Download 1 Biotechnology and Recombinant DNA

Biotechnology and Recombinant DNA
Recombinant DNA procedures - an overview
 Biotechnology: The use of microorganisms, cells, or cell
components to make a product.
 Foods, antibiotics, vitamins, enzymes
 Recombinant DNA technology = Genetic Engineering:
Insertion or modification of genes in an organism to produce
desired proteins/outcomes.
 e.g. gene(s) from one organism can be placed into another
organism’s DNA - including between species - to produce:
 recombinant proteins e.g. insulin, hepatitis B vaccine
 enough copies of the recombinant DNA for further studies
Recombinant DNA procedures - an overview
Recombinant DNA procedures - an overview
Clones
Clones
Vector must be
self-replicating
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Tools of Biotechnology
1. Selection:
 Natural selection - survival of the fittest
 Artificial selection - selection by humans of organisms with
desirable characteristic for breeding/cultivation
 e.g. Culturing of a naturally occurring microbe that produces a
desired product
2. Mutation (a change in the DNA):
 Mutagens cause mutations that might result in a microbe with a
desirable trait
 Select and culture microbe with a desired mutation
Tools of Biotechnology (cont.)
3. Restriction enzymes (RE)
 Cut specific sequences of DNA
 Occur naturally in some bacteria
 in vivo role - destroy bacteriophage DNA in bacterial cells
 Cannot digest (host) DNA with methylated cytosines
 Purified REs used in genetic engineering
 A specific RE always recognizes and cuts DNA at a very
specific DNA nucleotide sequence.
 e.g. enzyme EcoRI - GAATTC
 Site-directed mutagenesis: Change a specific DNA gene
Know what “DNA SEQUENCE” means
sequence to change a protein
Tools of Biotechnology (cont.)
3. Restriction enzymes (RE) - cont.
 100s of different REs
 The cuts made by some REs are staggered, producing
The role of restriction enzymes in making recombinant DNA
The sticky end of one DNA
fragment can hydrogen bond
with a complimentary base
sequence (sticky end) of
another fragment.
‘sticky ends’
 Note: recognition sites are
on both strands of DNA, but
DNA from another
source, cut with
the same RE
run in opposite directions
Covalent
linkage by
DNA ligase
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The role of restriction enzymes in making recombinant DNA
Recombinant DNA procedures - an overview
Vector must be
self-replicating
Tools of Biotechnology (cont.)
4. Vectors
Tools of Biotechnology (cont.)
4. Vectors (cont.)
 Role - transport recombinant DNA into the desired cell
 Example - plasmids, viruses
 Important characteristics:
 self-replicating
 small
 Shuttle vectors can exist in several different species
 Use of vectors in gene therapy:
 to insert functional genes into human cells with a defective gene
 resist destruction by the recipient cell
 Carry a marker gene: to make retrieval of recombinant clones
carrying the vector easy
 e.g. gene coding for antibiotic resistance, or a specific enzyme
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Vectors - example of a plasmid vector
Gene for
ampicillin
(antibiotic)
resistance
Gene for βgalactosidase
enzyme
Tools of Biotechnology (cont.)
5. Polymerase Chain Reaction (PCR)
 DNA "photocopier"
 Enzymatic technique to make multiple copies of (amplify) a
piece of DNA.
 Results in over a billion copies of the original target DNA!
Restriction
enzyme sites
 Used to:
 Clone DNA for recombination
 Amplify DNA to detectable levels
Origin of
replication
 Sequence DNA
 Diagnose genetic disease
 Detect pathogens
PCR
PCR
1st cycle
2nd cycle
1st cycle
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Recombinant DNA procedures - an overview
PCR
Vector must be
self-replicating
2nd cycle
3rd cycle
4th cycle… etc.. usually 25 - 35 cycles in total
Techniques of genetic engineering:
Inserting recombinant DNA into cells
 After recombinant (foreign) DNA is constructed in vitro (i.e.
outside of a cell), it needs to be inserted back into a cell
Techniques of genetic engineering:
Inserting recombinant DNA into cells
 Transformation:
 Few species are naturally “competent” (i.e. able to
spontaneously be transformed by DNA)
 Can be accomplished by:
 Transformation
 Electroporation
 Protoplast fusion
 Microinjection
 Gene gun
 Chemical treatment (e.g. CaCl2) can make them competent:
 e.g. treated E. coli mixed with DNA and heat shocked
 Electroporation:
 Electric current used - pores form in plasma membrane,
through which DNA enters
 If substantial cell wall present (e.g. G+) - need to first convert
cells to protoplasts by enzymatic treatment
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Techniques of genetic engineering:
Inserting recombinant DNA into cells
 Protoplast fusion:
 In solution, protoplasts fuse to form hybrids at a slow rate
 frequency increased by polyethylene glycol
 A fused hybrid cell contains
Protoplast fusion
with bacterial cells
two chromosomes that can
undergo natural recombination
Techniques of genetic engineering:
Inserting recombinant DNA into cells
 Microinjection:
 Glass micropipette used to inject DNA directly into a cell
Techniques of genetic engineering:
Inserting recombinant DNA into cells
 Gene gun:
 DNA used to coat microscopic gold or tungsten particles
 These then shot into a cell (eukaryotic) using a burst of helium
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Obtaining DNA

Gene/Genomic libraries
How do genetic engineers obtain the genes they are
interested in?

Two main sources of genes used by genetic engineers:
1.
Gene libraries: composed of pieces of an entire genome stored in
bacterial plasmids or in phages.
2. Synthetic DNA: DNA is synthesized by
a machine - but only short sequences (not
much > 100 nucleotides long).
The cloning of prokaryotic DNA is straightforward
The cloning of prokaryotic DNA is straightforward
Cloning of eukaryotic DNA is
generally more involved…
…this part is
more complicated
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Complementary DNA (cDNA)
– the cloning of eukaryotic DNA
Let's clone a gene!
cDNA is made from
mRNA by using the
enzyme reverse
transcriptase
Need a vector with an marker gene
(e.g. antibiotic resistance)
Selecting the desired
clone from a gene library
 Generally a two (or
more) step procedure:
FIRST STEP:
Blue-white screening
of the gene library
- selects for cells
containing foreign
http://oregonstate.edu/instruct/bb350/textmaterials/13/Slide15.jpg
(cloned) DNA
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Blue-white
screening
Blue-white screening
http://www.sigmaaldrich.com
http://oregonstate.edu/instruct/bb350/textmaterials/13/Slide19.jpg
http://oregonstate.edu/instruct/bb350/textmaterials/13/Slide19.jpg
Selecting the desired clone from a gene library
Colony hybridization
 We know which bacterial cells contain foreign DNA…
… but WHICH ones have the gene/DNA we are interested in??
 Uses DNA probes:
 Short pieces of single-stranded
DNA
 Complementary to the gene of
SECOND STEP:
 If an identifiable gene product produced:
 simple culturing required
 Otherwise.. gene identified by colony hybridization screening:
 Used to identify cells that carry a specific cloned gene
SDS
lysis of
cells
interest
 Can be labelled with fluorescent
Treat
with
NaOH
dye or radioactivity
 Will bind to the gene of interest
by complementary base pairing
 Are essentially “gene-seeking
missiles”
next slide
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Colony hybridization (cont.)
E. coli - the genetic engineering ‘workhorse’
 Advantages:
 easily grown, fast grower
 genomics well studied
 Disadvantages:
 Need to eliminate endotoxin from products
 Cells must be lysed to get product
 Alternatives:
 some G+ species (e.g. Bacillus subtilis)
 eukaryotes - yeast, mammalian and plant cells
Genetic engineering - therapeutic applications
 Subunit vaccines:
The Human Genome Project
 All 3 billion (!) nucleotides sequenced by 2001 - one of methods used
called random shotgun sequencing
 Production of only part of a pathogen - a protein
 e.g. nonpathogenic viruses carrying genes for pathogen's
antigens as vaccines
 DNA vaccines
 Gene therapy to replace defective or missing genes
 Gene silencing
www.lifesciencesfoundation.org
everydaybird.blogspot.com
 What do the results look like?????
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Sequencing of complete genomes by
random shotgun sequencing
The Human Genome Project
everydaybird.blogspot.com
Figure 9.14
Sequencing of complete genomes by
random shotgun sequencing
Sequencing of complete genomes
 Sequence data analysed,
and DNA sequences
assembled using computer
software... new field:
 Bioinformatics: the
science of analysing
genetic (and protein)
sequence data by
computer-assisted
analysis
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Pharmaceutical products of genetic engineering
Scientific applications of recombinant DNA technology
Apart from producing useful
products, other examples are:
1.
The study of DNA
2.
DNA sequencing - e.g. genes or complete
genomes of organisms; even ancient DNA
(neanderthal; mammoth)
Scientific applications of recombinant DNA technology
Agarose gel electrophoresis
Apart from producing useful
products, other examples are:
1.
The study of DNA
2.
DNA sequencing - e.g. genes or complete
genomes of organisms; even ancient DNA
(neanderthal; mammoth)
3.
Genetic screening: testing for the
presence of genetic diseases
4.
DNA fingerprinting (forensic microbiology):
- identification of pathogens
- epidemiology of infectious outbreaks
- forensic medicine
- parentage testing
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Genetic screening
Genetic screening
Genetic screening enables the
Genetic screening enables the
identification of a particular DNA
identification of a particular DNA
sequence among many others.
sequence among many others.
An example of a technique used
in genetic screening is
Southern Blotting and
subsequent hybridisation with a
probe targeted to the gene of interest:
NOTE: probe made from the DNA
sequence of the DEFECTIVE form
of the gene
Southern blotting and Hybridisation
Southern blotting and Hybridisation
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Southern blotting and Hybridisation
Southern blotting and Hybridisation
Southern blotting and Hybridisation
Southern blotting and Hybridisation
NOTE: In the case of
genetic screening
- the probe will bind
(hybridize) with
the defective gene
ONLY IF IT IS
PRESENT in the
sample tested
(= positive result).
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Genetic engineering:
safety issues and ethics
 Avoid accidental release of genetically engineered (modified)
organisms - GMOs
 Genetically modified crops must be safe for consumption and for
the environment
 Labelling of genetically modified food - proposition 522
 Who will have access to an individual's genetic information?
 What to do with a positive genetic test if there is no treatment or
cure?
 Biological weapons
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