Short Answer Type(a) Name the selectable markers in the cloning vector pBR322 ? Mention the role they play.
(b) Why is the coding sequence of an enzyme 
-galactosidase a preferred selectable marker in comparison to the ones named above ?
Suggest and describe a technique to obtain multiple copies of a gene of interest in vitro.
Long Answer TypeA. Describe the characteristics a cloning vector must possess.
B. Why DNA cannot pass through the cell membrane? Explain. How is a bacterial cell made competent to take up recombinant DNA from the medium?
A. Characteristics features of cloning vector
1. Presence of Origin of replication (ori) - The vector should have an ori which is a sequence from where replication starts. Any piece of DNA which is linked to the ori can be made to replicate within the host cells. The ori site also controls the copy number so cloning vectors having ori that support a high copy number are chosen.
2. Selectable marker – The vector should possess a selectable marker which helps to distinguish and identify the non-transformants from transformants and selectively permit the growth of transformants. Genes encoding for antibiotics like ampicillin, tetracycline, chloramphenicol or kanamycin are considered to be useful selectable markers for E.coli.
3. Cloning Sites – The cloning vector should contain a single recognition site for the restriction enzymes in order to link the alien DNA. The presence of more than one recognition sites generates several fragments and complicates the cloning process.
4. It should have a high copy number so that we obtain many copies of the DNA linked to it
5. They should be able to replicate independently.
6. They should help easy linking of alien DNA.
B. DNA cannot pass through the cell membranes as it is a hydrophilic molecule. The cell membrane is made up of lipid bilayer which makes it difficult for the hydrophilic DNA molecule to pass through it.
Bacterial cells are made competent to take up recombinant DNA from the medium in the following ways-:
1. Treating bacteria with specific concentration of divalent cation such as calcium that results in an increase in the efficiency with which DNA is taken up by the bacterial cell. Recombinant DNA can then be forced into such cells by incubating them with the recombinant DNA on ice followed by placing them briefly at 42°C (heat shock), and again back on the ice.
Some other methods available for introduction of desired DNA are:
1. Microinjection in which the recombinant DNA is directly injected into the nucleus of an animal cell.
2. Biolistics or gene gun method is suitable for plant cells, where cells are bombarded with high-speed micro-particles of gold or tungsten coated with DNA.
If a desired gene is identified in an organism for some experiments, explain the process of the following:
(i) Cutting this desired gene at specific location
(ii) Synthesis of multiple copies of this desired gene
Short Answer Type
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