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 micro- scope 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 poly- acrylamide matrix, allowing further enzymatic manip- ulations to be performed on all clones simul- taneously. We describe techniques to make replicas of these polony slides, and high throughput sequencing protocols for this technology. Other applications are also discussed.