Biology COMPETENT HOST AND RECOMBINANT DNA TECHNOLOGY

### KEY TOPICS

star Competent Host
star PROCESSES OF RECOMBINANT DNA TECHNOLOGY
star Isolation of the Genetic Material
star Cutting of DNA at Specific Locations
star Amplification of Gene of Interest using PCR

### COMPETENT HOST

● Since DNA is a color{Violet}"hydrophilic molecule", it cannot pass through color{Violet}"cell membranes".

● In order to color{Violet}"force bacteria" to take up the plasmid, the bacterial cells must first be color{Brown}"made ‘competent" to take up DNA.

● This is done by treating them with a specific concentration of a color{Violet}"divalent cation", such as color{Violet}"calcium", which increases the efficiency with which DNA enters the bacterium through color{Violet}"pores in its cell wall".

● Recombinant DNA can then be forced into such cells by color{Violet}"incubating the cells" with recombinant DNA on color{Violet}"ice", followed by placing them briefly at color{Violet}"42° C (heat shock)", and then putting them back on ice.

● This color{Violet}"enables the bacteria" to take up the color{Violet}"recombinant DNA".

● This is not the only way to color{Violet}"introduce alien DNA" into host cells.

● In a method known as color{Brown}"micro-injection", recombinant DNA is color{Violet}"directly injected" into the nucleus of an animal cell.

● In another method, color{Violet}"suitable for plants", cells are bombarded with color{Violet}"high velocity micro-particles" of color{Violet}"gold or tungsten" coated with DNA in a method known as color{Brown}"biolistics or gene gun".

● And the last method uses color{Violet}"disarmed pathogen" vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host.

### PROCESSES OF RECOMBINANT DNA TECHNOLOGY

color{Brown}"Recombinant DNA technology" involves color{Violet}"several steps" in color{Violet}"specific sequence" such as

● color{Violet}"Isolation" of DNA

● color{Violet}"Fragmentation" of DNA by color{Violet}"restriction endonucleases"

● color{Violet}"Isolation" of a desired DNA fragment

● color{Violet}"Ligation" of the DNA fragment into a vector

● color{Violet}"Transferring" the recombinant DNA into the host

● color{Violet}"Culturing" the host cells in a medium at large scale and color{Violet}"extraction" of the desired product.

### ISOLATION OF THE GENETIC MATERIAL (DNA)

● In majority of organisms the color{Violet}"nucleic acid" is color{Violet}"deoxyribonucleic acid" or DNA.

● In order to cut the DNA with restriction enzymes, it needs to be in color{Violet}"pure form", color{Violet}"free" from other macro-molecules.

● Since the color{Violet}"DNA is enclosed" within the membranes, we have to color{Violet}"break the cell open" to release DNA along
with other macromolecules such as color{Violet}"RNA", color{Violet}"proteins", color{Violet}"polysaccharides" and also color{Violet}"lipids".

● This can be achieved by color{Violet}"treating" the color{Violet}"bacterial cells"/color{Violet}"plant or animal tissue" with enzymes such as color{Violet}"lysozyme" (bacteria), color{Violet}"cellulase" (plant cells), color{Violet}"chitinase" (fungus).

● The genes are located on color{Violet}"long molecules of DNA" interwined with proteins such as color{Violet}"histones".

● The color{Violet}"RNA" can be removed by color{Violet}"treatment with ribonuclease" whereas color{Violet}"proteins" can be removed by treatment with color{Violet}"protease".

● Other molecules can be removed by color{Violet}"appropriate treatments" and purified DNA ultimately color{Violet}"precipitates out" after the addition of chilled ethanol.

● This can be seen as color{Violet}"collection of fine threads" in the suspension.

### CUTTING OF DNA AT SPECIFIC LOCATIONS

● color{Violet}"Restriction enzyme digestions" are performed by incubating purified DNA molecules with the color{Violet}"restriction enzyme", at the optimal conditions for that specific enzyme.

● color{Brown}"Agarose gel electrophoresis" is employed to check the color{Violet}"progression" of a restriction enzyme digestion.

● DNA is a color{Violet}"negatively charged molecule", hence it moves towards the color{Violet}"positive electrode" (anode).

● The process is repeated with the color{Violet}"vector DNA" also.

● The color{Violet}"joining of DNA" involves several processes.

● After having color{Violet}"cut the source DNA" as well as the color{Violet}"vector DNA" with a specific restriction enzyme, the cut out color{Violet}"Gene of interest" from the source DNA and the cut vector with space are mixed and ligase is added.

● This results in the preparation of color{Violet}"recombinant DNA".

### AMPLIFICATION OF GENE OF INTEREST USING PCR

● color{Violet}"PCR" stands for color{Brown}"Polymerase Chain Reaction".

● In this reaction, color{Violet}"multiple copies" of the gene (or DNA) of interest is color{Violet}"synthesised in vitro" using two sets of color{Violet}"primers" (small chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme color{Violet}"DNA polymerase".

● The enzyme color{Violet}"extends the primers" using the nucleotides provided in the reaction and the color{Violet}"genomic DNA as template".

● If the process of replication of DNA is color{Violet}"repeated many times", the segment of DNA can be amplified to approximately color{Violet}"billion times", i.e., 1 billion copies are made.

● Such color{Violet}"repeated amplification" is achieved by the use of a color{Violet}"thermostable DNA polymerase" (isolated from a bacterium, color{Violet}"Thermus aquaticus"), which remain active during the color{Violet}"high temperature" color{Violet}"induced denaturation" of double stranded DNA.

● The color{Violet}"amplified fragment" if desired can now be used to color{Violet}"ligate with a vector" for further cloning.