TheMcGraw-HillCompaniesChapter 10GeneticEngineeringARevolutioninFoundationsinMolecularBiologyMicrobiologyEIGHTHEDITIOKathleenParkTalaroBarryChessCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Chapter 10 Genetic Engineering: A Revolution in Molecular Biology Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Genetic Engineering: Basic knowledge is used to derive applied scienceor useful products Direct, deliberate modification of an organism'sgenome-BioengineeringBiotechnology - use of an organism'sbiochemical and metabolic pathways for industrialproduction2
2 Genetic Engineering • Basic knowledge is used to derive applied science or useful products • Direct, deliberate modification of an organism’s genome – Bioengineering • Biotechnology – use of an organism’s biochemical and metabolic pathways for industrial production
Tools and Techniques ofGenetic EngineeringPractical Properties ofDNAIntrinsic properties of DNA hold true even in a test tubeCopyright The McGraw -Hill Companies, Inc. Permission required for reproduction or dis play.DNA heated from 9o°C to95° C: the two strandsseparate.ThenucleotidesHeatingCoolingcan be identified, replicated,ortranscribed.Slowly cooling the DNAallows complementary(a)DNA heatingand cooling.DNAresponds to heat bydenaturing-losingnucleotides to hydrogenitshydrogenbondingand therebyseparating into itstwostrands.Whencooled,thetwostrandsrejoinatcomplementaryregions.Thetwostrandsbond and the DNA willneednotbefromthesameorganismsas long astheyhavematchingsitesregain double-stranded form3
3 Tools and Techniques of Genetic Engineering Practical Properties of DNA • Intrinsic properties of DNA hold true even in a test tube • DNA heated from 90°C to 95°C; the two strands separate. The nucleotides can be identified, replicated, or transcribed. • Slowly cooling the DNA allows complementary nucleotides to hydrogen bond and the DNA will regain double-stranded form Heating Cooling (a) DNA heating and cooling. DNA responds to heat by denaturing—losing its hydrogen bonding and thereby separating into its two strands. When cooled, the two strands rejoin at complementary regions. The two strands need not be from the same organisms as long as they have matching sites. Copyright © The McGraw -Hill Companies, Inc. Permission required for reproduction or display
EnzymesforDicing,Splicing,andReversingNucleicAcidsRestriction endonucleases - recognize specific sequencesof DNA and break phosphodiester bondsbetweenadjacent nucleotidesThe enzymes can be used to cleave DNA at desired sitesRecognize and clip the DNA at palindrome basesequencesUsed in the lab to cut DNA into smaller pieces -restrictionfragmentsCopyright The McGraw-HillCompanies, Inc. Permission required for reproduction or displayEcoRIHindlllEndonucleaseHaelllCuttingGAATTCGCTT.AGApatternCTTAAGT'TCGAACC4(b)Examplesofendonucleases,palindromes andcuttingpatterns.Thefirsttwo arestaggered cuts,andthethird isablunt cut
4 Enzymes for Dicing, Splicing, and Reversing Nucleic Acids Restriction endonucleases – recognize specific sequences of DNA and break phosphodiester bonds between adjacent nucleotides • The enzymes can be used to cleave DNA at desired sites • Recognize and clip the DNA at palindrome base sequences • Used in the lab to cut DNA into smaller pieces – restriction fragments (b) Examples of endonucleases, palindromes and cutting patterns. The first two are staggered cuts, and the third is a blunt cut. Cutting pattern Endonuclease G A A T T C C T T A A G EcoRI A A G C T T T T C G A A HindIII G G C C C C G G HaeIII Copyright © The McGraw -Hill Companies, Inc. Permission required for reproduction or display
MICRestrictionEndonucleasesawGAATTCDNADuplexCTTAAG国PlayPauseAudioTextRestrictionendonucleases areenzymesthat cleaveDNAatspecificnucleotidesequences.The sequencerecognizedis oftenfour to sixnucleotideslong.Forexample,therestrictionendonuclease EcoRI recognizesthesequence,GAATTCCopyright@TheMcGraw-Hill Companies,Inc
Restriction Fragment LengthPolymorphismsDNA sequences vary, even among members ofthe same speciesDifferences in the cutting pattern of specificrestriction endonucleases give rise to fragmentsof differing lengths (RFLPs)6
6 Restriction Fragment Length Polymorphisms • DNA sequences vary, even among members of the same species • Differences in the cutting pattern of specific restriction endonucleases give rise to fragments of differing lengths (RFLPs)
MICRestriction Fragment LengthawPolymorphisms(RFLPs)GAATTCGAATTCGAATTCCTTAAGCTTAAGCTTAAG品PlayPauseAudioTextRestriction enzymes recognize very specific sequences ofnucleotides in DNACopyright@TheMcGraw-Hill Companies,Inc
Enzymes forDicing,Splicing,andReversingNucleicAcidsLigase-rejoins phosphate-sugar bonds(stickyends)cutbyendonucleasesUsed for final splicing of genes into plasmidsandchromosomes8
8 Enzymes for Dicing, Splicing, and Reversing Nucleic Acids • Ligase – rejoins phosphate-sugar bonds (sticky ends) cut by endonucleases • Used for final splicing of genes into plasmids and chromosomes
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or displayFEXRestrictionendonucleasemakesstaggeredcutatpalindrome.ASite of cut(1)ICITAGGATICKTAGC+￥GAT★Stickyends(2)DNAOrganism1DNAOrganism2Action ofrestrictionendonucleases.(1)Arestrictionendonucleaserecognizesandcleaves DNAatthe siteofaspecificpalindromicsequence.Cleavageusuallyproducesstaggeredtailscalledstickyendsthataccept9complementarytailsforgenesplicing.ThispalindromeiscutbyAcil(2)Thestickyendscanbe usedto joinDNAfromdifferentorganismsbycuttingit withthesamerestrictionenzyme,ensuringthatbothfragmentshavetwocomplementaryends.ThispalindromeiscutbyTagl
9 DNA Organism 1 (2) DNA Organism 2 Action of restriction endonucleases. (1) A restriction endonuclease recognizes and cleaves DNA at the site of a specific palindromic sequence. Cleavage usually produces staggered tails called sticky ends that accept complementary tails for gene splicing. This palindrome is cut by Aci I. (2) The sticky ends can be used to join DNA from different organisms by cutting it with the same restriction enzyme, ensuring that both fragments have two complementary ends. This palindrome is cut by Taq I. Restriction endonuclease makes staggered cut at palindrome. Site of cut Sticky ends C T A G G A T C C T A G G A T C (1) Copyright © The McGraw -Hill Companies, Inc. Permission required for reproduction or display
EnzymesforDicing,Splicing,andReversingNucleicAcidsReversetranscriptase-makes a DNA copy of RNA-CDNAcDNAcan bemadefrommRNA,tRNA, orrRNAProvides a means of synthesizing eukaryotic genes frommRNA transcripts - synthesized gene is free of introns10
10 Enzymes for Dicing, Splicing, and Reversing Nucleic Acids • Reverse transcriptase – makes a DNA copy of RNA – cDNA • cDNA can be made from mRNA, tRNA, or rRNA • Provides a means of synthesizing eukaryotic genes from mRNA transcripts – synthesized gene is free of introns