Considerations of Solid-Phase DNA Amplification

Abstract

Solid-phase (SP) polymerase chain reaction (PCR) is an increasingly popular tool used to produce immobilized DNA for a variety of applications, including high-throughput DNA sequencing and SNP analysis. Despite its usefulness, the mechanism of DNA amplification using immobilized primers has not been thoroughly explored. Herein, we describe a SP-PCR process that was designed to explore and better understand some limitations of SP-DNA amplification. The rate of SP-DNA amplification was measured, and the ability to exponentially amplify DNA on a surface was demonstrated. Approximately 50 amol of DNA was amplified to detectable levels using SP-PCR. The mechanism and some limitations of the reaction were investigated by measuring the density of the primer on the surface prior to amplification and the amount of immobilized amplicon produced after SP-PCR. This enabled some of the practical limitations of the reaction to be addressed within a logical theoretical framework.