3 Why this name – PCR?Why do we use PCR? Short region of a DNA molecule a single gene for example, is copied many times by a DNA polymerase enzyme. At the end of PCR we get plenty of copies target DNA to analyze it by sing other methods – hybridisation, RFLP, sequencing and so on.
8 Primery krátké oligonukleotidy (18 – 30 nukleotidů) přímý primer x zpětný primerjsou komplementární k sekvencímna 3´konci odpovídajícího řetězce,ohraničujícím cílovou oblast DNA,kterou chceme amplifikovatBecause they correspond with the sequences flanking the target region on the template molecule, they delimit
10 Anelační teplota Tm = [4 x (G+C) + 2 x (A+T)] Tanneal. = [4 x (G+C) + 2 x (A+T) -5]5´ GAGACTCAAGAGAACCTA 3´Tanneal. = ?
11 The other components we will need for extension are free nucleotides and polymerase. This is enzyme that catalyze polymerisation of new strand. Polymerase needs to this matrice (this is template DNA), free nucleotides from them is the new strand created. ((The nucleosides enter the reaction initially as energy-rich nucleoside triphosphates, which provide the energy for the polymerisation reaction. The hydrolysis of one phosphoanhydride bond in the nucleoside triphosphate provides the energy for the condensation reaction that links the nucleoside monomer to the chain and releases pyrophosphate. The DNA polymeraze couples the release of this energy to the polymerization reaction. )) And primers, that are the start place, the beginning of polymerase work.Amplification is usually carried out by the DNA polymerase I enzyme from Thermus aquaticus, this is bacterium. This organism lives in hot springs and many of its enzymes, including Taq polymerase, are thermostable, meaning that they are resistant to denaturation by heat treatment. The thermostability of the Taq polymerase is an essential requirement in PCR methodology.
16 Modifikace PCR nested PCR multiplex PCR se sekvenčně specifickými primeryAsymetric PCR is PCR modification that prefers synthesis of only one strand od ds DNA. This is caused in the case where one of two primers is in abundance. The PCR product is ss DNA used f.e. for sequencing
17 Analýza PCR produktu PCR s obecnými primery : Hybridizace Sekvenování RFLP (Restriction Fragment LengthPolymorphism)RT-PCR (kvantitativní PCR)RNA => cDNA