Techniques In Biotechnology

Denaturation: In the first step, the DNA is heated to a high temperature (around 94-98°C). This separates the double-stranded DNA into single-stranded templates, and thus facilitates the process of amplification. Annealing: In the second step, the temperature is lowered (around 50-65°C) so that the primers can bind to the specific sequences flanking the target DNA sequence at the 3' ends of each of the templates. Extension: The temperature is then raised again to 72°C, the optimal temperature for the DNA polymerase enzyme to extend the primers. Here, the Taq DNA polymerase extends each primer using dNTPs, and replicates the DNA, producing a copy of the target region. Repeat Cycles: The entire cycle is repeated multiple times which amplifies the target DNA exponentially, thus producing millions of copies of the desired sequence.

Technique: Two-Dimensional Gel Electrophoresis Technique combined with Mass Spectrometry or Mass Spectrometry Technique can be used for analyzing the complete protein profile of a cell. Principle of Two-dimensional Gel Electrophoresis Technique: This method separates proteins based on their charge and size. In the first dimension, the proteins are separated using Isoelectric focusing (IEF) based on their isoelectric point. Then, the proteins are separated based on their molecular weight using SDS PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis). Principle of Mass Spectrometry Technique: This technique determines the molecular weight of a chemical compound or protein by separating the molecular ions according to their mass-to-charge (m/z) ratio.

Mass to Charge Ratio: The mass-to-charge ratio (m/z) is calculated by dividing the mass of an ion (in Daltons) by its charge. Given the molecular weight of the protein ion = 10,000 Daltons and the charge of the protein ion = 5+, the mass-to-charge ratio = (10,000 Daltons)/5 = 2000.