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Recombinant DNA Technology

Recombinant DNA Technology. Restriction endonucleases - Blunt ends and Sticky ends. Restriction endonuclease and DNA ligase yield Recombinant DNA. DNA cloning. Polylinker – multiple restriction sites. Selection of clones. Bacterial Artificial Chromosomes (BAC). Transformation:

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Recombinant DNA Technology

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  1. Recombinant DNA Technology

  2. Restriction endonucleases - Blunt ends and Sticky ends

  3. Restriction endonuclease and DNA ligase yield Recombinant DNA

  4. DNA cloning

  5. Polylinker – multiple restriction sites

  6. Selection of clones

  7. Bacterial Artificial Chromosomes (BAC) Transformation: 1. Heat shock: CaCl2 at 0oC then heat to 37-42oC 2. Electroporation – apply high voltage BAC – 5,000 to 400,000 bp insert

  8. Yeast Artificial Chromosomes (YAC)

  9. up to 150,000 bp insert

  10. Studying genes – cDNA library

  11. Polymerase Chain Reaction (PCR)

  12. Cloning of PCR products

  13. Hybridization allows the deletion of specific sequences

  14. DNA fingerprinting – RFLP (restriction fragment length polymorphism)

  15. DNA microarrays Any known DNA sequence from any source, can be used in microarray. Green spots – mRNA more abundant in single-cell stage Red spots – mRNA more abundant at later stages of development

  16. Cloned genes can be expressed – Expression vector

  17. Cloned genes can be altered • Site-directed mutagenesis • Oligonucleotide-directed mutagenesis

  18. Transgenic – cloning in mice for human growth hormone

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