Polony sequencing

Bologna sequence is inexpensive, but it can be used to “read” the millions of DNA sequences that are fixed in parallel, multiple sequence high-precision technology. This technique was developed by a group of Harvard University Medical School Dr. George Church first. Unlike sequencing technology other, Bologna sausage sequencing technology is an open platform and protocol download open source software freely. Further, it is possible to display on a computer controlled cell / fluid system and epifluorescence microscopy commonly available easily hardware of this technology. Generally, bologna sausages sequence, end tags of the genome of 17-18 two bases are separated by a common sequence comprises a each molecule of DNA template is sandwiched, the length is coupled to a library of 135 base pairs is performed by labeling the unit. Leaving a gap of 4-5 bases in each label, this reading of the length of this technology is the base multiplied by 13 and 26 base label.

The church of Bologna sausage sequencing Harvard University, which was developed in the laboratory of George M., was among the next generation of sequencing the first system that was used in the complete genome sequence of 2005. By binding to in vitro pair tag library by using a microscope emulsion PCR, an automated, it is that of about 9.1 Sanger sequencing cost genome of E. coli> and 99.9999% in accuracy for sequencing chemistry array of ligation-based. The after the technology has spun out the A Gen coat personal genomics, and licensed to A Gen Court Biosciences, Inc., participated in the robust platform of Applied Biosystems, which is owned by Life Technologies, Inc., a now in the end was.

This protocol is initiated by the shearing of genomic DNA testing in the particle size distribution tight randomly. Attached to the end of the repair then, DNA molecules of the truncated is line processing. At the end of the treatment, damaged or is oxidized 5′-phosphate is, a DNA processing of the A-line from the isolated and allows for the immediate blunt-end ligation, the protruding ends of compatibility and DNA blunt end DNA The repair added to the 3 ‘end of the. Is selected by the DNA molecules of a length of one kilobyte load this page gel 6% TBE. In the next step, two outwardly MmeI recognition site that is cyclized by T tail 30 base pairs, resulting circular DNA, comprises a rolling circle replication passage, DNA molecules, long synthetic oligonucleotide (T30),. Then, circular DNA molecule is amplified, digested with (Type II restriction endonuclease) MmeI of blocking releasing the T30 fragment sandwiched (≈ 70 BP Length) 17-18 bp tag, the recognition site are. The pair tag molecule, at both ends, you will need to be repaired before the end of the (RDV2 and FDV2) ligation EPCR (emulsion PCR) primer oligonucleotide. Library molecule of 135 bp to select the size as a result, and has been translated by the participants. Finally, the amplification of the end tag library molecule pair of 135 base pair PCR and to increase the size of the removal of by-product in a single ligation process and library materials. And because DNA template obtained consisting of the nucleotide sequence 44 FDV, 17-18 BP near sequence of T30, RDV sequence of 25 bp and 17-18 remote end.

I was pre-filled with a double forward primer biotin, a paramagnetic streptavidin-coated beads of mono size. Because streptavidin and biotin, the forward primer has a very strong affinity to bind firmly to the surface of the beads. I was prepared by the end of the associated tag library and reverse primer, pre-loaded beads, PCR mix, and forward the aqueous phase. Is mixed with the oil phase to create an emulsion, they were vortexed. Ideally, by performing PCR, droplets of water-in-oil emulsion has to allow one million volume scale 1ml per non-interacting amplification grain template DNA molecule. After amplification, Tris (10mM, pH7.5, of the EDTA pH8.0 1mM, an emulsion from the previous step, Triton X-100 NaCl in 100mM, 1% of (v / v) wash buffer and isopropanol, After a series of magnetic separation SDS that has been cut using mixed, and centrifuged. Each resulting solution, first a suspension of clones branched beads regardless emulsion droplets is zero, the presence of some empty obtained from the template DNA molecules of one or more. Reinforcing strips can be concentrated in the next step.

Concentration of hard particles is large, and is achieved by hybridization to the non-magnetic polystyrene beads preloaded (DNA sequence is amplicon EPCR sequence complementary) low density, biotinylated capture oligonucleotide. Was then centrifuged to separate the amplification and detection of the composite beads beads unamplified the mixture. Amplification, low density captured, thus bead complex remains liquid while the precipitate is formed beads that have not been amplified. Separating the complexes supernatant was collected This was treated with sodium hydroxide. Are separated by trapping non-magnetic beads by magnetic separation, paramagnetic beads that have been enhanced. This treatment protocol, enrichment of five times the beads can be hard. The purpose of the bead, there is a ceiling to be connected to “cap” the forward primer nucleotide non-stretch EPCR RDV two, the 3 ‘end of the segment of the template DNA. Using help the coupling of subsequent DNA to the matrix coating aminosilanated flow cell, at the same time, the cap is that it is an amino group to prevent the fluorescent probe ligation of these purposes.