Chapter 9Digital Analysis of Genomes9-1
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 9-1 Digital Analysis of Genomes Digital Analysis of Genomes Chapter 9
Sectionsto study9.1 Sequence-specificDNA fragmentation9.2CloningfragmentsofDNA9.3Hybridization9.4The polymerase chain reaction (PCR)9.5DNA sequenceanalysis9-2
9-2 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Sections to study Sections to study 9.1 Sequence 9.1 Sequence-specific DNA fragmentation specific DNA fragmentation 9.2 Cloning fragments of DNA 9.2 Cloning fragments of DNA 9.3 Hybridization 9.3 Hybridization 9.4 The polymerase chain reaction (PCR) 9.4 The polymerase chain reaction (PCR) 9.5 DNA sequence analysis 9.5 DNA sequence analysis 9.6 Bioinformatics: Informati 9.6 Bioinformatics: Information technology and genomes on technology and genomes 9.7 The hemoglobin genes: A comprehensive example 9.7 The hemoglobin genes: A comprehensive example
Tools of modern molecular analysisRecombinant DNA technology:atechnological revolution in themid-1970s.9-3
9-3 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Tools of modern molecular analysis Tools of modern molecular analysis Recombinant DNA technology Recombinant DNA technology: a technological revolution in the technological revolution in the mid-1970s
PlasmiddonorGeneOdonorRecombinantDNAtechnology: alsolationofbacterialplasmidtechnological revolutionin the mid-1970s.PlasmidopenedCIsolatedwithrestrictiongeneMolecularbiologyenzymeBiotechnologyDNAligasebindsendstogetherRecombinantDNAmoleculeTransformationoffreshbacteriumChromasomeODanorgeneTranscriptionTranslationmRNAProteinproduct
9-4 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Recombinant DNA Recombinant DNA technology technology: a technological revolution technological revolution in the mid in the mid-1970s. Molecular biology Molecular biology Biotechnology Biotechnology
Five basic operations in recombinant DNA technologyFragmentlong DNA into smallerfragments and separatethemthroughgelelectrophoresis.2Isolate, amplify, and purify the fragments through molecularcloning.3Use purified DNA fragments as probes toidentify similarsequences in DNA libraries by DNA (or RNA)hybridization.Rapidlyisolate and amplifyknown DNAorRNA sequencesthroughpolymerase chain reaction (PCR).5Determine the nucleotide seguence of DNA through DNAsequencing9-5
9-5 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Five basic operations in recombinant DNA technology Five basic operations in recombinant DNA technology 1. Fragment long DNA into smaller fragments and separate Fragment long DNA into smaller fragments and separate them through gel electrophoresis. them through gel electrophoresis. 2. Isolate, amplify, and purify the fragments through molecular Isolate, amplify, and purify the fragments through molecular cloning. cloning. 3. Use purified DNA fragments as probes to identify similar Use purified DNA fragments as probes to identify similar sequences in DNA libraries by sequences in DNA libraries by DNA (or RNA) hybridization. DNA (or RNA) hybridization. 4. Rapidly isolate and amplify known DNA or RNA sequences Rapidly isolate and amplify known DNA or RNA sequences through polymerase chain reaction (PCR). through polymerase chain reaction (PCR). 5. Determine the nucleotide sequence of DNA through DNA Determine the nucleotide sequence of DNA through DNA sequencing. sequencing
9.1 Sequence-specific DNAfragmentationRestrictionenzymes:Endonucleases made bybacteria that recognize specificshort nucleotide seguences andcutDNA at those sites9-6
9-6 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Restriction enzymes: Endonucleases made by bacteria that recognize specific, short nucleotide sequences and cut DNA at those sites. 9.1 Sequence 9.1 Sequence-specific DNA fragmentation specific DNA fragmentation
TABLE9.1TenCommonlyUsedRestrictionEnzymesSequenceofMicrobial OriginEnzymeRecognition Site4-8 base pairsTaqlThermusaquaticusYi198Palindromic symmetryCut both DNA strands atRsalRhodopseudomonassphaeroidesidentical base sequenceSau3AlStaphylococcusaureus3AECORIEscherichia coliBamHiBacillusamyloliquefaciensH.HindillHaemophilusinfluenzaeKpntKlebsielia pneumoniaeOK8ClalCaryophanon latumBacillusBssHII999999stearothermophilusGdadNotiNocardiaotitidiscaviarumodooacoccooaad
9-7 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 4-8 base pairs Palindromic symmetry Cut both DNA strands at identical base sequence
Restriction enzymes cut DNA molecules at specificlocations and produce either blunt or sticky endsa)Bluntends(Rsal)Sugar-phosphateRsalbackbone5AAASHASSSb)Sticky5ends(EcoRl)ECORI5-5overhangsc)Sticky3'ends(Kpnl)3'overhangsKpr2-TAAT59-8Fig.9.2
9-8 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Restriction enzymes cut DNA molecules at specific Restriction enzymes cut DNA molecules at specific locations and produce either blunt or sticky ends locations and produce either blunt or sticky ends Fig. 9.2
Restriction fragments:TheDNAfragmentsB-BamRrPlasmidE:EcoRrocahyrexgenerated by restrictionCenzymes.BamHLDigestion: The cutting ofDNA by restrictionEcoRtBulEEE1aenzymes.Lineanfnagmanty9-9
9-9 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Restriction fragments: The DNA fragments generated by restriction enzymes. Digestion: The cutting of DNA by restriction enzymes
Restriction enzymes are bacterial weaponsagainstbacteriophageattack1.Phage attachesProteincoattobacterium(host).DNACell bursts,清releasingnew限phage.39福Core2.PhageinjectsHostcell wallits genes intohostcellT2phageP4.Phageparticles3.PhageDNAreplicates:assemble.newphageproteinsaremadeFig.6.69-10
9-10 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Restriction enzymes are bacterial weapons Restriction enzymes are bacterial weapons against against bacteriophage bacteriophage attack Fig. 6.6