PowerPoint PresentationSlide 2NucleasesRestriction Endonucleases “Restriction Enzymes”Uses of Restriction EnzymesSlide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Lecture 26 (Chapter 41) Recombinant DNA: Cloning and Creation of Chimeric GenesLecture 26 (Chapter 41) Recombinant DNA: Cloning and Creation of Chimeric GenesOutlineOutline-Restriction Enzymes and their uses-Cloning•definition of plasmid•construction of a chimeric plasmid•transformation-DNA sequencing-Polymerase Chain Reaction-Microarrays-Restriction Enzymes and their uses-Cloning•definition of plasmid•construction of a chimeric plasmid•transformation-DNA sequencing-Polymerase Chain Reaction-MicroarraysNucleasesNucleases•Nucleases are enzymes that cut nucleic acids by hydrolyzing phosphodiester bonds•DNase - DNA; RNase - RNA; Some do both•Exonuclease - starts at an end•Endonuclease - cuts internally•Products may be oligonucleotides or nucleoside phosphates•Most are relatively nonspecific•Nucleases are enzymes that cut nucleic acids by hydrolyzing phosphodiester bonds•DNase - DNA; RNase - RNA; Some do both•Exonuclease - starts at an end•Endonuclease - cuts internally•Products may be oligonucleotides or nucleoside phosphates•Most are relatively nonspecificRestriction Endonucleases“Restriction Enzymes”Restriction Endonucleases“Restriction Enzymes”•Recognize sp ecific sequences in DNA•Many cut specifically at the recognition sequence•Products are relatively large polynucleotides•Some restriction enzymes produce products with “sticky ends”Uses of Restriction EnzymesUses of Restriction Enzymes•Cloning–Any two pieces of DNA with compatible ends can be easily ligated together.•Analysis–The relative position of restriction sites provides information about the sequence of the DNA.–Restriction analysis allows easy comparison of two different DNAs or confirmation of cloning events.•Cloning–Any two pieces of DNA with compatible ends can be easily ligated together.•Analysis–The relative position of restriction sites provides information about the sequence of the DNA.–Restriction analysis allows easy comparison of two different DNAs or confirmation of cloning events.Restriction Endonucleases: EcoRIRestriction Endonucleases: EcoRISticky ends can be ligatedSticky ends can be ligatedManManHorseHorseAAAABBBBAABBAABBOther common Restriction enzymesOther common Restriction enzymesSouthern blottingSouthern blotting5’3’EcoR1 sitesGene AGene ADNA probeDNA probeP32-5’3’-Southern blottingSouthern blotting5’3’EcoR1 sitesGene APink ChromosomePink ChromosomeBlue ChromosomeBlue ChromosomeGene ACut out from pink Chromosome Cut out from pink Chromosome Cut out from Blue Chromosome Cut out from Blue Chromosome 5’3’EcoR1 sitesGene ADNA FingerprintingDNA FingerprintingAll human DNA is 99.9% identical but 0.1% is variable. This usually occurs in non-protein coding regions. (Variable Number Tandem Repeat; VNTR)) All human DNA is 99.9% identical but 0.1% is variable. This usually occurs in non-protein coding regions. (Variable Number Tandem Repeat; VNTR)) suspectsCloningPicture of clone warsHow to CloneHow to CloneCloned Gene inserted into PlasmidCloned Gene inserted into PlasmidIn vivo synthesis of Recombinant proteins•Function •Structure•Pharmaceutical/biochemical use•Function •Structure•Pharmaceutical/biochemical useInserted into cells/organism•Function•Distribution•Effect of mutation•Gene therapy•Function•Distribution•Effect of mutation•Gene therapyComplementary DNAComplementary DNA+ antibiotic+ antibioticGenerate recombinant proteins or more plasmidsGenerate recombinant proteins or more plasmidsCloning (Transformation)Cloning (Transformation)Infect E. coliInfect E. coliAMPAMPAmpicllin resistance geneAmpicllin resistance geneEg. Human insulinEg. Human insulinHuman Proinsulin cDNABacterial DivisionBacterial DivisionDNA Sequencing: Sanger dideoxy methodDNA Sequencing: Sanger dideoxy methodControlled termination of replicationControlled termination of replicationStrand Elongation ReactionStrand Elongation ReactiondNTPdNTP3’3’5’5’*** *AAACCGGGGTTT3’-NNNNNNCTAAGCTCGACT-5’3’-NNNNNNCTAAGCTCGACT-5’templatePrimerdGTPdCTPdTTPdATPddATPdGTPdCTPdTTPdATP3’-NNNNNNCTAAGCTCGACT-5’3’-NNNNNNCTAAGCTCGACT-5’templatePrimer5’-NNNNNNGATTCG5’-NNNNNNGATTCG5’-NNNNNNG5’-NNNNNNGAAGAGAGGGATGATPolymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)3’3’5’5’dsDNA Template2 short synthetic DNA primers (15-20 bp) one to anneal to top strand and one for bottom strandDNA polymerasedNTPsdsDNA Template2 short synthetic DNA primers (15-20 bp) one to anneal to top strand and one for bottom strandDNA polymerasedNTPsThermocyclerPolymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)5’3’5’3’5’3’5’3’5’5’3’3’5’5’3’3’5’5’primers3’5’3’3’5’3’5’5’5’5’3’5’3’3’5’3’3’5’3’3’3’3’3’5’3’3’5’3’5’3’5’3’5’3’3’5’5’5’5’5’5’5’5’5’5’5’3’5’3’3’5’3’5’3’5’3’5’3’3’5’3’5’5’3’3’5’5’ 3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’qPCRqPCRmRNAmRNAmRNAcDNAcDNAcDNAReverse transcriptaseAmplification by qPCRReference mRNAcDNAqPCRCycle numberqPCRCycle numberof mRNAMicroarrays are used to investigate the transcriptomeMicroarrays are used to investigate the transcriptomeAdapted from: http://www.cs.wustl.edu/~jbuhler/research/array/Adapted from: http://www.cs.wustl.edu/~jbuhler/research/array/Control cellsControl cellsTreated or cancer cellsTreated or cancer cellsIsolate mRNAIsolate mRNAFluorescent labeled cDNAFluorescent labeled cDNAReverse transcriptaseReverse transcriptaseCombine equal amountsCombine equal amountshttp://en.wikipedia.org/wiki/File:NA_hybrid.svghttp://en.wikipedia.org/wiki/File:NA_hybrid.svg+Microarray chip/slideMicroarray chip/slide44Hybridization stepHybridization stepScan ResultsScan Resultsinductioninductionrepressionrepression•Understand how restriction enzymes work•Knowing what “to clone” means•Understanding the principles of Southern blotting, the polymerase
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