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Chapter 8
Recombinant DNA
Technology
10/1/11 1MDufilho
• Recombinant DNA Technology– Intentional modification of organisms’ genomes for
practical purposes
– Three goals – Eliminate undesirable phenotypic traits– Combine beneficial traits of two or more organisms– Create organisms that synthesize products humans
need
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Figure 8.1 Overview of recombinant DNA technologyBacterial cell
Bacterialchromosome
Plasmid
Gene of interest
DNA containinggene of interest
Isolate plasmid.
Enzymatically cleaveDNA into fragments.
Isolate fragmentwith the gene ofinterest.
Insert gene into plasmid.
Insert plasmid and gene intobacterium.
Culture bacteria.
Harvest copies ofgene to insert intoplants or animals
Harvest proteinscoded by gene
Eliminateundesirablephenotypictraits
Produce vaccines,antibiotics,hormones, orenzymes
Createbeneficialcombinationof traits
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The Tools of Recombinant DNA Technology
• Mutagens– Physical and chemical agents that produce
mutations– Scientists utilize mutagens to
– Create changes in microbes’ genomes to change phenotypes
– Select for and culture cells with beneficial characteristics
– Mutated genes alone can be isolated
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The Tools of Recombinant DNA Technology
• The Use of Reverse Transcriptase to Synthesize cDNA
– Isolated from retroviruses
– Uses RNA template to transcribe molecule of cDNA
– Easier to isolate mRNA molecule for desired protein first
– mRNA of eukaryotes has introns removed – Allows cloning in prokaryotic cells
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The Tools of Recombinant DNA Technology
• Synthetic Nucleic Acids– Molecules of DNA and RNA produced in cell-
free solutions
– Uses of synthetic nucleic acids– Elucidating the genetic code– Creating genes for specific proteins– Synthesizing DNA and RNA probes to locate
specific sequences of nucleotides– Synthesizing antisense nucleic acid molecules
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The Tools of Recombinant DNA Technology
• Restriction Enzymes– Bacterial enzymes that cut DNA molecules only at
restriction sites
– Categorized into two groups based on type of cut– Cuts with sticky ends– Cuts with blunt ends
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Figure 8.2 Actions of restriction enzymes-overview
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The Tools of Recombinant DNA Technology
ANIMATION Recombinant DNA Technology
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The Tools of Recombinant DNA Technology
• Vectors– Nucleic acid molecules that deliver a gene into
a cell
– Useful properties– Small enough to manipulate in a lab– Survive inside cells– Contain recognizable genetic marker– Ensure genetic expression of gene
– Include viral genomes, transposons, and plasmids
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Figure 8.3 Producing a recombinant vectorAntibioticresistancegene
Restrictionsite
mRNA for humangrowth hormone (HGH)
Reversetranscription
Plasmid (vector)
cDNA for HGH
Restrictionenzyme
Restrictionenzyme
Sticky ends
Gene for humangrowth hormone
Ligase
Recombinant plasmid
Introduce recombinantplasmid into bacteria.
Recombinantplasmid
Bacterialchromosome
Inoculate bacteriaon media containingantibiotic.
Bacteria containingthe plasmid withHGH gene survivebecause they alsohave resistance gene.10/1/11 11MDufilho
The Tools of Recombinant DNA Technology
• Gene Libraries– A collection of bacterial or phage clones
– Each clone in library often contains one gene of an organism’s genome
– Library may contain all genes of a single chromosome
– Library may contain set of cDNA complementary to mRNA
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Figure 8.4 Production of a gene library-overview
Genome
Isolate genomeor organism.
Generate fragments usingrestriction enzymes.
Insert each fragmentinto a vector.
Introduce vectorsinto cells.
Culture recombinant cells;descendants are clones.
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• Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)
– Large number of identical molecules of DNA produced in vitro
– Critical to amplify DNA in variety of situations– Epidemiologists use to amplify genome of
unknown pathogen – Amplified DNA from Bacillus anthracis spores in
2001 to identify source of spores
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Techniques of Recombinant DNA Technology
• Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)
– Repetitive process consisting of three steps– Denaturation– Priming– Extension
– Can be automated using a thermocycler
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Techniques of Recombinant DNA Technology
ANIMATION Polymerase Chain Reaction: Components
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Techniques of Recombinant DNA Technology
.
ANIMATION PCR: The Process
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Techniques of Recombinant DNA Technology
• Selecting a Clone of Recombinant Cells– Must find clone containing DNA of interest
– Probes are used
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Techniques of Recombinant DNA Technology
• Separating DNA Molecules: Gel Electrophoresis and the Southern Blot
– Gel electrophoresis– Separates molecules based on electrical charge, size,
and shape– Allows scientists to isolate DNA of interest– Negatively charged DNA drawn toward positive
electrode– Agarose makes up gel; acts as molecular sieve– Smaller fragments migrate faster than larger ones– Determine size by comparing distance migrated to
standards10/1/11 19MDufilho
Figure 8.6 Gel electrophoresis-overview
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Techniques of Recombinant DNA Technology
• Separating DNA Molecules: Gel Electrophoresis and the Southern Blot
– Southern blot– DNA transferred from gel to nitrocellulose membrane– Probes used to localize DNA sequence of interest– Northern blot: used to detect RNA
– Uses of Southern blots– Genetic “fingerprinting” – Diagnosis of infectious disease– Demonstrate incidence and prevalence of organisms
that cannot be cultured10/1/11 21MDufilho
Figure 8.7 The Southern blot technique-overview
DNA molecules
Restriction enzymes
Restriction fragments
Use gel electrophoresis to separatefragments by size; denature DNAinto single strands with NaOH.
Side view
DNA
DNA bandsGel
Nitrocellulosemembrane
Absorbentmaterial
The DNA fragmentsare invisible to theinvestigators atthis stage.
Electrophoresisgel
Nitrocellulosemembrane
Absorbentmaterial
Nitrocellulose membranewith DNA fragments atsame locations as in gel(still invisible) is baked topermanently affix DNA.
Add radioactive probescomplementary to DNAnucleotide sequenceof interest.
Probes bind to DNAof interest.
Incubate with film; radiation exposes film.Develop film.
Developed film
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Techniques of Recombinant DNA Technology
• DNA Microarrays– Consist of molecules of immobilized single-
stranded DNA
– Fluorescently labeled DNA washed over array will adhere only at locations where there are complementary DNA sequences
– Variety of scientific uses of DNA microarrays– Monitoring of gene expression– Diagnosis of infection– Identification of organisms in an environmental
sample10/1/11 23MDufilho
Figure 8.8 DNA microarray-overview
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Techniques of Recombinant DNA Technology
• Inserting DNA into Cells– Goal of DNA technology is insertion of DNA into cell
– Natural methods – Transformation– Transduction– Conjugation
– Artificial methods– Electroporation– Protoplast fusion– Injection: gene gun and microinjection
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Figure 8.9a Artificial methods of inserting DNA into cells: electroporation
Chromosome
Electroporation
Pores in wall and membrane
Competent cell
Electricalfield applied
DNA fromanother source
Cell synthesizesnew wall
Recombinant cell
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Figure 8.9b Artificial methods of inserting DNA into cells: protoplast fusion
Cell walls
Protoplast fusion
Polyethyleneglycol
Protoplasts
Enzymes removecell walls
Fused protoplasts
Recombinant cellNew wall
Cell synthesizesnew wall
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Figure 8.9c Artificial methods of inserting DNA into cells: gene gun
Gene gun
Protoplasts
Nylonprojectile
Nylonprojectile
Blank .22caliber shell
DNA-coated beads
Vent
Target cell
Plate to stopnylon projectile
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Figure 8.9d Artificial methods of inserting DNA into cells: microinjection
Microinjection
Target cell
Suction tubeto hold targetcell in place
Target cell’snucleus
Micropipettecontaining DNA
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Applications of Recombinant DNA Technology
• Genetic Mapping– Locating genes on a nucleic acid molecule
– Provides useful facts concerning metabolism, growth characteristics, and relatedness to others
• Locating Genes– Until 1970, genes identified by labor-intensive
methods
– Simpler and universal methods now available
– Restriction fragmentation
– Fluorescent in situ hybridization (FISH)10/1/11 30MDufilho
Figure 8.10 FISH
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Applications of Recombinant DNA Technology
• Environmental Studies– Most microorganisms have never been grown in a
laboratory
– Scientists know them only by their DNA fingerprints
– Allowed identification of over 500 species of bacteria from human mouths
– Determined that methane-producing archaea are a problem in rice agriculture
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Applications of Recombinant DNA Technology
• Pharmaceutical and Therapeutic Applications– Protein synthesis
– Creation of synthetic peptides for cloning
– Vaccines– Production of safer vaccines– Subunit vaccines– Genes of pathogens introduced into common fruits
and vegetables– Injecting humans with plasmid carrying gene from
pathogen– Humans synthesize pathogen’s proteins
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Applications of Recombinant DNA Technology
• Pharmaceutical and Therapeutic Applications– Genetic screening
– DNA microarrays used to screen individuals for inherited disease caused by mutations
– Can also identify pathogen’s DNA in blood or tissues
– DNA fingerprinting– Identifying individuals or organisms by their unique
DNA sequence
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Figure 8.12 DNA fingerprinting
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Applications of Recombinant DNA Technology
• Pharmaceutical and Therapeutic Applications– Gene therapy
– Missing or defective genes replaced with normal copies
– Some patients’ immune systems react negatively
– Medical diagnosis– Patient specimens can be examined for presence of
gene sequences unique to certain pathogens
– Xenotransplants– Animal cells, tissues, or organs introduced into
human body10/1/11 36MDufilho
The Ethics and Safety of Recombinant DNA Technology
– Supremacist view: humans are of greater value than animals
– Long-term effects of transgenic manipulations are unknown
– Unforeseen problems arise from every new technology and procedure
– Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms
– Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic
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The Ethics and Safety of Recombinant DNA Technology
• Studies have not shown any risks to human health or environment
• Standards imposed on labs involved in recombinant DNA technology
• Can create biological weapons using same technology
© 2012 Pearson Education Inc.
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The Ethics and Safety of Recombinant DNA Technology
• Ethical Issues– Routine screenings?
– Who should pay?
– Genetic privacy rights?
– Profits from genetically altered organisms?
– Required genetic screening?
– Forced correction of “genetic abnormalities”?
© 2012 Pearson Education Inc.
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