Introducing mutation in Saccharomyces cerevisiae gene SCW4 using PCR method

PCR (lančana reakcija polimeraze) je metoda koja omogućava selektivno, brzo i osjetljivo umnažanje željenog slijeda nukleotida DNA. Kroz tri stupnja reakcije dobije se kao produkt željena sekvenca DNA u velikom broju kopija. U ovom radu je PCR korišten za uvođenje mutacija u gen SCW4 kvasca Saccharomyces cerevisiae na način da su konstruirana dva fragmenta od kojih je jedan sadržavao sekvence uzvodno, a drugi nizvodno od mjesta mutacije. Mutacija se odnosila na deleciju dijela gena koji kodira za 126 aminokiseline na N-terminalnom kraju proteina Scw4. Dobiveni fragmenti su zatim drugim krugom PCR-a spojeni u jedan konstrukt budući da su međusobno komplementarni u dijelu gena neposredno uzvodno i nizvodno od deletiranog dijela u nativnom genu. Dobiveni konstrukt je zatim TA-ligacijom unesen u plazmid kojim je transformirana bakterija E. coli u kojoj je umnožen i iz koje je uspješno izoliran.PCR (polymerase chain reaction) is a method enabling rapid, selective and sensitive amplification of the DNA sequence. As a result, after three reaction steps, PCR product contains large number of copies of the target DNA sequence. In this experiment, PCR was used to introduce mutation in the SCW4 gene of the yeast Saccharomyces cerevisiae. First PCR was used for creating two fragments, one fragment contained sequence upstream of mutation position in gene sequence, and the second fragment contained sequence downstream of mutation position. Mutation consisted of deletion of the part of the gene encoding for 126 aminoacids in N-terminal part of Scw4 protein. Two products of first PCR were combined into one final construct, since they are complementary in sequence upstream and downstream of the deleted region. This fragment is then introduced into the plasmid using TA-ligation method and plasmid obtained was used for transformation of E. coli. In the end, the plasmid containing mutated form of SCW4 gene was successfully multiplied and isolated from transformed bacteria