Process of Recombination DNA Technology –

Recombination DNA technology involves the following steps:
1)Isolation of DNA.
2)Fragmentation of DNA by restriction of endonucleases.
3)Isolation of a desire DNA fragment.
4)Amplification of the gene of interest.
5)Ligation of the DNA fragment into a vector
6)Insertion of recombinant DNA into the host
7)Culturing the host cells on a suitable medium at higher at a large scale.
8)Extraction of the desired gene product.
9)Downstream processing of the product as finished product ready for marketing

Isolation of the genetic material (DNA)

1)RNA is removed by treatment with ribonuclease and proteins are removed by treatments with protease.
2)After several treatments, the purified DNA is precipitated by adding chilled ethanol.
3)The bacterial / plant / animal cell is broken down by enzymes to release DNA, along with RNA, proteins, and polysaccharides and lipids.
4)Bacterial cell is treated with enzyme lysozyme.
5)Plant cell is treated with enzyme cellulase.
6)Fungal cell is treated with chitinase.

Cutting of DNA at specific locations :-

• The DNA is using restrictions enzymes.
• The purified DNA is incubated, with the specific restriction editions optimum for the enzymes to act.

Isolation of desired DNA fragments :-

• Using agarose gel electrophoresis, the activity of the restriction enzymes an be checked.
• Since the DNA is negatively charged, it moves towards the positive electrodes or anode and in the process, DNA fragments separate our based on their sizes.
• The desired DNA fragments is eluted out.

Amplification of gene of interest using PCR :-

• The polymerase chain reaction (PCR) is reaction in which amplification of specific DNA sequence is carried out in vitro.
• This technique was developed by kary mullis in 1905 and for this he received nobel prize for chemistry in 1993.
• Requirements od PCR
  a)DNA template : – The double stranded DNA that needs to be amplified.
  b)Primers :- small chemically synthesized oligonucleotides of about 10-18 nucleotides that are complementary to a aquaticus and vent polymerase (isolated from thermococcus litoralis).