Cloning sites:
- In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites (palindromic site) for the commonly used restriction endonuclease.
- Commonly used vector is pBR322, for E.coli.
- The ligation of foreign DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.
- If a foreign DNA ligated or inserted at the Bam H I site of tetracycline resistance gene in the vector pBR322, the recombinant plasmid will lose tetracycline resistance. (insertional inactivation)
- The recombinant can be identified from the non-recombinant in following steps:
- All are grown in ampicilin medium
- One replica of above plate grown in ampicilin medium (control)
- Other replica grown in the medium containing both tetracycline and ampicilin.
- The colonies grows in plate-I but failed to grow in plate-II are identified as recombinants.
Alternative selectable marker:
- In E.coli a plasmid called PUK-18 is used as selectable marker, which is better than pBR322.
- The foreign DNA is introduced within the coding sequence of an enzyme β-galactosidase, which convert X-Gal (chromatogenic substrate) into Galactose and 5-bromo+4 chloro indigo (blue color)
- The non-recombinant produce enzyme and give blue colored colonies.
- The recombinant unable to produce β-galactosidase and does not produce blue colored colonies after addition of chromatogenic substrate i.e. X-Gal.
- This inactivation of insertion of foreign DNA called insertional inactivation.
Vectors for cloning genes in plants and animals:
- Agrobacterium tumefaciens, a pathogenic bacterium of several dicot plants.
- This bacterium contains a plasmid called Ti-plasmid.(tumor inducing)
- In natural condition the A.tumifaciens transfer the T-DNA into the plant which transform normal plant cells into a tumor and direct these tumor cells to produce the chemical required by the pathogen.
- Retroviruses in animals have the ability to transform normal cells into cancerous cells.
- The dis-armed retroviruses are being used to transfer gene into animals.
- In Ti-plasmid the T-DNA is replaced by the gene of interest, still A.tumifaciens able to transfer the gene into the plant without causing tumor in plants.
Competent Host (for transformation with recombinant DNA)
- DNA is a hydrophilic molecule; it cannot pass through cell membranes.
- In order to force bacteria to take-up the plasmid, the bacterial cells must first be made ‘competent’ to take up DNA.
- The bacterial cell is treated with divalent cations such as calcium, which increases the efficiency of DNA up take by the bacteria.
- Recombinant DNA and the bacterial cells are incubated in ice, followed by placing them briefly at 42oC (heat shock) and then putting them back in ice.
- By microinjection the recombinant DNA directly injected into the nucleus of the animal cell.
- Plant cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in a method known as biolistics or gene gun.
- The disarmed pathogen vectors which when allowed infecting the cell transfer the recombinant DNA into the host.
PROCESS OF RECOMBINANT TECHNOLOGY:
- Isolation of DNA ,
- Fragmentation of DNA by restriction endonuclease.
- Isolation of desired DNA fragment by gel electrophoresis.
- Ligation of DNA fragment with a vector by DNA ligase
- Transferring the recombinant DNA into the host
- Culturing the host cells in a medium at large scale in a bioreactor.
- Extraction of desired product by downstream processing.
Isolation of the Genetic material (DNA):
- Bacterial cell wall digested by Lysozyme.
- Plant cell wall is digested by cellulase and pectinase.
- Fungal cell wall is digested by chitinase.
- RNA of the cellular content is digested by ribonuclease.
- Proteins are removed by Proteases.
- Purified DNA ultimately precipitated out after addition of chilled ethanol.
- The precipitated DNA is separated and removed by spooling.
Amplification of Gene of Interest using PCR:
- PCR stands for Polymerase chain reaction:
- Multiple copy of gene of interest can be synthesized in vitro.
- PCR includes following steps:
Denaturation:
- Double stranded DNA made single stranded.
- It is done by heating the DNA at 94oC.
- Each single stranded DNA is called Template strand.
Annealing:
- Two sets of primer (small oligonucleotide chain that are complementary to the DNA at 3’ end of the DNA template) added to the medium.
- This is done at around 50oC.
Extension:
- Deoxyribonucleotides triphosphates are added in the medium.
- Taq polymerase catalyses the polymerization reaction using nucleotides extending from the primer towards 5’ end of the template.
- Taq polymerase is a thermostable polymerase isolated from a bacterium called Thermus aquaticus.
- It catalyses polymerization reaction at 74oC.
Obtaining the Foreign Gene product or Recombinant product:
- The protein encoding gene is expressed in a heterogeneous host is called a recombinant protein.
- The host is cultured in a continuous culture system provided in bioreactor.
- A bioreactor provides optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen)
- Bioreactor covert the raw materials into specific product, specific enzyme.
Downstream processing:
- After biosynthesis inside the bioreactor, the product has to be subjected through a series of processes before it is ready for marketing.
- The process includes separation and purification, which are collectively referred as downstream processing.
- The product has to be formulated in suitable preservatives.
- Such formulation has to undergo through clinical trials as in case of drugs.
CBSE Biology (Chapter Wise) Class XII ( By Mr. Hare Krushna Giri )
Email Id : [email protected]
