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Cloning by PCR

PCR. Cloning by PCR. Polymerase chain reaction If DNA sequence is known, amplify specific gene directly. RPE65 gene. human DNA. Cloning by PCR. Human DNA RPE65-specific 20-nt primers Taq DNA polymerase dNTPs. part of RPE65.

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Cloning by PCR

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  1. PCR Cloning by PCR • Polymerase chain reaction • If DNA sequence is known, amplify specific gene directly RPE65 gene human DNA

  2. Cloning by PCR • Human DNA • RPE65-specific 20-nt primers • Taq DNA polymerase • dNTPs part of RPE65 5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG 3′ TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCTCC heat heat primer 5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG TTTGACACCT 5′ 5′CCAGGTTGAG TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCT 5′ TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAAC primer CATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGA

  3. Cloning by PCR

  4. Cloning by PCR • Once amplified, ligate and transform as before + plasmid vector amplified copies of RPE65 gene

  5. Genetic engineering

  6. Genetic engineering • Modified microorganisms: • Insulin, growth hormone, clotting factors, EPO… • HPV vaccine • Ethanol from cellulose • Oil-eating bacteria • Modified plants and animals • BT corn • Roundup-ready soybeans • Golden rice • Modified humans • Gene therapy

  7. Growing Human Antithrombin • Previously derived from blood donations • First “pharmed” drug approved by FDA (Feb 6, 2009)

  8. Transgenic animals hAT PCR hAT Expression Vector hAT

  9. Transgenic animals hAT

  10. Transgenic animals • ATryn • 1 goat = 90,000 blood donations

  11. Transgenic plants • Pest resistance • Herbicide resistance • Delayed ripening • Increased nutritional values

  12. Transferring Genes to Plants • Electroporation • Agrobacterium tumefaciens

  13. Human Gene Therapy What diseases can we treat with Gene therapy? Replace genes, control expression

  14. Moving Genes around • Microinjecting DNA into Cells • Transfection hAT

  15. Moving Genes around • DNA viruses: Adenovirus • Retrovirus/Lentivirus

  16. Success? • First therapy experiment: X-SCID (1990) • First success: Metastatic melanoma (2006) • 2 of 15 Patients had tumors shrink/disappear • LCA2

  17. Almost perfect… • Adenovirus: elicits massive inflamation. • Retrovirus: insertional mutagenesis

  18. Embryonic Stem Cells • Embryonic stem cells • Embryonic germ cells

  19. Induced Pluripotent stem cells • Use of viral vectors • Inject fibroblasts transduced with OCT4, SOX2, KLF4, and c-MYC. 17.26

  20. Ethical considerations • Will we cause new disease using GMOs? • What is normal? • Somatic vs. Germline therapy? • Human cloning?

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