In biotechnology, scientists often need to amplify a small part of DNA or separate out different sized DNA fragments. How do scientists do this? They use specific techniques such as PCR and Gel electrophoresis that allow them to achieve these goals. Let’s learn about these techniques in detail.
Polymerase Chain Reaction (PCR)
In a breakthrough discovery in 1985, Kary B. Mullis invented the Polymerase Chain Reaction (PCR) technique. This technique allows scientists to make millions of copies of a very small amount of DNA. It is based on the natural process of DNA replication that occurs within a cell. Let’s learn about the components and steps of this technique. A PCR reaction needs the following components:
- DNA Template – The double-stranded DNA fragment of interest, that is to be amplified.
- Primers – Short, chemically synthesized, single-stranded pieces of DNA that are complementary to the DNA fragment of interest.
- DNA Polymerase – The enzyme that elongates the primers by adding nucleotides to it, using the desired DNA fragment as a template. This enzyme needs to be able to withstand high temperatures used in the PCR reaction. Therefore, scientists isolated a thermostable DNA polymerase from the bacterium –Thermus aquaticus. It is known as Taq polymerase.
- Nucleotides – Single bases A, T, C, and G are the building blocks of DNA synthesis in a PCR reaction.
- Buffer system – The buffer contains potassium and magnesium that are essential for the DNA denaturation and renaturation steps. It also contains other factors important for enzyme activity, fidelity and stability.
Steps in a PCR Reaction
In a PCR reaction, DNA polymerase synthesizes a new DNA strand complementary to the DNA template by adding nucleotides to the primers. The above components are mixed together in PCR tubes and run on a thermal cycler, where they go through multiple cycles involving the following reactions:
- Denaturation – This step involves heating the reaction mixture to a high temperature (94°C). This denatures the double-stranded DNA template into single-strands by breaking the weak hydrogen bonds between the two DNA strands.
- Annealing – In this step, the reaction mixture is brought to a temperature of 54-60°C, to allow the primers to bind (anneal) to their complementary sequence in the template DNA.
- Elongation/Extension – In this step, the DNA polymerase sequentially adds nucleotides to the primers and extends it in the 5′ to 3′ direction.
One cycle of denaturation, annealing, and elongation amplifies the double-stranded DNA template to give two pieces of double-stranded DNA. The cycles keep repeating to produce more and more copies, increasing the number of copies exponentially.Today, PCR is very important in many fields. In medicine, it is useful to detect genetic mutations and to detect HIV, malaria etc. in clinical samples.
This technique helps to separate DNA, RNA or proteins based on their size. For example, if you want to know the sizes of the DNA fragments after restriction enzyme digestion, you can use this procedure. Here, the DNA is run through a matrix or a gel and separated out. Agarose, a natural polymer from seaweeds is the most commonly used matrix. Let’s understand how this technique works.
Working of Agarose Gel Electrophoresis
The DNA to be separated is added to the wells of an agarose gel matrix. On the application of current, the negatively charged DNA moves to the positive electrode. Since the gel is difficult to move through, the DNA fragments travel at different speeds based on their size. Larger fragments move slower, while smaller fragments fit through the gel pores easily and move faster.The separated DNA fragments are not visible under normal light. Therefore, they are visualized after staining the bands with a compound – ethidium bromide and exposing them to UV radiation. Once they are stained and exposed to UV light, you can see the separated DNA bands and determine their sizes using a DNA marker. In this way, we can cut out the desired size of DNA fragment, extract it and further ligate it with cloning vectors. The techniques of PCR and gel electrophoresis are crucial in the field of forensics, for genetic fingerprinting and identifying crime suspects.
Solved Example for You
Question: The process of PCR is based on which natural process within a cell?
Solution: The answer is ‘a’. The basic principle of PCR is replication.