{"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\/sk\/blog\/news\/umer-nobelevskij-laureat-keri-mullis-izobretatel-polimeraznoj-tsepnoj-reaktsii-dnk","title":{"rendered":"Zomrel nobelovsk\u00fd laure\u00e1t Kary Mullis \u2014 vyn\u00e1lezca polymer\u00e1zovej re\u0165azovej reakcie DNA","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"

\"Zomrel V Kalifornii vo veku 74 rokov zomrel americk\u00fd nobelovsk\u00fd laure\u00e1t v oblasti ch\u00e9mie Kary Mullis. Pod\u013ea jeho man\u017eelky smr\u0165 nastala 7. augusta. Pr\u00ed\u010dina \u2014 srdcov\u00e1 a d\u00fdchac\u00eda nedostato\u010dnos\u0165 sp\u00f4soben\u00e1 pneum\u00f3niou.<\/p>\n

O tom, ak\u00fd pr\u00ednos mal pre bioch\u00e9miu a za \u010do z\u00edskal Nobelovu cenu, n\u00e1m povie samotn\u00fd James Watson \u2014 objavite\u013e molekuly DNA. <\/p>\n

\u00daryvok z knihy Jamesa Watsona, Andrewa Berryho, Kevina Davisa <\/p>\n

DNA. Hist\u00f3ria genetickej revol\u00facie<\/h2>\n

Kapitola 7. Gen\u00f3m \u010dloveka. Scen\u00e1r \u017eivota<\/h3>\n

\n<\/a><\/noindex>
\n\u2026
\nPolymer\u00e1zov\u00e1 re\u0165azov\u00e1 reakcia (PCR) bola vyn\u00e1jden\u00e1 v roku 1983 bioch\u00e9mikom Kary Mullisom, ktor\u00fd pracoval v spolo\u010dnosti Cetus. Objav tejto reakcie bol ve\u013emi pozoruhodn\u00fd. Nesk\u00f4r Mullis spom\u00ednal: \u201eRaz v piatok v apr\u00edli 1983 som bol akosi osvieten\u00fd. Viedol som auto, i\u0161iel po osvetlenej mesa\u010dn\u00fdm svetlom zvl\u0148enej horskej ceste v Severnej Kalifornii, na okraji sekvojov\u00fdch lesov.\u201d Je p\u00f4sobiv\u00e9, \u017ee pr\u00e1ve v takej situ\u00e1cii ho nav\u0161t\u00edvila in\u0161pir\u00e1cia. A nie je to tak, \u017ee na severe Kalifornie s\u00fa \u0161peci\u00e1lne cesty, ktor\u00e9 napom\u00e1haj\u00fa osvieteniu; jednoducho jeho priate\u013e raz videl, ako Mullis bezhlavo uh\u00e1\u0148a po z\u013eadovatelej ceste s obojsmern\u00fdm pohybom a v\u00f4bec ho to neznepokojovalo. Priate\u013e povedal New York Times: \u201eMullisovi sa zdalo, \u017ee zomrie, naraziac do sekvoje. Preto sa neboj\u00ed jazdi\u0165, ak pozd\u013a\u017e cesty nerast\u00fa sekvoje.\u201d Pr\u00edtomnos\u0165 sekvoj\u00ed pozd\u013a\u017e cesty don\u00fatila Mullisa s\u00fastredi\u0165 sa a\u2026 aha, osvietenie. Za svoj vyn\u00e1lez z\u00edskal v roku 1993 Mullis Nobelovu cenu za ch\u00e9miu a odvtedy sa stal e\u0161te podivnej\u0161\u00edm vo svojich \u010dinostiach. Napr\u00edklad je z\u00e1stancom revizionistickej te\u00f3rie, \u017ee AIDS nie je spojen\u00fd s HIV, \u010d\u00edm zna\u010dne podkopal svoju vlastn\u00fa reput\u00e1ciu a znepr\u00edjemnil \u017eivot lek\u00e1rom.<\/p>\n

PCR je pomerne jednoduch\u00e1 reakcia. Na jej vykonanie potrebujeme dva chemicky syntetizovan\u00e9 primery, komplement\u00e1rne k opa\u010dn\u00fdm koncom r\u00f4znych re\u0165azcov po\u017eadovan\u00e9ho fragmentu DNA. Primery s\u00fa kr\u00e1tke \u00faseky jednovl\u00e1knovej DNA, ka\u017ed\u00fd s d\u013a\u017ekou pribli\u017ene 20 p\u00e1rov b\u00e1z. Osobitos\u0165 primerov spo\u010d\u00edva v tom, \u017ee zodpovedaj\u00fa \u00fasekom DNA, ktor\u00e9 je potrebn\u00e9 amplifikova\u0165, teda DNA materskej. <\/p>\n

\"Zomrel<\/a><\/noindex>
\n(Obr\u00e1zok je klikate\u013en\u00fd) Kary Mullis, vyn\u00e1lezca PCR<\/i><\/p>\n

\u0160pecifickos\u0165 PCR je zalo\u017een\u00e1 na tvorbe komplement\u00e1rnych komplexov medzi matricou a primerami, kr\u00e1tkymi syntetick\u00fdmi oligonukleotidmi. Ka\u017ed\u00fd z primerov je komplement\u00e1rny jednej z re\u0165azcov dvojz\u00e1vitovej matice a obmedzuje za\u010diatok a koniec amplifikovanej oblasti. Fakticky z\u00edskan\u00e1 \u201ematrika\u201c predstavuje cel\u00fd gen\u00f3m a na\u0161\u00edm cie\u013eom je izolova\u0165 z nej fragmenty, ktor\u00e9 n\u00e1s zauj\u00edmaj\u00fa. Na tento \u00fa\u010del sa dvojz\u00e1vitov\u00e1 DNA matica zahreje na 95 \u00b0C na nieko\u013eko min\u00fat, aby sa re\u0165azce DNA rozdelili. T\u00e1to f\u00e1za sa naz\u00fdva denatur\u00e1cia, preto\u017ee sa ni\u010dia vod\u00edkov\u00e9 v\u00e4zby medzi dvoma re\u0165azcami DNA. Ke\u010f sa re\u0165azce rozdelia, teplota sa zn\u00ed\u017ei, aby sa primery mohli viaza\u0165 na jednov\u00e4zobn\u00fa maticu. DNA polymer\u00e1za za\u010d\u00edna replik\u00e1ciu DNA, pri\u010dom sa via\u017ee na \u00fasek re\u0165azca nukleotidov. Enzym DNA polymer\u00e1za replikujte maticov\u00fd re\u0165azec a pou\u017e\u00edva primer ako n\u00e1stroj alebo vzor na kop\u00edrovanie. V\u00fdsledkom prv\u00e9ho cyklu je opakovan\u00e9 sekven\u010dn\u00e9 zdvojenie konkr\u00e9tneho \u00faseku DNA. Potom t\u00fato proced\u00faru opakujeme. Po ka\u017edom cykle z\u00edskavame cie\u013eov\u00fd \u00fasek v dvojn\u00e1sobnom mno\u017estve. Po dvadsiatich piatich cykloch PCR (t.j. za menej ako dve hodiny) m\u00e1me \u00fasek DNA, ktor\u00fd n\u00e1s zauj\u00edma, v mno\u017estve 225-kr\u00e1t vy\u0161\u0161om ako p\u00f4vodn\u00e9 (t.j. amplifikovali sme ho pribli\u017ene 34 mili\u00f3nov kr\u00e1t). Fakticky sme na vstupe mali zmes primerov, materskej DNA, enz\u00fdmu DNA polymer\u00e1zy a vo\u013en\u00fdch b\u00e1z A, C, G a T, mno\u017estvo \u0161pecifick\u00e9ho produktu reakcie (obmedzen\u00e9ho primerami) rastie exponenci\u00e1lne, zatia\u013e \u010do mno\u017estvo \u201edlh\u00fdch\u201c k\u00f3pi\u00ed DNA rastie line\u00e1rne, preto v produktoch reakcie dominuje.<\/p>\n

\"Zomrel
\nAmplifik\u00e1cia po\u017eadovan\u00e9ho \u00faseku DNA: polymer\u00e1zov\u00e1 re\u0165azov\u00e1 reakcia<\/i><\/p>\n

Na za\u010diatku pou\u017e\u00edvania PCR bola hlavn\u00e1 probl\u00e9m v tom, \u017ee po ka\u017edom cykle zahriatia a ochladenia bolo potrebn\u00e9 prida\u0165 do reak\u010dnej zmesi DNA polymer\u00e1zu, preto\u017ee sa inaktivovala pri teplote 95 \u00b0C. Preto bolo potrebn\u00e9 ju op\u00e4tovne prid\u00e1va\u0165 pred ka\u017ed\u00fdm z 25 cyklov. Proced\u00fara vykon\u00e1vania reakcie bola relat\u00edvne neefekt\u00edvna, vy\u017eadovala ve\u013ea \u010dasu a enz\u00fdmu polymer\u00e1zy, \u010do nie je lacn\u00fd materi\u00e1l. Na\u0161\u0165astie pr\u00edroda pri\u0161la na pomoc. Mnoho zvierat sa c\u00edti pohodlne pri teplot\u00e1ch, ktor\u00e9 s\u00fa ove\u013ea vy\u0161\u0161ie ako 37 \u00b0C. Pre\u010do sa pre n\u00e1s stala t\u00e1to teplota d\u00f4le\u017eitou? Stalo sa to preto, \u017ee t\u00e1to teplota je optim\u00e1lna pre E. coli, z ktorej sa p\u00f4vodne z\u00edskavala enz\u00fdm polymer\u00e1zy pre PCR. V pr\u00edrode sa nach\u00e1dzaj\u00fa mikroorganizmy, ktor\u00fdch prote\u00edny sa v priebehu mili\u00f3nov rokov prirodzen\u00e9ho v\u00fdberu stali odolnej\u0161\u00edmi vo\u010di vysok\u00fdm teplot\u00e1m. Bolo navrhnut\u00e9 pou\u017e\u00edva\u0165 DNA polymer\u00e1zy z termofiln\u00fdch bakt\u00e9ri\u00ed. Tieto enz\u00fdmy sa uk\u00e1zali ako termostabiln\u00e9 a boli schopn\u00e9 zvl\u00e1dnu\u0165 mnoho cyklov reakcie. Ich pou\u017eitie umo\u017enilo zjednodu\u0161i\u0165 a automatizova\u0165 vykon\u00e1vanie PCR. Jedna z prv\u00fdch termostabiln\u00fdch DNA polymer\u00e1z bola izolovan\u00e1 z bakt\u00e9rie Thermus aquaticus, ktor\u00e1 \u017eije v hor\u00facich prame\u0148och N\u00e1rodn\u00e9ho parku Yellowstone, a nazvala sa Taq polymer\u00e1zou.<\/p>\n

PCR sa r\u00fdchlo stala hlavnou pracovnou silou projektu \"Gen\u00f3m \u010dloveka\". V podstate je proces rovnak\u00fd ako ten, ktor\u00fd vyvinul Mullis, len bol automatizovan\u00fd. U\u017e sme sa nespoliehali na skupinu slep\u00fdch postgradu\u00e1lnych \u0161tudentov, ktor\u00ed starostlivo prelievali kvapky tekutiny do plastov\u00fdch sk\u00famaviek. V modern\u00fdch laborat\u00f3ri\u00e1ch, ktor\u00e9 vykon\u00e1vaj\u00fa molekul\u00e1rno-genetick\u00fd v\u00fdskum, sa t\u00e1to pr\u00e1ca vykon\u00e1va na robotizovan\u00fdch link\u00e1ch. PCR roboty zapojen\u00e9 do tak\u00e9hoto rozsiahleho projektu sekvenovania ako \"Gen\u00f3m \u010dloveka\" ne\u00faprosne pracuj\u00fa s obrovsk\u00fdmi objemami termostabilnej polymer\u00e1zy. Niektor\u00ed vedci zapojen\u00ed do projektu \"Gen\u00f3m \u010dloveka\" boli pob\u00faren\u00ed neod\u00f4vodnen\u00fdmi vysok\u00fdmi poplatkami, ktor\u00e9 majite\u013e patentu na PCR, eur\u00f3psky priemyseln\u00fd farmaceutick\u00fd gigant Hoffmann-LaRoche, prid\u00e1va k n\u00e1kladom na spotrebn\u00fd materi\u00e1l.<\/p>\n

Druh\u00fdm \"hnac\u00edm motorom\" bol samotn\u00fd met\u00f3da sekvenovania DNA. Chemick\u00fd z\u00e1klad tejto met\u00f3dy u\u017e vtedy nebol novinkou: Medzin\u00e1rodn\u00fd projekt \"Gen\u00f3m \u010dloveka\" (HGP) prijal rovnak\u00fd vynaliezav\u00fd pr\u00edstup, ktor\u00fd vyvinul Fred Sanger u\u017e v polovici 70. rokov. Inov\u00e1cia spo\u010d\u00edvala v rozsahu a \u00farovni automatiz\u00e1cie, ktor\u00fdch sa podarilo dosiahnu\u0165 pri sekvenovan\u00ed.<\/p>\n

Automatick\u00e9 sekvenovanie sa p\u00f4vodne vyv\u00edjalo v laborat\u00f3riu Li Huda na Kalifornskom technologickom in\u0161tit\u00fate. Ukon\u010dil \u0161kolu v \u0161t\u00e1te Montana a hral americk\u00fd futbal na poz\u00edcii \u00fato\u010dn\u00edka; v\u010faka Hudovi t\u00edm nieko\u013ekokr\u00e1t vyhral \u0161t\u00e1tny \u0161ampion\u00e1t. Zru\u010dnosti t\u00edmovej spolupr\u00e1ce mu pomohli aj v jeho vedeckej kari\u00e9re. V Hudovom laborat\u00f3riu pracoval pestrofarebn\u00fd t\u00edm chemikov, biol\u00f3gov a in\u017einierov, a \u010doskoro sa jeho laborat\u00f3rium stalo l\u00eddrom v oblasti technologick\u00fdch inov\u00e1ci\u00ed.<\/p>\n

Met\u00f3da automatick\u00e9ho sekvenovania bola v skuto\u010dnosti vyn\u00e1jden\u00e1 Lloydom Smithom a Mikeom Hunkapillerom. Mike Hunkapiller, ktor\u00fd vtedy pracoval v Hudovej laborat\u00f3rii, oslovil Lloyda Smitha s n\u00e1vrhom na zdokonalen\u00fa met\u00f3du sekvenovania, v ktorej by boli b\u00e1zy ka\u017ed\u00e9ho typu sfarben\u00e9 do r\u00f4znych farieb. Tak\u00fdto koncept mohol zv\u00fd\u0161i\u0165 efektivitu Sangerovho procesu a\u017e \u0161tvorn\u00e1sobne. Pri sekvenovan\u00ed od Sangeru sa v ka\u017edej zo \u0161tyroch sk\u00famaviek (pod\u013ea po\u010dtu b\u00e1z) za \u00fa\u010dasti DNA polymer\u00e1zy vytv\u00e1ra jedine\u010dn\u00fd s\u00fabor oligonukleotidov r\u00f4znej d\u013a\u017eky, ktor\u00fd zah\u0155\u0148a prim\u00e9rov\u00fa sekvenciu. N\u00e1sledne sa do sk\u00famaviek prid\u00e1val formamid na rozpad re\u0165azcov a vykon\u00e1val sa elektrofor\u00e9za v polyakrylamidovom g\u00e9li na \u0161tyroch dr\u00e1hach. V variante Smitha a Hunkapillera s\u00fa didenzoxynukleotidy ozna\u010den\u00e9 \u0161tyrmi r\u00f4znymi farbivami a PCR sa vykon\u00e1va v jednej sk\u00famavke. Potom, po\u010das elektrofor\u00e9zy na polyakrylamidovom g\u00e9li, l\u00fa\u010d laseru na ur\u010denom mieste g\u00e9lu aktivuje farbiv\u00e1 a detektor ur\u010duje, ktor\u00fd nukleotid moment\u00e1lne migruje cez g\u00e9l. Sprvu bol Smith pesimisticky naladen\u00fd \u2014 ob\u00e1val sa, \u017ee pou\u017eitie ultra mal\u00fdch d\u00e1vok farbiva povedie k tomu, \u017ee nukleotidov\u00e9 \u00faseky bud\u00fa nezrozumite\u013en\u00e9. Av\u0161ak v\u010faka vynikaj\u00facemu ovl\u00e1daniu laserov\u00fdch technol\u00f3gi\u00ed \u010doskoro na\u0161iel rie\u0161enie, ke\u010f pou\u017eil \u0161peci\u00e1lne fluorescen\u010dn\u00e9 farbiv\u00e1, ktor\u00e9 fluorescuj\u00fa pri p\u00f4soben\u00ed laserov\u00e9ho \u017eiarenia.<\/p>\n

\"Zomrel
\n(\u00dapln\u00e1 verzia po kliknut\u00ed<\/a><\/noindex> \u2014 4,08 MB) Mal\u00fdm p\u00edsmom: sekvencia DNA, ktor\u00fa dek\u00f3doval automatick\u00fd sekven\u00e1tor, z\u00edskan\u00e1 z pr\u00edstroja na automatick\u00e9 sekvenovanie. Ka\u017edej farbe zodpoved\u00e1 jedna z \u0161tyroch b\u00e1z.<\/i> <\/p>\n

V klasickej variante met\u00f3dy Sanger sa jedna z re\u0165azcov analyzovanej DNA pou\u017e\u00edva ako matrica na synt\u00e9zu komplement\u00e1rneho re\u0165azca enz\u00fdmom DNA polymer\u00e1zou, potom sa sekvencia fragmentov DNA triedi v g\u00e9li pod\u013ea ve\u013ekosti. Ka\u017ed\u00fd fragment, ktor\u00fd sa zah\u0155\u0148a do DNA po\u010das synt\u00e9zy a umo\u017e\u0148uje n\u00e1sledne vizualizova\u0165 produkty reakcie, je ozna\u010den\u00fd fluorescen\u010dn\u00fdm farbivom, ktor\u00e9 zodpoved\u00e1 termin\u00e1lnej b\u00e1ze (o tom sa hovorilo na str. 124); preto fluorescencia tohto fragmentu bude identifik\u00e1torom pre dan\u00fa b\u00e1zu. Potom sta\u010d\u00ed len vykona\u0165 detekciu a vizualizova\u0165 produkty reakcie. V\u00fdsledky analyzuj\u00fa pomocou po\u010d\u00edta\u010da a prezentuj\u00fa vo forme sekvencie r\u00f4nofarebn\u00fdch \u0161pic, ktor\u00e9 zodpovedaj\u00fa \u0161tyrom nukleotidom. \u010ealej sa inform\u00e1cie pren\u00e1\u0161aj\u00fa priamo do informa\u010dn\u00e9ho syst\u00e9mu po\u010d\u00edta\u010da, \u010d\u00edm sa vylu\u010duje \u010dasovo n\u00e1ro\u010dn\u00fd a niekedy mu\u010div\u00fd proces zad\u00e1vania \u00fadajov, ktor\u00fd ve\u013emi komplikoval sekvenovanie.<\/p>\n

\u00bb Podrobnej\u0161ie inform\u00e1cie o knihe n\u00e1jdete na webovej str\u00e1nke vydavate\u013ea<\/a><\/noindex>
\n\u00bb Obsah<\/a><\/noindex>
\n\u00bb \u00daryvok<\/a><\/noindex><\/p>\n

Pre Habr \u010ditate\u013eov z\u013eava 25% s kup\u00f3nom \u2014 PCR<\/b>
\n
Zdroj: habr.com<\/a><\/p>","protected":false,"gt_translate_keys":[{"key":"rendered","format":"html"}]},"excerpt":{"rendered":"

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