CGDP - DNA Amplification|
DNA amplification by the polymerase chain reaction (PCR) is schematically outlined in the diagram to the left. There are 3 general steps to the process that are repeated for a number of cycles to exponentially increase the number of copies of a specific target region. Genomic DNA is normally double-stranded (DS-DNA).
STEP 1 is to first unzip the DS-DNA, also called denaturation, into two complementary single strands of DNA by heating the reaction mix to 95 degrees Celsius. STEP 2 isolates the target region of the Genomic DNA by landing 2 primers (P1 & P2) which exactly match two 20-30 unique base pair regions that bookend the target region. This is called annealing. Once time is allowed for the primers to land on the sites, STEP 3 invloves heating the mix to 72 degrees at which point a special polymerase builds the DNA strand starting at the primers and continuing in the 5 prime direction. This is called extension.
These three steps are repeated 25-40 times to produce millions of exact copies of the target region of DNA. Because during the second cycle of this process, extension can occur on both the original copy of genomic DNA and the newest pieces (the colored ones in the diagram) subsequent extensions are quickly limited precisely to the target region (A).
Now we must Visualize the reactions in order to determine whether these PCR reactions worked and decide whether we can go on to sequencing.