biotechnology fate acquired by roche in 2007 and shut down by roche in 2013 founded 2000 headquarters branford connecticut usa products genome sequencers reagents services sequencing of genetic samples website www.454.com 454 life sciences was a biotechnology company based in branford connecticut that specialized in high throughput dna sequencing . it was acquired by roche in 2007 and shut down by roche in 2013 when its technology became noncompetitive . 1 contents 1 history s 2 technology 2.1 dna library preparation and empcr 2.2 sequencing 3 see also 4 notes history s edit 454 life sciences was founded by jonathan rothberg and was originally known as 454 corporation a subsidiary of curagen . for their method for low cost gene sequencing 454 life sciences was awarded the wall street journal s gold medal for innovation in the biotech medical category in 2005 . 2 the name 454 was the code name by which the project was referred to at curagen and the numbers have no known special meaning . 3 in november 2006 rothberg michael egholm and colleagues at 454 published a cover article with svante p bo in nature describing the first million base pairs of the neanderthal genome and initiated the neanderthal genome project to complete the sequence of the neanderthal genome by 2009 . citation needed in late march 2007 roche diagnostics acquired 454 life sciences for us 154.9 million . 4 it will remain a separate business unit . 5 in october 2013 roche announced that it will shut down 454 and stop supporting the platform by mid 2016 . 6 in may 2007 454 published the results of project jim the sequencing of the genome of james watson co discoverer of the structure of dna . 7 8 technology edit 454 sequencing used a large scale parallel pyrosequencing system capable of sequencing roughly 400 600 megabases of dna per 10 hour run on the genome sequencer flx with gs flx titanium series reagents . 9 the system relied on fixing nebulized and adapter ligated dna fragments to small dna capture beads in a water in oil emulsion . the dna fixed to these beads was then amplified by pcr . each dna bound bead was placed into a 29 m well on a picotiterplate a fiber optic chip . a mix of enzymes such as dna polymerase atp sulfurylase and luciferase was also packed into the well . the picotiterplate was then placed into the gs flx system for sequencing . 454 released the gs20 sequencing machine in 2005 the first next generation dna sequencer on the market . in 2008 454 sequencing launched the gs flx titanium series reagents for use on the genome sequencer flx instrument with the ability to sequence 400 600 million base pairs per run with 400 500 base pair read lengths . in late 2009 454 life sciences introduced the gs junior system a bench top version of the genome sequencer flx system . 10 dna library preparation and empcr edit genomic dna was fractionated into smaller fragments 300 800 base pairs and polished made blunt at each end . short adaptors were then ligated onto the ends of the fragments . these adaptors provided priming sequences for both amplification and sequencing of the sample library fragments . one adaptor adaptor b contained a 5 biotin tag for immobilization of the dna library onto streptavidin coated beads . after nick repair the non biotinylated strand was released and used as a single stranded template dna sstdna library . the sstdna library was assessed for its quality and the optimal amount dna copies per bead needed for empcr is determined by titration . 11 the sstdna library was immobilized onto beads . the beads containing a library fragment carried a single sstdna molecule . the bead bound library was emulsified with the amplification reagents in a water in oil mixture . each bead was captured within its own microreactor where pcr amplification occurs . this resulted in bead immobilized clonally amplified dna fragments . sequencing edit single stranded template dna library beads were added to the dna bead incubation mix containing dna polymerase and were layered with enzyme beads containing sulfurylase and luciferase onto a picotiterplate device . the device was centrifuged to deposit the beads into the wells . the layer of enzyme beads ensured that the dna beads remained positioned in the wells during the sequencing reaction . the bead deposition process was designed to maximize the number of wells that contain a single amplified library bead . the loaded picotiterplate device were placed into the genome sequencer flx instrument . the fluidics sub system delivered sequencing reagents containing buffers and nucleotides across the wells of the plate . the four dna nucleotides were added sequentially in a fixed order across the picotiterplate device during a sequencing run . during the nucleotide flow millions of copies of dna bound to each of the beads were sequenced in parallel . when a nucleotide complementary to the template strand was added into a well the polymerase extended the existing dna strand by adding nucleotide s . addition of one or more nucleotide s generated a light signal that was recorded by the ccd camera in the instrument . this technique was based on sequencing by synthesis and called pyrosequencing . 12 the signal strength was proportional to the number of nucleotides ; for example homopolymer stretches incorporated in a single nucleotide flow generated a greater signal than single nucleotides . however the signal strength for homopolymer stretches was linear only up to eight consecutive nucleotides after which the signal fell off rapidly . 13 data were stored in standard flowgram format sff files for downstream analysis . see also edit dna sequencing notes edit hollmer mark october 17 2013 . roche to close 454 life sciences as it reduces gene sequencing focus . fierce biotech . totty michael october 24 2005 . a better idea . the wall street journal . pollack andrew august 22 2003 . company says it mapped genes of virus in one day . the new york times . http www.roche.com med cor 2007 03 29 roche snares 454 s sequencing tech in buyout . fiercebiotech . march 28 2007 . http www.genomeweb.com sequencing following roches decision shut down 454 customers make plans move other platform wheeler d. a. ; srinivasan m. ; egholm m. ; shen y. ; chen l. ; mcguire a. ; he w. ; chen y. j. ; makhijani v. ; roth g. t. ; gomes x. ; tartaro k. ; niazi f. ; turcotte c. l. ; irzyk g. p. ; lupski j. r. ; chinault c. ; song x. z. ; liu y. ; yuan y. ; nazareth l. ; qin x. ; muzny d. m. ; margulies m. ; weinstock g. m. ; gibbs r. a. ; rothberg j. m. 2008 . the complete genome of an individual by massively parallel dna sequencing . nature . 452 7189 872 876 . bibcode 2008natur .452 . .872 w. doi 10.1038 nature06884 . pmid 18421352 . project jim watson s personal genome goes public . karl v ; et al. 2009 . next generation sequencing from basic research to diagnostics . clinical chemistry . 55 4 41 47 . doi 10.1373 clinchem .2008.112789 . pmid 19246620 . 454 life sciences unveils new bench top sequencer significant improvements to the genome sequencer flx system including 1 000 bp reads for 2010 press release . 454 life sciences . november 19 2009 . retrieved november 19 2009 . zheng z ; et al. 2010 . titration free massively parallel pyrosequencing using trace amounts of starting material . nucleic acids res . 38 13 e137 . doi 10.1093 nar gkq332 . pmc 2910068 . pmid 20435675 . king c ; scott horton t. 2008 . pyrosequencing a simple method for accurate genotyping . j vis exp 11 . doi 10.3791 630 . pmc 2582836 . pmid 19066560 . margulies marcel ; michael egholm ; 54 additional coauthors september 15 2005 . genome sequencing in open microfabricated high density picoliter reactors . nature . nature publishing group . 437 7057 376 380 . bibcode 2005natur .437 . .376 m. doi 10.1038 nature03959 . pmc 1464427 . pmid 16056220 . retrieved from https en.wikipedia.org w index.php title 454 life sciences & oldid 743660669 categories biotechnology 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