BIO Tech Final Term Past Papers

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Q No1: Define DNA Ligase and briefly explain its function and way of working.
DNA Ligase: Ligase means ‘’ joining enzyme’’ it can be defined as an enzyme which attach one DNA strand to another strand by creating the phosphodiester bond between them.
Function: It name indicate that it ligate two DNA sequences together by creating a phosphodiester bond. During the replication the Okazaka fragments are formed at the one strand of DNA molecule. This Okazaka fragment are not joined with each other due to absence of phosphodiester bond, these fragments have single stranded DNA gaps which are known as ‘’Nicks’’. DNA ligase are utilized are joined to these Okazaka fragment together by creating phosphodiester bond. The Two DNA molecules are joined by this type of enzyme and processes are called ligation.
Working: The ligase enzyme is used to attach two nucleotides of DNA molecules by creation phosphodiester bond. It create phosphodiester bond between 3OH/ group of one nucleotide to 5PO4 groups of other nucleotide with the help of ATP and NAD+ with the elimination of H2O molecule. There are usually two molecule are utilized in this reaction. AMP is required for the ligation process. This take place in four steps.
a)   This is the first step is to reorganization of Okazaka fragment or nicks.
b)  The second step is adenylation which mean addition of AMP to the lysine and in the reaction center causes a released a specific enzyme pyrophosphate.
c)   Transfer the AMP to the 5 PO4 so this is called a donor and that accept which are called acceptor and formed pyrophosphate bond.
d)  This last step to create a phosphodiester bond between the 3OH/ group of one nucleotide and 5PO4 groups of other nucleotide.

DNA extraction by natural strategy

Where does DNA originate from in blood?

'Red platelets don't have any DNA, as they lose their cores (the compartment in a cell that contains the DNA) as they develop. So the DNA in your blood is in your white platelets.'

Guideline of PCI strategy:

'The essential guideline of phenol-chloroform DNA extraction technique depends on the fluid extraction of biomolecules.

The protein segments of the cell are denatured and expelled by isolating DNA into the solvent stage. The whole system of division depends on the dissolvability of the biomolecules. Water is a polar dissolvable and phenol is a non-polar dissolvable. Likewise, phenol is denser than water.

Then again, DNA is a polar particle with a net negative charge on its spine and protein is non-polar. As we realize that the polar atom can break down in polar arrangements in this way DNA disintegrates in water yet not in phenol. Furthermore, water can not blend in with the phenol so phenol stays at the base because of its higher thickness. In the mean time, DNA breaks up in water/chloroform and stays on the highest point of the phenol as a watery layer, fluid stage.

At the point when we blend phenol in with the cell suspension, the protein part of the cells get processed or denatured and on centrifugation, the denatured protein subsided into the base of the cylinder alongside the phenol.'

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