Staff Site Universitas Negeri Yogyakarta
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
GANTC
Enzyme digestion (HinfI)
CTNAG
CC (wild type)G A G C C
198 bp
Ala
TT
(mutant)
GAGTC
Val
~23 bp
~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)
CGGCCG
GCCGGC
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 replicationdefective, 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
• 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 b-carotene
• 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
• 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
•
•
•
•
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
• 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
GANTC
Enzyme digestion (HinfI)
CTNAG
CC (wild type)G A G C C
198 bp
Ala
TT
(mutant)
GAGTC
Val
~23 bp
~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)
CGGCCG
GCCGGC
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 replicationdefective, 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
• 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 b-carotene
• 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
• 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
•
•
•
•
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