BIOTEK
• BIOTECHNOLOGY – technologies that involve
the use of living organisms or products from
living organisms in order to benefit humans
usually for medical, agricultural and industrial
application
Science based technologies, such as
recombinant DNA techniques, biochemistry,
molecular and cellular biology, microbiology,
and genetics
Impact on society
• Biotechnology has made it possible to detect,
and in some cases treat, diseases such as
sickle-cell anemia, and cystic fibrosis
• Genetic tests used to track down criminals in
assault cases based on the uniqueness of their
DNA
• Genetic counselling provides advice on
heritable diseases, and genetic screening of
workers in possible risk industries
Concerns about biotechnology and genetic
engineering
– Novel DNA
– Antibiotic resistance genes
– New proteins
– Escape of genes in the environment
– Reduction in biodiversity
• Many countries are actively reviewing the
safety and ethics of biotechnology research
and its applications
• Some countries have established research
guidelines, biosafety framework
• Ban work on stem cell research
Biotech and health
PCR – RFLP (Restriction Fragment Length Polymorphisms)
A combination of PCR – restriction method to detect SNP
(single nucleotide polymorphsim)
The sample is first run in a restriction digest to cut the DNA, then
gel electrophoresis is performed on this digest. In the case of
MTHFR C677T polymorphism, single band of 198 bp denotes CC
genotype, two bands of 198 and 175 bp denote CT genotype
and single band of 175 bp denotes TT genotype.
After gel electrophoresis, DNA can be visualized by staining
with ethidium bromide, an intercalating agent and fluorescent
dye.
PCR amplification of MTHFR exon 4
G A N T C
C T N A G
198 bp
G A G C C
Ala
Enzyme digestion (HinfI)
CC (wild type)
TT (mutant)
~23 bp
G A G T C
Val
~175 bp
Gel Electrophoresis
M
CC
CT
TT
198 bp
175 bp
PCR-RFLP untuk polimorfisme G135A gena RET
PCR amplification of RET exon 2
Enzyme digestion (EagI)
C G G C C G
G C C G G C
294 bp
~87 bp
~207 bp
Gel Electrophoresis
Vietnamese SMA Patients
Mk
A
3
5
7
8
9
11 19 20 21
22 23
24 C+ CSMN1 Exon 7
SMN2 Exon 7
B
C
SMN2 Exon 8
SMN2 Exon 8
SMN2 Exon 8
NAIP Exon 5
Insulin
Cloning organism
Human Gene Therapy
• repair the damage caused by a genetic
deficiency through introduction of a functional
version of the defective gene.
• a cloned variant of the gene must be
incorporated into the organism in such a
manner that it is expressed only at the proper
time and only in appropriate cell types.
• A basic strategy in human gene therapy
involves incorporation of a functional gene
into target cells.
• The gene is typically in the form of an
expression cassette consisting of a cDNA
version of the gene downstream from a
promoter that drives expression of the gene.
• A vector carrying such an expression cassette
is introduced into target cells, either
– ex vivo via gene transfer into cultured cells in the
laboratory and administration of the modified
cells to the patient
– or in vivo via direct incorporation of the gene into
the cells of the patient.
• A replication-deficient version of Maloney murine
leukemia virus can serve as a vector for
expression cassettes up to 9 kb in size.
• Recombinant, replication-deficient adenoviruses
enter target cells via specific receptors on the
target cell surface; the transferred genetic
information is expressed directly from the
adenovirus recombinant DNA and is never
incorporated into the host cell genome.
Retrovirus-mediated gene delivery ex vivo
• Retroviruses are RNA viruses that replicate
their RNA genome by first making a DNA
intermediate.
• The Maloney murine leukemia virus (MMLV) is
the retrovirus used in human gene therapy.
• Deletion of the essential genes gag, pol, and
env from MMLV makes it replication-deficient
(so it can’t reproduce)
• (a) creates a space for insertion of an expression cassette
• (b) The modified MMLV acts as a vector for the expression
cassette; although replication-defective, it is still infectious.
Infection of a packaging cell line that carries intact gag, pol,
and env genes allows the modified MMLV to reproduce
• (c), and the packaged retroviral viruses can be collected
and used to infect a patient (d).
• In the cytosol of the patient’s cells, a DNA copy of the viral
RNA is synthesized by viral reverse transcriptase, which
accompanies the viral RNA into the cells. This DNA is then
randomly integrated into the host cell genome, where its
expression leads to production of the expression cassette
product.
Adenovirus-mediated gene delivery in vivo.
• Adenoviruses are DNA viruses.
• The adenovirus genome (36 kb) is divided into early genes
(E1 through E4) and late genes (L1 to L5)
• (a) Adenovirus vectors are generated by deleting gene E1
(and sometimes E3 if more space for an expression cassette
is needed)
• (b)deletion of E1 renders the adenovirus incapable of
replication unless introduced into a complementing cell line
carrying the E1 gene
• (c)Adenovirus progeny from the complementing cell line
can be used to infect a patient. In the patient, the
adenovirus vector with its expression cassette enters the
cells via specific receptors
• (d) Its linear dsDNA ultimately gains access to the cell
nucleus, where it functions extra chromosomally and
expresses the product of the expression cassette (e).
DNA fingerprinting
• DNA fingerprinting is used to link small amounts
of biological material
• human genome contains non-coding repetitive
DNA sequences, the length of which varies from
individual to individual.
• Short tandem repeats (STRs) thus exist in which
dinucleotides (e. g., -T-X-) are frequently
repeated.
• Each STR can occur in five to 15 different lengths
(alleles), of which one individual possesses only
one or two.
Biotechnology in agriculture
Reasons for developing transgenic plants
– To improve agricultural, horticultural and
ornamental value of a crop plant
– Can act as a living bioreactor
– Means of studying the action of genes
Crops and traits
• Canola Herbicide tolerance; modified seed fatty acid content
• Maize Herbicide tolerance; resistance to corn root worm; resistance
to European corn borer
• Melon Delayed ripening
• Papaya Resistance to viral infection
• Potato Resistance to Colorado potato beetle; resistance to potato
leafroll luteovirus
• Rice Herbicide tolerance; Beta-carotene
• Soya bean Herbicide tolerance; modified seed fatty acid content
• Squash Resistance to viral infection
• Tomato Delayed ripening; delayed softening
Delayed ripening in Tomato
• Introduction of a gene
that results in degradation
of a precursor of the plant
hormone, ethylene
• Production of tomatoes
for human consumption,
either fresh or processed
• Agritope Inc. USA
Resistance to Colorado potato beetle
• Introduction of a toxin
gene from a bacteria that
results in insect resistance
in potato
• Production of potatoes for
human consumption and
livestock feed including
potato process residue
• Monsanto Company, USA
Herbicide tolerance Corn
• Glyphosate herbicide
Tolerance
• Production of Z. mays
for human consumption
(wet mill or dry mill or
seed oil), and meal and
silage for livestock feed
• Monsanto Company,
USA
Papaya Resistant to viral infection
• Resistance to papaya
ringspot virus (PRSV)
• Production of papaya for
human consumption,
either fresh or processed
• Cornell University, USA &
UWI, Jamaica
Virus resistant tomato
• Insertion of a mutant
gene from the virus to
prevent replication
• Production of
tomatoes for human
consumption, either
fresh or processed
• UW-Madison, Hebrew
Univ., UWI
Benefits of the New Technology
• Higher yields & lower pesticide usage
– Provide indirect benefits for consumers and
the environment through lower usage of
pesticides and there are higher yields due to
reduced pest losses.
• More Nutritious Foods
– increasing the levels of essential amino acids,
vitamins, bio-available iron and to reduce
toxicity
– more nutritious harvested products that keep
much longer in storage and transport.
• Utilization of marginal lands
– produce plants that are more tolerant to
drought, salt and heat stresses, toxic heavy
metals
Problems with rDNA technology
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Instability in transgene expression
Interruption or silencing of existing genes
Activation of silent genes
Expression of anti-nutrients
Unanticipated effects
in transgenic crops
• Canola – overexpression of phytoene synthase
resulted in X500 increase in levels of a and bcarotene
• Maize – the stems of Bt maize contain more
lignin
• Potato – expressing kanamycin showed
changes in phynotypic and yield performance
Concerns
• Food safety
• Allergic reaction – Cry9C protein in Starlink
corn
• Use of antibiotic marker genes
• Negative impact non-target species – Bt corn
• Creation of super weeds - gene flow
• Creation of new viruses - recombination
Food safety
• Unsafe for human consumption ?
– Allergic reaction to new proteins
– 60% of processes foods in supermarkets in the
USA contain a GM ingredient
– Soy, corn, canola and some fresh vegetables
Allergic reaction
• Cry9C a protein in Starlink corn
• Insecticidal protein shares properties with
proteins that are known food allergen
• Not licensed for human consumption
• “Taco Bell Home originals”
Antibiotic marker genes
• Antibiotics – kanamycin, hygromycin,
tetracycline, ampicillin
• Used to identify plants carrying the transgene
• Presence of the gene in the gut could enter
gut organism, increase resistance
• WHO report that antibiotic genes are safe
Impact on non-target species
• Pollen from GM corn contains the insecticidal
Bt toxin thought to be a danger to the
monarch butterfly
• Milkweed with pollen from Bt corn plants
could kill monarch catapillars that fed on them
• Six recent studies finds that pollen from
varieties of Bt corn most commonly grown
inthe USA do not contain enough toxin to
harm monarchs.
Super weeds
• Herbicide resistant GM crops
• Insect resistant GM crops
• Gene flow between trangenics and traditional
maize in Mexico
• Gene flow - natural process
New viruses
• Recombination through horizontal gene
transfer
• Naturally occurring viruses and spread to new
hosts
Regulating Biotechnology
• Cartagena Protocol
• CODEX Alimentarius
• National Biosafety Committee-NCST
Cartagena Protocol
• Trade related treaty
• Regulation on the transboundary movement
of living modified organisms
• Risk assessment system based on science
intended to assure the safety of products to
be imported
CODEX Alimentarius
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Human health risk analysis of GM foods
Case-by-case assessment
Evaluate direct and unintentional effects
Toxicity
Allergenicity
Changes in nutritional properties
Gene stability
NBC - NCST
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Permission to import GMOs
Risk assessment
Environmental impact assessment
Monitor imports and uses of transgenic crops
European Union vs. USA
• EU ban the importation of GM foods for sale
• Strong support by consumers
• Call for labeling of products derived from
GMO
• Label if there is >1% GM material in the foods