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DNA Technology and DNA Technology and GenomicsGenomics
Chapter 15 Chapter 15
Learning Objective 1Learning Objective 1
• How does a typical How does a typical restriction enzymerestriction enzyme cut cut DNA molecules?DNA molecules?
• Give examples of the ways in which these Give examples of the ways in which these enzymes are used in enzymes are used in recombinant DNA recombinant DNA technologytechnology
Recombinant DNA TechnologyRecombinant DNA Technology
• Isolates and amplifiesIsolates and amplifies• specific sequences of DNAspecific sequences of DNA• incorporates them into incorporates them into vectorvector DNA molecules DNA molecules
• Resulting Resulting recombinant DNArecombinant DNA • is propagated and amplified is propagated and amplified (cloned)(cloned)• in organisms such as in organisms such as E. coliE. coli
Restriction EnzymesRestriction Enzymes
• Recognize and cut DNARecognize and cut DNA• at highly specific base sequencesat highly specific base sequences
• May produce complementary, single-May produce complementary, single-stranded sticky endsstranded sticky ends
Restriction EnzymesRestriction Enzymes
Fig. 15-1, p. 324
Plus HindIII restriction enzyme
Sticky ends
KEY CONCEPTSKEY CONCEPTS
• Recombinant DNARecombinant DNA techniques allow techniques allow scientists to scientists to cloneclone many copies of specific many copies of specific genes and gene productsgenes and gene products
Recombinant DNA VectorsRecombinant DNA Vectors
• Naturally occurring circular bacteria DNA Naturally occurring circular bacteria DNA molecules molecules (plasmids)(plasmids)
• Bacterial viruses Bacterial viruses (bacteriophages)(bacteriophages)
Recombinant DNA MoleculesRecombinant DNA Molecules
• ConstructionConstruction• ends of DNA fragment and vectorends of DNA fragment and vector• cut with same restriction enzymecut with same restriction enzyme• associate by complementary base pairing associate by complementary base pairing
• DNA ligaseDNA ligase • covalently links DNA strandscovalently links DNA strands• forms stable recombinant moleculeforms stable recombinant molecule
Fig. 15-2, p. 325
Plasmid from a bacterium
DNA of interest from another organism
1 Plasmid and DNA from another organism are cut by the same restriction enzyme (in this example, Hin dIII). This produces molecules with complementary single-stranded ends.
Clonable DNA fragment
2 Mix two types of molecules so their sticky ends pair. DNA ligase then forms covalent bonds at junctions, linking fragments.
Recombinant DNA
3 Transfer recombinant DNA molecule to host cell, where it is copied and turned on to produce gene product.
PlasmidsPlasmids
Fig. 15-3a, p. 326
AatI
XbaI
HpaI
PvuII ClaI
SalIBamHI SmaI
URA-3
Ampicilli
n
resista
nce
E. coliorigin of replication
Tetracycline
resistance
Yeast origin
of replication
Fig. 15-3bc, p. 326
Main bacteria DNABacterium
Plasmid
0.5 μ m
Learning Objective 2Learning Objective 2
• What is the difference between a What is the difference between a genomic genomic DNA libraryDNA library, a , a chromosome librarychromosome library, and a , and a complementary DNA (cDNA)complementary DNA (cDNA) library? library?
• Why would one clone the same eukaryotic Why would one clone the same eukaryotic gene from both a genomic DNA library and gene from both a genomic DNA library and a cDNA library?a cDNA library?
Libraries (1)Libraries (1)
• Genomic DNA libraryGenomic DNA library• thousands of DNA fragmentsthousands of DNA fragments• all DNA of an organismall DNA of an organism
• Chromosome libraryChromosome library• all DNA fragments of a specific chromosome all DNA fragments of a specific chromosome
Libraries (2)Libraries (2)
• Genomic DNAGenomic DNA and and chromosome librarieschromosome libraries• DNA fragments stored in specific bacterial DNA fragments stored in specific bacterial
strainsstrains• Provide information about genes and encoded Provide information about genes and encoded
proteinsproteins
Chromosome Chromosome LibraryLibrary
Fig. 15-4, p. 327
Sites of cleavageFragment
1Fragment
2Fragment
3Fragment
4
Human DNA1 Cut with a restriction enzyme
Produce recombinant DNA
2 2 2 2
Gene for resistance to antibiotic Transformation 3
R R R R
Plate with antibiotic- containing medium
Bacteria with plasmid live and multiply 4 Bacteria without
plasmid fail to grow
Stepped Art
Fig. 15-4, p. 327
Human DNA 1 Cut with a restriction enzyme
4
Sites of cleavage
Fragment 1
Fragment 2
Fragment 3
Fragment 4
Plate with antibiotic- containing medium
Bacteria with plasmid live and multiply
3Transformation
Bacteria without plasmid fail to grow
Produce recombinant DNA
2 2 2 2
Gene for resistance to antibiotic
R R R R
cDNA LibrarycDNA Library
• Complementary DNA (cDNA)Complementary DNA (cDNA)• produced using produced using reverse transcriptasereverse transcriptase• makes DNA copies of eukaryotic mRNAmakes DNA copies of eukaryotic mRNA
• Copies are incorporated into recombinant Copies are incorporated into recombinant DNA DNA vectorsvectors
cDNAcDNA
Fig. 15-6a, p. 328
Exon Intron Exon Intron Exon
DNA in a eukaryotic chromosome Transcription
Pre-mRNARNA processing (remove introns)
Mature mRNA
Formation of cDNA relies on RNA processing that occurs in the nucleus to yield mature mRNA.
Fig. 15-6b, p. 328
Reverse transcriptase
1 mRNA
cDNA copy of mRNA
Degraded RNA2
cDNA
3DNA polymerase
4Double-stranded cDNA
Mature mRNA is extracted and purified.
Introns (1)Introns (1)
• Genes regions that do not code for protein Genes regions that do not code for protein • present in eukaryote genomic DNA and present in eukaryote genomic DNA and
chromosome librarieschromosome libraries
• Genes with Genes with intronsintrons• can be amplified in bacteriacan be amplified in bacteria• but protein is not properly expressedbut protein is not properly expressed
Introns (2)Introns (2)
• Eukaryotic genes in cDNA libraries Eukaryotic genes in cDNA libraries • can be expressed in bacteria to produce can be expressed in bacteria to produce
functional protein productsfunctional protein products• because because intronsintrons have been removed from have been removed from
mRNA moleculesmRNA molecules
Learning Objective 3Learning Objective 3
• What is the purpose of a What is the purpose of a genetic probegenetic probe??
Genetic ProbeGenetic Probe
• Radioactive DNA or RNA sequenceRadioactive DNA or RNA sequence• used to screen recombinant DNA molecules used to screen recombinant DNA molecules
in bacterial cellsin bacterial cells• to find specific colony with DNA of interestto find specific colony with DNA of interest
Genetic ProbeGenetic Probe
Fig. 15-5, p. 328
Bacterial colonies Transfer cells from colonies to nitrocellulose filter
1
Radioactively labeled nucleic acid probe is added
Filter with bacteria from colonies; cells are lysed and DNA denatured
2
3 Some radioactive nucleic acid probe molecules become hybridized to DNA of some colonies
4 Exposed X-ray film; dark spots identify colonies with desired DNA
Animation: Use of a Radioactive Animation: Use of a Radioactive ProbeProbe
CLICKTO PLAY
Learning Objective 4Learning Objective 4
• How does the How does the polymerase chain reactionpolymerase chain reaction amplify DNA amplify DNA in vitroin vitro??
Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)
• Automated Automated in vitroin vitro technique technique• targets a particular DNA sequence by specific targets a particular DNA sequence by specific
primers primers • clones it using heat-resistant DNA clones it using heat-resistant DNA
polymerasepolymerase
• Used to analyze tiny DNA samplesUsed to analyze tiny DNA samples• from crime scenes, archaeological remainsfrom crime scenes, archaeological remains
Fig. 15-7, p. 329
Learning Objective 5Learning Objective 5
• What is the difference between DNA, What is the difference between DNA, RNA, and protein RNA, and protein blottingblotting??
Southern BlotSouthern Blot
• Detects Detects DNA fragmentsDNA fragments• separates using separates using gel electrophoresisgel electrophoresis• transfer to nitrocellulose or nylon membranetransfer to nitrocellulose or nylon membrane
• Probe is Probe is hybridizedhybridized • by complementary base pairing to DNA bound by complementary base pairing to DNA bound
to membraneto membrane• bands of DNA identified by autoradiography bands of DNA identified by autoradiography
or chemical luminescenceor chemical luminescence
Gel Gel ElectrophoresisElectrophoresis
Fig. 15-8a, p. 330
Fig. 15-8a, p. 330
DNA
Cut with restriction enzyme
100 base pairs 200 base pairs 300 base pairs
Mixture placed in well Standards of
known size
+ –Origin
Direction of movement 200 base pairs
300 base pairs
100 base pairs
Gel
Fig. 15-8b, p. 330
Southern Southern BlotBlot
Fig. 15-9, p. 332
1 Digest DNA with restriction enzymes. 2
Load DNA fragments on gel for electrophoresis.
– +
DNA DNA fragments
Buffer solution Agarose gel
5
Fig. 15-9, p. 332
4Buffer solution moves upward, transferring DNA fragments to a DNA-binding filter.
5DNA fragments are in same location as those on gel.
3 Separate DNA by electrophoresis.
Weight
Absorbent paper
Longer DNA fragments
Nitrocellulose filter
GelWickBuffer
Shorter DNA fragments6 7
Fig. 15-9, p. 332
6
Place filter and radioactively labeled probe together in sealed bag so it can hybridize.
7
Wash filter to remove excess probe and then expose filter to X-ray film; resulting autoradiograph shows hybridized DNA fragments.
Radioactive probe solution
RNA and ProteinsRNA and Proteins
• Northern BlotNorthern Blot • RNA moleculesRNA molecules separated by electrophoresis separated by electrophoresis• transferred to a membranetransferred to a membrane
• Western BlotWestern Blot• Proteins or polypeptidesProteins or polypeptides previously separated previously separated
by gel electrophoresisby gel electrophoresis
Learning Objective 6Learning Objective 6
• What is the What is the chain termination methodchain termination method of of DNA sequencingDNA sequencing??
DNA SequencingDNA Sequencing
• Yields information about gene structureYields information about gene structure• and amino acid sequence of encoded proteinsand amino acid sequence of encoded proteins
• Geneticists compare DNA sequencesGeneticists compare DNA sequences• with other sequences stored in databaseswith other sequences stored in databases
Automated DNA SequencingAutomated DNA Sequencing
• Based on Based on chain termination methodchain termination method• uses dideoxynucleotidesuses dideoxynucleotides• tagged with colored fluorescent dyestagged with colored fluorescent dyes• terminates elongation during DNA replicationterminates elongation during DNA replication
• Gel electrophoresisGel electrophoresis• separates resulting fragmentsseparates resulting fragments• laser identifies nucleotide sequencelaser identifies nucleotide sequence
DideoxynucleotideDideoxynucleotide
Fig. 15-10, p. 333
Dideoxyadenosine triphosphate (ddATP)
Chain Termination MethodChain Termination Method
Fig. 15-11a, p. 334
Single-strand DNA fragment to be sequenced
+ddATP +ddCTP +ddGTP +ddTTP
Fig. 15-11b, p. 334
Radioactive primer
Direction of synthesis+ddATP
Reaction products from mixture containing dideoxyATP
Fig. 15-11c, p. 334
Larger fragments
Smaller fragments
Fig. 15-11d, p. 334
CA TG
Automated DNA SequenceAutomated DNA Sequence
KEY CONCEPTSKEY CONCEPTS
• Biologists study DNA using Biologists study DNA using gel gel electrophoresis, DNA blotting, automated electrophoresis, DNA blotting, automated sequencing,sequencing, and other methods and other methods
Animation: Automated DNA Animation: Automated DNA SequencingSequencing
CLICKTO PLAY
Learning Objective 7Learning Objective 7
• What are the three main areas of interest What are the three main areas of interest in genomics?in genomics?
Genomics (1)Genomics (1)
Field of biology that studies the entire DNA Field of biology that studies the entire DNA sequence of an organism’s sequence of an organism’s genomegenome
1. Structural genomics1. Structural genomics• mapping and sequencing genomesmapping and sequencing genomes
Genomics (2)Genomics (2)
2. Functional genomics2. Functional genomics• functions of genes and nongene sequences in functions of genes and nongene sequences in
genomesgenomes
3. Comparative genomics3. Comparative genomics• comparing genomes of different speciescomparing genomes of different species• understanding evolutionary relationshipsunderstanding evolutionary relationships
KEY CONCEPTSKEY CONCEPTS
• GenomicsGenomics is an emerging field that is an emerging field that comprises the structure, function, and comprises the structure, function, and evolution of genomesevolution of genomes
Learning Objective 8Learning Objective 8
• What does a What does a DNA microarrayDNA microarray do? do?
• Give an example of its research and Give an example of its research and medical potentialmedical potential
DNA Microarrays (1)DNA Microarrays (1)
• Used in diagnostic testsUsed in diagnostic tests• different DNA molecules placed on glass chip different DNA molecules placed on glass chip
• Enable researchers to compareEnable researchers to compare• many genes in normal and diseased cells many genes in normal and diseased cells
DNA Microarrays (2)DNA Microarrays (2)
• Cancer and other diseases exhibit altered Cancer and other diseases exhibit altered patterns of gene expressionpatterns of gene expression
• DNA microarrays DNA microarrays identify disease-causing identify disease-causing genes (or the proteins they code for)genes (or the proteins they code for)
DNA DNA MicroarrayMicroarray
Fig. 15-13, p. 336
1 Prepare microarray. Each microdot contains multiple copies of a specific single-stranded cDNA.
Treated cell Untreated (control) cell2 Prepare cDNA from
two cell populations (treated and control).
Mature mRNA Mature mRNAReverse transcriptase
Reverse transcriptase
cDNA copy of mRNA
cDNA copy of mRNA
3 Tag each cDNA with different fluorescent dye.
cDNA mRNA (discard) cDNA mRNA (discard)
Fig. 15-13, p. 336
4 Hybridize two cDNA populations to array.
Laser 1 Scan array to identify fluorescence where hybridization has occurred.
5Laser 2
Gene that was inactive in both treated and untreated cells
Emissions6 Computer analysis
produces color-coded readout.
Gene in treated cell that increased activity, compared to controlGene in treated cell that decreased activity, compared to controlGene that was active in both treated and untreated cells
Learning Objective 9Learning Objective 9
• What are What are pharmacogeneticspharmacogenetics and and proteomicsproteomics??
PharmacogeneticsPharmacogenetics
• Science of gene-based medicineScience of gene-based medicine• analyzes individual’s genetic makeupanalyzes individual’s genetic makeup• customizes drugs to matchcustomizes drugs to match
ProteomicsProteomics
• Study of all proteins encoded by genomeStudy of all proteins encoded by genome• Try to identify all proteins made by a cellTry to identify all proteins made by a cell• Harder than sequencing the human genomeHarder than sequencing the human genome
Learning Objective 10Learning Objective 10
• Describe at least one important application Describe at least one important application of of recombinant DNA technologyrecombinant DNA technology in each of in each of the following fields: medicine and the following fields: medicine and pharmacology, DNA fingerprinting, and pharmacology, DNA fingerprinting, and transgenic organismstransgenic organisms
Genetically Altered BacteriaGenetically Altered Bacteria
• Produce important human protein productsProduce important human protein products• insulininsulin• growth hormonegrowth hormone• tissue plasminogen activator (TPA)tissue plasminogen activator (TPA)• tissue growth factor-beta (TGF- tissue growth factor-beta (TGF- ββ))• clotting factor VIIIclotting factor VIII• Dornase Alpha (DNase)Dornase Alpha (DNase)
DNA FingerprintingDNA Fingerprinting
• Analysis of individual’s DNAAnalysis of individual’s DNA• based on based on short tandem repeats (STRs)short tandem repeats (STRs)
(molecular markers, highly (molecular markers, highly polymorphicpolymorphic))
• Applications inApplications in• law enforcementlaw enforcement• disputed parentagedisputed parentage• tracking tainted foodstracking tainted foods
Fig. 15-14, p. 339
1 2 3 4 5 6 7From blood at
crime scene
Animation: DNA FingerprintingAnimation: DNA Fingerprinting
CLICKTO PLAY
Transgenic OrganismsTransgenic Organisms
• Foreign DNAForeign DNA• incorporated into genetic material incorporated into genetic material
• Transgenic livestockTransgenic livestock• produce foreign proteins in milkproduce foreign proteins in milk
• Transgenic plantsTransgenic plants• have great potential in agriculturehave great potential in agriculture
Fig. 15-15, p. 340
Fig. 15-16, p. 341
Fig. 15-17, p. 342
Fig. 15-17a, p. 342
Fig. 15-17b, p. 342
KEY CONCEPTSKEY CONCEPTS
• DNA technology and genomics have wide DNA technology and genomics have wide applications, from medical to forensic to applications, from medical to forensic to agriculturalagricultural
Learning Objective 11Learning Objective 11
• Describe at least two safety issues Describe at least two safety issues associated with associated with recombinant DNA recombinant DNA technologytechnology
• How are these issues being addressed?How are these issues being addressed?
Safety ConcernsSafety Concerns
• Genetically engineered organismsGenetically engineered organisms • Scientists have specific safety guidelines for Scientists have specific safety guidelines for
using recombinant DNA technology using recombinant DNA technology
• Introduction of Introduction of transgenictransgenic plants and plants and animalsanimals into the natural environment into the natural environment• may spread in an uncontrolled mannermay spread in an uncontrolled manner
Genetically Engineered PlantGenetically Engineered Plant
Animation: Gene Transfer Using a Ti Animation: Gene Transfer Using a Ti PlasmidPlasmid
CLICKTO PLAY
Animation: Base-pairing of DNA Animation: Base-pairing of DNA FragmentsFragments
CLICKTO PLAY
CLICKTO PLAY
Video: Cloned PoochVideo: Cloned Pooch
• From ABC News, Biology in the Headlines, 2005 DVD.From ABC News, Biology in the Headlines, 2005 DVD.