Sanger's Method and the Sequencing by Synthesis Through Replacing Extension

摘要

The present paper describes a feasible protocol enabling a merge of the classical Sanger sequencing method with the sequencing by DNA synthesis: The cycled proofreading sequencing. This new protocol involves partial chain termination and replacing extension. The first step is done with mixed ddNTP/dNTP targeting one type of nucleotide per subcycle and its relevant replacing extension with unlabeled phosphorothioate-modified dNTP of the same nucleotide type used in partial chain termination. Replacing extension serves as the key step to eliminate fluorescent signals integrated at the previous step, proofread errors, reactivate and synchronize the 3' termini of extended DNA molecules. This microchip-based sequencing method thus inherits advantages from "sequencing-by-termination" and "sequencing-by-extension"; ddNTP is used and reactions are cycled. This microchip-based sequencing method is applicable for reaching the