● 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.

`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.

● 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.

● `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"`.

● `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.