Biotechnology

Bui Hong Thuy, Ph.D.

School of Biotechnology,
International University
Email: bhthuy@hcmiu.edu.vn

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 Mutations are changes in the
DNA sequence
Some mutations are caused by pieces of
DNA that can jump around the genome
Such jumping DNA is called a transposon or
transposable element
Transposons exist in both prokaryotes and
eukaryotes
For most their normal function (if any) is
unknown, but some larger ones can provide
benefits by moving copies of useful genes
with them

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Missense Mutation Example: Sickle-Cell Anemia

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 Missense Mutation Example: Sickle-Cell Anemia
• Missense at 6th codon in hemoglobin b chain
(counted after protein processing)
• In DNA a T is replaced with an A; this leads to valine
instead of glutamic acid in the protein
• Resulting hemoglobin is “sticky” with other
hemoglobin chains, crystallizing easily
Normal hemoglobin b chain
DNA: CAC GTG GAC TGA GGA CTC CTC
RNA: GUG CAC CUG ACU CCU GAG GAG-
Protein: val-his-leu-thr-pro-glu-glu-

Sickle cell anemia hemoglobin b chain

DNA: CAC GTG GAC TGA GGA CAC CTC
RNA: GUG CAC CUG ACU CCU GUG GAG-
Protein: val-his-leu-thr-pro-val-glu-
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Mutations

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6
 Biotechnology
KEY CONCEPTS
1. DNA cloning yields multiple copies of
a gene or other DNA segment
2. DNA technology allows us to study the
sequence, expression, and function of a
gene
3. Cloning organisms may lead to
production of stem cells for research and
other applications
4. The practical applications of DNA
technology affect our lives in many ways

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1. DNA cloning yields multiple copies of a
gene or other DNA segment

 Gene cloning involves using bacteria to

make multiple copies of a gene
 Foreign DNA is inserted into a plasmid,
and the recombinant plasmid is inserted
into a bacterial cell
 Reproduction in the bacterial cell results in
cloning of the plasmid including the
foreign DNA
 This results in the production of multiple
copies of a single gene

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A preview of gene cloning and some
uses of cloned genes
Bacterium Cell containing gene
1 Gene inserted into of interest
plasmid

Bacterial Plasmid
chromosome Gene of
Recombinant interest DNA of
DNA (plasmid) 2 Plasmid put into chromosome
bacterial cell
Recombinant
bacterium
Host cell grown in culture
3 to form a clone of cells
containing the “cloned”
gene of interest
Gene of Protein expressed
Interest by gene of interest
Copies of gene Protein harvested
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Basic Basic research and Basic
research various applications research
on gene on protein

Gene for pest Gene used to alter Protein dissolves Human growth hor-
resistance inserted bacteria for cleaning blood clots in heart mone treats stunted 9
into plants up toxic waste attack therapy growth
 A preview of gene cloning and some
uses of cloned genes
Bacterium Cell containing gene
of interest
1 Gene inserted into
plasmid

Bacterial Plasmid
chromosome
Gene of
Recombinant interest
DNA of
DNA (plasmid) 2 chromosome
2 Plasmid put into
bacterial cell
Recombinant
bacterium

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 A preview of gene cloning and some
uses of cloned genes
Host cell grown in
Recombinant 3 culture to form a clone
bacterium of cells containing the
“cloned” gene of
interest

Gene of
Interest Protein expressed
by gene of interest
Copies of gene
Protein harvested

4 Basic research
Basic and various Basic
research research
on gene
applications on protein

Gene for pest Gene used to alter Protein dissolves Human growth hor-
resistance inserted bacteria for cleaning blood clots in heart mone treats stunted
into plants up toxic waste attack therapy growth 11
Using Restriction Enzymes to Make
Recombinant DNA
 Bacterial restriction enzymes cut DNA
molecules at specific DNA sequences called
restriction sites
 A restriction enzyme usually makes many cuts,
yielding restriction fragments
 The most useful restriction enzymes cut DNA in
a staggered way, producing fragments with
“sticky ends” that bond with complementary
sticky ends of other fragments
 DNA ligase is an enzyme that seals the bonds
between restriction fragments
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 Restriction site
Using a
restriction DNA 5 3
3 5
1
enzyme and Restriction enzyme
DNA ligase to cuts sugar-phosphate
make backbones.
recombinant
DNA 2 Sticky end
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.

3 One possible combination

DNA ligase
seals strands.

Recombinant DNA molecule 13

 TECHNIQUE Hummingbird
cell
Bacterial cell
lacZ gene

Restriction Sticky Gene of interest

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments

Nonrecombinant
plasmid
Recombinant plasmids

Bacteria carrying

Cloning a
plasmids

Eukaryotic
Gene in a RESULTS

Bacterial Colony carrying non- Colony carrying recombinant

Plasmid
recombinant plasmid plasmid with disrupted lacZ gene
with intact lacZ gene

One of many
bacterial
clones
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 DNA in

Making complementary nucleus

mRNAs in
DNA (cDNA) for a cytoplasm

eukaryotic gene
Reverse
transcriptase Poly-A tail
 A complementary DNA (cDNA) mRNA

library is made by cloning DNA

made in vitro by reverse Degraded
DNA Primer
strand
transcription of all the mRNA mRNA

produced by a particular cell

 A cDNA library represents only
part of the genome—only the
subset of genes transcribed
into mRNA in the original cells DNA
polymerase

cDNA 15
 5 3
TECHNIQUE

Amplifying DNA in Vitro: Target

sequence

The Polymerase Chain Genomic DNA 3 5

Reaction (PCR) 1 Denaturation 5 3

 The PCR can produce 3 5

many copies of a specific 2 Annealing

target segment of DNA Cycle 1

yields
2
Primers

 A three-step cycle— molecules

heating, cooling, and 3 Extension

replication—brings about New

nucleo-

a chain reaction that tides

produces an
exponentially growing Cycle 2
yields
population of identical 4
molecules
DNA molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
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 5 3
TECHNIQUE
Target
sequence

Genomic DNA 3 5

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 1 Denaturation 5 3

3 5
2 Annealing

Cycle 1
yields Primers
2
molecules

3 Extension

New
nucleo-
tides

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 Cycle 2
yields
4
molecules

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 Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence

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2. DNA technology allows us to study the
sequence, expression, and function of a gene

 DNA cloning allows researchers to

– Compare genes and alleles between
individuals
– Locate gene expression in a body
– Determine the role of a gene in an organism
 Several techniques are used to analyze the
DNA of genes

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 Gel Electrophoresis TECHNIQUE

Mixture of
DNA mol-
Power
source
ecules of – Cathode Anode +
different
sizes
 One indirect method of rapidly
analyzing and comparing
Gel
1

genomes is gel electrophoresis Power

source

 This technique uses a gel as a Longer

– +

molecular sieve to separate molecules

nucleic acids or proteins by size 2 Shorter

molecules

 A current is applied that causes RESULTS

charged molecules to move

through the gel
 Molecules are sorted into
“bands” by their size

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 TECHNIQUE

Mixture of Power
source
DNA mol-
ecules of – Cathode Anode +
different
sizes

Gel
1

Power
source
– +
Longer
molecules

2 Shorter
molecules 23
RESULTS

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 Restriction fragment analysis

 DNA fragments produced by restriction

enzyme digestion of a DNA molecule are
sorted by gel electrophoresis
 Restriction fragment analysis is useful for
comparing two different DNA molecules,
such as two alleles for a gene
 The procedure is also used to prepare pure
samples of individual fragments

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Using restriction fragment analysis to distinguish the
normal and sickle-cell alleles of the β-globin gene
Normal -globin allele Normal Sickle-cell
allele allele

175 bp 201 bp Large fragment

DdeI DdeI DdeI DdeI Large

fragment
Sickle-cell mutant -globin allele

376 bp
376 bp Large fragment 201 bp
175 bp
DdeI DdeI DdeI

(a) DdeI restriction sites in (b) Electrophoresis of

normal and sickle-cell restriction fragments from
alleles of -globin gene normal and sickle-cell alleles

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 DNA Sequencing
• Many copies of a single strand of DNA are placed in a
test tube
• DNA polymerase is added
• A mixture of nucleotides is added, some of which have
dye molecules attached
• Each base (A,T,C,G) has a different color dye.
• By chance, some dyed nucleotides & some regular ones
are added
• Dye molecules are large and stop the chain from growing
DNA Primer Deoxyribonucleotides Dideoxyribonucleotides
(template strand) (fluorescently tagged)

dATP ddATP
dCTP ddCTP

DNA dTTP ddTTP

polymerase dGTP ddGTP

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TECHNIQUE
DNA Sequencing
DNA (template Labeled strands
strand)

Shortest Longest

Direction
of movement Longest labeled strand
of strands

Detector

Laser
Shortest labeled strand
RESULTS Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand 28
 DNA Sequencing

 The result is DNA fragments

of multiple sizes with colors
that can be identified.
 After the gel separates the
resulting fragments by size,
we 'read' the sequence from
bottom to top.

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 Analyzing Gene Expression
 Nucleic acid probes can hybridize with
mRNAs transcribed from a gene
 Probes can be used to identify where or
when a gene is transcribed in an organism
 Reverse transcriptase-polymerase chain
reaction (RT-PCR) is added to mRNA to
make cDNA, which serves as a template for
PCR amplification of the gene of interest
 The products are run on a gel and the mRNA
of interest identified

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 TECHNIQUE
1 cDNA synthesis mRNAs

cDNAs
Primers
2 PCR amplification

-globin
gene
3Gel electrophoresis

RESULTS Embryonic stages

1 2 3 4 5 6

RT-PCR analysis of
expression of single genes
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 In situ hybridization uses fluorescent
dyes attached to probes to identify the
location of specific mRNAs in place in the
intact organism

Determining where genes are expressed by in situ

hybridization analysis 32
DNA microarray assay of TECHNIQUE
gene expression levels Tissue sample

1 Isolate mRNA.

2 Make cDNA by reverse mRNA molecules

transcription, using
fluorescently labeled
nucleotides.

Labeled cDNA molecules

3 Apply the cDNA mixture to a (single strands)
DNA fragments
microarray, a different gene in representing
each spot. The cDNA hybridizes specific genes
with any complementary DNA on
the microarray.
DNA microarray

4 Rinse off excess cDNA; scan DNA microarray

microarray for fluorescence. with 2,400
human genes
Each fluorescent spot represents a
gene expressed in the tissue sample. 33
3. Cloning organisms may lead to production
of stem cells for research and other applications

Cloning Plants: Single-Cell Cultures

 One experimental approach for testing

genomic equivalence is to see whether a
differentiated cell can generate a whole
organism
 A totipotent cell is one that can generate
a complete new organism

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EXPERIMENT Can a differentiated plant cell RESULTS
develop into a whole plant?
Transverse
section of
carrot root

2-mg
fragments

Fragments were Single Embryonic Plantlet was A single

cultured in nu- cells plant developed cultured on somatic
trient medium; free in from a cultured agar medium. carrot cell
stirring caused suspension single cell. Later it was developed
single cells to began to planted into a mature
shear off into divide. in soil. carrot plant.
the liquid. 35
 Cloning Animals:
Nuclear Transplantation
 In nuclear transplantation, the nucleus of an
unfertilized egg cell or zygote is replaced
with the nucleus of a differentiated cell
 Experiments with frog embryos have shown
that a transplanted nucleus can often
support normal development of the egg
 However, the older the donor nucleus, the
lower the percentage of normally developing
tadpoles

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37
 Stem Cells of Animals
 A stem cell is a relatively unspecialized cell
(Undifferentiated cell) that can reproduce
itself indefinitely and differentiate into
specialized cells of one or more types
 Stem cells isolated from early embryos at
the blastocyst stage are called embryonic
stem cells; these are able to differentiate
into all cell types
 The adult body also has stem cells, which
replace nonreproducing specialized cells

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The aim of stem cell research is to supply cells
for the repair of damaged or diseased organs
Embryonic stem cells Adult stem cells
(Pluripotent) (Multipotent)

Early human embryo From bone marrow

at blastocyst stage in this example

Cells generating Cells generating

all embryonic some cell types
cell types

Cultured
stem cells

Different
culture
conditions

Different Liver cells Nerve cells Blood cells

types of
differentiated
cells

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4. The practical applications of DNA
technology affect our lives in many ways
Medical Applications
Diagnosis of Diseases
 Scientists can diagnose many human genetic
disorders by using PCR and primers
corresponding to cloned disease genes, then
sequencing the amplified product to look for
the disease-causing mutation
 Genetic disorders can also be tested for
using genetic markers that are linked to the
disease-causing allele

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 Human Gene Therapy
 Gene therapy is the alteration of an afflicted 
individual’s genes
 Gene therapy holds great potential for 
treating disorders traceable to a single 
defective gene
 Vectors are used for delivery of genes into 
specific types of cells, for example bone 
marrow
 Gene therapy raises ethical questions, such 
as whether human germ‐line cells should be 
treated to correct the defect in future 
generations
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Cloned Gene therapy using a retroviral
gene vector
1 Insert RNA version of normal allele
into retrovirus.
Viral RNA

2 Let retrovirus infect bone marrow cells

Retrovirus that have been removed from the
capsid patient and cultured.

3 Viral DNA carrying the normal

allele inserts into chromosome.

Bone
marrow
cell from
patient

4 Inject engineered Bone

cells into patient. marrow 42
 Pharmaceutical Products
Protein Production in Cell Cultures
 Host cells in culture can be engineered
to secrete a protein as it is made
 This is useful for the production of insulin,
human growth hormones, and vaccines

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 Protein Production by “Pharm”
Animals and Plants
 Transgenic animals are pharmaceutical
“factories,” producers of large amounts of
otherwise rare substances for medical use
 “Pharm” plants are also being developed to
make human proteins for medical use

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Produce human therapeutic protein
Dairy cow cells
DNA Transfection
or Electroporation
Human proteins Positive, negative selection
producing genes

Positive
Negative (GFP+)
(GFP-)

Cloned
transgenic
embryo
Cloning animal Embryos
transfer

Human Transgenic cow

proteins 45
 Great business?
Therapeutic Proteins Animal $/animal/year
Cow
AAT (alpha-1-antitrysin) 150.000 USD/cow/year
tPA (Tissue plasminogen Cow
700.000 USD/cow/year
activator, chống đông máu)
Factor VIII or IX or (Blood Cow
200.000 -300.000
clotting factors, điều trị chống
chảy máu). USD/cow/year

Lactoferrin 25.000 USD/cow/year

CFTR (Cystic fibrosis Cow
800.000 USD/cow/year
transmembrane conductance
regulator) Supper dairy cow
Human Protein C (Chống đông Cow 10 milions
máu) USD/cow/year 200.000.000
Cow

USD/cow
Fibrinogen (điều trị vết thương) 400.000 USD/cow/year
Glutamic acid decarboxylase Cow
200.000 USD/cow/year
(Điều trị tiểu đường type 1)
Anpha-lactalbumin (điều trị Cow
500.000 USD/cow/year
nhiễm trùng)
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 Erythropoietin (Epoetin Alfa or EPO)

• Many mammals are being genetically

engineered to produce Erythropoietin, like
sheep or cows. EPO is used to treat anemia or
given to patients after chemotherapy. “EPO is
a glycoprotein (protein-sugar conjugate) that
serves as the primary regulator of red blood
cells (erythrocytes) in mammals” (Ritter).
Recombinant DNA isolates the human gene
erythropoietin and is put into the mammal to
target a more modified and better version of
erythropoietin. EPO is a natural substance
produced from the kidneys in our bodies but
transporting the gene to a mammalian animal,
such as a sheep, causes a large quantity and
quality of the drug.
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Recombinant DNA of EPO
 • “Erythropoietin [epoetin alfa (Epogen, Procrit)]
is used in many installation-fitting clinic. The
Erythropoietin most common use is in people with anemia
(Epoetin Alfa or associated with abnormal function
EPO) (dysfunction) kidney. When the kidneys are not
functioning properly, they produce less than
normal amounts of erythropoietin, which can
lead to the production of red blood cells are
low, or anemia. Therefore, by replacing
erythropoietin with an injection of
erythropoietin from a genetically engineered
animal, anemia associated with kidney disease
may be treated” (World of Health).

• Many athletes, such as cyclists and runners,

use EPO because the increasing number of red
blood cells can increase oxygen capacity and
produce improved physical activity. Creating
Erythropoietin through genetic engineering, it
is much more efficient than what humans can
produce alone. 48
 Future: pig produce human organs?
Organ related genes
Pig enucleated
knockout cloned pig
blastocyst
oocyte
Embryo
transfer
Pig fibroblasts cells

siRNA
Transfection Lentiviral
vector

Cloned
pig with
Pig knockout human
pancreas, heart, Human organs
iPS cells
? ????
liver, kidney related
genes cells

? ?
? Humans
organs
?
?
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 Agricultural Applications
Genetic Engineering in Plants
 Agricultural scientists have endowed a number
of crop plants with genes for desirable traits
 The Ti plasmid is the most commonly used
vector for introducing new genes into plant
cells
 Genetic engineering in plants has been used
to transfer many useful genes including those
for herbicide resistance, increased resistance
to pests, increased resistance to salinity, and
improved nutritional value of crops
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TECHNIQUE

Agrobacterium tumefaciens

Ti
plasmid

Site where
restriction
enzyme cuts
T DNA
DNA with RESULTS
the gene
of interest

Recombinant
Ti plasmid

Plant with new trait51

Biotechnology Breakthroughs
 Insulin (1982)
– First commercial biotech product
– Reliable, inexpensive source of insulin
 Rice
– Enriched with beta-carotene and iron
 Bananas
– Containing edible hepatitis vaccine
 Potatoes with higher solid content
 Garlic that lowers cholesterol
 Fruits and vegetables that reduce the risks of cancer
and heart disease

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 Environmental Benefits
• Reduced pesticide use
• Lower energy requirements
• Cleaner water
• Less soil erosion

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