Download Chap 8 Recombinant DNA technology Fall 2012

Chapter 8
Recombinant
DNA
Technology
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• 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 technology
Bacterial cell
DNA containing
gene of interest
Bacterial
chromosome
Plasmid
Isolate plasmid.
Gene of interest
Enzymatically cleave
DNA into fragments.
Isolate fragment
with the gene of
interest.
Insert gene into plasmid.
Insert plasmid and gene into
bacterium.
Culture bacteria.
Harvest copies of
gene to insert into
plants or animals
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Eliminate
undesirable
phenotypic
traits
Harvest proteins
coded by gene
Create
beneficial
combination
of traits
Produce vaccines,
antibiotics,
hormones, or
enzymes
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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 cellfree 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 vector
mRNA for human
growth hormone (HGH)
Antibiotic
resistance
gene
Restriction
site
Reverse
transcription
cDNA for HGH
Plasmid (vector)
Restriction
enzyme
Restriction
enzyme
Sticky ends
Gene for human
growth hormone
Ligase
Recombinant plasmid
Introduce recombinant
plasmid into bacteria.
Bacterial
chromosome
Recombinant
plasmid
Inoculate bacteria
on media containing
antibiotic.
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Bacteria containing
the plasmid with
HGH gene survive
because they also
have resistance gene.
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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 genome
or organism.
Generate fragments using
restriction enzymes.
Insert each fragment
into a vector.
Introduce vectors
into 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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.
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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
standards
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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
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– Genetic “fingerprinting”
– Diagnosis of infectious disease
– Demonstrate incidence and prevalence of organisms
that cannot be cultured
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Figure 8.7 The Southern blot technique-overview
DNA molecules
Restriction enzymes
Restriction fragments
Use gel electrophoresis to separate
fragments by size; denature DNA
into single strands with NaOH.
DNA
DNA bands
The DNA fragments
are invisible to the
investigators at
this stage.
Gel
Nitrocellulose
membrane
Absorbent
material
Side view
Electrophoresis
gel
Nitrocellulose
membrane
Absorbent
material
Nitrocellulose membrane
with DNA fragments at
same locations as in gel
(still invisible) is baked to
permanently affix DNA.
Add radioactive probes
complementary to DNA
nucleotide sequence
of interest.
Probes bind to DNA
of 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 singlestranded 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
sample
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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
Pores in wall and membrane
Chromosome
Cell synthesizes
new wall
Electrical
field applied
Recombinant cell
Competent cell
DNA from
another source
Electroporation
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Figure 8.9b Artificial methods of inserting DNA into cells: protoplast fusion
Cell synthesizes
new wall
Cell walls
Enzymes remove
cell walls
Polyethylene
glycol
Recombinant cell
New wall
Protoplasts
Fused protoplasts
Protoplast fusion
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Figure 8.9c Artificial methods of inserting DNA into cells: gene gun
Blank .22
Nylon
caliber shell projectile
Vent
Plate to stop
nylon projectile
Target cell
DNA-coated beads
Protoplasts
Gene gun
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Nylon
projectile
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Figure 8.9d Artificial methods of inserting DNA into cells: microinjection
Micropipette
containing DNA
Target cell’s
nucleus
Target cell
Suction tube
to hold target
cell in place
Microinjection
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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)
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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 body
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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
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© 2012 Pearson Education Inc.
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The Ethics and Safety of Recombinant DNA Technology
• Ethical Issues
–
–
–
–
–
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Routine screenings?
Who should pay?
Genetic privacy rights?
Profits from genetically altered organisms?
Required genetic screening?
Forced correction of “genetic abnormalities”?
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© 2012 Pearson Education Inc.
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