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