2. The Role of Recombinant DNA Technology in
Biotechnology
• 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
3. Overview of recombinant DNA technology
Bacterial cell
Bacterial
chromosome
Plasmid
Gene of interest
DNA containing
gene of interest
Isolate plasmid.
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
Harvest proteins
coded by gene
Eliminate
undesirable
phenotypic
traits
Produce vaccines,
antibiotics,
hormones, or
enzymes
Create
beneficial
combination
of traits
4. 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
5. 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
6. 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
7. 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
9. 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
10. Producing a recombinant vector
Antibiotic
resistance
gene
Restriction
site
mRNA for human
growth hormone (HGH)
Reverse
transcription
Plasmid (vector)
cDNA for HGH
Restriction
enzyme
Restriction
enzyme
Sticky ends
Gene for human
growth hormone
Ligase
Recombinant plasmid
Introduce recombinant
plasmid into bacteria.
Recombinant
plasmid
Bacterial
chromosome
Inoculate bacteria
on media containing
antibiotic.
Bacteria containing
the plasmid with
HGH gene survive
because they also
have resistance gene.
11. 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
12. 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.
13. Techniques of Recombinant DNA Technology
• 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
14. 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
15. The use of PCR to replicate DNA, steps 1-3
3´
Original DNA
molecule
5´
5´
3´
Heat to 94°C
DNA primer
DNA polymerase
Deoxyribonucleotide
triphosphates
Denaturation
Priming
Cool to 65°C
DNA polymerase
DNA primer
Extension
3´
72°C
3´ 5´
5´
5´
5´
16. The use of PCR to replicate DNA, step 4
Repeat
First cycle
2 DNA
molecules
4 DNA
molecules
Second cycle Third cycle Fourth cycle
8 DNA
molecules
16 DNA
molecules
17. Techniques of Recombinant DNA Technology
• Selecting a Clone of Recombinant Cells
– Must find clone containing DNA of interest
– Probes are used
18. 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
20. 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 cultured
21. 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.
Side view
DNA
DNA bands
Gel
Nitrocellulose
membrane
Absorbent
material
The DNA fragments
are invisible to the
investigators at
this stage.
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
22. 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
sample
24. 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
25. Artificial methods of inserting DNA into cells: electroporation
Chromosome
Electroporation
Pores in wall and membrane
Competent cell
Electrical
field applied
DNA from
another source
Cell synthesizes
new wall
Recombinant cell
26. Artificial methods of inserting DNA into cells: protoplast fusion
Cell walls
Protoplast fusion
Polyethylene
glycol
Protoplasts
Enzymes remove
cell walls
Fused protoplasts
Recombinant cell
New wall
Cell synthesizes
new wall
27. Artificial methods of inserting DNA into cells: gene gun
Gene gun
Protoplasts
Nylon
projectile
Nylon
projectile
Blank .22
caliber shell
DNA-coated beads
Vent
Target cell
Plate to stop
nylon projectile
28. Artificial methods of inserting DNA into cells: microinjection
Microinjection
Target cell
Suction tube
to hold target
cell in place
Target cell’s
nucleus
Micropipette
containing DNA
29. 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)
32. 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
33. 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
34. 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
36. 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
38. Applications of Recombinant DNA Technology
• Agricultural Applications
– Herbicide tolerance
– Gene from Salmonella conveys resistance to
glyphosate (Roundup™)
– Farmers can kill weeds without killing crops
– Salt tolerance
– Scientists have inserted gene for salt tolerance into
tomato and canola plants
– Transgenic plants survive, produce fruit, and
remove salt from soil
39. Applications of Recombinant DNA Technology
• Agricultural Applications
– Freeze resistance
– Crops sprayed with genetically modified bacteria
can tolerate mild freezes
– Pest resistance
– Bt toxin
– Naturally occurring toxin harmful only to insects
– Organic farmers used to reduce insect damage to
crops
– Gene for Bt toxin inserted into various crop plants
– Genes for Phytophthora resistance inserted into
potato crops
40. Applications of Recombinant DNA Technology
• Agricultural Applications
– Improvements in nutritional value and yield
– Tomatoes allowed to ripen on vine and shelf life
increased
– Gene for enzyme that breaks down pectin
suppressed
– BGH allows cattle to gain weight more rapidly
– Produce meat with lower fat content and produce
10% more milk
– Gene for β-carotene (vitamin A precursor) inserted
into rice
– Scientists considering transplanting genes coding
for entire metabolic pathways
41. 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
42. 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
43. 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”?
