CHP.11 - Principles & Process CLASS XII BIOTECHNOLOGY CHP.11 - Principles & Process CLASS XII

PRICIPLES OF BIOTECHNOLOGY (i) Genetic Engineering-to alter the chemistry of DNA & RNA-introduced in host organism-change in phenotype of the host organism. (ii) Maintenance of sterile ambience.

Techniques of genetic engineering: 1.Creation of recombinant DNA 2.Gene cloning 3.Gene transfer -alien piece of DNA attached with host chromosome at ORIGIN OF REPLICATION. -alien piece of DNA replicate and multiply itself in the host organism. CLONING)

Construction of an artificial r-DNA molecule Stanley Cohen & Herbert Boyer 1972-isolated antibiotic resistant gene and link it to PLASMID (self replicating circular extra-chromosomal DNA) of Salmonella typhimurium. Cutting of DNA at specific locations- restriction enzymes (molecular scissors). Cut DNA piece linked with plasmid DNA- these plasmid DNA act as VECTORS. Linking of antibiotic resistant gene with the plasmid vector-DNA Ligase enzyme. r-DNA transferred in E.coli –a no. of copies formed by replication (CLONING)

Three basic steps - 1.identification of DNA with desirable genes. 2.identified DNA introduced into host. 3.maintenence of introduced DNA in the host and transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY 1.Restriction Enzymes- restriction endonuclease- cut DNA at a particular point. First restriction endonuclease- Hind II. Exonucleases- remove nucleotides from the ends of DNA. Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA. Restriction enzymes cut the strand of DNA a little away from the centre of the palindromic site leaving single stranded streches called STICKY ENDS. Sticky ends are joined together by DNA ligase.

Separation & isolation of DNA fragments(Gel Electrophoresis) DNA fragments are –vely charged molecule. They are forced towards anode through Agarose matrix. Separated fragments are stained with Ethidium Bromide & exposed to UV radiation. We can see bright Orange coloured bands of DNA. Separated bands of DNA are cut out from agarose gel & extracted from gel piece.(ELUTION) Now DNA fragments are used in constructing r-DNA by joining them with cloning vectors..

CLONING VECTORS (i)Origin of replication-(ori)-sequence from where replication starts,also responsible for controlling copy number of the linked DNA. (ii)Selectable marker-helps in identifying & removing non transformants. (iii) Genes encoding resistance to antibiotics like Ampicillin,chloramphenicol,Tetracycline or Kanamycin are useful selectable markers for E.coli.

Hind III Cla I EcoR I Pvu I tet R BamH I amp R Pst I pBR322 Sal I Ori rop Pvu II

(iv)Cloning Sites Ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes. EXAMPLE-A foreign DNA is ligated at the Bam H I site of tetracycline resistance gene in the vector pBR322. Recombinant plasmids lose tetracycline resistance & can be selected from non-recombinants by plating the transformants on ampicillin containing medium & then transferring on a medium containing tetracycline. Recombinants will grow in ampicillin containing medium but not on tetracycline medium. Non recombinants will grow on the medium containing both the antibiotics.

Presently alternative selectable markers are developed which differentiate recombinants from non recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. A recombinant DNA is inserted within the coding sequence of an enzyme â-galactosidase. Enzyme gets inactivated. (INSERTIONAL INACTIVATION). Colonies do not produce any colour & identified as recombinant colonies.

(iv)Vectors for cloning genes in plants & animals Agrobacterium tumifaciens a pathogen of dicot plants deliver T-DNA which transform normal plant cells into a tumor. The Tumor inducing plasmid (Ti-plasmid) of Agrobacterium is now modified into a CLONING VECTOR. RetroViruses in animals transform normal cells into Cancerous cells.Now they are disarmed & used as CLONING VECTORS.

Competent Host(for transformation with Recombinant DNA) DNA is a hydrophillic molecule,it cannot pass through cell membranes. Bacterial cells are treated with divalent Cation (like Ca). Cells are incubated with r-DNA on ice, then placed briefly at 420C (Heat Shock) & placed back on ice. MICROINJECTION-r-DNA is directly injected into the nucleus of an animal cell. BIOLISTICS (Gene Gun)-In plants cells are bombarded with high velocity micro-particles of Gold or Tungsten coated with DNA.

Processes of Recombinant DNA Technology 1.Isolation of DNA- Cells are treated with enzymes such as LYSOZYME(Bacteria), CELLULASE(plant cells), CHITINASE (Fungi) to break the cell wall. RNA is removed by RIBONUCLEASE. Proteins are removed by PROTEASE. Purified DNA precipitates out after the addition of chilled ETHANOL.

2.Cutting of DNA at specific locations By Agarose gel electrophoresis DNA undergoes restriction enzyme digestion. Cutting of Gene of interest & addition of Ligase. Recombinant DNA is formed.

3.Amplification of Gene of Interest using PCR PCR(Polymerase chain reaction)- Multiple copies of the gene of interest is synthesized in vitro using two sets of primers & the enzyme DNA polymerase. Repeated amplification is achieved by using Taq-polymerase( which is isolated from a bacteria Thermus aquaticus) which remains active during the high temperature required for denaturation of double stranded DNA.

4.Insertion of recombinant DNA into host Cell/organism. 5.Obtaining the foreign gene product- BIOREACTORS These are vessels in which raw materials are biologically converted to specific products, individual enzyme etc. It provides optimal conditions of growth (temperature, pH,substrate,salts,Vitamins,Oxygen ).

DONSTREAM PROCESSING It includes separation & purification of product. Product is formulated with suitable preservatives. Clinical trials of formulation. Strict quality control testing for each product.