Lecture'26'(Chapter'41)'Recombinant'DNA:''Cloning'and'Crea?on'of'Chimeric'Genes!Outline'D Restric?on'Enzymes'and'their'uses'D Cloning'• ' defini?on'of'plasmid'• construc?on'of'a'chimeric'plasmid'• ' transforma?on'D DNA'sequencing'D Polymerase'Chain'Reac?on'D Microarrays'Nucleases • 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” • Recognize specific 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 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.Restric?on'Endonucleases:''EcoRI'S?cky'ends'can'be'ligated'Man'Horse'A!A!B!B!A!B!A!B!Other'common'Restric?on'enzymes'Southern'bloPng'5’'3’'EcoR1'sites'Gene'A'DNA'probe'P32D5’'3’D'Southern'bloPng'5’'3’'EcoR1'sites'Gene'A'Pink(Chromosome(Blue(Chromosome(Gene!A!Cut'out'from'pink'Chromosome'è'Cut'out'from''Blue'Chromosome'è'5’'3’'EcoR1'sites'Gene'A'DNA'Fingerprin?ng'All'human'DNA'is'99.9%'iden?cal'but'0.1%'is'variable.''This'usually'occurs'in'nonDprotein'coding'regions.''(Variable'Number'Tandem'Repeat;'VNTR))''Mr.'X'Mr.'Y'Mr.'Z'Mr.'T'vic?m'Fragment!size!DNA'found'at'the'crime'scene'Mr.'G'suspects'Cloning'Picture'of'clone'wars'How'to'Clone'Cloned'Gene'inserted'into'Plasmid'In'vivo'synthesis'of'Recombinant'proteins'• Func?on''• Structure'• Pharmaceu?cal/biochemical'use'Inserted'into'cells/organism'• Func?on'• Distribu?on'• Effect'of'muta?on'• Gene'therapy'Complementary'DNA'+'an?bio?c'Generate'recombinant'proteins'or'more'plasmids'Cloning'(Transforma?on)'Infect'E.'coli'AMP'Ampicllin'resistance'gene'Eg.'Human'insulin'Human'Proinsulin'cDNA'Bacterial'Division'DNA'Sequencing:'Sanger'dideoxy'method'Controlled'termina?on'of'replica?on'Strand'Elonga?on'Reac?on'dNTP'3’'5’'*'*'*' *'A!A!A!C!C!G!G!G!G!T!T!T!3’DNNNNNNCTAAGCTCGACTD5’'template'Primer'dGTP'dCTP'dTTP'dATP'ddATP'dGTP'dCTP'dTTP'dATP'3’DNNNNNNCTAAGCTCGACTD5’'template'Primer'5’DNNNNNNGATTCG'5’DNNNNNNG'A'A'GA!G!GAT!Polymerase'Chain'Reac?on'(PCR)'3’!3’!5’!5’!Ø dsDNA'Template''Ø 2'short'synthe?c''DNA'primers'(15D20'bp)'one'to'anneal'to'top'strand'and'one'for'bogom'strand''Ø DNA'polymerase''Ø dNTPs'Thermocycler'Polymerase'Chain'Reac?on'(PCR)'5’!3’!5’!3’!5’!3’!5’!3’!5’!5’!3’!3’!5’!5’!3’!3’!5’!5’!primers'3’!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’!qPCR'mRNA!mRNA!mRNA!cDNA!cDNA!cDNA!Reverse'transcriptase'Amplifica?on'by'qPCR'Reference!mRNA!cDNA!qPCR'Cycle'number'of!mRNA!Microarrays'are'used'to'inves?gate'the'transcriptome'Adapted'from:'hgp://www.cs.wustl.edu/~jbuhler/research/array/'Control'cells'Treated'or'cancer'cells'Isolate'mRNA'Fluorescent'labeled'cDNA'Reverse'transcriptase'Combine'equal'amounts'hgp://en.wikipedia.org/wiki/File:NA_hybrid.svg'+'Microarray'chip/slide'4'Hybridiza?on'step'Scan'Results'induc?on'repression'• Understand'how'restric?on'enzymes'work'• Knowing'what'“to'clone”'means'•
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