LSM1301

L7: Biotechnology
A/P Henry Mok Yu Keung
Office at S3-03-01d
dbsmokh@nus.edu.sg 1
Biotechnology
In its broadest sense, biotechnology is any use or alteration
of organisms, cells or biological molecules to achieve
specific practical goals.”
Ancient biotechnology:
Fermentation (beer, bread, wine),
domestication, selective breeding
a model of a bakery before
Modern biotechnology: century around 1782

≈ Genetic engineering
manipulates genetic information in an
organism

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 Ancient
biotechnology
• Use of biology and living
organisms for practical
purposes

• Traditional examples
– Beer-brewing
– Wine-making
– Animal breeding
– Plant breeding

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Thinking

What is the limitation for the traditional

biotechnology?

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Food Security: The Challenge of Feeding 9.8 Billion People by 2050

Changes in the relative global production of crops

and animals since 1961
 5
Outline of Modern biotechnology
Recombinant DNA PCR & DNA agarose
Technology gel electrophoresis
– Plasmids
– Enzymes DNA Chips/Genetic
– Transgenic Screening
organisms – Prenatal genetic
screening
DNA Fingerprinting – Gene therapy
– short tandem repeats Political and Ethical
– Gel electrophoresis Issues
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Recombinant DNA Technology

(DNA Cloning/Engineering)
• Recombinant DNA (rDNA)
– Contains DNA (usually not found together in nature) from two or
more different organisms
• Requires
– Vector: to introduce recombinant DNA into host cell
• Plasmids are common vectors (for bacteria and plants)
• Viruses are vectors for human and animals
– Enzymes: to introduce foreign DNA into vector DNA
• Restriction enzyme – to cleave DNA
• DNA ligase – to link two genes together

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Plasmids
Small circle DNA in bacterial cell
– Not essential for bacterial growth
– Capable of autonomous replication
– Can move from one bacterium to another
– Can deliver DNA into another cell

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PCR
• A cycled reaction that uses a heat-tolerant form of DNA
polymerase (Taq polymerase) to produce billions of copies of
a DNA fragment
• Requires knowledge of nucleotide sequence
– For design of a pair of short pieces of DNA (called primers)
complementary to targeted sequence of DNA for amplification
• DNA replication in test tube
– Reaction mix consists of DNA polymerase, template DNA, primer, and
nucleotides

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PCR Replicates DNA in a Test Tube
Denaturation: Targeted DNA
sequence to be amplified
heated to 90-95°C to
separate DNA to single
strands
Annealing: Temperature
lowered to 50°C to allow
primers to bind to single
DNA strands
Extension: Temperature
raised to 70-72°C for DNA
polymerase to uses free
nucleotides to synthesize
complementary strands
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PCR begins with a mixture containing a DNA template, a pair of short ssDNA
oligonucleotide primers, a pool of the four dNTPs, and a heat-resistant DNA
polymerase, Taq Enzyme
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 The locations of the 2 primers
Amplify the region between
determine the final size of the
bp 20 to 80 of a 100 bp DNA
PCR product

2 template strands

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4 template strands

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8 template strands

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16 template strands

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Polymerase Chain Reaction

• Invented by Kary B. Mullis in 1985

• Awarded the Nobel Prize in
Chemistry 1993
– “for his invention of the PCR method”
• Website
– http://nobelprize.org/nobel_prizes/
chemistry/laureates/1993/mullis-
lecture.html

How can PCR detect the presence of Covid-19 virus from nose scrub?
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DNA Agarose Gel Electrophoresis

• Technique to separate out mixture of DNA and/or RNA

fragments based on their lengths (number of base pairs)

• Steps
– DNA mixtures placed at one end of gel
– Electric current applied through gel
– Negatively-charged DNA fragments move toward positive end of gel
– Short DNA fragments move faster than long fragments
– Separated bands of DNA/RNA made visible by stains or DNA probes

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Gel casting
Anode Cathode

molten agarose

(Agarose) Jelly Food

Visualizing DNA by
staining
• Ethidium Bromide (EtBr)
• SYBR® Safe

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Enzymes
• Restriction enzymes
– Cut DNA at specific nucleotide sequences
– Number of cuts in DNA depends on number of times
“target” sequence (restriction site) occurs
– Some restriction enzymes cut straight across the
double helix and producing blunt ends
– Some restriction enzymes make a staggered cut,
snipping the DNA in a different location on each of the
two strands and producing sticky ends
• DNA ligase
– Produce covalent bond between two DNA ends

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 3’ overhang with 2 bases

Making recombinant DNA EcoRI: GAATTC PvuI: CGATCG

BamHI: GGATCC EcoRV: GATATC

5’ overhang with 4 bases
Blunt end
5’
5’ 3’
3’ 5’restriction
5’ mix DNA ligase
enzyme (cut)
(paste)

A A restriction B Researchers use C Matching sticky D DNA ligase joins

enzyme recognizes restriction enzymes to ends of different the fragments of
a specific base sequence cut DNA from different fragments base-pair DNA where they
in DNA (red boxes). For sources into fragments. with each other, overlap. Molecules
this and many other Fragments with identical regardless of the of recombinant DNA
enzymes, the sequence is sticky ends are mixed source of the DNA. are the result.
the same in the 5’ to 3’ together.
direction on both strands.

The sticky ends generated by staggered cuts must be complementary to each

other, i.e. the nucleotides must be able to pair with each other according to
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Chargaff’s rule.
 Restriction Endonucleas

Model image of the restriction enzyme EcoRI (red

and yellow) binding to DNA (blue). The enzyme is
made up of two symmetrical units (dimer) that
correspond to the double helix structure of DNA.
Transgenic Organisms
• Transgenic organisms contain foreign DNA that has been
introduced using biotechnology. The terms transgenic organism
and genetically modified organism (GMO) are generally
synonymous.

• Genetic modification differs from selective breeding (“traditional

biotechnology”) by
– Genetic engineering is much more rapid
– Genetic engineering can transfer genes between species
– Genetic engineering can produce novel genes never seen before on Earth

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Transgenic Bacteria

• Used to produce wide

range of recombinant
DNA products
– Human insulin
– Human growth
hormone
– Interferon
– Vaccines

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Thinking What must you consider when you want to get
a functional human protein from E. coli?

Reverse transcription

mature mRNA cDNA

from eukaryotes (Complementary DNA)

E.Coli cells of rDNA

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Transgenic Animals

• New DNA inserted into fertilised egg

– During development, all cells in body will have the
new gene
• Inefficient process
– Success rate of about 1%
• Requires normal expression
– Must not disrupt other essential genes
– Must be expressed at right cells and right time

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Gene pharming

tPA is Involved in the breakdown

of blood clots
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Tissue plasminogen activator (tPA)
 Scientists breed goats that produce spider silk

Goats that produce spider silk protein in their milk could enable researchers to collect large quantities of
the silk proteins for a variety of applications. For instance, due to its strength and elasticity, spider silk
fiber could be used for making artificial ligaments and tendons, for eye sutures, and for jaw repair. The
silk could also have applications in bulletproof vests and improved car airbags.

Read more at: http://phys.org/news194539934.html#jCp 28

Transgenic Animals
GM salmon inventor
• First transgenic
animals in 1974
– Mouse with a virus
gene
• Today
– Pigs, goats, sheep,
monkeys, etc
– Even fishes (Salmon
with growth hormone)

AquAdvantage Salmon is genetically engineered for rapid growth with

two stretches of foreign DNA, a growth hormone gene with an antifreeze
protein promoter.
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 GloFish: pollution monitors
Small Fish Detect Big Problems
Environmental Scientists Use Fish Behavior To Monitor Water Quality

Gene for fluorescence protein

inserted downstream of stress
response gene.

GloFish acts like a biosensor to

detect water pollution.

Fish glowing fluorescent under stress 30

Transgenic Plants

• Easier to modify plants than animals

• Ti (Tumour-inducing) plasmid
– From plant-infecting bacterium Agrobacterium tumefaciens

• When A. tumefaciens infects plant cells

– Ti plasmid is inserted into plant chromosome in nucleus

• Researchers replace plant tumour-causing genes with

beneficial genes (e.g. insect resistance)
– Genes therefore transferred into plant chromosome when A.
tumefaciens with modified Ti plasmid infects plant cells

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 Many Crops Are
Genetically
Modified

Bt gene (from Bacillus thuringiensis

bacterium) can be inserted into
plants to produce insect-killing
protein in crops 32
In 2008, about 80% of the corn, 86% of the cotton, and 92% of the
soybeans grown in the U.S. were transgenic; that is, they
contained the genes from other species
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 Supplementary Information

Cloning of an Organism

Human cloning could help infertile parents have children and could be used
to harvest embryonic stem cells. But ethical issues surround cloning of our
own species.

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Cloning of an Organism (video)

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 Supplementary Information

Stem Cells Divide into Multiple Cell Types

Embryonic stem
cells give rise to all
cell types in the
body.

- Replace beta-pancreatic cell in

diabetes patient to produce insulin?

- Generate neurons and transplant into

patients with Parkinson’s disease?

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 Supplementary Information

Stem Cells Divide into Multiple Cell Types

Adult stem cells

differentiate into a
limited number of cell
types. Stem cells in bone
marrow, for example,
differentiate into all
blood cell types.

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DNA Fingerprinting/Forensic Science

• Forensic scientists have found that small, repeating segments of DNA,

called short tandem repeats (STRs), can be used to identify people
• STRs
– STR is short (consisting of 2 to 5 nucleotides), repeated (as many as 5 to 50
times), and tandem
– STR does not code for proteins
– Many different STRs in genome
– Vary greatly between different people, i.e. each person carries unique combination
of STRs like genetic fingerprints
• The U.S. Department of Justice established a standard set of 13 STRs,
each four nucleotides long (but 5 to 38 repeats), to identify individuals
by DNA samples
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DNA Fingerprinting

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Non-Coding Regions and STRs

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DNA Fingerprinting
• DNA from sample (e.g. crime scene)
– First amplified by polymerase chain reaction
– Separated by gel electrophoresis
– STRs in gel identified by DNA probes/staining

• Match of 13 different STRs between suspect’s and crime

scene DNA virtually proves that suspect was at crime
scene

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 Unrelated people almost never have identical DNA
profiles
 The positions of the bands on the gel are determined
by the numbers of repeats in each STR
 Using the standard array of STRs, technicians
determine the criminal’s DNA profile, which is coded
by the number of repeats of each STR found in the
criminal’s DNA
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Link to Life
Daily
Life

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Who is the child’s father?

A. Male 1
B. Male 2

Kinship Testing
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45
Who is the criminal?

A. Mr. Blonde
B. Miss Red
C. Mr. Mustache

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DNA Barcoding

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DNA Chips/Genetic Screening

• Contains up to thousands of DNA fragments

arranged on a glass plate or slide
• Also known as DNA microarray
– To analyse genetic profile of individuals
– To study expression of thousands of genes at one time
– To screen for genetic abnormalities, pathogens, or
cancer

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DNA Chips for diagnosis
Steps of analysis
1) Isolate DNA from cells
2) Amplify by polymerase chain reaction
3) Cut into fragments
4) Tag with fluorescent dye
5) Apply tagged fragments to DNA chip
6) Detect the fluorescent signal

Binds to cannot
the DNA base-pair

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Detect Existing Disease

Remain
binding after
wash

Washed
away

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DNA microarray technique
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 Microarray for
Disease Screen

affects the exocrine (mucus) glands

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Prenatal Genetic Screening

Down Syndrome is due to

The presence of 3 copies
of chromosome 21
(Trisomy 21)

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Thinking

The
world's
most
precious
genes?

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http://www.ebi.ac.uk/fgpt/gwas/#timeseriestab https://www.23andme.com/
 Gene Therapy
An approach to treat disease by either modifying the expressions of
an individual’s genes or correction of abnormal genes. One of
potential approaches is

Targeted gene delivery using viral vector

Watch the above video on Gene Transfer using viral vector

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Gene Therapy Replaces Faulty Genes

Gene therapy may someday

provide new treatment options
for genetic diseases by
replacing a faulty gene in a
person’s cells.

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COVID-19
SARS-CoV-2

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60
Safety and Ethics

Thinking

• Safety issues
– Could ingestion of Bt protein in insect-resistant plants be dangerous to
humans?
– Are transgenic fish producing extra growth hormone dangerous to eat?
– Could GM crops cause allergic reactions?
• Legal issues
– Who has access to my genetic information?
– Should animals be modified to provide organs for human transplants?
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• Environmental issues
– Could herbicide resistance genes be transferred to weed
species creating superweeds?
– Could GM fish reduce biodiversity in wild population if they
escape?
• Reduced diversity in wild makes population of fish more
susceptible to catastrophic disease outbreaks

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 Ethical issues

– Should parents be given

information about the genetic
health of an unborn foetus?
• Should parents be allowed to
design or correct the genomes of
their offspring?
– Who decides what should be
“corrected”?
• Who gets well and who gets
enhanced?
– Should humans be cloned?

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 When do we need human gene engineering?

(Modify genetic material of an organism, usually using

recombinant DNA technology)
Case 1

Will you select genetic engineering to treat a gene

defects in your baby?

Pentagon’s DARPA (Defense Advanced Research Projects Agency)

Case 2
Genetically engineered super blood to boost your
baby’s physical strength?
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 Key Terms and Key Concepts

Key Terms

Plasmid, Restriction enzymes, recombinant DNA, STR, genetically modified organism

(GMO),

Key Concepts

PCR and DNA agarose gel electrophoresis

How is recombinant DNA made?
How do scientists make GMO?
What are the limitation and ethical issues of GMO?
How is STR used for identification in forensic science?

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Please complete the quiz on Canvas for lecture 7

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 efore the o n-l ine tu torial
Try quizzes b

tr y of Lif e, C e ll s tr uc ture,
Review: Chemis Gene
life, DN A & h e re d it y,
Energy of m m a ry an d
n olo g y ; S u
expression, Biotech
Make Connections

th Sep): 8am to 10am

Wed (1 8
r ep ar e you r qu estions !)
(p
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