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

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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

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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

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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

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Animation: Base-pairing of DNA Animation: Base-pairing of DNA FragmentsFragments

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Video: Cloned PoochVideo: Cloned Pooch

• From ABC News, Biology in the Headlines, 2005 DVD.From ABC News, Biology in the Headlines, 2005 DVD.