• Artificial selectionArtificial selection

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

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)

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)

Genentech- Humulin 1982

Restriction EnzymesRestriction Enzymes

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

Gen reportero

Gen de interéscDNA

Vector Adenovirall

Fig. 15-15, p. 340

Fig. 15-16, p. 341

Fig. 15-17b, p. 342

Genetically Engineered PlantGenetically Engineered Plant

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

Chromosome Chromosome LibraryLibrary

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

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

Animation: Use of a Radioactive Animation: Use of a Radioactive ProbeProbe

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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

Gel Gel ElectrophoresisElectrophoresis

Fig. 15-8a, p. 330

Fig. 15-8a, p. 330

DNACut 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

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

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

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

DideoxynucleotideDideoxynucleotide

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

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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

Animation: DNA FingerprintingAnimation: DNA Fingerprinting

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Fig. 15-14, p. 339

1 2 3 4 5 6 7From blood at

crime scene

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