(1) DNA fragments are separated by forcing them to move towards anode under an electric field through a medium. Agarose gel is used as medium.
(2) Ethidium bromide is used as stain for DNA.
(3) Then on exposure to UV-light appear as orange coloured bands.
(4) Separated bands of DNA are cut out from agrose gel, this is called elution.
(5) These DNA fragments are used in recombinant DNA by joining them with cloning vectors.
(ii) Cutting of DNA at specific location : The purified DNA is cut by use of restriction enzymes. Agarose gel electrophoresis is used to check the progression of restriction enzymes digestion.
(iii) Amplification of gene of interest using PCR : Amplification is the process of making multiple copies of desired DNA segment in vitro. Polymerase chain reaction involves three steps :
(a) Denaturation : The target DNA is heated to high temperature (94°C), resulting the separation of two strands of DNA. Each strand acts as template.
(b) Annealing : Two oligonucleotide primers anneal to each of the single stranded DNA template.
(c) Extension of Primers : DNA polymerase (Taq polymerase) extends the primers using the nucleotides provided in the reactions.
Taq polymerase is a heat stable (Thermostable) DNA polymerase which is isolated from thermophilic bacterium named Thermus aquaticus.
(iv) Ligation : The cut out gene of interest from the source of DNA and cut vector with appropriate space, are mixed and ligase enzyme is added. This results recombinant DNA (r-DNA).
(v) Transfer of recombinant DNA into the host : the ligated DNA is introduced into the recipient cell makes itself competent to receive and take up DNA present in the surrounding.
(vi) Obtaining the foreign gene product : The cell containing the foreign gene is cultured on suitable medium and the product can be extracted from the medium.
Bioreactors are used for processing large volume of culture for obtaining products of interest in sufficient quantities. Bioreactor is a large vessel in which raw material is biologically converted into specific product under optimal condition.
