Polony sequencing

The paired-tag molecules need to be end-repaired prior to the ligation of ePCR (emulsion PCR) primer oligonucleotides (FDV2 and RDV2) to their both ends.

Next, an aqueous phase is prepared with the pre-loaded beads, PCR mixture, forward and reverse primers, and the paired end-tag library.

Ideally, each droplet of water in the oil emulsion has one bead and one molecule of template DNA, permitting millions of non-interacting amplification within a milliliter-scale volume by performing PCR.

The resulted solution is a suspension of empty, clonal and non-clonal beads, which arise from emulsion droplets that initially have zero, one or multiple DNA template molecules, respectively.

The cap that being use is an amino group that prevents fluorescent probes from ligating to these ends and at the same time, helping the subsequent coupling of template DNA to the aminosilanated flow cell coverslip.

First, the coverslips are washed and aminosilane-treated, enabling the subsequent covalent coupling of template DNA on it and eliminating any fluorescent contamination.

The amplified, enriched beads are mixed with acrylamide and poured into a shallow mold formed by a Teflon-masked microscope slide.

First, a series of anchor primers are flowed through the cells and hybridize to the synthetic oligonucleotide sequences at the immediate 3’ or 5’ end of the 17-18 bp proximal or distal genomic DNA tags.

Next, an enzymatic ligation reaction of the anchor primer to a population of degenenerate nonamers that are labeled with fluorescent dyes is performed.

[1] The sequencing instrument used in this technique could be set up by the commonly available fluorescence microscope and a computer controlled flowcell.

[citation needed] A dedicated polony sequencing machine, Polonator, was developed in 2009 and sold at US$170,000 by Dover.

Furthermore, the polony sequencing technique is emphasized as an open system that shares everything including the software that have been developed, protocol and reagents.

[5] Methods were developed in 2003 to sequence in situ polonies using single-base extension which could achieve 5-6 bases reads.