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Polony sequencing is an inexpensive but highly accurate multiplex sequencing technique that can be used to “read” millions of immobilized DNA sequences in parallel. This technique was first developed by Dr. George Church's group at Harvard Medical School. Unlike other sequencing techniques, Polony sequencing technology is an open platform with freely downloadable, open source software and protocols. Also, the hardware of this technique can be easily set up with a commonly available epifluorescence microscopy and a computer-controlled flowcell/fluidics system. Polony sequencing is generally performed on paired-end tags library that each molecule of DNA template is of 135 bp in length with two 17–18 bp paired genomic tags separated and flanked by common sequences. The current read length of this technique is 26 bases per amplicon and 13 bases per tag, leaving a gap of 4–5 bases in each tag. ==Workflow== The protocol of Polony sequencing can be broken into three main parts, which are the paired end-tag library construction, template amplification and DNA sequencing. ;Paired end-tag library construction This protocol begins by randomly shearing the tested genomic DNA into a tight size distribution. The sheared DNA molecules are then subjected for the end repair and A-tailed treatment. The end repair treatment converts any damaged or incompatible protruding ends of DNA to 5’-phosphorylated and blunt-ended DNA, enabling immediate blunt-end ligation, while the A-tailing treatment adds an A to the 3’ end of the sheared DNA. DNA molecules with a length of 1 kb are selected by loading on the 6% TBE PAGE gel. In the next step, the DNA molecules are circularized with T-tailed 30 bp long synthetic oligonucleotides (T30), which contains two outward-facing MmeI recognition sites, and the resulting circularized DNA undergoes rolling circle replication. The amplified circularized DNA molecules are then digested with MmeI (type IIs restriction endonuclease) which will cuts at a distance from its recognition site, releasing the T30 fragment flanked by 17–18 bp tags (≈70 bp in length). 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. The resulting 135 bp library molecules are size-selected and nick translated. Lastly, amplify the 135 bp paired end-tag library molecules with PCR to increase the amount of library material and eliminate extraneous ligation products in a single step. The resulted DNA template consists of a 44 bp FDV sequence, a 17–18 bp proximal tag, the T30 sequence, a 17-18 bp distal tag, and a 25 bp RDV sequence. ;Template amplification * Emulsion PCR The mono-sized, paramagnetic streptavidin–coated beads are pre-loaded with dual biotin forward primer. Streptavidin has a very strong affinity for biotin, thus the forward primer will bind firmly on the surface of the beads. Next, an aqueous phase is prepared with the pre-loaded beads, PCR mixture, forward and reverse primers, and the paired end-tag library. This is mixed and vortexed with an oil phase to create the emulsion. 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. * Emulsion breaking After amplification, the emulsion from preceding step is broken using isopropanol and detergent buffer (10 mM Tris pH 7.5, 1 mM EDTA pH 8.0, 100 mM NaCl, 1% (v/v) Triton X‐100, 1% (w/v) SDS), following with a series of vortexing, centrifuging, and magnetic separation. 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 amplified bead could be enriched in the following step. • 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Polony sequencing」の詳細全文を読む スポンサード リンク
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