PCR (Polymerase Chain Reaction)
PCR is a process of DNA amplification (replication). Polymerase is an enzyme responsible for DNA synthesis, while "Chain Reaction" means exponential growth (at the rate of 2n where n denotes the number of generation) - hence the PCR name. The process is essential for sequencing unique piece of DNA such as those from the genomic specimen, the fossil, or a single hair in a crime scene. Here's a summary of each step in running the PCR (see Figure 07): 1. A target sequence is chosen on the DNA. The sequence had to be known, or at least its two termini. 2. Since DNA replication always runs from the 5' end to the 3' end, two DNA primers were synthesized for each strand. The primer is a small complementary to the 3' end of the original DNA about 20 bp long, it acts as an anchor | point for DNA polymerase and as initiator of the copying process. 3. Modern technique uses the Taq polymerase (from a microbe in hot springs) to add nucleotide to the new strand. It has the advantage of tolerance to heating up to 94oC - well beyond the 72oC for the extension phase. 4. All four DNA nucleotide building blocks are added in sufficient quantity. 5. The sample is heated to a temperature of up to 98oC to separate the complementary strands. This step is called denaturation. 6. Then the sample is cooled. During the cooling stage, the synthetic primers found complementary sites on the separated DNA strands, whereas the two long DNA strands were unable to find each other because they were present in minute concentration. This process is called primer annealing. 7. The polymerase extended the two primers in opposite directions. As a result, two daughter DNA appeared. 8. Initially, the new DNA carries long single-stranded tail. Only at the 3rd cycle |
Figure 07 PCR Process | of denaturation-annealing-extension do first authentic copy of the molecules appear. The process can make billion copies by the 30th cycle. |
