Solid-phase PCR in a picowell array for immobilizing and arraying 100,000 PCR products to a microscope slide

Abstract

We present a method for performing highly parallel PCR reactions in a picowell array (PWA) simultaneously immobilizing generated PCR products in a covalent and spatially-resolved manner onto a microscope slide via solid-phase PCR (SP-PCR). This so called PWA-SP-PCR was performed in picowell arrays featuring 100,000 wells cm(-2) of 19 pL reaction volumes with a surface-to-volume ratio of 0.2 μm(-1). Positive signals were obtained in 97.2% of the 110,000 wells in an area of 110 mm(2). Immobilized DNA was either indirectly detected using streptavidin-Cy5 or directly by molecular hybridisation of Cy3- and/or Cy5-labelled probes. Amplification and immobilization was demonstrated for template DNA ranging from 100 bp up to 1513 bp lengths. Even single DNA molecules were successfully amplified and immobilized demonstrating digital solid-phase PCR. Compared to widely established emulsion based PCR (emPCR) approaches, leading to PCR products immobilized onto bead surfaces in a highly parallel manner, the novel technique results in direct spatial registration of immobilized PCR products in a microarray format. This enables the subsequent use for massively parallel analysis similar to standard microarrays.