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DNA cloning

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DNA cloning

  1. 1. By – AKSHAY PAREEK M.Sc. Biotech. III sem Dept. of Biotechnology NIET, NIMS University
  2. 2. • Clones – Genetically identical molecules, cells, or organisms all derived from a single ancestor • Cloning – The production of identical copies of molecules, cells, or organisms from a single ancestor
  3. 3. Why cloning - • DNA clones are used to find genes, map them, and transfer them between species • Cloning technology is used to find carriers of genetic disorders, perform gene therapy, and create disease-resistant plants
  4. 4. Cloning Cell based PCR based
  5. 5. Requirements • A way to cut DNA at specific sites • A carrier molecule to hold DNA for cloning • A place where the DNA can be copied (cloned)
  6. 6. 1 •Extraction 2 •Isolation 3 •Ligation 4 •Insertion 5 •Selection 6 •Storation
  7. 7. Isolation of desired gene • Using restriction endonuclease • Mostly RE II used Gene of interest
  8. 8. Ligation
  9. 9. Overall process of cloning
  10. 10. Insertion (Transformation) Vectors used – 1. Plasmid 2. Bacteriophage 3. Cosmid 4. YACs 5. BACs
  11. 11. Techniques for Transformation Transformation using CaCl2
  12. 12. Electroporation
  13. 13. Microinjection
  14. 14. Selection • Blue White Screening - –Only plasmids with functional lacZ gene can grow on Xgal lacZ functional => polylinker intact => nothing inserted, no clone lacZ(-) => white colonies polylinker disrupted => successful insertion & recombination!
  15. 15. A Revolution in Cloning : Polymerase Chain Reaction Requirement 1. Target DNA 2. Two Primers 3. Four dNTPs 4. DNA Polymerase
  16. 16. Natural habitat of Thermus aquaticus • Thermostable DNA Polymerase is used like- Taq DNA Polymerase from Thermus aquaticus Pfu DNA Polymerase from Pyrococcus furiosus
  17. 17. Steps in PCR Denaturation Annealing Renaturation
  18. 18. Application of Cloning • Development of methods for cloning higher plants and animals represents a significant advance in genetic technology – Improving crops – Producing domestic animals
  19. 19. • Cloned plants and animals are used in research, agriculture, and medicine • Single base changes then detected by one or more of following: -dot blot technique -Restriction enzyme analysis (RFLP). -direct sequencing of DNA. • PCR to detect HIV
  20. 20. • Crucial forensic evidence may be present in very small quantities often too little material for direct DNA analysis. • PCR also possible on extensively degraded DNA examples include DNA from single dried blood spot, saliva (on cigarette butt), semen, tissue from under fingernails, hair roots.

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