Introduction to Polonies (go back)
Polonies (paw-lo-nees) are colonies of PCR amplicons derived from a single molecule of nucleic acid. Thousands to millions of spatially distinct polonies, each with an effective reaction size on the order of nanoliters to femtoliters, can be amplified simultaneously within an acrylamide gel. Enzymatic reactions on polonies (e.g. in situ hybridization; mini-sequencing) can be subsequently performed in parallel. A concise schematic description of how polony amplification and sequencing are performed, can be found here. Applications for the polony technology include genotyping, haplotyping, quantification of the transcriptome of tissues or single cells, quantification of exon combinatorics on alternatively spliced loci, in situ sequencing and genomic sequencing. Some of these technologies are past the proof-of-concept phase whereas others are still in development. Links to publications utilizing polonies can be found on the Polony Technology Menu.
The purpose of this manual is to concisely provide all of the necessary information to get the polony protocol up and running for individuals or laboratories new to the technology. It also provides a working document that we use as a reference for "what works" but simultaneously allow to evolve as protocols and software develop. We are committed to keeping all protocols and software open-source, and will endeavor to use off-the-shelf equipment and reagents whenever possible. Contributions of anecdotes, as well as suggestions for modifications and clarifications are by all means encouraged and will be incorporated into future versions of this document. We have tried to be very specific about the reagents and equipment that are currently used in the Church & Mitra Labs. We strongly urge users to stick to this reagent and equipment list, and to only make substitutions after having clearly established a working implementation of the protocol.