Pcr Rflp Report Pcr - 1166 Words

PCR-RFLP Report PCR What is it: The Polymerase Chain Reaction is a method that uses the capability of DNA polymerase to synthesize to new DNA strands which are matching to the template strand. A primer needs to be added to the first nucleotide due to the fact that DNA polymerase only can add a nucleotide only onto a 3 -OH group that already exists. Because of this condition, we are able to define a chosen region of template sequence which we can then generate millions to billions of copies. This technique was developed by Kary Mullis in 1983 and is a very common indispensable technique which has a variety of uses such as DNA cloning for sequencing, genetic fingerprints, detection and diagnosis of infectious disease (often cancer) etc...†¦show more content†¦To achieve this, the reaction must be heated to 94-98 Degrees Celsius for about 20 to 30 seconds. Annealing Steps: Annealing must take place in order for the hybridization of primers to the single stranded DNA template. This temperature must be high enough in order for the hybridizations to bind specifically and low enough that it is possible for hybridization of primers to occur. Annealing temperature is usually 3-5 Degrees Celsius lower than the melting temperature of the primers. Extension/elongation step: This step consists of DNA polymerase synthesizing with a new DNA strand which is complementary to the DNA template strand. This is done by adding dNTP s which complement the template in the 5 -3 strand, condensing the 5 -Phosphate group of the dNTP along with the 3 -hydroxyl group at the end of the nascent extending DNA strand. The temperature at which the reaction is treated depends upon the primer used. The duration of this step also depends on the DNA polymerase used also, and the length of DNA fragment used. Under optimal required temperature of the primer, the DNA polymerase will polymerize a thousand base pairs per minute. Final elongation: After the last PCR cycle is completed, the reaction is cooled to a temperature of 70-74 Degrees Celsius (optimal temperature needed for activity for most polymerases