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Recombinant DNA technology and its applications

recombinant DNA technology

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Recombinant DNA technology and its applications

  1. 1. Recombinant DNA Technology and its Applications. Pragati Randive Reg No.0025 Sr.M.Sc.Agril.Biotechnology
  2. 2. Basic Steps of Genetic Engineering Step 1: DNA Extraction Step 2 : Gene Cloning Step 3 : Gene Design Step 4 : Transformation Step 5 : Backcross Breeding Recombinant DNA (or rDNA) is made by combining DNA from two or more sources. Introduction
  3. 3. What is Gene Cloning? Gene cloning is a technique, where an isolated single copy of a gene is cloned to obtain its indefinite identical copies. There are two types of gene cloning: In vivo, which involves the use of restriction enzymes and ligases using vectors and cloning the fragments into host cell. The other type is in vitro which is using the polymerase chain reaction (PCR) method to create copies of fragments of DNA.
  4. 4. A. Steps in molecular cloning 1. Formation of recombinant DNA molecule includes: (A) Selecting the host organism and cloning vector, (B) preparation of DNA to be cloned, (C) preparation of vector DNA and (D) creation of recombinant DNA. 2. Transfer of recombinant DNA into host organism. 3. Selection and screening for positive clones.
  5. 5. Fig: Different steps involved in procedure to clone a gene
  6. 6. Creating Recombinant DNA This involves joining of the broken ends of the vector DNA with the two ends of DNA sequences to be cloned. There are 3 methods of joining DNA fragments : DNA ligase- joins the cohesive ends T4 Ligase- forms phosphodiester bonds between blunt ended fragments. Terminal deoxynucleotidyl transferase- synthesizes homopolymeric tails at the ends of fragments.
  7. 7. B. Confirmation of Cloned Gene 1. By Gel Electrophoresis
  8. 8. 2. Southern Blot - Specific genes can be identified with DNA or RNA probes using the Southern Blot technique
  9. 9. Gene Cloning in Plants by Agrobacterium mediated gene transfer.
  10. 10. Fig: Agrobacterium transfers T-DNA into plant cell and induces tumour
  11. 11. Polymerase Chain Reaction
  12. 12. Applications of rDNA Technology in Crop Improvement Distant Hybridization Development of Transgenic Plants Development of Root Nodules in Cereal Crops Development of C4 Plants
  13. 13. Case Studies: Recombinant DNA Technology
  14. 14. Case Study 1: Golden Rice
  15. 15. Case Study 2: Bt Cotton
  16. 16. Case Study 3: Flavr Savr Tomato
  17. 17. References Fundamentals of Recombinant DNA Technology Molecular Biotechnology-Principles and Practices- Channarayappa (https://ag4impact.org/sid/genetic-intensification/)
  18. 18. Thank You

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