Question #18fd9
1 Answer
DNA polymerase is important because its discovery allowed the invention of PCR (Polymerase Chain Reaction). PCR is an important tool that revolutionized the study of DNA and molecular biology. It allows the duplication of thousands copies of target loci, which are localized strands of DNA, either coding or non-coding.
These regions are targeted usually using a couple of primers, small strands composed usually by 18-21 nucleotides that are complementary to opposite regions adjacent to our target sequence on the two single strands of the DNA molecule. During a PCR, the Polymerase "fills" this gap millions of times. This "amplification" facilitates the sequencing and consequent study of these target loci.
To understand a PCR you must consider
A) The reagents involved. Apart from the DNA, in the reaction mix you will find:
- Free nucleotides, called dNTPs, that work as "building blocks" for your reaction
- Primers, already explained
- cofactors which "enhance" the enzyme performance, such as Magnesium Chloride
- the DNA polymerase.
B) The temperature cycles
A PCR usually occurs in a number of repeated cycles. Each cycle contain three main phases with different temperatures and duration;
- Denaturation phase, where the DNA is exposed at high temperatures (usually ~95˚) for 20-30s, where the two DNA strands separate;
- Annealing phase, where temperature lowers to 50-60˚ for 20-30s, the primers find their complementary regions and attach to them
- elongation step, where the polymerase finds the primer-DNA coupled strands and starts the amplification.
The most common polymerase used is Taq polymerase, which comes from a bacterium (Thermus aquaticus) that live in very warm places; this enzyme works optimally at 75-80˚! The duration of this step depends on the length of the target sequence.
The video I am linking should help to understand how a PCR works.
Polymerase Chain Reaction
