In situ localized amplification and contact replication of many individual DNA molecules

We describe a method to clone and amplify DNA by performing the polymerase chain reaction (PCR) in a thin polyacrylamide film poured on a glass microscope slide. The polyacrylamide matrix retards the diffusion of the linear DNA molecules so that the amplification products remain localized near their respective templates. At the end of the reaction, a number of PCR colonies, or 'polonies', have formed, each one grown from a single template molecule. As many as 5 million clones can be amplified in parallel on a single slide. If an Acrydite modification is included at the 5' end of one of the primers, the amplified DNA will be covalently attached to the polyacrylamide matrix, allowing further enzymatic manipulations to be performed on all clones simultaneously. We describe techniques to make replicas of these polony slides, and high throughput sequencing protocols for this technology. Other applications are also discussed.