Polymerase chain reaction (PCR), a breakthrough in scientific research, led to the evolution of modern science. PCR technique was invented by Kary Mullis who was later awarded the Noble Prize in Chemistry in 1993. For most biological research conducted in various laboratories, the starting material is DNA or gene, which might be required in large quantities. Thus, amplification of DNA becomes a prerequisite. PCR enables multiple DNA copies in a very small amount of time.
PCR for instance is just like a recipe that involves specific ingredients tossed in the right amount.
Before understanding PCR let’s look into some basic terminology –
DNA – DNA or deoxyribonucleic acid stores all the genetic information that is passed from one generation to another (parent to child). This information includes features such as skin color, behavior, immunity, and also specific species identity (i.e., Human). DNA is found in almost all cells of our body (there are always exceptions in biology) and is the same. DNA is a polymer that is composed of basic building units (known as nucleotides) – adenine (A), thymine (T), cytosine (C) and guanine (G). In general, the information stored in DNA is essential for basic cellular physiology.
Nucleotides – These are four codes that form the DNA (ATGC). It is made of a sugar moiety, attached with three phosphate molecules and a base. This base distinguishes the four codes and is formed of Carbon, Hydrogen, Oxygen and Nitrogen.
Gene – It is a part of DNA that is the basic and functional unit of hereditary. A gene may or may not code for a protein but is essential for the normal behavior of the cell. Every person has two copies of the gene, each derived from one of the parents. Approximately, 99.9% of genes are similar between humans. The rest 0.1% makes us different from one another.
Enzyme – Enzyme is a catalytic protein that accelerates cellular reactions. For all the physiological processes a bunch of enzymes is involved. Thus, the enzyme becomes an essential component of the cell or body.
Ingredients required –
1. DNA polymerase – The most important part of this reaction is DNA polymerase. DNA polymerase is an enzyme that adds nucleotides to a growing strand of DNA. DNA polymerase used in PCR is mostly extracted from various thermostable organisms. An optimum polymerase for PCR reaction is judged on the following characteristics –
Thermostability – Stability at a higher temperature
Extension rate – Speed at which nucleotides are added in a growing DNA chain
Fidelity – Accuracy by which correct nucleotides are added sequentially
Processivity – Number of nucleotides are added by the polymerase in a single binding event (or before getting detached from the DNA chain)
2. Template – The DNA strand (a small part of the whole DNA that contains our gene of interest) to be amplified.
3. dNTPs – It stands for deoxynucleoside triphosphates. These are monomers that are joined to a growing DNA chain amplifying it to a DNA strand.
4. Primers – These are customized small DNA strands that usually get attached to the flanking portion of our gene of interest. Primers are required for two reasons –
a. It ensures that a specified region containing our gene of interest is only amplified.
b. It helps in the initiation of DNA strand formation by providing a base to which DNA polymerase can attach nucleotides.
5. Buffer – It serves to maintain the optimum reaction condition for the DNA polymerase to function. These can contain salts and ions that assist polymerase.
STAGE I – DENATURATION
PCR mix is heated at a temperature of 98°C. This allows the separation of DNA strands. Separation is important as it gives access to primer binding.
STAGE II – ANNEALING
In the next step, the temperature is decreased to a specified value. Later depends on the primer used. This temperature allows the primer to bind to the separated DNA strands.
STAGE III – EXTENSION
This is the final step of PCR in which a newly formed DNA strand is obtained. Here, DNA polymerase at a specified temperature starts adding nucleotides to the growing DNA chain.
**Temperature and time are already set in the PCR machine before the start and therefore this is a totally automated process.
Edited by - Ms. Neha Mishra