Razlika između inačica stranice »Lančana reakcija polimeraze«

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uklonjena promjena suradnika Drdroga (razgovor), vraćeno na posljednju inačicu suradnika Amherst99
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Oznake: VisualEditor vandalizam? Uklonjeno uređivanje
m (uklonjena promjena suradnika Drdroga (razgovor), vraćeno na posljednju inačicu suradnika Amherst99)
Oznake: brzo uklanjanje SWViewer [1.4]
{{nedostaju izvori}}
'''Lančana reakcija polimeraze''' poznata i pod [[engleski jezik|engleskom]] kraticom '''PCR''' (''polymerase chain reaction'') metoda je koja omogućuje kopiranje DNA dijelova pomoću enzima DNA polimeraze. Ovom metodom se klonira
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[[DNK]] bez potrebe za živim stanicama. Metodu je 1983. razvio američki biokemičar Kary Mullis za koju je 1993. dobio [[Nobelova nagrada za kemiju|Nobelovu nagradu za kemiju]].<ref>https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/|Polymerase Chain Reaction (PCR)</ref>
 
Bit [[kloniranje|kloniranja]] DNK je u izolaciji jednog lanca DNA iz složene smjese molekula DNK. Kloniranje je postiglo velike korake u razumijevanju sastava i funkcije gena i genoma. Klasično kloniranje ima veliki nedostatak, naime da je izuzetno dugotrajno i tehnički zahtjevno. Klasično kloniranje fragmenata DNK može potrajati nekoliko dana, jer su za umetanje u vektor kloniranja potrebne opsežne promjene DNK. Nakon ove faze slijedi prijenos vektora u stanicu domaćina, a kasnije i odabir odgovarajućih rekombinanta. PCR usavršava kloniranje dopuštajući pročišćavanje određenog fragmenta DNK u samo nekoliko sati. Ipak, PCR ima određena ograničenja. Što je najvažnije, moramo znati barem jedan dio fragmenta koji želimo klonirati.<ref>https://hr.iliveok.com/health/lancana-reakcija-polimeraze-pcr-u-dijagnostici-zaraznih-bolesti_75249i15983.html|Lančana reakcija polimeraze (PCR) u dijagnostici bolesti</ref>
 
Kopiranje segmenata DNA vrši se korištenjem dvije kratke molekule DNA koje su komplementarne početnom i završnom dijelu segmenta koji se kopira, dodavanjem enzima, fiziološke otopine i deoksiribonukleotida (blokovi DNK). Reakcija se odvija u lancu; odnosno ponavljamo nekoliko puta. Sa svakom ponavljanjem broj kopija pomnoženog odjeljka eksponencijalno se povećava; tako se nakon 20 ponavljanja reakcije teoretski može dobiti više od 1 milijun kopija iz 1 kopije DNA. Temperatura reakcije mora se kontrolirati na kontroliran način. Da bi se kratke molekule DNK (početni [[oligonukleotid]]i) vezale za matricu koju želimo umnožiti, potrebno je da je matrica u jednolančanom obliku (DNK je obično dvolančana molekula), a to se postiže kratkotrajnim zagrijavanjem DNA na 92 ​​°C. ili više (ovaj stupanj naziva se denaturacija DNA). Da bi se početni oligonukleotidi prilijepili na uzorak, temperatura se mora ponovno spustiti (na 37 °C – 55 °C), a zatim ponovno prilagoditi uvjetima koji su optimalni za djelovanje DNK polimeraze.
 
Nekoliko godina nakon što je Mullis opisao PCR metodu, drugi su je autori usavršili uporabom polimeraze [[Escherichia coli]] (koja ima optimalnu radnu temperaturu od 37 °C i razgrađuje se pri visokim temperaturama denaturacije) umjesto DNA polimeraze. bakterija Thermus aquaticus, pronađena u vrućim izvorima u američkom nacionalnom parku [[Yellowstone]]. Ova [[polimeraza]] je termostabilna: optimalno djeluje na 72 °C i može podnijeti kratkotrajno zagrijavanje iznad 90 °C. Stoga više nije bilo potrebno dodavati svježi enzim u svaki ciklus, što je omogućilo automatizaciju postupka.
 
PCR se izvodi u precizno programabilnim termostatima. Možemo odrediti temperature, trajanje [[inkubacija|inkubacije]], broj ponavljajućih [[ciklus]]a i slično. Reakcije se izvode u količinama od 20 μl do 100 μl. U klasičnoj izvedbi, koja je i dalje najčešća, proizvodi se analiziraju elektroforezom u agaroznom gelu nakon završetka reakcije. Novija implementacija, PCR u stvarnom vremenu, omogućuje praćenje količine nastalog proizvoda u stvarnom vremenu i stoga je brža, ali omogućuje i kvantificiranje određene DNK u početnom uzorku.
[[Datoteka:Polymerase chain reaction-en.svg|frameless|center|Prikaz ciklusa PCR|1024x438px]]
== Vanjske poveznice ==
{{Commons|Polymerase chain reaction|Lančana reakcija polimeraze}}