2_genomics.ppt

/news_detail.php?news_id=1406 * /nature/journal/v443/n7109/full/443258a.html * www.liv.ac.uk/agf/454sequencing.html 454 Sequencing Facts ?How the 454 System works Attach DNA fragments to beadsThe system relies on fixing nebulized and adapter-ligated DNA fragments to small DNA-capture beads in a water-in-oil emulsion. Amplify the DNAThe DNA fixed to these beads is then amplified by PCR. Place beads in microscopic wellsEach DNA-bound bead is placed into a ~44 μm well on a PicoTiterPlate, which is a fiber optic chip. A mix of enzymes such as polymerase, ATP sulfurylase, and luciferase are also packed into the well. The PicoTiterPlate is then placed into the GS-FLX for sequencing. Add nucleotidesAt this stage, the four nucleotides (TAGC) are washed in series over the PicoTiterPlate. During the nucleotide flow, each of the hundreds of thousands of beads with millions of copies of DNA is sequenced in parallel.If a nucleotide complementary to the template strand is flowed into a well, the polymerase extends the existing DNA strand by adding nucleotide(s). Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal Read the light signal from each wellThe light signals are recorded by the CCD digital camera in the instrument. This technique is an example of pyrosequencing. The signal strength is proportional to the number of nucleotides, for example, homopolymer stretches, incorporated in a single nucleotide flow. However, the signal strength for homopolymer stretches is linear up to eight consecutive nucleotides; signal fall-off is rapid after a stretch of more than eight identical nucleotides. * / * * The direction of biological databases Genomes on the Web http://www.ebi.ac.uk/2can/genomes/genomes.html The genome of P. decumbens 真兽次亚纲 北方兽类 灵长总目 哺乳类门 Database search tools Entrez SRS http://www.ncbi.nlm.nih.giv/Entrez/ SRS (Sequence Retrieval System ) SRS是欧洲分子生物学网EMBnet的主要检索工具。 SRS, Sequence Retrieval System, is a power