How Does Polony Sequencing Work?

Polony sequencing is performed in three steps:

Step 1: Make library of linear DNA molecules with universal priming sites (see figure at right).
Each molecule in the library will contain a variable region flanked by two constant regions. The constant regions contain primer-binding sites to allow universal amplification by PCR. This type of library was first created to perform SELEX experiments.

Step 2: Amplify polymerase colonies (polonies) in a polyacrylamide gel (see figure at right).
A thin polyacrylamide gel is poured on a glass microscope slide. The library made in step 1 is amplified in this gel by performing PCR. At the end of the reaction, each single template molecule gives rise to a polony. As many as 15 million polonies can be amplified on a single slide. An acrydite modification is included at the 5' end of one of the primers, so that one strand of the amplified DNA is covalently attached to the polyacrylamide matrix. This allows further enzymatic manipulations and washing to be performed on the attached DNA. The details of polony amplification have been previously described.

Step 3: Sequence polonies by fluorescent in situ sequential quantitation (FISSEQ). (see figure at right).
The immobilized DNA is denatured, the unattached strand is washed away, and a universal primer is hybridized to the template. A primer extension reaction is performed. The gel is then scanned using a scanning fluorescence microscope. If a polony has incorporated the added base, it will fluoresce, revealing the identity of the template base immediately 3' of the annealed primer. The fluorescence is then removed by cleaving the linker between the fluorophore and the nucleotide, and washing away the fluorophore. The cycle is repeated by adding a different dye labeled base, washing away unincorporated nucleotide and scanning the gel. In this way, the sequence of every polony on the gel can be determined in parallel.