Rdt (recombinant dna technology)
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Recombinant DNA technology (RDT) involves the direct manipulation of genes to create recombinant DNA for various applications. The process includes obtaining and inserting a desired gene into a vector, introducing it to host cells, and selecting transformed cells. Techniques for transformation include methods like calcium chloride treatment, microinjection, and electroporation, along with various cloning vectors used for gene transfer.
Rdt (recombinant dna technology)
- 2. 2 RECOMBINANT DNA TECNOLOGY Genetic engineering is defined as the direct manipulation of an organism's genes including heritable and nonheritable recombinant DNA constructs. Recombinant DNA Technology (RDT)/ Genetic Engineering is the technique of joining together distinct/different DNA molecules to produce new genetic combinations as Recombinant DNA(rDNA).
- 3. 3 First discovered RDT in 1977 when they first successfully expressed somatostatin in bacteria. STEPS INVOLVED IN RDT:
- 4. 4 1. Obtaining a copy of desired gene by cleaving the DNA with Restriction Endonuclease. 2. Inserting the gene in a suitable Vector. 3. Introducing the vector with gene in a host cell. 4. Selection of the transformed host cell. 5. Cloning of Gene.
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- 6. 6 TOOLS AND TECHNIQUES OF RECOMBINANT DNA TECHNOLOGY (RDT) PLASMID BACTERIOPHAGE COSMIDS PHAGEMIDS ARTIFICIAL CHROMOSOMES BAC PAC YAC TOOLS OF RDT RESTRICTION ENZYME CLONING VECTORS DNA LIGASE
- 7. 7 TECHNIQUES OF RDT TECHNIQUES FOR SLECTION OF TRANSFORMED CELLS CLONING VECTORS Vectors are those, that transfer donar DNA fragment with gene of interest to host cell (recipient) and are capable of replicating in the host cell. Cloning vectors include plasmids, bacteriophages, cosmids, phasmids, Bacterial Artificial Chromosomes (BACs) &Yeast Artificial Chromosomes (YACs). CHARACTERISTIC FEATURES OF CLONING VECTORS CLONING STRATEGIES TECHNIQUES TO TRANSFER CLONED VECTORS INTO HOST CELLS
- 8. 8 1. An Ori (Origin of Replication) sequence. 2. A selectable marker. 3. One or more restriction sites 4. Antibiotic resistance genes
- 9. 9 VARIOUS TYPES OF VECTORS AND THEIR INSERT SIZE CLONING VECTORS INSERT SIZE (kb) Plasmid 0.5-8 Bacteriophage 5-25 Cosmids 35-45 BAC 50-300 YAC 200-1000 CLONING STRATEGIES TECHNIQUES TO TRANSFER CLONED VECTORS INTO HOST CELLS
- 10. 10 The process of uptaking foreign DNA by the host cell is known as transformation. The transforming ability can be induced artificially by various methods depending upon the host cell discussed below: Calcium chloride (CaCl2) method : In this, bacterial cells are treated with cold CaCl2 which make the cell competent for transformation. After addition, cells are given heat shock treatment at 42°C for 90s which transiently create pores in the cell membrane allowing entry of foreign DNA. (A) Bacterial cells Transfection : It is the transfer of foreign DNA into cultured host cell mediated through charged chemicals (cationic liposomes, calcium phosphate, DEAE dextran) which are taken & mixed with DNA molecules. The recipient host cell is overlayered by this mixture & foreign DNA is taken up by the host cell.
- 11. 11 Microinjection : A technique of delivering foreign DNA into a living cell ( cell, egg, oocyte, embryos of animals) through a glass micropipette. (also used in plant cell) (B) Animal cells Direct transformation : A foreign DNA fragment is precipitated with calcium phosphate & mix with the cells to be transformed. DNA molecule passes through the cell membrane & integrates with the mammalian chromosome. Vector-mediated or indirect gene transfer method: This is mediated by Ti plasmid Agrobacterium tumefaciens which is known as natural genetic engineer of plants. (C) Plant cells Particle bombardment method (biolistics) :The foreign DNA is coated onto the surface of minute gold or tungsten particles & bombarded (shot) onto the target tissue/ cells using a particle gun (gene gun/ shot gun/ microprojectile gun). Electroporation :This involves application of a pulse of high voltage (~350 V) to protoplasts/ cells/ tissues which induce the formation of temporary pores in the plasma membrane facilitating the uptake of foreign DNA.
- 12. 12 TECHNIQUES FOR SELECTION TRANSFORMED CELLS: I. Using Selectable Marker by Insertional Inactivation Method:
- 13. 13 II.Blue and White Method of selection:
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