{"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\/ro\/blog\/news\/umer-nobelevskij-laureat-keri-mullis-izobretatel-polimeraznoj-tsepnoj-reaktsii-dnk","title":{"rendered":"A murit laureatul Nobel Carey Mullis \u2014 inventatorul reac\u021biei de polimerizare a lan\u021bului de ADN","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"

\"A \u00cen California, la v\u00e2rsta de 74 de ani, a murit laureatul american al Premiului Nobel pentru chimie, Carey Mullis. Potrivit so\u021biei sale, decesul a avut loc pe 7 august. Cauza a fost insuficien\u021b\u0103 cardiac\u0103 \u0219i respiratorie din cauza pneumoniei.<\/p>\n

Despre contribu\u021bia sa \u00een biochimie \u0219i motivul pentru care a primit Premiul Nobel ne va povesti chiar James Watson \u2014 descoperitorul moleculei de ADN. <\/p>\n

Fragment din cartea lui James Watson, Andrew Berry, Kevin Davis <\/p>\n

ADN. Istoria revolu\u021biei genetice<\/h2>\n

Capitolul 7. Genomul uman. Scenariul vie\u021bii<\/h3>\n

\n<\/a><\/noindex>
\n\u2026
\nReac\u021bia de polimerizare a lan\u021bului (PCR) a fost inventat\u0103 \u00een 1983 de biochimistul Carey Mullis, care lucra pentru compania Cetus. Descoperirea acestei reac\u021bii a fost deosebit de notabil\u0103. Ulterior, Mullis a rememorat: \u201e\u00centr-o sear\u0103 de vineri, \u00een aprilie 1983, m-a lovit ca un flash. Eram la volan, rul\u00e2nd pe un drum de munte \u00eentortocheat, luminat de lun\u0103, \u00een California de Nord, \u021bara p\u0103durilor de secuoya.\u201d Este impresionant c\u0103 inspira\u021bia l-a vizitat \u00een acea situa\u021bie. \u0218i nu este vorba c\u0103 drumurile din California de Nord sunt speciale, favorabile inspira\u021biei; pur \u0219i simplu, un prieten de-al s\u0103u a v\u0103zut odat\u0103 cum Mullis se \u00eendrepta f\u0103r\u0103 grij\u0103 pe o \u0219osea \u00eenghe\u021bat\u0103 cu circula\u021bie \u00een ambele sensuri \u0219i acest lucru nu-l deranja deloc. Prietenul a spus ziarului New York Times: \u201eMullis a avut viziunea c\u0103 va muri \u00eentr-un accident cu o secuoya. A\u0219a c\u0103 nu-i este fric\u0103 la volan, at\u00e2ta timp c\u00e2t pe marginea drumului nu cresc secuoyas.\u201d Prezen\u021ba secuoyas pe marginea drumului \u00eel obliga pe Mullis s\u0103 se concentreze \u0219i\u2026 a\u0219a a venit inspira\u021bia. Pentru inven\u021bia sa, \u00een 1993, Mullis a primit Premiul Nobel pentru chimie, iar de atunci a devenit \u0219i mai excentric \u00een comportamentul s\u0103u. De exemplu, el sus\u021bine teoria revizionist\u0103 c\u0103 SIDA nu este legat\u0103 de HIV, ceea ce i-a subminat semnificativ propria reputa\u021bie \u0219i a \u00eempiedicat medicii.<\/p>\n

PCR este o reac\u021bie destul de simpl\u0103. Pentru a o efectua, avem nevoie de doi primers sintetiza\u021bi chimic, complementari la capetele opuse ale diferitelor lan\u021buri ale fragmentului de ADN necesar. Primerele sunt secven\u021be scurte de ADN monocatenar, fiecare av\u00e2nd aproximativ 20 de perechi de baze \u00een lungime. Caracteristica primelor este c\u0103 acestea corespund secven\u021belor de ADN care trebuie amplificate, adic\u0103 matricei ADN. <\/p>\n

\"A<\/a><\/noindex>
\n(Imaginea este clicabil\u0103) Carey Mullis, inventatorul PCR<\/i><\/p>\n

Specificitatea PCR se bazeaz\u0103 pe formarea complexelor complementare \u00eentre matrice \u0219i primeri, scurte oligonucleotide sintetice. Fiecare dintre primeri este complementar\u0103 uneia dintre lan\u021burile matricei cu dou\u0103 fire \u0219i delimiteaz\u0103 \u00eenceputul \u0219i sf\u00e2r\u0219itul secven\u021bei amplificabile. Practic, matricea ob\u021binut\u0103 reprezint\u0103 un genom \u00eentreg, iar scopul nostru este de a extrage fragmentele de interes din ea. Pentru aceasta, matricea de ADN cu dou\u0103 fire este \u00eenc\u0103lzit\u0103 la 95 \u00b0C timp de c\u00e2teva minute pentru a separa lan\u021burile de ADN. Aceast\u0103 etap\u0103 se nume\u0219te denaturare, deoarece leg\u0103turile de hidrogen dintre cele dou\u0103 lan\u021buri de ADN sunt distruse. Dup\u0103 ce lan\u021burile sunt separate, temperatura este redus\u0103 pentru ca primerii s\u0103 se poat\u0103 lega de matricea cu un singur fir. ADN-polimeraza \u00eencepe replicarea ADN-ului, leg\u00e2ndu-se de segmentul lan\u021bului de nucleotide. Fermentul ADN-polimeraza replic\u0103 lan\u021bul matricei, folosind primerul ca model sau ca exemplu pentru copiere. Ca urmare a primului ciclu, ob\u021binem o dublare secven\u021bial\u0103 a unei anumite sec\u021biuni de ADN. Apoi repet\u0103m aceast\u0103 procedur\u0103. Dup\u0103 fiecare ciclu, ob\u021binem sec\u021biunea \u021bint\u0103 \u00eentr-o cantitate dubl\u0103. Dup\u0103 dou\u0103zeci \u0219i cinci de cicluri PCR (adic\u0103 \u00een mai pu\u021bin de dou\u0103 ore) avem sec\u021biunea de ADN dorit\u0103 \u00eentr-o cantitate de 225 de ori mai mare dec\u00e2t ini\u021bial (adic\u0103 am amplificat-o de aproximativ 34 de milioane de ori). Practic, la intrare aveam un amestec de primeri, ADN matrice, ferment ADN-polimeraza \u0219i baze libere A, C, G \u0219i T, iar cantitatea de produs specific al reac\u021biei (limitata de primeri) cre\u0219te exponen\u021bial, \u00een timp ce cantitatea de copii \u201elungi\u201d de ADN cre\u0219te linear, astfel c\u0103 \u00een produsele reac\u021biei predomin\u0103.<\/p>\n

\"A
\nAmplificarea sec\u021biunii dorite de ADN: reac\u021bia de polimeraz\u0103 \u00een lan\u021b<\/i><\/p>\n

La \u00eenceputul utiliz\u0103rii PCR, principala problem\u0103 consta \u00een faptul c\u0103, dup\u0103 fiecare ciclu de \u00eenc\u0103lzire-r\u0103cire, trebuia ad\u0103ugat \u00een amestecul de reac\u021bie ADN polimeraza, deoarece aceasta se inactivaba la temperatura de 95 \u00b0C. De aceea, era necesar s\u0103 o ad\u0103ug\u0103m din nou \u00eenainte de fiecare dintre cele 25 de cicluri. Procedura de realizare a reac\u021biei era relativ ineficient\u0103, necesita mult timp \u0219i polimeraza era un material destul de costisitor. Din fericire, natura ne-a venit \u00een ajutor. Multe animale se simt bine la temperaturi mult mai mari de 37 \u00b0C. \u0218i de ce a devenit important\u0103 pentru noi cifra de 37 \u00b0C? Acest lucru s-a \u00eent\u00e2mplat deoarece aceast\u0103 temperatur\u0103 este optim\u0103 pentru E. coli, din care ini\u021bial se ob\u021binea enzima polimerazic\u0103 pentru PCR. \u00cen natur\u0103 exist\u0103 microorganisme ale c\u0103ror proteine, dup\u0103 milioane de ani de selec\u021bie natural\u0103, au devenit mai rezistente la ac\u021biunea temperaturilor ridicate. S-a propus utilizarea ADN polimerazelor din bacterii termofile. Aceste enzime s-au dovedit a fi termostabile \u0219i au fost capabile s\u0103 reziste la numeroase cicluri de reac\u021bie. Utilizarea lor a simplificat \u0219i automatizat realizarea PCR-ului. Una dintre primele ADN polimeraze termostabile a fost izolat\u0103 din bacteria Thermus aquaticus, care tr\u0103ie\u0219te \u00een izvoarele termale ale Parcului Na\u021bional Yellowstone, \u0219i a fost numit\u0103 polimeraza Taq.<\/p>\n

PCR a devenit rapid calul de b\u0103taie principal al proiectului \u201eGenomul uman\u201d. \u00cen esen\u021b\u0103, procesul nu difer\u0103 de cel dezvoltat de Mullis, doar c\u0103 a fost automatizat. Nu mai depindeam de o mul\u021bime de studen\u021bi robi, care turnau cu grij\u0103 pic\u0103turi de lichid \u00een eprubete de plastic. \u00cen laboratoarele moderne care efectueaz\u0103 cercet\u0103ri moleculare \u0219i genice, aceast\u0103 munc\u0103 este realizat\u0103 pe linii de produc\u021bie robotic\u0103. Robo\u021bii PCR, implica\u021bi \u00eentr-un astfel de proiect de amploare cum este \u201eGenomul uman\u201d, lucreaz\u0103 neobosi\u021bi cu volume imense de polimeraz\u0103 rezistent\u0103 la c\u0103ldur\u0103. Unii cercet\u0103tori care lucreaz\u0103 \u00een proiectul \u201eGenomul uman\u201d au fost indigna\u021bi de redeven\u021bele nejustificat de mari pe care le adaug\u0103 la costul materialelor consumabile de\u021bin\u0103torul patentului pentru PCR, gigantul european din industria farmaceutic\u0103 Hoffmann-LaRoche.<\/p>\n

Un alt \u201emotor\u201c a fost metoda de secven\u021biere a ADN-ului. Fundamentul chimic al acestei metode nu mai era o nou\u0103 descoperire: proiectul interna\u021bional \u201eGenome uman\u201d (HGP) a \u00eembr\u0103\u021bi\u0219at aceea\u0219i metod\u0103 ingenioas\u0103 dezvoltat\u0103 de Fred Sanger \u00een mijlocul anilor 1970. Inova\u021bia consta \u00een amploarea \u0219i gradul de automatizare realizate \u00een procesul de secven\u021biere.<\/p>\n

Secven\u021bierea automat\u0103 a fost ini\u021bial dezvoltat\u0103 \u00een laboratorul lui Lee Hood de la California Institute of Technology. El a absolvit liceul \u00een Montana \u0219i a jucat fotbal american \u00een pozi\u021bia de atacant; datorit\u0103 lui Hood, echipa a c\u00e2\u0219tigat de mai multe ori campionatul statului. Abilit\u0103\u021bile sale de colaborare \u00een echip\u0103 s-au dovedit utile \u0219i \u00een cariera sa \u0219tiin\u021bific\u0103. \u00cen laboratorul lui Hood lucra o echip\u0103 diversificat\u0103 de chimi\u0219ti, biologi \u0219i ingineri, iar \u00een cur\u00e2nd laboratorul s\u0103u a devenit lider \u00een inova\u021bii tehnologice.<\/p>\n

De fapt, metoda de secven\u021biere automat\u0103 a fost inventat\u0103 de Lloyd Smith \u0219i Mike Hunkapiller. Mike Hunkapiller, care lucra la laboratorul Hood, i-a propus lui Lloyd Smith o metod\u0103 \u00eembun\u0103t\u0103\u021bit\u0103 de secven\u021biere, \u00een care baza fiec\u0103rui tip era colorat\u0103 diferit. Aceast\u0103 idee ar fi putut cre\u0219te eficien\u021ba procesului Sanger de patru ori. La Sanger, \u00een timpul secven\u021bierii, \u00een fiecare din cele patru eprubete (num\u0103rul bazelor) cu ajutorul ADN-polimerazei se formeaz\u0103 un set unic de oligo-nucleotide de lungimi diferite, care includ secven\u021ba primarului. Ulterior, \u00een eprubete se ad\u0103uga formamid\u0103 pentru a desface lan\u021burile \u0219i se realiza electroforez\u0103 \u00eentr-un gel de poliacrilamid\u0103 pe patru benzi. \u00cen varianta lui Smith \u0219i Hunkapiller, didesoxinucleotizii sunt marca\u021bi cu patru coloran\u021bi diferi\u021bi \u0219i se realizeaz\u0103 PCR \u00eentr-o singur\u0103 eprubet\u0103. Apoi, \u00een timpul electroforezei \u00eentr-un gel de poliacrilamid\u0103, un fascicul laser \u00eentr-un anumit loc al gelului excit\u0103 activitatea coloran\u021bilor, iar detectorul stabile\u0219te ce nucleotide migreaz\u0103 \u00een prezent prin gel. La \u00eenceput, Smith era pesimist \u2014 se temea c\u0103 utilizarea unor doze extrem de mici de colorant va face ca regiunile nucleotide s\u0103 fie indistincte. Cu toate acestea, fiind bine familiarizat cu tehnologiile laser, el a g\u0103sit repede o solu\u021bie, folosind coloran\u021bi fluorescen\u021bi speciali care fluoresceaz\u0103 sub ac\u021biunea radia\u021biei laser.<\/p>\n

\"A
\n(Versiunea complet\u0103 la clic<\/a><\/noindex> \u2014 4,08 MB) Cu litere mici: secven\u021ba ADN-ului, decodificat\u0103 folosind un secven\u021bator automat, ob\u021binut\u0103 dintr-un aparat de secven\u021biere automat\u0103. Fiec\u0103rui culoare \u00eei corespunde una dintre cele patru baze.<\/i> <\/p>\n

\u00cen varianta clasic\u0103 a metodei Sanger, una dintre lan\u021burile ADN-ului analizat ac\u021bioneaz\u0103 ca matrice pentru sinteza unui lan\u021b complementar de c\u0103tre enzima ADN-polimeraz\u0103, apoi secven\u021ba fragmentelor ADN este sortat\u0103 \u00een gel \u00een func\u021bie de dimensiune. Fiecare fragment care se integreaz\u0103 \u00een ADN-ul sintetic \u0219i permite ulterior vizualizarea produselor reac\u021biei este marcat cu un colorant fluorescent, corespunz\u0103tor bazei terminale (acest lucru a fost men\u021bionat pe p. 124); prin urmare, fluorescen\u021ba acestui fragment va fi un identificator pentru baza respectiv\u0103. Apoi, trebuie doar s\u0103 se efectueze detectarea \u0219i vizualizarea produselor reac\u021biei. Rezultatele sunt analizate cu ajutorul unui calculator \u0219i prezentate sub form\u0103 de secven\u021b\u0103 de v\u00e2rfuri colorate, corespunz\u0103toare celor patru nucleotide. Informa\u021bia este apoi transmis\u0103 direct \u00een sistemul informatic al computerului, elimin\u00e2nd procesul consumator de timp \u0219i adesea chinuitor de introducere a datelor, care \u00eengreuna semnificativ secven\u021bierea.<\/p>\n

\u00bb Pute\u021bi g\u0103si mai multe detalii despre carte pe site-ul editurii<\/a><\/noindex>
\n\u00bb Cuprins<\/a><\/noindex>
\n\u00bb Fragment<\/a><\/noindex><\/p>\n

Pentru utilizatorii Habr, discount de 25% cu codul \u2014 PCR<\/b>
\n
Sursa: habr.com<\/a><\/p>","protected":false,"gt_translate_keys":[{"key":"rendered","format":"html"}]},"excerpt":{"rendered":"

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