11- AGE Polymerase Chain Reaction -PCR.ppt

Gel Electrophoresis & PCR 1 Learning objectives - To be introduced to Gel Electrophoresis - Understand the importance of gel electrophoresis and southern blotting in DNA extraction and isolation - Learn about Polymerase Chain Reaction (PCR) technique. 2 Agarose Gel Electrophoresis 3 ** Restr...

Gel Electrophoresis & PCR 1 Learning objectives - To be introduced to Gel Electrophoresis - Understand the importance of gel electrophoresis and southern blotting in DNA extraction and isolation - Learn about Polymerase Chain Reaction (PCR) technique. 2 Agarose Gel Electrophoresis 3 ** Restriction fragment analysis by Southern blotting 4 Polymerase Chain Reaction (PCR) 5 • A very quick, easy, automated method used to make copies of a specific segment of DNA • What is needed…. 1- DNA primer constructs that “bracket” the desired sequence to be cloned 2- Heat-resistant DNA polymerase 3- DNA nucleotides 4- Thermocycler 6 * Before initiating the PCR experiment : → Before initiating the PCR experiment you must have a “DNA primer construct”: is a singlestranded oligonucleotides, usually 20–35 nucleotides long, which is complementary to the respective flanking sequences of the target DNA. These synthetic oligonucleotides function as primers in PCR reactions. For a PCR experiment two constructs must be used. **Flanking sequences, are short segments on each side of the DNA fragment which bracket the DNA sequence of interest. 7 → A PCR cycle consist of the following consecutive steps (Slide 10): 1- Denature the DNA: The DNA to be amplified is heated to separate the doublestranded target DNA into single strands. 2- Annealing (binding) of primers to ssDNA: The separated strands are cooled and allowed to anneal to the two primers (one for each strand). 8 3- Chain extension: DNA polymerase and deoxyribonucleoside triphosphates (in excess) are added to the mixture to initiate the synthesis of two new chains complementary to the original DNA chains. DNA polymerase adds nucleotides to the 3′-hydroxyl end of the primer, and strand growth extends across the target DNA, making complementary copies of the target. At the completion of one cycle of replication, the reaction mixture is heated again to denature the DNA strands (of which there are now four). Each DNA strand binds a complementary primer, and the cycle of chain extension is repeated. Typically 20– 30 cycles are run during this process, amplifying the DNA by a million-fold to a billion-fold. 4- Stop the reaction by adding Taq antibody. 9 10 * Notes regarding PCR: • By using a heat-stable DNA polymerase (for example, Taq polymerase) from a bacterium (for example, Thermus aquaticus) that normally lives at high temperatures (a thermophilic bacterium), the polymerase is not denatured and, therefore, does not have to be added at each successive cycle. • Thermus aquaticusis a bacteria specie which was discovered in the hot springs of Yellowstone National Park (USA) by a microbiologist named Thomas Brock in 1969. 11 * Outcome of a PCR experiment 12