Stage Five - Growth/cloning of the population of host cells

• The polymerase chain reaction (PCR) is a method of copying fragments of DNA. This process is automated, making it both rapid and efficient.
• The polymerase chain reaction requires five different elements : a DNA fragment (to be copied), DNA polymerase enzyme, primers, nucleotides and a thermocycler.
• The DNA polymerase enzyme is capable of joining together many nucleotides rapidly to form a new DNA strand. In PCR, taq polymerase is used because it is highly thermostable and doesn't denature under high temperatures. This is because this enzyme is obtained from bacteria living in hot springs (extremophiles).
• Primers are short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments.
• Nucleotides contain each of the four bases found in DNA.
• A thermocycler is a computer-controlled machine that varies temperature precisely over a period of time.
• There are three major stages within the polymerase chain reaction (PCR): separation of the DNA strand, annealing of the primers and synthesis of DNA.
• During the separation of the DNA strand in PCR, the DNA fragments, primers and DNA polymerase are placed into a thermocycler vessel. The temperature is increased to 95 degrees, causing the two strands of DNA to separate due to breaking of the hydrogen bonds between the complementary base pairs.
• After separation of the DNA strands, the mixture is cooled to 55 degrees, causing the primers in the mixture to anneal to their complementary bases at the end of the DNA fragment.
• The primers provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain. Primers also prevent the two separate strands from simply rejoining.
• Once the primers have annealed, the temperature is increased to 72 degrees, which is the optimum temperature for the action of DNA polymerase to add complementary nucleotides along each of the separated DNA strands. It begins at the primer on both strands and adds nucleotides in sequence until it reaches the end of the chain.
• In PCR, both the separated strands are copied simultaneously, so there are now two copies of the original fragment. Once the two DNA strands are complete, the process is repeated by repeating the temperature cycle, resulting in four strands. Each temperature cycle takes approximately 2 minutes, and this is repeated many times.