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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. - PowerPoint PPT Presentation
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Polymerase chain reaction If DNA sequence is known, amplify specific gene directly
Cloning by PCR
humanDNA
RPE65gene
PCR
Cloning by PCR• Human DNA• RPE65-specific 20-nt primers• Taq DNA polymerase• dNTPs
5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG3′ TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCTCC
part of RPE65
heat
5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG TTTGACACCT
5′primer
TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAAC
heat
5′ CCAGGTTGAG TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCT
5′
primerCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGA
Cloning by PCR
Once amplified, ligate and transform as before
Cloning by PCR
+amplified copiesof RPE65 gene
plasmidvector
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
Genetic engineering
Previously derived from blood donations First “pharmed” drug approved by FDA (Feb 6, 2009)
Growing Human Antithrombin
Transgenic animals
Expression Vector
hAT
hAT
PCR
hAT
Transgenic animals
hAT
Transgenic animals ATryn 1 goat = 90,000 blood
donations
Transgenic plants Pest resistance Herbicide resistance Delayed ripening Increased nutritional values
Electroporation Agrobacterium tumefaciens
Transferring Genes to Plants
Human Gene Therapy
What diseases can we treat with Gene therapy?Replace genes, control expression
Microinjecting DNA into Cells
Transfection
Moving Genes around
hAT
DNA viruses: Adenovirus Retrovirus/Lentivirus
Moving Genes around
First therapy experiment: X-SCID (1990) First success: Metastatic melanoma (2006)
2 of 15 Patients had tumors shrink/disappear LCA2
Success?
Almost perfect… Adenovirus: elicits massive inflamation. Retrovirus: insertional mutagenesis
Embryonic Stem Cells Embryonic stem cells Embryonic germ cells
Induced Pluripotent stem cells Use of viral vectors Inject fibroblasts transduced with OCT4, SOX2, KLF4, and c-
MYC.
17.26
Will we cause new disease using GMOs? What is normal? Somatic vs. Germline therapy? Human cloning?
Ethical considerations