Biochemical Techniques and Instrumentation

6.1.2. Technique

PCR technique is used to amplify a specific region of a DNA strand known as the target DNA. It amplifies DNA fragments of up to ~10 kilo base pairs (kb). There are some techniques that can amplify DNA fragments upto 40 kb in size. A heat-stable DNA polymerase known as "Taq polymerase" is used for the amplification the target DNA into millions of copies. This enzyme is obtained from the thermophilic bacterium, Thermus aquaticus, which occurs naturally in hot environments of 50 to 80°C. This enzyme can withstand high temperatures of >90 °C which is required for separation of the two DNA strands. The enzyme also assembles a new DNA strand by using single-stranded DNA as a template and DNA oligonucleotides as the primers. Primers are short (oligo) DNA fragments that contain sequences complementary to the target DNA. Primers and dNTPs (deoxyribo nucleotide triphosphates) are required for the synthesis of DNA. Amplification depends on thermal cycling process which consists of cycles of repeated heating and cooling. As the cycle progresses, the DNA generated itself acts as a template for replication, and sets a motion for the chain reaction.