Question

Discuss 1) The Sanger method and 2) Affinity chromatography

Answer 1

1. The Sanger method is a method of sequencing DNA developed by the famous Frederick Sanger in 1977. It is a chain termination based sequencing method which involves in vitro DNA synthesis with incorporation of modified di-deoxynucleotidetriphosphates (ddNTPs) which terminate further elongation of the DNA strand. Because the modified di-deoxynucleotidetriphosphates (ddNTPs) do not have a free 3'-OH group that is needed for the formation of a phosphodiester bond between two nucleotides, they hinder the chain elongation process causing DNA polymerase to halt its action. In recent times however this method has been limited only for smaller scale sequecing as next generattion sequecing has taken over the market.

2. Affinity Chromatography: The technique of chromatography is used to separate biological molecules on the basis of their nature. On the basis of parameters that is/are used for separation many different kinds of chromatography have been devised. One such is affinity choromotagraphy that separates biomolecules on the basis of their high and specific affinity or attractiveness towards a particular moiety like ligand, antibody, antigen, etc. A simple procudure just like any other chromatography involves a stationary and a mobile phase. The statonary phase is made from the affinity molecules (like antibody if we are separting some antigens) while the mobile phase contains mixture of biomolecules to be separated. After the two phases are allowed to sit together for a certain period of time for interaction to occur the column is washed using specific buffers. Here the one which interact strongly will be eluted last whie the ones bounf weakly will be eluted out fast.