American Nobel laureate muchemistry Kary Mullis akafa muCalifornia aine makore makumi manomwe nemana. Maererano nemudzimai wake, rufu rwakaitika musi waAugust 74. Chikonzero chacho ndechekutadza kwemoyo uye kufema nekuda kwemabayo.
James Watson pachake, muvambi wemorekuru weDNA, achatiudza nezve mupiro wake kubiochemistry uye kwaakawana Mubairo weNobel.
Kubva mubhuku raJames Watson, Andrew Berry, Kevin Davis
DNA. Nhoroondo yeGenetic Revolution
Chitsauko 7. Genome yevanhu. Mamiriro ezvinhu ehupenyu
...
Iyo polymerase chain reaction (PCR) yakagadzirwa muna 1983 nebiochemist Carey Mullis, aishanda kuCetus. Kuwanikwa kwekuita uku kwaishamisa. Mullis akazoyeuka kuti: βMamwe manheru eChishanu muna April 1983, ndakava neshungu. Ndakanga ndiri seri kwevhiri, ndichidzika nomugwagwa unovhenekera mwedzi, unomonereka womugomo muNorthern California, nyika yemasango eredwood.β Zvinoshamisa kuti maive mumamiriro ezvinhu akadaro kuti kufemerwa kwakamurova. Uye hakusi kuti kuchamhembe kweCalifornia kune migwagwa yakakosha inokurudzira njere; kungoti shamwari yake yakamboona Mullis achimhanya zvisina tsarukano munzira yengoro ine chando uye hazvina kumunetsa. Shamwari yakaudza The New York Times, kuti: βMullis akava nechiono chokuti aizofa kupfurikidza nokurovera mumuti weredwood. Naizvozvo, haatyi chero chinhu paanenge achityaira, kunze kwekunge paine miti yeredwood iri kukura munzira. Kuvapo kweredwoods munzira yakamanikidza Mullis kuisa pfungwa uye ... apa yaive, nzwisiso. Mullis akagamuchira Mubairo weNobel muChemistry nekuda kwekugadzirwa kwake muna 1993 uye kubva zvino ave mutorwa muzviito zvake. Semuenzaniso, mutsigiri wedzidziso yekudzokorora kuti AIDS haina hukama neHIV, izvo zvakanyanya kukanganisa mukurumbira wake uye kupindira kune vanachiremba.
PCR iri nyore kuita. Kuti zviitike, tinoda maprimers maviri akagadzirwa nemakemikari anopindirana kune akapesana emhando dzakasiyana dzechidimbu chinodiwa cheDNA. Maprimers zvikamu zvipfupi zveDNA ine tambo imwe chete, chimwe nechimwe chingangoita 20 base pairs pakureba. Iyo peculiarity yemaprimers ndeyekuti inoenderana nezvikamu zveDNA zvinoda kukwidziridzwa, kureva, iyo DNA template.
(Mufananidzo unodzvanya) Kary Mullis, muvambi wePCR
Iyo chaiyo yePCR yakavakirwa pakuumbwa kweanopindirana macomplexes pakati pe template uye primers, pfupi synthetic oligonucleotides. Imwe neimwe yeprimers inopindirana kune imwe yetambo yekaviri-yakasungwa template uye inodzikamisa kutanga uye kupera kwenzvimbo yakasimudzwa. Kutaura zvazviri, "matrix" inoguma igenome yose, uye chinangwa chedu ndechekuparadzanisa zvidimbu zvekufarira kwatiri kubva mairi. Kuti uite izvi, template yakapetwa kaviri yeDNA inopisa kusvika 95 Β° C kwemaminetsi akati wandei kuparadzanisa tambo dzeDNA. Danho iri rinonzi denaturation nekuti mabhondi ehaidhirojeni ari pakati pematambo maviri eDNA akatyoka. Kana tambo dzaparadzana, tembiricha inodzikiswa kuitira kuti maprimers asunge kune imwechete-stranded template. DNA polymerase inotanga kudzokorora DNA nekusunga kune yakatambanudzwa ye nucleotide cheni. Iyo enzyme DNA polymerase inodzokorora template strand ichishandisa primer seprimer kana muenzaniso wekukopa. Nekuda kwekutenderera kwekutanga, tinowana kutevedzana kwakapetwa kaviri kwechimwe chikamu cheDNA. Zvadaro tinodzokorora nzira iyi. Mushure mekutenderera kwega kwega tinowana nzvimbo inotarirwa muhuwandu hwakapetwa. Mushure memakore makumi maviri neshanu ePCR (kureva kuti, mumaawa asingasviki maviri), tine dunhu reDNA rekuda kwatiri muhuwandu 225 nguva yakakwirira kudarika yepakutanga (kureva kuti, takaiwedzera kanokwana mamiriyoni makumi matatu nemana). Muchokwadi, pakuiswa kwatakagamuchira musanganiswa wemaprimers, template DNA, iyo DNA polymerase enzyme uye mabhesi emahara A, C, G uye T, huwandu hwechimwe chinhu chinoitwa chigadzirwa (chinogumira nemaprimers) chinokura zvakanyanya, uye nhamba. e "refu" makopi eDNA ane mutsara, saka mune reaction zvigadzirwa zvinotonga.
Kukwidziridzwa kwechikamu chinodiwa cheDNA: polymerase chain reaction
Mumazuva ekutanga ePCR, dambudziko guru raive rinotevera: mushure mekudziya kwega kwega-kutonhora kutenderera, DNA polymerase yaifanira kuwedzerwa kumusanganiswa wekuita, sezvo yaive isina kuvharwa patembiricha ye95 Β° C. Naizvozvo, zvaive zvakafanira kuiwedzera zvakare isati yasvika yega yega makumi maviri neshanu. Maitiro ekuita akange asina kunyatsoshanda, aida nguva yakawanda uye polymerase enzyme, uye zvinhu zvacho zvaidhura zvakanyanya. Sezvineiwo, Amai Nature vakauya kuzobatsira. Mhuka zhinji dzinonzwa dzakasununguka patembiricha dzakakwirira kudarika 25 Β°C. Nei nhamba 37 Β°C yakave yakakosha kwatiri? Izvi zvakaitika nekuti tembiricha iyi ndiyo yakakwana yeE. coli, kubva mairi enzyme yepolymerase yePCR yakatanga kuwanikwa. Muzvisikwa kune ma microorganisms ayo mapuroteni, pamusoro pemamiriyoni emakore ekusarudzwa kwechisikigo, ave achinyanya kupikisa kutonhora kwakanyanya. Yakakurudzirwa kushandisa DNA polymerases kubva kune thermophilic mabhakitiriya. Aya maenzayimu akachinja kuva anopisa uye akakwanisa kutsungirira maitiro akawanda ekuita. Kushandisa kwavo kwakaita kuti zvive nyore kurerutsa uye otomatiki PCR. Imwe yekutanga thermostable DNA polymerases yakaparadzaniswa nebhakitiriya Thermus aquaticus, inogara muzvitubu zvinopisa zveYellowstone National Park, uye yakanzi Taq polymerase.
PCR yakakurumidza kuita bhiza reHuman Genome Project. Kazhinji, maitiro acho haana kusiyana neaya akagadzirwa naMullis, achangove otomatiki. Takanga tisisiri kuvimba nemhomho yevadzidzi vakapedza kudzidza vari mapofu vachidurura madonhwe emvura mumachubhu epurasitiki. Mumarabhoritari emazuva ano anoita mamolecular genetic research, basa iri rinoitwa pamarobhoti conveyors. Marobhoti ePCR anobatanidzwa muchirongwa chekutevedzana chakakura seHuman Genome anoshanda asingaregi aine mavhoriyamu makuru ekupisa-kugadzikana kwepolymerase. Mamwe masayendisiti anoshanda paHuman Genome Project akatsamwiswa nemitengo yakakwira zvisingaite yakawedzerwa kumutengo wezvinodyiwa nemuridzi wePCR patent, hofori yemishonga yekuEurope yeindasitiri Hoffmann-LaRoche.
Imwe "musimboti wekutyaira" yaive iyo DNA yekuteedzera nzira pachayo. Kemikari yenzira iyi yakanga isisiri itsva panguva iyoyo: Interstate Human Genome Project (HGP) yakatora nzira yehungwaru yakafanana neyakaitwa naFred Sanger kare pakati pemakore ekuma1970. Ihwo hutsva hwakanga huri muchiyero uye chiyero che automation icho kutevedzana kwakakwanisa kuwana.
Kutevedzanisa otomatiki kwakagadzirwa murabhoritari yaLee Hood kuCalifornia Institute of Technology. Akapinda chikoro chesekondari muMontana uye akatamba nhabvu yekoreji se quarterback; Kutenda kuna Hood, timu yakahwina mukombe wehurumende kanopfuura kamwe. Unyanzvi hwake hwekubatana hwakauyawo zvakanaka mubasa rake resainzi. Hood's laboratory yakaitwa nemotley crew yemakemikari, biologist, uye mainjiniya, uye marabhoritari yake yakakurumidza kuva mutungamiriri muhunyanzvi hwekuvandudza tekinoroji.
Muchokwadi, iyo otomatiki yekutevedzana nzira yakagadzirwa naLloyd Smith naMike Hunkapiller. Mike Hunkapiller, akazoshanda murabhoritari yeHood, akaenda kuna Lloyd Smith nechikumbiro chenzira yakagadziridzwa yekutevedzana umo rudzi rwega rwegasi rwaizove neruvara zvakasiyana. Pfungwa yakadai inogona kupeta kugona kweiyo Sanger maitiro. MuSanger, pakutevedzana mune imwe neimwe yemachubhu mana (maererano nenhamba yezvigadziko), nekutora chikamu kweDNA polymerase, yakasarudzika seti ye oligonucleotides yehurefu hwakasiyana inoumbwa, kusanganisira yekutanga kutevedzana. Zvadaro, formamide yakawedzerwa kumachubhu ekuparadzanisa cheni uye polyacrylamide gel electrophoresis yakaitwa panzira ina. Mushanduro yaSmith naHunkapiller, dieoxynucleotides akanyorwa nedhayi ina dzakasiyana uye PCR inoitwa muchubhu imwe. Zvadaro, panguva yepolyacrylamide gel electrophoresis, danda relaser pane imwe nzvimbo pane gel rinonakidza basa remadhayi, uye detector inotarisa kuti ndeipi nucleotide iri kufamba nejeri. Pakutanga, Smith akanga asina tariro - aitya kuti kushandisa madosi edhayi edhayi zvakanyanya zvaizoita kuti matunhu aite nucleotide asaonekwe. Nekudaro, aine nzwisiso yakanakisa yelaser tekinoroji, akakurumidza kuwana nzira yekubuda mumamiriro ezvinhu nekushandisa chaiwo madhayi efluorochrome ayo anovhenekera kana akafumurwa nemwaranzi yelaser.
(Full version - 4,08 MB) Kudhinda zvakanaka: Kutevedzana kweDNA kunotevedzana pachishandiswa otomatiki sequencer, inowanwa pamuchina wekutevedzanisa. Ruvara rumwe norumwe rwunoenderana neimwe yezvigadziko zvina
Muchinyakare vhezheni yenzira yeSanger, imwe yetambo yeakaongororwa DNA inoita setemplate yekubatanidzwa kweiyo tambo yekuwedzera neiyo enzyme DNA polymerase, ipapo kutevedzana kwezvimedu zveDNA kunorongwa mujeri nehukuru. Chidimbu chimwe nechimwe, icho chinosanganiswa muDNA panguva yekubatanidza uye chinobvumira kutaridzika kunotevera kwezvigadzirwa zvemaitiro, yakanyorwa nedhayi yefluorescent inoenderana neiyo terminal base (izvi zvakakurukurwa pap. 124); saka, fluorescence yechimedu ichi chichava chiziviso chehwaro hwakapihwa. Ipapo chasara kuita yekuona uye kuona maitiro ekuita zvigadzirwa. Mhedzisiro yacho inoongororwa nekombuta uye inoratidzwa senhevedzano yemakomo ane mavara mazhinji anoenderana nemanucleotides mana. Ruzivo rwunobva rwaendeswa zvakananga kuhurongwa hweruzivo rwekombuta, zvichibvisa iyo inopedza nguva uye dzimwe nguva inorwadza yekupinda data iyo yakaita kuti kutevedzana kuve kwakaoma.
Β» Mamwe mashoko pamusoro pebhuku anogona kuwanikwa pa
Β»
Β»
For Khabrozhiteley 25% dhisikaundi uchishandisa kopani - PCR
Source: www.habr.com