{"id":37174,"date":"2019-10-31T22:16:11","date_gmt":"2019-10-31T19:16:11","guid":{"rendered":"https:\/\/prohoster.info\/blog\/umer-nobelevskij-laureat-keri-mullis-izobretatel-polimeraznoj-tsepnoj-reaktsii-dnk\/"},"modified":"2019-10-31T22:16:11","modified_gmt":"2019-10-31T19:16:11","slug":"umer-nobelevskij-laureat-keri-mullis-izobretatel-polimeraznoj-tsepnoj-reaktsii-dnk","status":"publish","type":"post","link":"https:\/\/prohoster.info\/et\/blog\/novosti-interneta\/umer-nobelevskij-laureat-keri-mullis-izobretatel-polimeraznoj-tsepnoj-reaktsii-dnk","title":{"rendered":"Surm on leidnud Nobeli auhinna laureaat K\u00e6ri Mullis - DNA pol\u00fcmeraas-ahelreaktsiooni leiutaja","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"<p><img decoding=\"async\" alt=\"Surm on leidnud Nobeli auhinna laureaat K\u00e6ri Mullis - DNA pol\u00fcmeraas-ahelreaktsiooni leiutaja\" src=\"\/wp-content\/uploads\/2019\/08\/bbb8f4620ea2a5301d5f6285a639ce6d.jpg\" style=\"display:block;margin: 0 auto;\" \/> California's 74-year-old American Nobel laureate in Chemistry, Cary Mullis, has passed away. According to his wife, he died on August 7 due to heart and respiratory failure caused by pneumonia.<\/p>\n<p>James Watson, the co-discoverer of the DNA molecule, will tell us about his contributions to biochemistry and why he received the Nobel Prize. <\/p>\n<p>Excerpt from the book by James Watson, Andrew Berry, Kevin Davis <\/p>\n<h2>DNA. The Story of the Genetic Revolution<\/h2>\n<h3>Chapter 7. The Human Genome. The Script of Life<\/h3>\n<p>\n<noindex><a rel=\"nofollow\" name=\"habracut\"><\/a><\/noindex><br \/>\n\u2026<br \/>\nPol\u00fcmeraasi ahelreaktsioon (PCR) leiutati 1983. aastal biokeemiku Kary Mullis'i poolt, kes t\u00f6\u00f6tas ettev\u00f5ttes Cetus. Selle reaktsiooni avastus oli v\u00e4ga t\u00e4helepanuv\u00e4\u00e4rne. Hiljem meenutas Mullis: \"Kord aprilli 1983. aasta reede\u00f5htul tajusin \u00e4kki. Ma s\u00f5itsin, liikudes m\u00f6\u00f6da heledalt kuuvalguses j\u00e4rske m\u00e4giteid P\u00f5hja-Californias, sekvoiadest metsade \u00e4\u00e4res.\" Muljetavaldav on, et just sellistes tingimustes leidis ta inspiratsiooni. Ja see ei seisne \u00fcldsegi selles, et P\u00f5hja-Californias oleks eredad teed, mis inspireerivad; hoopis tema s\u00f5ber n\u00e4gi kord, kuidas Mullis muretu peaga kihutas libeda kahe suuna teel ega polnud sellest sugugi h\u00e4iritud. S\u00f5ber r\u00e4\u00e4kis New York Times'ile j\u00e4rgmist: \"Mullisele j\u00e4i mulje, et ta sureb sekvoia sisse s\u00f5ites. Seet\u00f5ttu ei karda ta s\u00f5ites, kui teed m\u00f6\u00f6da ei kasva sekvoiad.\" Sekvoiate kohalolek tee \u00e4\u00e4res sundis Mullist keskenduma ja... voil\u00e0, inspiratsioon oli kohal. Oma leiutise eest sai Mullis 1993. aastal keemia Nobeli auhinna ning p\u00e4rast seda muutus ta oma k\u00e4itumises veelgi erilisemaks. N\u00e4iteks toetab ta revizionistlikku teooriat, et AIDSi ei seostata HIV-iga, mis on t\u00f5siselt kahjustanud tema mainet ja seganud arste.<\/p>\n<p>PCR on \u00fcsna lihtne reaktsioon. Selle l\u00e4biviimiseks on meil vaja kahte keemiliselt s\u00fcnteesitud primerit, mis on komplementaarsed erinevate DNA fragmentide vastassuundadele. Primerid on l\u00fchikesed \u00fcheastmelised DNA osad, iga\u00fcks umbes 20 aluspaari pikk. Primerite erip\u00e4ra on see, et need sobivad DNA osadega, mida tuleb amplifitseerida, st DNA-materjaliga. <\/p>\n<p><noindex><a rel=\"nofollow\" href=\"https:\/\/habrastorage.org\/webt\/uj\/gu\/49\/ujgu49z-nrw9zkcbru0kobwf6-k.jpeg\"><img decoding=\"async\" alt=\"Surm on leidnud Nobeli auhinna laureaat K\u00e6ri Mullis - DNA pol\u00fcmeraas-ahelreaktsiooni leiutaja\" src=\"\/wp-content\/uploads\/2019\/08\/b324c9f97ad73d5708a423f91e2584d8.jpg\" style=\"display:block;margin: 0 auto;\" \/><\/a><\/noindex><br \/>\n<i>(Pilt on klikatav) Kary Mullis, PCR-i leiutaja<\/i><\/p>\n<p>PCR spetsiifilisus p\u00f5hineb t\u00e4iendavate komplekside loomisel matriitsi ja primereid, l\u00fchikesed s\u00fcnteetilised oligonukleotiidid. Iga primer on komplementaarne \u00fche keera topeltheliks matriitsiga ja piirab amplifitseeritava ala algust ja l\u00f5ppu. Tegelikult saadud \"matriits\" esindab tervet genoomi ning meie eesm\u00e4rk on eraldada sealt meid huvitavad fragmendid. Selleks kuumutatakse kaheastmelise DNA matriitsi 95 \u00b0C juures mitmeks minutiks, et DNA ahelad eralduksid. Seda etappi nimetatakse denaturatsiooniks, kuna vesiniksidemed kahe DNA ahela vahel purunevad. Kui ahelad on eraldatud, alandatakse temperatuuri, et primerid saaksid seonduda \u00fcherhelise matriitsiga. DNA pol\u00fcmeraasi k\u00e4ivitab DNA replikatsiooni, seondudes nukleotiidide ahela l\u00f5iguga. DNA pol\u00fcmeraasi ens\u00fc\u00fcm replitseerib matriitsiahela, kasutades primerit koopia n\u00e4itena. Esimese ts\u00fckli tulemuseks on teatud DNA osa korduv ja j\u00e4rjestikune kahekordistamine. Edasi kordame seda protseduuri. Iga ts\u00fckli j\u00e4rel saame sihtala kahekordse koguse. Kaksk\u00fcmmend viis PCR ts\u00fcklit hiljem (ehk v\u00e4hem kui kahe tunni p\u00e4rast) saame meile huvipakkuva DNA osa koguses, mis \u00fcletab algse 225 korda (s.o. meil on see ligikaudu 34 miljonit korda amplifitseeritud). Tegelikult saime me alguses segu primeritest, matriitse, DNA pol\u00fcmeraasi ens\u00fc\u00fcmist ja vabade alus A, C, G ja T, see konkreetse reaktsiooni toote kogus (mis on piiratud primeritega) kasvab eksponentsiaalselt, samas kui \"pikad\" DNA kopeerimine lineaarne, seega domineerivad reaktsiooni produktides.<\/p>\n<p><img decoding=\"async\" alt=\"Surm on leidnud Nobeli auhinna laureaat K\u00e6ri Mullis - DNA pol\u00fcmeraas-ahelreaktsiooni leiutaja\" src=\"\/wp-content\/uploads\/2019\/08\/2f4e0fc328c01fd93323c7128b30d265.jpg\" style=\"display:block;margin: 0 auto;\" \/><br \/>\n<i>DNA vajaliku osa amplifikatsioon: pol\u00fcmeraasi ahelreaktsioon<\/i><\/p>\n<p>PCR-i varajamisel seisnes peamine probleem j\u00e4rgmis: iga kuumutamists\u00fckli j\u00e4rel tuli reaktsioonisegusse lisada DNA-pol\u00fcmeraasi, kuna see inaktiveerus temperatuuril 95 \u00b0C. Seega tuli see iga 25 ts\u00fckli eel uuesti lisada. Reaktsiooni l\u00e4biviimise protseduur oli suhteliselt ebaefektiivne, n\u00f5udis palju aega ja pol\u00fcmeraasi fermenti, mis on suhteliselt kallis materjal. \u00d5nneks tuli appi ema-loodus. Paljud loomad tunnevad end mugavalt temperatuuridel, mis on palju k\u00f5rgemad kui 37 \u00b0C. Miks on meie jaoks t\u00e4htis just number 37 \u00b0C? See tuleneb sellest, et see temperatuur on ideaalne E. coli jaoks, millest algselt saadi pol\u00fcmeraasi ferment PCR-i jaoks. Looduses leidub mikroorganisme, kelle valgud on miljoneid aastaid loodusliku selektsiooni t\u00f5ttu muutunud k\u00f5rgematele temperatuuridele vastupidavamaks. Pakuti v\u00e4lja kasutada termofiilsete bakterite DNA-pol\u00fcmeraase. Need ens\u00fc\u00fcmid osutusid termostabiilseteks ja suudavad taluda mitmeid reaktsiooni ts\u00fckleid. Nende kasutamine lihtsustas ja automatiseeris PCR-i l\u00e4biviimist. \u00dcks esimesi termostabiilseid DNA-pol\u00fcmeraase eraldati bakterist Thermus aquaticus, mis elab Yellowstone'i rahvuspargi kuumaveeallikates, ja seda nimetati Taq-pol\u00fcmeraasiks.<\/p>\n<p>PCR on kiiresti muutunud projekti \"Inimese genoom\" peamiseks t\u00f6\u00f6riistaks. \u00dcldiselt ei erine protsess M\u00fcllise v\u00e4lja t\u00f6\u00f6tatud meetodist, lihtsalt see on automatiseeritud. Me ei s\u00f5ltunud enam pimedatest doktorantidest, kes valasid tihedalt vedelikku plastikutesse proovidesse. Kaasaegsetes laborites, kus teostatakse molekulaargeneetilisi uuringuid, teeb seda t\u00f6\u00f6d robotiseeritud konveier. PCR-robotid, mis osalevad nii ulatuslikus projekti nagu \"Inimese genoom\", t\u00f6\u00f6tavad vankumatult suurte temperatuurikindlate DNA-pol\u00fcmeraaside kogustega. M\u00f5ned teadlased, kes t\u00f6\u00f6tavad projekti \"Inimese genoom\" kallal, olid n\u00f6rdinud \u00fclem\u00e4\u00e4ra k\u00f5rgetest tasudest, mille lisab tarvikute maksumusele PCR-i patendi omanik, Euroopa t\u00f6\u00f6stus- ja farmaatsiahiiglane Hoffmann-LaRoche.<\/p>\n<p>Teiseks \u201eliikuvaks alguseks\u201c sai DNA j\u00e4rjestamise meetod. Selle meetodi keemiline alus ei olnud sel ajal enam uudne: rahvusvaheline projekt \u201eInimese genoom\u201c (HGP) v\u00f5ttis kasutusele sama nutika meetodi, mille t\u00f6\u00f6tas v\u00e4lja Fred Sanger juba 1970. aastate keskel. Uudsus seisnes mastaabis ja automatiseerimise astmes, mida oli v\u00f5imalik saavutada j\u00e4rjestamisel.<\/p>\n<p>Automaatne j\u00e4rjestamine t\u00f6\u00f6tati algselt v\u00e4lja Lee Hudi laboratooriumis California Tehnoloogiainstituudis. Ta l\u00f5petas kooli Montanas ja m\u00e4ngis Ameerika jalgpalli r\u00fcndajana; t\u00e4nu Hudile v\u00f5itis meeskond korduvalt osariigi meistriv\u00f5istlused. Meeskonnat\u00f6\u00f6 oskused olid talle kasuks ka teaduskarj\u00e4\u00e4ris. Hudi laboratooriumis t\u00f6\u00f6tas mitmekesine seltskond keemikuid, biolooge ja insenere ning varsti t\u00f5usis tema laboratoorium tehnoloogiliste uuenduste osas esirinda.<\/p>\n<p>Tegelikult leiutasid automaatse sekveneerimise meetodi Lloyd Smith ja Mike Hunkapiller. Mike Hunkapiller, kes t\u00f6\u00f6tas sel ajal Hudi laboris, p\u00f6\u00f6rdus Lloyd Smithi poole, pakkudes v\u00e4lja t\u00e4iustatud sekveneerimismeetodi, kus iga t\u00fc\u00fcpi nukleotiid v\u00e4rvitaks eri v\u00e4rviga. Selline idee suutis nelja korra v\u00f5rra t\u00f5sta Sangeri protsessi efektiivsust. Sangeril sekveneerimise k\u00e4igus luuakse igas neljas katseklaasis (mille arv vastab nukleotiidide arvule) ainulaadne nukleotiidide komplekt, mille pikkused varieeruvad ning sisaldavad primerj\u00e4rjestust, millega osaleb DNA-pol\u00fcmeraas. Seej\u00e4rel lisatakse katseklaasidesse formamiid, et ahelad lahkuda, ja tehakse elektroforees pol\u00fcakreklaamidi geelil neljal radadel. Smithi ja Hunkapilleri variandis v\u00e4rvitakse didesoks\u00fcnukleotiidid nelja erineva v\u00e4rviga ja PCR viiakse l\u00e4bi \u00fches katseklaasis. Seej\u00e4rel elektroforeesi ajal pol\u00fcakreklaamidi geelil aktiveerib laserkiir geeli kindlas kohas v\u00e4rvide aktiivsuse, ning detektor m\u00e4\u00e4rab, milline nukleotiid parasjagu geelil r\u00e4ndab. Algselt oli Smith pessimistlik \u2014 ta kartis, et kasutamine \u00fcliv\u00e4ikestes annustes v\u00e4rvi viib selleni, et nukleotiidide osad on eristamatud. Kuid olles suurep\u00e4raselt valgustehnoloogiate teadmisega, leidis ta peagi lahenduse, kasutades spetsiaalseid fluorofoorseid v\u00e4rve, mis fluoresseeruvad laserkiirguse m\u00f5jul.<\/p>\n<p><img decoding=\"async\" alt=\"Surm on leidnud Nobeli auhinna laureaat K\u00e6ri Mullis - DNA pol\u00fcmeraas-ahelreaktsiooni leiutaja\" src=\"\/wp-content\/uploads\/2019\/08\/2a6898bf2f81ec7b15edb365879f840c.jpg\" style=\"display:block;margin: 0 auto;\" \/><br \/>\n<i>(T\u00e4ielik versioon <noindex><a rel=\"nofollow\" href=\"https:\/\/habrastorage.org\/webt\/hi\/kv\/pj\/hikvpjrv-nfqqjr_kpeflbmkg-0.jpeg\">kl\u00f5psamisel<\/a><\/noindex> \u2014 4,08 MB) V\u00e4ikese kirjat\u00e4htedega: DNA j\u00e4rjestus, mis de\u0161ifreeriti automaatse sekveneerimise seadmega, saadud automaatse sekveneerimise seadme abil. Iga v\u00e4rv vastab \u00fchele neljast alusest<\/i> <\/p>\n<p>Sangeri meetodi klassikalises variandis on \u00fcks anal\u00fc\u00fcsitava DNA ahelatest matriiks, mille alusel s\u00fcntesitakse komplementaarne ahel DNA-pol\u00fcmeraasens\u00fc\u00fcmi abil. Seej\u00e4rel sorteeritakse DNA fragmentide j\u00e4rjestused geelil suuruse j\u00e4rgi. Iga fragment, mis kaasatakse DNA koosseisu s\u00fcntesi k\u00e4igus ja v\u00f5imaldab hiljem reaktsiooni tooteid visualiseerida, m\u00e4rgistatakse fluoressentsv\u00e4rviga, mis vastab terminalile alusele (millest r\u00e4\u00e4giti lk 124); seega on selle fragmendi fluoressents antud aluse identifikaator. Seej\u00e4rel tuleb vaid tuvastamine ja reaktsiooni toodete visualiseerimine. Tulemusi anal\u00fc\u00fcsitakse arvuti abil ja esitatakse neljakordsete nukleotiide vastavate eriv\u00e4rviliste tippudena. Seej\u00e4rel antakse teave otse arvuti infos\u00fcsteemi, mis v\u00e4listab ajakuluka ja tihti vaevarikka andmete sisestamise protsessi, mis v\u00e4ga raskendas sekveneerimist.<\/p>\n<p>\u00bb Raamatu kohta on t\u00e4iendavat teavet saadaval <noindex><a rel=\"nofollow\" href=\"https:\/\/www.piter.com\/product\/dnk-istoriya-geneticheskoy-revolyutsii\">v\u00e4ljaandja veebisaidil<\/a><\/noindex><br \/>\n\u00bb <noindex><a rel=\"nofollow\" href=\"https:\/\/storage.piter.com\/upload\/contents\/978544610549\/978544610549_X.pdf\">Sisukord<\/a><\/noindex><br \/>\n\u00bb <noindex><a rel=\"nofollow\" href=\"https:\/\/storage.piter.com\/upload\/contents\/978544610549\/978544610549_p.pdf\">L\u00f5ik<\/a><\/noindex><\/p>\n<p>Habr'i lugejatele 25% allahindlus kupongiga \u2014 <b>PCR<\/b><br \/>\n<br \/>Allikas: <a content=\"nofollow\" rel=\"nofollow\" href=\"https:\/\/habr.com\/ru\/company\/piter\/blog\/463873\/\">habr.com<\/a><\/p>","protected":false,"gt_translate_keys":[{"key":"rendered","format":"html"}]},"excerpt":{"rendered":"<p>\u0412 \u041a\u0430\u043b\u0438\u0444\u043e\u0440\u043d\u0438\u0438 \u0432 \u0432\u043e\u0437\u0440\u0430\u0441\u0442\u0435 74 \u043b\u0435\u0442 \u0443\u043c\u0435\u0440 \u0430\u043c\u0435\u0440\u0438\u043a\u0430\u043d\u0441\u043a\u0438\u0439 \u043d\u043e\u0431\u0435\u043b\u0435\u0432\u0441\u043a\u0438\u0439 \u043b\u0430\u0443\u0440\u0435\u0430\u0442 \u043f\u043e \u0445\u0438\u043c\u0438\u0438 \u041a\u044d\u0440\u0438 \u041c\u0443\u043b\u043b\u0438\u0441. \u041f\u043e \u0441\u043b\u043e\u0432\u0430\u043c \u0435\u0433\u043e \u0441\u0443\u043f\u0440\u0443\u0433\u0438, \u0441\u043c\u0435\u0440\u0442\u044c \u043d\u0430\u0441\u0442\u0443\u043f\u0438\u043b\u0430 7 \u0430\u0432\u0433\u0443\u0441\u0442\u0430. \u041f\u0440\u0438\u0447\u0438\u043d\u0430 \u2014 \u0441\u0435\u0440\u0434\u0435\u0447\u043d\u0430\u044f \u0438 \u0434\u044b\u0445\u0430\u0442\u0435\u043b\u044c\u043d\u0430\u044f \u043d\u0435\u0434\u043e\u0441\u0442\u0430\u0442\u043e\u0447\u043d\u043e\u0441\u0442\u044c \u0438\u0437-\u0437\u0430 \u043f\u043d\u0435\u0432\u043c\u043e\u043d\u0438\u0438. \u041e \u0442\u043e\u043c, \u043a\u0430\u043a\u043e\u0439 \u0432\u043a\u043b\u0430\u0434 \u043e\u043d \u0432\u043d\u0435\u0441 \u0432 \u0431\u0438\u043e\u0445\u0438\u043c\u0438\u044e \u0438 \u0437\u0430 \u0447\u0442\u043e \u043f\u043e\u043b\u0443\u0447\u0438\u043b \u041d\u043e\u0431\u0435\u043b\u0435\u0432\u0441\u043a\u0443\u044e \u043f\u0440\u0435\u043c\u0438\u044e, \u043d\u0430\u043c \u0440\u0430\u0441\u0441\u043a\u0430\u0436\u0435\u0442 \u0441\u0430\u043c \u0414\u0436\u0435\u0439\u043c\u0441 \u0423\u043e\u0442\u0441\u043e\u043d \u2014 \u043f\u0435\u0440\u0432\u043e\u043e\u0442\u043a\u0440\u044b\u0432\u0430\u0442\u0435\u043b\u044c \u043c\u043e\u043b\u0435\u043a\u0443\u043b\u044b 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