Genetics and cloning

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Genetics and cloning, is all about modifying genes,more like evolution of genes.

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Genetics and cloning

  1. 1. Genetics and Cloning. Presented By Nkosi K.T B.eD
  2. 2. Contents  PCR Definition  Components of the reaction mixture  PCR primer design guidelines  Steps in PCR  Variations on the basic PCR technique  Comparison of PCR and Gene cloning  Applications of PCR  Problems related to PCR
  3. 3. What is PCR? Polymerase Chain Reaction is an in vitro technique for the amplification of a specific sequence of DNA Which is used for further testing.
  4. 4. Components of the reaction mixture Template DNA. Primers (forward and reverse) dNTPs Taq DNA Polymerase Buffer solution Divalent cations Sterile deionized water
  5. 5. Template DNA It contains the DNA region to be amplified Range - 1-2 µl ( for a total reaction mixture of 10 µl)
  6. 6. Hairpins Intramolecular interaction within the primer
  7. 7. dNTPs De oxy nucleotide triphosphate (dATP, dGTP, dTTP, dCTP) They are the building blocks from which the DNA polymerases synthesizes a new DNA strand. • Range - 0.5 µl (for 10µl reaction mixture)
  8. 8. dNTPs in the reaction mix
  9. 9. Steps in PCR Initialization Denaturation Annealing Extension / Elongation Final elongation Final hold Initialization step Heating the reaction to a temperature of • 94-96°C for 1-9 minutes.
  10. 10. Application of PCR Cloning a Gene encoding a known protein Amplification of old DNA Amplifying cloned DNA from Vectors Rapid Amplification of cDNA ends Detecting Bacterial or Viral Infection ● AIDS infection ●Tuberculosis (Mycobacterium tuberculosis)
  11. 11. PCR Product at different Temperature.
  12. 12. DNA STRUCTURE • DNA molecules are polymers called polynucleotides. • Each polynucleotide is made of monomers called nucleotides. • Each nucleotide consists of : • a nitrogenous base (Adenine, Thymine, Cytosine or Guanine) • a pentose sugar (DNA = Deoxyribose sugar), • and a phosphate group.
  13. 13. CHROMOSOME DUPLICATION AND DISTRIBUTION
  14. 14. Cloning process.
  15. 15. Modification of cDNA for cloning
  16. 16. Directional cloning of foreign DNA
  17. 17. Production of transgenic animal
  18. 18. Screening genomic libraries in bacteriophage
  19. 19. REFERENCE • http://www.slideshare.net/alokbharti18/pcr-4529063 • Dr.Zeyad Akawi Jreisat M.D, M.A. Ph.D • Ms. J. Williamson (2014).

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