Dna cloning


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DNA cloning is the starting point for many genetic engineering approaches to biotechnology research.

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Dna cloning

  1. 1. DNA Cloning
  2. 2. DNA Cloning • • • • • Isolation of DNA Ligating the dna into a vector Transformation of a host cell with the recombinant dna Selection of host cells harboring the recombinant dna Screening of cells for those harboring the recombinant dna or producing the appropriate protein product
  3. 3. Cloning vectors • Transport recombinant dna into a host cell • a DNA molecule that carries foreign DNA into a host cell, replicates and produces many copies of itself and the foreign DNA
  4. 4. What cloning vectors must have • 1. have an origin of replication • 2. be small • 3. have several unique restriction sites • 4. Have selectable markers
  5. 5. Types of cloning vectors • 1. bacterial vectors Greatest variety of cloning vectors ( e. coli)
  6. 6. Plasmids- circular double strands that are extra chromosomal • Functions: 1. Encode substance s for antibiotic resistance 2. Bacteriocins 3. Physiological functions 4. Toxin- producing 5. Virulence plasmids
  7. 7. Role of plasmids in dna cloning • Engineered with unique restriction sites for insertion of foreign dna fragments
  8. 8. Types of cloning vectors • 2. bacteriophage • A virus that infects a bacterium • Derived from a double strand genome that has a single strand complementary ends of 12 nucleotides that can base pair which forms a circular dna molecule once inside the host cell
  9. 9. Types of cloning vectors • 3. Cosmids • Engineered hybrids of phage dna and plasmids • Plasmids with small portion of bacteriophage vectors and replication origin for replication in bacterial hosts • Used for cloning large dna fragments
  10. 10. Types of cloning vectors (for other organisms) • • • • 4. Yeast artificial chromosomes (YAC) For eukaryotic molecular studies Has the following necessary components: A. centromere-distributes the chromosome to the daughter cells • B. telomere- ensures that the end is correctly replicated and protects against degradation • C. autonomously replicating sequence- specific dna that enable the molecule to replicate
  11. 11. Types of cloning vectors (for other organisms) • 5. Bacterial artificial chromosomes (bac) • Synthetic vectors for large genome project and complex genomes
  12. 12. Types of cloning vectors (for other organisms) • • • • • • • 6. Plant cloning vectors Purposes: A. generate resistance to disease, pests and herbicides B. improve crop yield C. quality and nutritional value D. development of new ornamental plant characteristics E. increase shelf life of common fruits and vegetables
  13. 13. • Commonly used plant vectors • 1. tobacco mosaic virus • 2. Ti or tumor inducing plasmid- agrobacterium tumafaciens
  14. 14. Types of cloning vectors (for other organisms) • 7. Mammalian cell vectors • First eukaryotic infecting virus to be used • Can infect the cells of several mammalian species including monkey cells
  15. 15. What is your stand on cloning?