Cloning and recombinant dna


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Cloning and recombinant dna

  1. 1. An Introduction to Cloning andRecombinant DNA Ankit Bhardwaj
  2. 2. What Are Clones? 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. Cloning Higher Plants and Animals Development of methods for cloning higher plants and animals represents a significant advance in genetic technology • Improving crops • Producing domestic animals
  4. 4. Plants Can Be Clonedfrom Single Cells 1950s: Charles Steward grew individual carrot cells in the laboratory by using special nutrients Single cells grew and divided to form a ball of undifferentiated cells (callus) Calluses transferred to a different medium grew into full-size carrots (clones)
  5. 5. A Cloned Plant Single cells grow and divide to form a callus
  6. 6. Animals Can Be Clonedby Several Methods Embryo splitting • After in vitro fertilization, early embryonic cells are divided and grown into clones Nuclear transfer (cell fusion) • Enucleated eggs are fused with embryonic or adult cells and grown into clones • Dolly the sheep
  7. 7. Why is DNA Cloning Important? 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
  8. 8. Cloning GenesIs a Multistep Process Technology was developed to clone segments of DNA molecules, based on enzymes (restriction endonucleases) that recognize and cut DNA at specific nucleotide sequences
  9. 9. Recombinant DNA Technology Recombinant DNA technology • Techniques in which DNA fragments are linked to self-replicating vectors to create recombinant DNA molecules which are replicated in a host cell
  10. 10. What’s Needed to Clone DNA? 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)
  11. 11. DNA Can Be Cut at Specific SitesUsing Restriction Enzymes Bacteria produce restriction enzymes to protect themselves from viral infections Restriction enzymes • Bacterial enzymes that cut DNA at specific sites
  12. 12. Vectors are Carriers of DNA to be Cloned Linking DNA segments produced by restriction- enzymes with vectors (plasmids or engineered viral chromosomes) produces recombinant DNA Vectors • Self-replicating DNA molecules used to transfer foreign DNA segments between host cells
  13. 13. Cloning Recombinant DNA Recombinant DNA molecules are transferred into host cells; cloned copies are produced as the host cells grow and divide Most common host cell: the bacterium E. coli Cloned DNA molecules can be recovered from the host cells and purified for further use
  14. 14. E. coli The recognition and cutting site for EcoRI
  15. 15. Plasmids Plasmids used as vectors for cloning DNA
  16. 16. Steps in the Process of Cloning DNA is cut with a restriction enzyme • Fragments produced end in specific sequences Fragments are mixed with vector molecules cut by the same enzyme • DNA ligase joins recombinant DNA molecules
  17. 17. Steps in the Process of Cloning Plasmid vectors with inserted DNA fragments are transferred into bacterial cells • Recombinant plasmids replicate and produce many clones of the recombinant DNA molecule • Colonies carrying cloned recombinant DNA molecules are identified, collected, and grown • Host cells are broken open and recombinant plasmids are extracted
  18. 18. Cloning
  19. 19. Cloning Bacteria on Petri Plates Each colony is a clone, descended from a single cell
  20. 20. Introduction in host cell
  21. 21. Identifying ColoniesWith Recombinant DNA Plasmid pBR322 has been engineered to carry two antibiotic-resistance genes with restriction sites, one for tetracycline, one for ampicillin Colonies with human DNA inserted into the tetracycline gene will not grow on tetracycline plates, but will grow on ampicillin plates
  22. 22. 13.5 A Revolution in Cloning:The Polymerase Chain Reaction Polymerase chain reaction (PCR) • A method for amplifying DNA segments using cycles of denaturation, annealing to primers, and DNA polymerase-directed DNA synthesis PCR copies a DNA molecule without restriction enzymes, vectors, or host cells • Faster and easier than conventional cloning
  23. 23. First Step in PCR: Denaturation1. DNA is heated to break the hydrogen bonds between the two polynucleotide strands • Two single-stranded DNA molecules serve as templates
  24. 24. Second Step in PCR: Annealing2. Short nucleotide sequences (primers for DNA replication) are mixed with the DNA and bind to complementary regions on single-stranded DNA • Takes place at lower temperature • Primers are 20-30 nucleotides long, synthesized in the laboratory
  25. 25. Third Step in PCR: DNA Synthesis3. The enzyme Taq polymerase is added to synthesize a complementary DNA strand • Taq is a DNA polymerase from a bacterium found in hot springs These three steps make up one PCR cycle
  26. 26. Many Uses for PCR DNA to be amplified by PCR does not have to be purified and can be present in small amounts • Used in clinical diagnosis, forensics, conservation • Samples can be small or old (insects in amber)