Isothermal amplification method for next-generation sequencing

Abstract

We report an approach for generating immobilized monoclonal templates for next- generation sequencing applications. Our isothermal amplification method is based on a template walking mechanism using a pair of low-melting temperature (Tm) solid-surface homopolymer primers and a low-Tm solution phase primer. The method can generate more than one billion submicrometer-sized colonies in a single lane of a next-generation sequencing flowchip. An alternative paired-end sequencing method using interstrand DNA photo cross-linking to covalently link the complementary strands of the original templates to the solid surface is also demonstrated.