1.1 Techniques in Biotech

What students think I am doing What I am actually doing
26/11/2018
来自中国深圳的科学家贺建奎说，他对这对名为露露和娜娜的双胞胎的一
个基因进行了修改，使她们出生后即能天然抵抗艾滋病。
据贺建奎介绍，他的团队采用了一项名为“CRISPR/Cas9”基因编辑技术，
这种技术能够精确定位并修改基因，也被称为“基因手术刀”。
基因编辑技术被认为能够让人类对目标基因进行“编辑”，实现对特定
DNA片段的敲除、加入等，有其它基因编辑技术无可比拟的优势，能够在
活细胞中最有效、最便捷地“编辑”任何基因。这一技术不仅能修改胚胎
、消除遗传病，理论上甚至能够改变人的外貌，让父母“设计婴儿”。
CCR5-δ32—prevents HIV from infecting these cells
and thus confers resistance infection.
However, epidemiological studies of West Nile disease
subsequently found that humans homozygous for the
CCR5 mutation suffer more serious disease and higher
rates of death from that virus.
Pre-Confirmation studies?
-32/-23/-15/-9/-1/+4
Introduction to
modern biotechnology
Introduction of
biotechnology – oldies
 Biotechnology: the use of biological
processes, biological systems or organisms to
produce goods or provide services to humans
 has been in practice long before there was a term
for it, examples include:
 selective breeding
 fermentation
 known as traditional biotechnology
Examples of traditional biotechnology
Cows with a high milk production Maize with a high sugar content
are selected for breeding. is selected for seed production.
Bread-making involves The production of cheese involves

fermentation by yeast. fermentation by bacteria.
Cow with good quality of meat will be selected for
breeding
STUD cow…
Male
Female
Also…Dogs! Yes Dogs!
Artificial
selection of
wild mustard
(Brassica sp.)
Artificial
selection of
wild Teosinte
(Zea sp.)
Introduction to modern
biotechnology
 Modern biotechnology refers to various
techniques or processes that manipulate DNA,
cells or tissues. (unlike traditional, the breeding
is being manipulated, or make use of whole
individual to perform)
 Two examples of it are:

 genetic engineering
 cloning
Genetic engineering
 Genetic engineering (genetic modification) is
the changing of an organism’s genetic make-up.
 done by directly manipulating the genetic material
of the organism
 For example, genes from one organism can be
introduced into another organism.
 The latter organism is a genetically modified
organism (GMO) and, it possesses new
characteristics.
Some applications of genetic engineering
The bacteria E. coli can be genetically Tomatoes can be genetically modified

modified to produce human insulin for to give them resistance to diseases
medical use. and a longer shelf life.
Salmon can be genetically modified Genetically modified microorganisms (e.g.

to have a higher growth rate. yeast and bacteria) can be used in sewage
treatment.
Genetic engineering
 selective breeding:
 time-consuming
 can be carried out to animals or plants of the
same species only
 involves sexual reproduction
• genetic variations
• desirable characters may not appear or
undesirable characters may be resulted
in the offspring
Genetic engineering
 genetic engineering:
 quicker
 a more precise way of modifying the genetic
make-up of organisms so as to produce
desirable characteristics
 scientists can transfer genes to an organism
of a completely non-related species so that it
can acquire new characteristics
Cloning
 Cloning is the production of genetically
identical copies of a gene, a cell, or even a
whole organism.
 The copy, which has the same genetic make-up
as the original, is called a clone.
Dolly the sheep, the first mammal

successfully cloned from an adult cell
Learning Tip
The details about animal and plant cloning will be
discussed in section 41.3.
But it will die Cloning
soon ><
Selective
YES breeding
Do you like its
genome?
NO
Changing the
genome!
Selective Genetic
breeding engineering
Quick Practice
Which of the following is/are application(s) of

biotechnology?
(1) production of genetically modified crops
(2) sewage treatment using microorganisms
(3) making of compost using kitchen waste
A. (1) only
B. (1) and (2) only
C. (2) and (3) only
D
D. (1), (2) and (3)
Genetic engineering
Recombinant DNA technology
 Recombinant DNA: DNA which contains
genetic materials from two different organisms
or cells (DNA mixture)
 It involves donor cell & receiver cell (the mixing
process usually perform inside a cell)
Involving
 Cut or copy DNA from the donor
 Put it on a vector
 Paste on receiver
 Finish!
Vectors as the vehicle to transfer the
foreign DNA into the cell
 DNA containing the target gene is inserted into
vector to transfer the target gene into a host cell
for replication and expression.
 The two most commonly used vectors:
 Viruses (in form of viral plasmid) (useful in
animals & plants)
 Plasmids (useful in bacteria & yeast)
Contain antibiotic resistance genes
that is advantageous to survival.
Contain multiple cloning sites

for the insertion of DNA
containing gene of interest
Contain origin of replication

for instructing the bacterial cell
to replicate the plasmid DNA
A plasmid is an (artificial) circular strand of

extrachromosomal
dsDNA containing gene(s) of interest
Concept clarifying: Plasmid vs
Plasmid
Naturally occurring
plasmid in bacteria
Modified into
Engineered Plasmid in engineering

Obtained from company/scientists
(with very few resemblance to its “ancestor” except the origin)
 Plasmids can present in multiple copies in a
single bacterial cell as they are capable of
replicating independently of the bacterial
chromosome.
How plasmid work as a vehicle?
 Get DNA with target DNA with whatever means
 Transfer the DNA to the plasmid
 The recombinant plasmid is transferred to

bacteria with whatever means
How to get that DNA?
 Extract DNA from a cell (Strawberry DNA
extraction?)
 Multiply a specific portion of DNA by PCR
 Cut the donor DNA by two specific restriction

enzymes (restriction endonucleases).
Learning Tip
Restriction enzymes are naturally found in many strains of bacteria. It
is thought that bacteria produce these enzymes to recognise and cut
up foreign DNA in order to defend against invading viruses.
When you obtain the DNA
This is the target…

PCR is a specific amplification
technique (will teach later)
Allow multiplication
of specific DNA
fragment
technique (enough?)
technique (should be enough)
How restriction enzymes cut DNA
restriction enzyme
cuts here restriction enzyme
cuts here
restriction enzyme restriction site restriction enzyme

restriction site cuts here cuts here
DNA fragments DNA fragments

with blunt ends with sticky ends
Restriction enzymes work like scissors
 They cut small DNA fragments (not
chromosome level)
 They are able to recognize specific base

sequences and cut DNA at specific points.
 restriction sites
 Restriction enzymes produce DNA fragments
with unpaired, single-stranded cut ends, which
are called sticky ends. (Why blunt end is bad?)
A DNA with “HindIII” cut
ends can only stick to
another DNA with
“HindIII” cut ends
The principle of recombinant
DNA technology
 A sticky end can pair up with another sticky end
containing the complementary base sequence.
 Hence, two DNA fragments with complementary

sticky ends, which are often produced by using
the same restriction enzyme, can then be joined
together.
A RE recognize site of and cut at
the 2/4 position
The main steps of recombinant DNA technology
donor cell bacterium
DNA containing bacterial
the target gene plasmid chromosome
Obtain DNA fragments

containing the target gene. restriction
enzyme
plasmid
Cut the DNA
fragments and
the plasmid using
the same sticky ends
sticky ends restriction
enzymes.
cut plasmid
target gene
Insert the target gene into the
plasmid and join them using
DNA ligase. The process of
recombinant plasmid joining the two pieces of DNA
is called ligation.
WS1
-Function of A?
-What is required in process X?
WS1
WS1
Technical precautions
 Two restriction enzymes are commonly used in
cutting plasmid and DNA fragments
 Avoid self ligation of plasmid
 Ensure directional transfer of DNA
fragments
Meat  Taem
 It is a must to cut the DNA fragments with RE

 Uncut DNA fragments cannot ligate to the
blunt end of the plasmid
Single Possible
RE cut rejoin
Double
RE cut
 You can basically ignore the by-products of the
RE cuts (DNA fragments beyond the RE cut
site)
 It is a basically a must for both plasmid and

DNA fragments to be cut by the same Res
 To generate compatible ends
Ligation by ligases
Ligase catalyze the

formation of
phosphodiester bond
 Merge the sugar
backbone together
Reality: How to do it?
 Starting from sufficient insulin gene DNA sample and
plasmid DNA
 Mix DNA and RE 1 solution, 37°C, 2-3 hours,
(individual tube)
 DNA purification (~15mins)
(individual tube)
 DNA quality checking and quantification (~30mins)
 Prepare cut insulin DNA and plasmid DNA in
appropriate ratio (~3:1 molecule) (~10mins)
 Mix cut DNA(s) and ligase, 16°C, O/N
x
Major method of obtaining the target gene

The synthesis of DNA from mRNA is an alternative
method of obtaining the target gene. This method is
particularly useful for a target gene which is actively
expressed in a certain cell type because a large
amount of its mRNA can be obtained.
x

Using the mRNA as a template, a single strand of
DNA, called complementary DNA (cDNA), is
synthesised. This process is called reverse
transcription, and is catalysed by the enzyme
reverse transcriptase. The cDNA can then be used
as a template for the synthesis of the second DNA
strand. The two strands of DNA finally make up a
double-stranded DNA molecule, which contains the
target gene.
x

cell actively producing the target gene product
Obtain mRNA of the target gene

mRNA
Synthesise cDNA from mRNA
mRNA
cDNA Remove the mRNA with alkali
DNA containing Synthesise the second DNA strand from

the target gene the cDNA
Production of human insulin using
recombinant DNA technology
 With recombinant DNA technology, scientists
can now produce useful biological molecules in
large quantities.
 e.g. the human insulin
Having a plasmid containing insulin gene
 Plasmid can ask the cell to produce that gene

product (insulin protein)
 Transform cell with that plasmid
 A lot of gene product (insulin protein) will be
synthesized
 Break down the cell and let those proteins
released from the cell
 Isolate the insulin protein from others
Common “machine” used to
produce proteins
 Bacteria (Prokaryotes, common)
 Mass production
 Yeast (Eukaryotes, common)
 Mass production
 Mammalian cancer cell (Eukaryotes)

 Low yield production
 Mostly for other purposes
bacterium
bacterial
Learning Tip DNA
Obtain chromosome
fragments encoding
The process
human ofinsulin
producing recombinant human insulin
shown inthrough
Fig. proper
processes. is a simplified one so as toplasmid
41.8 facilitate
Cut the
understanding. In fact, human DNA is a protein
insulin
fragments and antibiotic resistance gene
composed of two polypeptide chains.using
the plasmid The DNA fragments
encodingsticky
the ends
two polypeptidethe same
chains are inserted into two
restriction
groups of plasmids separately. The plasmids are
enzyme. then
cut plasmid
introduced into two groups of E. coli and cloned. As a
result, each group of bacteria can produce one of the two
kinds of polypeptide chains. The two kinds Insert the DNA fragment
of polypeptide
into the plasmid and
chains are then extracted and purified. Finally, join themthey are
using DNA
linked to form recombinant human insulin.
ligase. Cont’d
recombinant plasmid
recombinant plasmid
Introduce the
recombinant plasmid into
transformed bacterium a bacterium (e.g. E. coli).
(bacterium that has taken up the
recombinant plasmid)
Select the transformed bacteria
and culture them on a large scale.
Recombinant plasmids replicate inside the bacteria and gene expression is

induced for protein synthesis. Pure and functional human insulin can be obtained
after further processing of the gene products.
 In the production process of human insulin,
bacteria serve as the host cells in which the
genes encoding human insulin replicate and
express.
 When bacteria are mixed with recombinant
plasmids, some of them will take up the
recombinant plasmids.
 This process is called transformation.
 transformed bacteria: the bacteria that have
taken up the recombinant plasmids
resistant gene
 In order to select the transformed bacteria, the
plasmids used in the process carry an
antibiotic resistance gene, which acts as a
selection marker.
Learning Tip
There are various methods to select the transformed
bacteria. Making use of antibiotic resistance genes is only
one of them.
Selection of the transformed bacteria using
antibiotic resistance gene as a marker
transformed bacterium bacterium that has not
possesses both the gene taken up the recombinant
encoding human insulin plasmid does not possess
and the antibiotic the antibiotic resistance
resistance gene gene
visible colonies
incubation
at 37°C
The bacteria are spread onto the Only the transformed bacteria can
agar plate with an antibiotic. survive and reproduce. (Some bacteria
may take up the plasmid without the
gene encoding human insulin.)
Bacteria transformed by vectors with
ampicillin resistant gene grown on
ampicillin containing plate
Case Colonies?
The bacteria uptake an empty plasmid Yes
The bacteria uptake an plasmid with Yes
desirable gene
The bacteria cannot uptake a plasmid No
The bacteria uptake an plasmid with Yes
undesirable gene
You transform the bacteria with No
plasmid containing another antibiotic
resistant gene
The bacteria uptake a broken plasmid No
 The transformed bacteria are then selected and
cultured on a large scale.
 As the transformed bacteria reproduce, the
recombinant plasmids replicate, and the DNA
encoding human insulin is passed to the
daughter cells.
 numerous identical copies of the target gene
can be obtained as:
 plasmids are able to replicate independently
 plasmid may exist in multiple copies in each
bacterium
Pick a handsome colony (Don’t pick two!)
Transfer the colony to liquid culture medium
with antibiotics for expansion, 37°C, O/N
The liquid becomes blurred, indicating
the presence of vast amount of bacterial
cells
Reality: How to do it? (day1)
 Starting from sufficient insulin gene DNA sample and
plasmid DNA
(individual tube)
(individual tube)
 DNA quality checking and quantification (~30mins)
 Prepare cut insulin DNA and plasmid DNA in
appropriate ratio (~3:1 molecule) (~10mins)
 Mix cut DNA(s) and ligase, 16°C, O/N
Continue to do it…(day2)
 Transformation of bacteria (~15mins)
 Mix the ligated DNA mix with ice cold bacterial
cells in a tube (5mins)
 Put the bacterial cells in 42°C water bath for 1 min
 Put the bacteria cells back to ice (10mins)
 Add culture medium to transformed bacteria for
recovery, 37°C, 1 hour
 Spread the recovered bacteria on an agar plate with
antibiotics (~10mins)
 Grow the agar plate with bacteria, 37°C, O/N
Hopefully!
One colony
One cell origin
Continue to do it (day3)…
 Check which bacterial colonies contain plasmid with
desirable genes
 Pick some colonies (1-100, depending on how
hopeless are you) on the agar plate and transfer to
another clean agar plate with antibiotics (labelling)
 Transfer the colonies to individual tubes with liquid
culture medium
 Grow both new agar plate and liquid culture
medium tubes, 37°C, O/N
Continue to do it (day4)…
 Check which bacterial colonies contain plasmid with
desirable genes
 Put the agar plate back to the fridge (1min)
 Isolate plasmid DNA from various (~36) bacterial
cultures (commercial method: <1 hour; traditional
method: <3 hours)
 RE cutting and subsequent checking (~3hours)
 Successful: continue; Failed: start from day1
 Identify the good colony, grow a new, bigger
volume of liquid cultural medium (~200ml – 1L)
from the labelled agar plate. 37°C, O/N
 gene cloning: the process of producing
identical copies of a gene
 Finally, gene expression is induced in the
transformed bacteria to synthesise the gene
products.
 After further processing of the gene products,
pure and functional human insulin can be
obtained.
Human insulin produced using
Development of recombinant DNA technology
Recombinant DNA technology was developed in

the early 1970s by two American scientists:
Herbert Boyer (1936– ) and Stanley Cohen
(1935– ). Boyer discovered a restriction enzyme
in the bacterium E. coli that cut DNA and
produced sticky ends. Cohen developed a
method that enabled the bacteria to take up
plasmids.
Development of recombinant DNA technology
In 1973, they successfully

inserted an amphibian
gene into a bacterial
Herbert Boyer
plasmid. The recombinant
plasmid was introduced
into bacteria and multiple
copies of the amphibian
gene were found.
Stanley Cohen
Desired gene DNA Plasmid DNA
Cut with same REs

Ligation
Bacteria Recombinant plasmid DNA

(assume successful)
Transformation
Antibiotic selection
Transformed bacteria
From transformed bacteria, we
can collect
 Plasmid DNA
 A stable storage form of DNA for that gene
 For further transformation of other cells
 Proteins
 For pharmaceutical purpose (make drugs)
 For functional and structural analyses
 Whole transformed cell
 Agrobacterium can further transform plant
cells!
Key Point
1. Recombinant DNA technology is a set of
techniques that isolate a fragment of DNA
from an organism or cell (donor) and insert
it into the genetic material of another
organism or cell (host).
2. Plasmids and virus are two most

commonly used vectors in recombinant DNA
technology.
Key Point
3. Plasmids have the following characteristics:
• They are small circular pieces of DNA. Bacteria
can transfer plasmids with one another.
• They carry genes that are not necessary for the
bacteria to survive, but some genes (e.g.
antibiotic resistance genes) may be
advantageous to survival.
• They are separated from the bacterial
chromosomes, and are capable of replicating
independently. They are often present in
multiple copies in a single bacterial cell.
Key Point
4. The basic steps of recombinant DNA technology include:
Step 1: Obtaining DNA fragments containing the target gene
Step 2: Cutting the DNA fragments and plasmids

(vectors) using the same restriction enzyme
Step 3: Inserting the target gene into the plasmid

and join them using DNA ligase
5. One application of recombinant DNA technology is the

production of useful biological molecules (e.g. human
insulin) in large quantities.
A successful ligation is pretty much
chance based
DNA sample is usually scare in
amount
 DNA number must be multiplied for many times
(i.e. 100000) for minimal success of lab work
DNA sample could contain
multiple genes
 But you only want to transform one of them
 The gene of interest must be specifically
amplified for many times for successful lab
work
How cell copy DNA?
Polymerase chain reaction
 Polymerase chain reaction (PCR) is a
technique for replicating specific DNA
sequences outside cells.
The principle of PCR

 In a cell, DNA replication begins when the DNA
double helix unwinds and the two DNA strands
separate.
Learning Tip
To revise the process of DNA replication, refer to Book 4,
Ch 27 (p.27-7).
Recipe
 A template to copy: DNA template
 Copy machine: DNA polymerase
 Raw materials to copy: dATP, dTTP, dCTP,

dGTP
 A guide to tell where to copy: DNA primer pair

 The principle of PCR is similar to that of DNA
replication. But instead of taking place inside
cells, PCR is performed in a reaction mixture,
which consists of the following components:
 a DNA template - template that contains the
target DNA sequence that needed to be
amplified (i.e. target sequence)
 primers - short sequences of synthetic single-
stranded DNA which are complementary to
specific regions of the DNA template. They
mark the regions where the synthesis of new
DNA strands starts.
 a heat-stable DNA polymerase - an

enzyme that synthesises new DNA strands.
The most commonly used DNA polymerase
in PCR is the Taq DNA polymerase. It is
extracted from Thermus aquaticus, a
bacterium found in hot springs.
 Deoxyribonucleotides (dNTPS) - the
building blocks of the new DNA strands.
 3 steps
Learning Tip in each PCR cycle: denaturation,
The DNAprimer annealing
polymerase used in PCR and extension
must be heat-stable because the
reaction mixture will be heated to various temperatures.
Step 1: Denaturation
 The reaction mixture is first heated to about 95

°C. At this temperature, the hydrogen bonds
between nucleotides are broken and thus the
DNA denatures (i.e. the double helix unwinds and
separates into two single strands).
double-stranded DNA
denaturation at about 95 °C
two pieces of single-

Denaturation of DNA stranded DNA
Step 2: Primer annealing
 The reaction mixture is cooled to between 50 °C

and 65 °C. This allows primers to anneal (bind)
to the single-stranded DNA by complementary
base pairing. One primer binds to one DNA
strand at one end of the target sequence, while
another primer binds to another DNA strand at
the other end.
primer 1
primer 2
Step 3: Extension
 The temperature at this step depends on the

DNA polymerase used.
 After the enzymes have attached to the ends of
the primers, free nucleotides begin to pair up
with the base sequences of the two DNA strands
(or templates).
 DNA polymerase catalyses the formation of
bonds between nucleotides. Through the
extension of primers, two new strands of DNA
are synthesised.
Step 3: Extension
Taq DNA polymerase
primer 2
primer 1
free DNA nucleotides
synthesis of new DNA strands
Extension of primers to synthesise new DNA strands

Step 3: Extension
 The cycle is then repeated about 30 to 40 times.
 At the end of each cycle, the number of DNA
strands is doubled.
 Each strand acts as a template in the next cycle.
DNA copies
template DNA At the 1
beginning
1st cycle 2
2nd cycle 4
3rd cycle 8 Amplification of

DNA by PCR
4th cycle 16
 The whole cycling process of PCR can now be

performed automatically in a thermal cycler.
 30 cycles of PCR can be completed in only
a few hours
 There are considerable amounts of reagents in
the products of PCR.
 products need to be purified before using
Thermal cycler
EXTRA:
Process DNA replication PCR
Separation of double Enzyme catalyzed Denaturation by heat
stranded DNA (helicase/topoisomerase)
Primer RNA primer DNA primer
Primer synthesis and Enzyme catalyzed Order and made from
binding (Primase) company
Random binding at
annealing temperature
Elongation Enzyme catalyzed Enzyme catalyzed
(DNA pol. III) (Taq DNA pol. III)
Direction of new DNA Unidirectional (5’-3’) Unidirectional (5’-3’)
synthesis
DNA template Always the same old Old DNA and newly made
DNA in one cell DNA as template
Temperature Always physiological 95C (Denaturation)
temperature (~37C) 60C (Annealing)
72C (Extension)
The invention of PCR
In the early days of DNA research, DNA
amplification was a time-consuming and
expensive procedure. One reason was that the
enzyme DNA polymerase is easily denatured at
high temperatures. Therefore, it was necessary to
add the enzyme to the reaction mixture after
every denaturation step.
In the 1980s, the American
biochemist Kary Mullis (1944– )
invented PCR. He started to use
Taq DNA polymerase, a DNA
polymerase extracted from the
bacterium Thermus aquaticus. This
bacterium lives in very hot
environments such as hot springs.
Kary Mullis
Taq DNA polymerase extracted from this bacterium
is not easily denatured by heat, and thus it does
not have to be replaced every reaction cycle. This
greatly reduces the cost and labour of the DNA
amplification process. It also allows PCR to be
performed in automated thermal cyclers, so that
the whole reaction can be completed within a few
hours. For his invention of PCR method, Mullis was
awarded the Nobel Prize for Chemistry in 1993.
Old PCR way
-DIY
zzZZzzZZ
95°C 60°C 72°C

Applications of PCR
 The advantages of PCR:

 PCR is specific. It can target and amplify
one specific segment of DNA (or a gene).
 PCR is highly sensitive. It can amplify even
a very small amount of DNA to a significant
amount that allows analysis.
 PCR is fast. It can be completed within a
few hours.
Applications of PCR
Scientists can amplify the DNA extracted PCR can be used to amplify DNA from
from a cell of a foetus, which can be blood, other body fluids or tissues
found in the amniotic fluid, to detect found at a crime scene for further
genetic diseases. analysis (e.g. DNA fingerprinting).
PCR can be used to amplify DNA Scientists can amplify genes from an
found in the remains of dead organism for scientific research.
organisms for archaeological studies.
Cut with same REs

Ligation

(assume successful)
Transformation
Raw DNA sample
PCR
Cut with same REs

Ligation

(assume successful)
Transformation
Connections
Testing for GM foods by PCR

 PCR primers are highly specific and can only
bind to a complementary DNA sequence.
 Based on this property of primers, GM food
tests may involve the use of a specially
designed primer that binds to a particular
DNA sequence present in GM foods.
 If the DNA sequence is present in a food
sample, it is amplified by the PCR.
Connections
Testing for GM foods by PCR

151
101
GM maize
151
Non-GM maize
Time
An analysis of the DNA of a GM maize and a non-GM maize. The GM
maize contains a marker with 101 base pairs (bp), which allows it to be
differentiated from non-GM maize.
Practical example
 A diploid GM crop has a BT toxin gene inserted
in its genome as below
BT toxin A 2000 base pair long DNA (transgene) inserted to the crop genome,
gene accompanied by the deletion of some original DNA (400 base pair long)
GM region
BT toxin
gene1 gene2 gene3 transgene gene5 gene6
non-GM region
gene1 gene2 gene3 gene5 gene6
1. Design a pair of primers
 Amplify the region that able to distinguish the
GM and non-GM DNA by the same pair of
primers
 The primers (at least one) should bind to
both type of DNA
GM region BT toxin
transgene
gene1 gene2 gene3 (2000bp) gene5 gene6
non-GM region
400bp
Strategy 1: To include the whole GM region
GM region BT toxin
transgene
non-GM region
400bp
GM DNA: A 2000bp long DNA will be amplified

Non GM DNA: A 400bp long DNA will be amplified
Adv: Position of transgene can be confirmed; non-GM DNA as control

Disadv: PCR might fail if transgene is too long (>5000bp)
Strategy 2: To include the partial GM region
GM region BT toxin
transgene
non-GM region
400bp
GM DNA: A 1000bp long DNA will be amplified

Non GM DNA: No DNA region will be amplified, as one of the primers nowhere
to bind
Adv: Position of transgene can be confirmed; less restricted by transgene

length
Disadv: Lack of control
Strategy 3: To include the GM region only
GM region BT toxin
transgene
non-GM region
400bp
GM DNA: A short DNA (~300bp) will be amplified

Non GM DNA: No DNA region will be amplified, as both primers nowhere to
bind
Adv: Not restricted by transgene length. Fast (a shorter DNA means a shorter
PCR time!)
Dsiadv: Lack of control, position of transgene cannot be confirmed
Using strategy 1
GM region BT toxin
transgene
non-GM region
400bp
Since PCR reaction occurred inside a tube, you can never “see” a 2000bp &
400bp long DNA fragments by naked eye
Use gel electrophoresis!

Gel electrophoresis
 Gel electrophoresis is method for separating

charged molecules, such as DNA, based on
their size.
 The DNA fragments are placed at the
negatively charged (cathode) end of the gel
plate.
 DNA is negatively charged due to the
presence of the phosphate groups.
Gel electrophoresis
 When an electric current is applied, the DNA

fragments move towards the positively
charged (anode) end of the gel.
 The smaller fragments can pass through the
gel more easily and they travel faster than the
larger ones.
 After some time, DNA fragments are separated
into bands according to their molecular size.
Separation of DNA fragments using gel electrophoresis
 Load samples of DNA fragments into wells of the gel.

samples
-
row of wells
gel +
 Apply an electric current to drive the DNA

fragments across the gel.
direction of movement
+
 DNA fragments move different distances according to their

size. (Very small DNA fragments will “swim” eventually
-
larger fragments
smaller fragments
+
Gel electrophoresis
 After the electrophoresis is complete, the DNA

bands on the gel can be visualised by staining
them with special dyes.
DNA bands stained with

fluorescent dye are revealed
under ultraviolet light.
Making prediction based on strategy 1
GM region BT toxin
transgene
non-GM region
400bp
A GM crop will show DNA bands of what size(s)?

And a non-GM crop?
Hints: Remember the crop is diploid.

Limitation of gel electrophoresis
• A 5000bp & 6000bp DNA looks the same at 1.5%

• A 3000bp & 100bp DNA loaded to a 1.5% gel, when 3000bp band is okay to
see, the 100bp DNA is swimming happily
Key Point
1. Polymerase chain reaction (PCR) is a
technique for replicating specific DNA
sequences outside cells.
2. A reaction mixture of PCR containing a DNA

template , primers , heat-stable DNA
polymerase and nucleotides undergoes
many cycles of polymerase chain reaction.
Each cycle consists of three steps: Cont’d
Key Point
2.
Step 1: Denaturation
Heat the reaction mixture to cause DNA to unwind and separate into
two single strands.
Step 2: Primer annealing

Cool the reaction mixture to allow primers to anneal to the single-
stranded DNA.
Step 3: Extension
Raise the temperature for DNA polymerase to catalyse the
synthesis of new DNA strands.
Key Point
3. At the end of each cycle of PCR, the number of
DNA strands is doubled .
4. PCR can amplify DNA for detection of genetic

disorders , DNA fingerprinting ,
archaeological studies , and
scientific research .
Quick Practice
Which of the following is not present in the reaction

mixture of polymerase chain reaction?
A. DNA template
B. DNA ligase
C. DNA polymerase
D. nucleotides B
DNA fingerprinting
 DNA fingerprinting (DNA profiling) is a
technique for identifying individuals using
their DNA profiles.
The principle of DNA fingerprinting
 only less than 5% of human DNA sequence

codes for functional proteins
 the remaining DNA sequence has no obvious
function, known as non-coding DNA
 At different positions (or loci) of the non-coding
DNA, there are variable number tandem
repeats (VNTRs), which are repetitive
sequences arranged in a head-to-tail manner
and show great variations in the number of
repeats among individuals.
 For a given VNTR, the number of repeats varies

from one individual to another in a population.
 genetic polymorphism
 Each individual has two alleles of the VNTR –
one from each parent.
 The chance of having two individuals with
the same set of VNTRs is very low (except
identical twins).
 This forms the basis of DNA fingerprinting.
VNTR at a given locus on a pair of
homologous chromosomes
VNTR with 16 repeats
maternal chromosome
alleles of
a VNTR paternal chromosome
VNTR with 24 repeats

GGCGGTGCTGGTGTGGG
 Various methods of DNA fingerprinting have

been developed, such as:
 Restriction fragment length polymorphism
(RFLP) analysis
• involves restriction fragment
preparation, gel electrophoresis and
DNA hybridisation
 short tandem repeat (STR) analysis
DNA fingerprinting using RFLP analysis
DNA extraction
DNA is extracted from the sample
(e.g. hair, blood, saliva or semen).
Restriction fragment preparation

samples from Restriction enzymes are used to cut the
different individuals DNA at specific sites, producing a large
number of DNA fragments. Some of these
fragments contain VNTRs.
DNA extracted DNA cut by restriction

enzymes
Cont’d
Gel electrophoresis
DNA fragments are then Denaturation of DNA fragments
separated by gel electrophoresis and transfer of DNA fragments to
according to their molecular size. a membrane
cathode (-) anode (+) The gel is immersed in an alkaline
solution so that the separated DNA
fragments are denatured and
gel become single-stranded DNA. The
DNA fragments are then
transferred from the gel to a nylon
movement of DNA fragments membrane.
nylon membrane
gel with DNA
fragments Cont’d
Incubation of the membrane Production of DNA fingerprints
with radioactive DNA probes The unbound DNA probes are
(DNA hybridisation) washed off. An X-ray film is then
The nylon membrane is incubated placed against the nylon
with radioactive DNA probes membrane in the dark. The
(single-stranded DNA fragments radioactive probes on the DNA
with nucleotide sequences that fragments expose the film, giving a
are complementary to the VNTRs pattern of dark bands when the
at several different loci). The DNA film is developed. The pattern
probes bind to the DNA makes up the DNA fingerprint
fragments containing the VNTRs (DNA profile).
being examined. X-ray film
radioactive
DNA probes
nylon membrane bound DNA fingerprint
with DNA probes
Restriction fragment length
polymorphism (RFLP) analysis
 RFLP analysis is time-consuming as the entire
analysis may require several weeks for
completion.
 It also requires relatively large amounts of
DNA samples.
Short tandem repeat (STR) analysis
 Nowadays, DNA fingerprinting is mostly carried

out using STR analysis.
 STR, which stands for short tandem repeat, is
a kind of repetitive sequence.
 It is similar to VNTR in that it is located in the
non-coding DNA and the number of repeats
varies among individuals.
 its repetitive sequence is shorter in length
Short tandem repeats

Short tandem repeat (STR) analysis
 STR analysis does not require DNA

hybridisation
 faster than RFLP
 often coupled with PCR and requires only small
amounts of samples
 works even with partially degraded DNA
DNA fingerprinting using STR analysis
DNA extraction
DNA is extracted from the sample
(e.g. hair, blood, saliva or semen). DNA extracted
PCR
The STR loci are targeted with specific
primers and amplified using PCR. A large
amount of DNA fragments containing the
STRs are obtained. These DNA fragments
vary in molecular size, depending on the sequence containing
number of repeats of the STRs. the STR amplified
Production of DNA fingerprints
The amplified DNA fragments are
separated according to their molecular
size. The result is then detected and
becomes the DNA fingerprint. DNA fingerprint
Applications of DNA fingerprinting
(i) Forensic science
 DNA fingerprinting is useful in forensic science.
 DNA from small samples of cells found at the
scene of the crime can be extracted for analysis.
 To prepare a DNA fingerprint, only a very
small amount of sample is required
 PCR can be used to amplify the extracted
DNA
 The DNA fingerprints of the samples obtained in
the crime scene are then compared with those of
the victims and the suspects.
 If two samples match, it suggests that the two
samples come from the same individual.
Investigation into a crime using DNA fingerprints
hair from victim’s suspect’s blood on
crime scene blood blood suspect’s shoe
To whom do the hair

obtained from the
crime scene and the
blood found on the
shoe belong?
The hair obtained from the crime scene

belongs to the suspect, while the blood found
on the shoe belongs to the victim.
 The DNA fingerprint of the hair found at the
crime scene matches the DNA fingerprint of the
suspect.
 This suggests that the hair belongs to the
suspect.
 However, it should be noted that this result does
not prove the suspect committed the crime.
 In the courts, other types of evidence are also
taken into consideration.
Practical 41.1
Crime scene investigation using DNA

fingerprinting
SBA Practical Workbook 5, Ch 41, p.1–9
Objective
To simulate a crime scene investigation by
analysing DNA samples using DNA fingerprinting.
Practical 41.1
Introduction
PCR is a technique used to amplify specific DNA
sequences outside cells. Provided with a sufficient
supply of free nucleotides, a single piece of DNA
segment can be amplified into billions of copies by
undergoing cycles of PCR. In the laboratory, the
process of PCR can be performed automatically in a
thermal cycler. Programmes can be set to alter the
temperature required by the different steps of PCR.
Practical 41.1
Introduction
In this practical, you are going to
perform DNA fingerprinting which
is commonly used in crime scene
investigation. PCR will be used to
amplify the DNA samples
collected from a crime scene and
from a suspect. The amplified A thermal cycler used
DNA will then be separated by gel for polymerase chain
electrophoresis for analysis. reaction (PCR)
Practical 41.1
Materials and apparatus (per group, for PCR)

PCR tubes (200 μL) 4
marker pen 1
micropipette (2-20 μL) 1
micropipette tips (2-20 μL) 1 box
ice bucket (with ice) 1
capless PCR tube adaptors 4
PCR master mix with primers
crime scene DNA sample (20 μL)
suspect DNA sample (20 μL)
Practical 41.1
Materials and apparatus (per group, for PCR)

distilled water
disposable gloves
Note
•Crime scene DNA and suspect DNA samples are included
in some PCR kits, produced by some laboratory
equipment suppliers.
•The PCR master mix with primers is a mixture that
contains Taq DNA polymerase, primers, nucleotides,
magnesium ions and a salt buffer.
Practical 41.1
Materials and apparatus
(per group, for gel electrophoresis)
spatula 1
conical flask (250 cm3) 1
measuring cylinder (100 cm3) 1
thermometer 1
casting tray 1
gel comb 1
micropipette (2-20 μL) 1
micropipette tips (2-20 μL) 1 box
electrophoresis tank 1
power supply 1
Practical 41.1
connecting wires 2
plastic boxes (with lids) 2
white tile 1
ice bucket (with ice) 1
agarose powder
adhesive tape
1X TBE buffer solution
5X loading dye
DNA marker
1X methylene blue solution
Practical 41.1
distilled water
safety goggles
disposable gloves
heat-resistant gloves
Note
The DNA samples, PCR master mix with primers, PCR
products and the DNA marker should be kept on ice.
Practical 41.1
Materials and apparatus (per class, for PCR)

vortex mixer
microcentrifuge
thermal cycler

(per class, for gel electrophoresis)
electronic balance
hotplate
digital camera
Practical 41.1
Safety precautions
1. Wear safety goggles.
2. Cover any wounds in your hands with sterile
dressings and wear disposable gloves.
3. Methylene blue can trigger acute haemolytic
anaemia in G6PD deficient individuals. Students
with G6PD deficiency are recommended to use
other stains such as SYBR Safe stain and silver
stain to perform this practical.
Practical 41.1
Safety precautions
4. After the practical:
• All waste materials should be immersed in
disinfectant for one hour before disposal.
• Dispose of the gloves properly.
• Wash hands thoroughly with soap and water.
Practical 41.1
Procedure Using a micropipette
A. Amplification of DNA fragments using PCR

1. Label three PCR tubes A, B and C on the side
of the tubes.
2. You are provided with a DNA sample collected
from the crime scene and a DNA sample
collected from a suspect. Use a micropipette
to transfer the DNA samples and other
reagents to the PCR tubes according to the
following table.
Cont’d
Practical 41.1

PCR tube Reagents
20 μL of crime scene DNA sample +
A 20 μL of PCR master mix with primers
20 μL of suspect DNA sample +
B
20 μL of PCR master mix with primers
20 μL of distilled water +
C
20 μL of PCR master mix with primers
Note
•The DNA samples and the reagents should be kept on ice.
•To prevent contamination of the sample, use a new micropipette tip
each time for transferring samples or reagents.
Practical 41.1

3. Close the cap of the PCR tubes and mix the
reagents using a vortex mixer.
PCR tube with reagents
vortex mixer
Practical 41.1

4. Pulse spin the reagents in the PCR tubes
using a microcentrifuge.
Note
•Pulse spinning can bring the contents to the
bottom of the PCR tubes as some contents
may be found underneath the caps or on the
inner sides of the tubes after mixing.
PCR tube with •The rotor in the microcentrifuge should be
reagents (in balanced. Otherwise, the rotor may be
capless PCR damaged. This can be achieved by placing
tube adaptor) PCR tubes A, B, C and another PCR tube
across each other. This PCR tube should
contain distilled water that has the same mass
microcentrifuge as those reagents in PCR tube A, B or C.
Practical 41.1

5. Place the PCR tubes into the thermal cycler. Run
the PCR according to the following thermal cycle.
Tempera- Time Number
Step Process
ture(°C) (min) of cycles Note
1
Initial DNA
94 2 1 •Preheat the thermal cycler
denaturation to 94 °C before placing the
Denaturation 94 0.5 PCR tubes in it.
Primer •Keep the PCR tubes on
2 52 0.5 35
annealing ice until the thermal cycler
Extension 72 1 is ready for PCR.
3 Final extension 72 10 1
Practical 41.1

6. Remove the PCR tubes from the thermal cycler
and place them on ice or at 4 °C after the
reaction.
B. Gel electrophoresis
1. Prepare a 3% agarose gel for electrophoresis by
carrying out the following steps:
a) Add 1.8 g of agarose powder and 60 cm3 of
buffer solution into a conical flask. Swirl the
flask gently.
Note
The concentration of the agarose solution depends on the size of the
DNA fragments to be separated by gel electrophoresis.
Practical 41.1
b) Heat the mixture on a hotplate until the agarose
powder dissolves completely.
Caution
•Do not touch the surface of a hotplate.
•Do not overheat the gel solution.
Otherwise, it may boil violently.
•Handle the hot agarose solution with
heat-resistant gloves.
Practical 41.1
c) Leave the agarose solution at room temperature
and allow it to cool down to 50 to 60 °C.
Note
•Hot agarose solution (over 60 °C) may
cause the deformation of the casting tray.
•Remove any bubbles formed in the
agarose solution with a micropipette.
Practical 41.1
d) Seal the two open ends of a casting tray with
adhesive tape. Pour the agarose solution into the
casting tray slowly to prevent the formation of
bubbles.
agarose solution
casting tray
adhesive tape
Practical 41.1
e) Insert a gel comb into the casting tray
casting tray at one end to gel comb
create wells for loading the
gel solution
samples.
f) Allow the agarose solution Note

to solidify at room Do not leave the gel at
temperature for about 30 room temperature for too
long. Otherwise, it may
minutes. dry up and then crack.
Practical 41.1
2. Perform gel electrophoresis by carrying out the
following steps:
a) After the gel has completely solidified, gently
remove the gel comb. A row of wells is then
formed in the gel.
Practical 41.1
b) Remove the adhesive tape. Place the gel with the
casting tray in the electrophoresis tank. Add
buffer solution to the electrophoresis tank to
cover the gel to a depth of about 1 mm.
c) Add 10 μL of loading dye to each of the three
PCR products A to C obtained in Part A. Pipette
up and down to mix the PCR products and the
loading dye.
Note
The TBE buffer solution conducts electricity and provides a constant
pH medium for electrophoresis.
Practical 41.1
B. Gel electrophoresis Note

•DNA marker consists of a
d) Load 20 μL DNA marker into range of DNA fragments of
the leftmost well of the gel known sizes. It can be used
slowly, using a micropipette. Do as a reference to estimate
not puncture the well in the gel. the size of DNA fragments in
samples.
micropipette
•Avoid overloading of the
samples as it will lead to
cathode anode smearing of the DNA bands.
right side •Use a new micropipette tip
electro- for loading each DNA sample.
DNA phoresis tank
marker
gel left side buffer solution
Practical 41.1
e) Load 20 μL of each of the samples from PCR
products A to C into separate wells of the gel from
the left to the right respectively.
f) Cover the lid of the electrophoresis tank. Connect
the electrodes to the power supply. Switch on the
power supply and apply a 100 V d.c. voltage to
the electrodes. Make sure that the side of the gel
containing the DNA samples is at the cathode (-)
side of the tank. Run the gel for about 60 minutes.
Cont’d
Practical 41.1
power supply
Note
•DNA samples may diffuse 100V
away from the wells if gel

electrophoresis is not run
immediately after loading the cathode anode
samples.
•Check that bubbles appear at
both electrodes after
switching on the power PCR
supply. products
DNA marker lid
Practical 41.1
g) When the tracking dyes Note
have reached about half to The tracking dyes in the
two-thirds of the length of samples indicate the progress
the gel, turn off the power of electrophoresis.
supply.
100V power supply
cathode anode
tracking dyes
Practical 41.1
3. Stain the gel by carrying out
the following steps: Caution
a) Take out the casting tray Methylene blue solution is
together with the gel from harmful. Avoid contact with skin.
the electrophoresis tank
carefully. Transfer the gel
into a plastic box containing Note
methylene blue solution. Do not leave the gel at
b) Cover the box with a lid and room temperature for too
leave it overnight. long. Otherwise, it may
dry up and then crack.
Practical 41.1
c) If the gel is overstained, remove the gel from
the methylene blue solution and destain it in
distilled water for one hour.
d) Place the gel on a white tile for observation.
4. Take a photograph of the stained gel with DNA

bands.
Practical 41.1
Results
Stick and label a photograph of the stained gel in

the space below.
(Results vary with students.)

Practical 41.1
Discussion
1. What is the function of Taq DNA polymerase in
PCR?
Taq DNA polymerase is the enzyme that
catalyses the synthesis of new DNA strands in
the extension step.
Practical 41.1
Discussion
2. How should the primers be designed to enable
them to anneal to the target sequence of the
DNA molecule that is to be amplified?
Each of the primers has to be complementary to
one of the ends of the target DNA region.
Practical 41.1
Discussion
3. Why should nucleotides be included in the
master mix for the PCR?
Nucleotides are used in the extension step for
the synthesis of new DNA strands.
Practical 41.1
Discussion
4. Why should the PCR products be kept on ice?
To prevent the PCR products from degrading.
Practical 41.1
Discussion
5. Why should the DNA marker be used in gel
electrophoresis?
DNA marker consists of a range of DNA
fragments of known sizes. It can be used as a
reference to estimate the size of DNA fragments
in samples.
Practical 41.1
Discussion
6. Based on the results, determine whether the
suspect’s DNA matches with the DNA sample
collected from the crime scene. Explain your
answer.
Yes, all the DNA bands of the suspect match
with the corresponding DNA bands from the
crime scene. /
No, (some of) the DNA bands of the suspect do
not match with the corresponding DNA bands
from the crime scene.
(ii) Parentage testing
 DNA fingerprinting can be used to determine
whether two or more individuals are biologically
related.
 It is most commonly done in parentage tests to
prove whether parents are biologically related to
their children.
Activity 41.1 Handling Data
Using DNA fingerprinting in a parentage test

The following shows the DNA fingerprints of a boy,
his mother and two individuals who both claim to
be his biological father.
mother boy individual 1 individual 2
DNA fingerprints
obtained for a
parentage test
Activity 41.1 Handling Data
Using DNA fingerprinting in a parentage test
1. Study the DNA fingerprints, and circle the DNA

bands shared by the boy and his mother.
2. Compare the remaining DNA bands of the boy
and the DNA bands of individuals 1 and 2.
Deduce who is likely to be the biological father of
the boy. (Hint: the remaining DNA bands should
come from his biological father.)
individual 1
Surf the net
Conduct a virtual parentage test using DNA
fingerprinting at
http://cd1.edb.hkedcity.net/cd/science/biology
/resources/animation/Topic03.swf
(iii) Diagnosis of genetic diseases
 DNA fingerprinting is now used to diagnose
genetic diseases in both foetuses and newborn
babies in hospitals around the world.
 These diseases may include: cystic fibrosis,
haemophilia, Huntington’s disease, sickle-
cell anaemia, thalassaemia and many others.
(iv) Evolution studies
 helps scientists identify the evolutionary
relationships among different groups of
organisms
 It is assumed that the closer the evolutionary
relationship of two groups of organisms, the
more bands they have in common in their
DNA fingerprints.
DNA fingerprinting has been used to study the DNA
recovered from the remains of a 40,000-year-old
woolly mammoth frozen in a glacier.
Key Point
1. DNA fingerprinting, also called DNA
profiling, is a technique for identifying
individuals using their DNA profiles.
2. Restriction fragment length polymorphism

(RFLP) analysis and short tandem repeat
(STR) analysis are two methods of DNA
fingerprinting.
Key Point
3. RFLP analysis involves the following main steps:
a) Extract DNA from the samples.
b) Cut the DNA samples with restriction
enzymes into DNA fragments .
c) Perform gel electrophoresis to separate
the DNA fragments according to their
molecular size.
d) Separate the double-stranded DNA into
single strands (denaturation) and transfer
the DNA fragments to a nylon membrane.
Key Point
3. RFLP analysis involves the following main steps:
e) Incubate the nylon membrane with
radioactive DNA probes (DNA
hybridisation).
f) Place an X-ray film against the nylon
membrane in the dark to obtain the DNA
fingerprints.
4. DNA fingerprinting is used in forensic science,
parentage testing, diagnosis of genetic
diseases and evolution studies.
Quick Practice
Which of the following is an application of DNA

fingerprinting?
A. to separate DNA fragments

B. to amplify DNA samples
C. to sequence DNA from bacteria
D. to identify suspects involved in a crime
D
Genetically modified organisms
 Genetically modified organisms (GMOs) are
organisms whose genetic compositions have
been altered using genetic engineering
techniques.
 e,g. the bacterium that has been genetically
modified to produce human insulin
 Scientists have also created genetically
modified animals and genetically modified
plants.
How can microorganisms be genetically modified?
 bacteriophages (viruses that infect bacteria)
can also be used as vectors to transfer target
genes into bacteria
 Bacteriophages attach to the surface of bacteria
and inject their genetic materials into the
bacterial cells for replication
 This natural property makes bacteriophages
useful as vectors.
 The target gene is first inserted into the DNA
molecule of a bacteriophage.
 When the bacteriophage infects a bacterium, the
target gene is injected into the bacterium along
with the bacteriophage DNA.
bacteriophages
Bacteriophages attach to and

inject genetic materials into an
E. coli host
x
Life cycle of a bacteriophage

A bacteriophage attaches to a
host cell (a bacterium) and bacteriophage
injects its DNA into the host cell.
bacteriophage DNA
bacterial chromosome
x

The viral genetic material takes
control of the host cell’s activities to bacteriophage protein
produce a large number of the viral
nucleic acids and proteins.
bacteriophage DNA
bacterial chromosome breaks down

x

The viral nucleic acid and proteins assemble into new
bacteriophages.
x

The cell lyses and new bacteriophages are released. Each
of these new viruses can then infect another host cell.
 Genetically modified bacteria can pass the target
gene from one generation to the next.
 useful in biotechnology
 The high reproductive rate of bacteria allows
scientists to produce large quantities of the
desired gene products within a short period of
time.
Learning Tip
Yeast can also be genetically modified for research purposes
and the production of useful products. Specific vectors and
gene transfer techniques have been developed for use in yeast.
How can animals be genetically modified?
 Animal cells can be genetically modified by
transferring target genes into them.
 An animal that has acquired foreign genes is
called a genetically modified animal or a
transgenic animal.
 If all the cells of the transgenic animal should
contain the target gene, it has to be introduced
into a fertilised egg cell.
 This is usually done by a method known as
microinjection.
The procedure of creating a genetically
modified sheep by microinjection
Egg cells are removed
from a female sheep.
sperm These are fertilised by
sperms in vitro.
female sheep egg cell from the DNA containing
female sheep the target gene
fertilised egg
Learning Tip
A pronucleus is the nucleus of a Foreign DNA containing the
sperm or an egg cell during the target gene is injected directly
process of fertilisation, after the into one of the pronuclei of
sperm enters the ovum, but before fertilised eggs using a very fine
the zygote nucleus is formed. needle-like pipette. Cont’d
The procedure of creating a genetically
modified sheep by microinjection
The genetically modified fertilised
eggs are then implanted into the
uterus of a female sheep (the
surrogate mother) for development.
surrogate mother
The surrogate mother gives

birth to offspring containing
the target gene.
genetically modified sheep
Extras — Do you know?
Genetically modified zebra fish
When creating GMOs, scientists often include a
gene that encodes a fluorescent protein in the
vector. One example is the green fluorescent
protein found in some jellyfish. This helps the
scientists to identify more easily which organism
has been successfully modified.
Genetically modified zebra fish
A similar technique has been
applied to create genetically
modified pets. In 2003,
genetically modified zebra fish
called ‘Glofish’ were introduced
to the market. They can glow
GM zebra fish with
in the dark with bright red, fluorescent colours
green, orange-yellow, blue and
purple fluorescent colours.
How can plants be genetically modified?
 A plant that has acquired foreign genes is called
a genetically modified plant or transgenic
plant.
 One of the most effective methods of transferring
target genes into plants makes use of the soil
bacterium Agrobacterium.
 contains the Ti plasmid, which can be used to
carry the target gene
 infect most dicotyledonous plants
 widely used in modifying these plants
The procedure of creating genetically modified
plants using Agrobacterium
Ti plasmid
Extract Ti plasmid
and cut it using a
cut plasmid restriction enzyme.
Agrobacterium
target gene Insert the target gene

into the plasmid and
join them using DNA
ligase.
recombinant plasmid
Cont’d
 Introduce the
recombinant plasmid into
the Agrobacterium.
transformed Agrobacterium
Infect plant tissues with the

transformed Agrobacterium.
infected plant tissues
containing the target gene
Cont’d
Transfer the infected plant

tissues to an agar plate
containing nutrients for growth.
genetically modified plant

containing the target gene
How can plants be genetically modified?
 This technique also works well for tomato, potato
and many other plants.
 Another method of transferring target genes into
plant cells is to use a gene gun.
 DNA containing the target gene is first obtained
and coated onto the surface of gold or tungsten
beads.
 The beads are then physically forced into the
plant cells by a gene gun.
A gene gun being used to transfer
foreign DNA into a plant
Key Point
1. Genetically modified organisms (GMOs)
are organisms whose genetic compositions
have been altered using genetic engineering
techniques.
2. Plasmids and bacteriophages can be

used as vectors to transfer the target gene into
bacteria to create genetically modified bacteria.
Key Point
3. Genetically modified animals (transgenic
animals) can be created by introducing the
target gene into a fertilised egg by
microinjection .
4. Genetically modified plants (transgenic plants)

can be created by transferring the target gene
into the plant using the soil bacterium
Agrobacterium or gene gun .
Quick Practice
1. Which of the following is not a genetically

modified organism?
A. Agrobacterium with a pesticide resistance gene

B. a mouse with the gene coding for human insulin
C. a tomato plant with an antifreeze gene from fish
D. a maize plant produced by selective breeding
for its high sugar content D
Quick Practice
2. Production of GM animals by microinjection

involves
(1) in vitro fertilisation.
(2) the insertion of the target gene with a gene gun.
(3) transferring the genetically modified fertilised eggs into
a surrogate mother.
A. (1) and (2) only
B. (1) and (3) only B
C. (2) and (3) only
D. (1), (2) and (3)
Benefits and potential risks
of genetic engineering
 Benefits offered by genetic engineering:
 providing scientists with new means to study
organisms at the molecular level
 speeding up research in medical science and
bringing advances in medicine
 opening up new possibilities for solving
food shortage and providing ways to
improve food quality
 Potential risks of genetic engineering:
 Antibiotic resistance genes are often used as
selection markers in genetic engineering. If
these genes were accidentally transferred to
pathogens, the problem of antibiotic
resistance would be worsened, and
‘superbugs’ which are resistant to most
antibiotics could be resulted.
 New genes are transferred to GMOs. Some
people may have allergies after having food
or drugs made from GMOs.
 GM food and recombinant drugs have a
relatively short history, and their long-term
effects on consumers’ health have not
been very well studied.
 GMOs, if released to the environment, may
breed with wild types and transfer their
genes to the offspring. This may cause
unknown or adverse effects on the species.
GMOs may also outcompete the wild types
and result in a loss of biodiversity.
Learning Tip
Genetic engineering also brings about other ethical, social
and legal concerns, which will be discussed in Ch 43.
Key Point
Genetic engineering offers many benefits, yet it
also causes potential hazards to our health and
the environment:
Benefits
• Providing scientists with new tools to study organisms
at the molecular level
• Speeding up research in medical science and bringing
advances in medicine
• Opening up new possibilities for solving food
shortage and providing ways to improve food quality
Cont’d
Key Point
Potential hazards
•The use of antibiotic resistance genes raises concerns in
creating antibiotic-resistant ‘superbugs’.
•GM foods and recombinant drugs may cause allergies
and their long-term effects on our health are still
unknown.
•GMOs may breed with wild types and transfer their
genes to the offspring. GMOs may also out-compete
wild types and result in a loss of biodiversity.
Animal and plant
cloning
Animal and plant cloning
 Cloning of organisms is the production of
genetically identical individuals.
 With the advances in biotechnology, scientists

have successfully cloned many species of
animals and plants.
Animal cloning
 Embryo splitting involves isolating individual
cells from an embryo at an early stage and
growing them independently.
 Each of these cells develops into an embryo.
 The embryos obtained are then transferred into

the uteri of surrogate mothers for development.
Animal cloning by embryo splitting
embryo at an
early stage
individual cells isolated
from the embryo
embryos grown
from individual cells
surrogate mothers
clones
Animal cloning
 nuclear transfer is another cloning method
 The cloning process involves the following steps:
1. A mammary gland cell was obtained from an
adult sheep (nucleus donor).
2. An egg cell was obtained from another sheep
(egg donor). The nucleus of the egg cell was
then removed.
Learning Tip
The nucleus of an ovum is haploid (n). It cannot be used to develop a new
individual because it only has half the chromosomes of a normal body cell. An
ordinary diploid cell, even though it has all the chromosomes, is specialised.
Transferring a diploid (2n) nucleus into an egg cell that has its nucleus
removed results in a cell that is capable of developing into a new individual.
Animal cloning
3. The mammary gland cell and the egg cell fused
together. The nucleus from the mammary gland
cell, containing a full set of chromosomes, was
transferred to the egg cell. An electric shock
was applied to make the fused cell begin to
divide. The cell developed into an embryo.
4. The embryo was transferred into the uterus of a
surrogate mother for development.
5. The surrogate mother gave birth to Dolly.
Cloning of Dolly
nucleus donor
donor nucleus
electric shock
mammary gland cell

embryo
egg cell with its fused cell

nucleus removed
nucleus surrogate mother
egg donor
egg cell
Dolly
Animal cloning
Clear your concepts
It should be noted that a clone does not
necessarily look the same as the nucleus donor.
Although the genetic make-up of the clone is
identical to the nucleus donor, some genes may be
expressed differently in the clone. In addition, the
expression of a phenotype can also be affected by
environmental factors.
Applications and limitations of animal cloning
 potential applications of animal cloning:
 be used to propagate animals of considerable
economic importance or save endangered
animals
 be used as models for studying diseases
and testing drugs
 Cloned GM animals could serve as ‘biological
factories’ to produce pharmaceutical
products or other useful chemicals.
 Cloning early embryos could provide stem
cells for use in research or medical treatment.
Learning Tip
The uses of stem cells in medicine will be
discussed in Ch 42.
Applications and limitations of animal cloning
 limitations of animal cloning:

 The success rate of animal cloning using
nuclear transfer is still low and the cost is
high.
 Cloned animals may also age faster and

have a shorter lifespan.
The death of Dolly
Dolly was born in 1996 in the United Kingdom. She
lived in the laboratory all her life and gave birth to
six lambs. She began to suffer from various
diseases from the age of five and was put to death
in 2003, at the age of six. The lifespan of a sheep
like Dolly is usually about 12 years. Some think that
Dolly’s poor health was a result of staying indoors
and overfeeding from visitors.
The death of Dolly
Others believe Dolly’s early death was due to the
fact that her DNA was already six years old (the age
of the nucleus donor at the time of the nuclear
transfer) when she was born.
Dolly was preserved

after her death and is
displayed in a museum.
Tissue culture
 Traditionally, plant cloning has been practiced
through artificial propagation methods.
 e.g. cutting
 Modern methods involving tissue culture

(micropropagation) are commonly used.
Tissue culture
 During tissue culture, tissue samples are taken
from a parent plant and cultured in a sterile
culture medium containing essential nutrients
and plant hormones.
 The cultured tissues will eventually develop into
whole plants.
Learning Tip
Meristematic tissues from the shoot tip are preferable for tissue
culture. These consist of undifferentiated cells undergoing active cell
division. Furthermore, they are usually free of diseasecausing
organisms that may be present in the rest of the plant.
The main steps involved in tissue culture
Tissue samples are taken from
the parent plant. These samples
are called explants.
callus
Each tissue sample is placed on a sterile

culture medium containing essential
nutrients and plant hormones. A mass of
undifferentiated cells called callus is
Cont’d formed by mitotic cell division.
The main steps involved in tissue culture
Small pieces of the callus are
transferred to different culture
media, which contain plant
hormones that promote the
growth of shoots and roots.
The plantlets formed can
be planted into the soil.
plantlet
Applications and limitations of tissue culture
 advantages of tissue culture:

 a faster method to propagate plants
 only takes up little space
 Since plants produced by this method are
genetically identical to the parent plant,
they also possess the desirable features of
the parent plant.
 some applications of plant cloning using tissue
culture:
 To propagate endangered plants (e.g.
orchids), plants of considerable economic
importance, or plants that are difficult to
grow by conventional propagation methods.
 To produce plants which are free of
disease-causing organisms by growing
them under sterile conditions.
 To mass produce GM plants from cells
with target genes inserted.
 limitation of plant cloning using tissue culture:
 a much higher cost than the conventional
methods as it requires sterile conditions
and is labour-intensive
 due to the lack of genetic variations, the
clone population may have a reduced
ability to adapt to changes in the
environment
Key Point
1. Cloning of organisms is the production of
genetically identical individuals.
2. Animals can be cloned by embryo splitting
or nuclear transfer .
3. Animal cloning by nuclear transfer involves the
following steps:
Step 1: A body cell is obtained from an animal.
Step 2: An egg cell is obtained from another

animal and its nucleus is then removed.
Cont’d
Key Point
3. Animal cloning by nuclear transfer involves the
following steps:
Step 3: The body cell fuses with the egg cell lacking its
nucleus. The fused cell develops into an embryo.
Step 4: The embryo is transferred into the

uterus of a surrogate mother.
Step 5: The surrogate mother gives

birth to the cloned animal.
Key Point
4. Animal cloning has many potential applications,
but there are some limitations:
Potential applications
• It can be used to propagate animals of considerable
economic importance or save endangered animals.
• Cloned animals can be used as models for studying
diseases and testing drugs.
• Cloned GM animals could serve as ‘biological factories’
to produce pharmaceutical products or other useful
chemicals.
• Cloning early embryos could provide stem cells for use
in research or medical treatment.
Cont’d
Key Point
4. Animal cloning has many potential applications,
but there are some limitations:
Limitations
•The success rate of animal cloning using nuclear transfer
is low and the cost is high.
•Animal clones may age faster and have a shorter
lifespan.
Key Point
5. Plant cloning can be achieved by tissue
culture, which involves the following steps:
Step 1: Tissue samples are taken from the parent plant.
Step 2: The sample is placed in a sterile culture

medium to allow the formation of callus.
Step 3: Small pieces of the callus are transferred to different

culture media to promote the growth of shoots and roots.
Step 4: The plantlets formed can be planted into the soil.

Key Point
6. Plant cloning culture has many potential
applications, yet there are some limitations:
Potential applications
• To propagate plants that are endangered (e.g. orchids),
difficult to grow by conventional propagation, or plants
that have economic values.
• To produce plants which are free from disease by
growing them under sterile conditions.
• To mass produce GM plants from cells inserted with
target genes.
Cont’d
Key Point
6. Plant cloning culture has many potential
applications, yet there are some limitations:
Limitations
• The cost is much higher than artificial propagation
methods as tissue culture requires sterile conditions
and is labour-intensive.
• A lack of genetic variations in the clone population
reduces its ability to adapt to changes in the
environment.
Quick Practice
1. The genetic material of Dolly the sheep came from

(1) the sheep from which the mammary gland cells was
obtained.
(2) the egg donor.
(3) its surrogate mother.
A. (1) only
B. (2) only
C. (1) and (2) only
D. (1), (2) and (3)
A
Quick Practice
2. A plant can be cloned using tissue culture. Arrange the
following steps of tissue culture in the correct order.
(1) Allow plantlets to grow.
(2) Obtain tissue samples from a plant.
(3) Transfer pieces of callus to different culture media to
promote the growth of shoots and roots.
(4) Place tissue samples in a sterile culture medium.
A. (1), (2), (4), (3)

B. (2), (4), (3), (1)
C. (3), (2), (1), (4)
D. (4), (2), (1), (3) B
41 Techniques in modern biotechnology  Key terms
Key terms
 biotechnology (生物工程)
 selective breeding (選擇育種)
 fermentation (發酵)
 genetic engineering (遺傳工程)
 cloning (克隆)
 genetically modified organism (GMO)
(基因改造生物)
 recombinant DNA technology
(重組 DNA 技術)
41 Techniques in modern biotechnology  Key terms
Key terms
 recombinant DNA (重組 DNA)

 vector (載體)
 plasmid (質粒)
 restriction enzyme (限制酶)
 sticky end (黏端)
 DNA ligase (DNA 連接酶)
 recombinant plasmid (重組質粒)
 polymerase chain reaction (PCR)
(聚合酶鏈反應)
 primer (引物)
 DNA fingerprinting (DNA 指紋分析)
 variable number tandem repeat (VNTR)
(可變數目銜接重複)
 short tandem repeat (STR) (短銜接重複)
 bacteriophage (噬菌體)
 embryo splitting (胚分割)
 nuclear transfer (核移植)
 tissue culture (組織培養)
 callus (胼胝體)

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What students think I am doing What I am actually doing

Bread-making involves The production of cheese involves

 Two examples of it are:

The bacteria E. coli can be genetically Tomatoes can be genetically modified

Salmon can be genetically modified Genetically modified microorganisms (e.g.

Dolly the sheep, the first mammal

Which of the following is/are application(s) of

Contain multiple cloning sites

Contain origin of replication

A plasmid is an (artificial) circular strand of

Engineered Plasmid in engineering

 Get DNA with target DNA with whatever means

 Transfer the DNA to the plasmid

 The recombinant plasmid is transferred to

 Multiply a specific portion of DNA by PCR

 Cut the donor DNA by two specific restriction

This is the target…

restriction enzyme restriction site restriction enzyme

DNA fragments DNA fragments

 They are able to recognize specific base

 Hence, two DNA fragments with complementary

Obtain DNA fragments

 It is a must to cut the DNA fragments with RE

 It is a basically a must for both plasmid and

Ligase catalyze the

Major method of obtaining the target gene

Major method of obtaining the target gene

Major method of obtaining the target gene

Obtain mRNA of the target gene

DNA containing Synthesise the second DNA strand from

 Plasmid can ask the cell to produce that gene

 Mammalian cancer cell (Eukaryotes)

Recombinant plasmids replicate inside the bacteria and gene expression is

Recombinant DNA technology was developed in

In 1973, they successfully

Cut with same REs

Bacteria Recombinant plasmid DNA

2. Plasmids and virus are two most

Step 2: Cutting the DNA fragments and plasmids

Step 3: Inserting the target gene into the plasmid

5. One application of recombinant DNA technology is the

The principle of PCR

 Copy machine: DNA polymerase

 Raw materials to copy: dATP, dTTP, dCTP,

 A guide to tell where to copy: DNA primer pair

 a heat-stable DNA polymerase - an

 The reaction mixture is first heated to about 95

two pieces of single-

 The reaction mixture is cooled to between 50 °C

 The temperature at this step depends on the

Taq DNA polymerase

synthesis of new DNA strands

Extension of primers to synthesise new DNA strands

3rd cycle 8 Amplification of

 The whole cycling process of PCR can now be

95°C 60°C 72°C

 The advantages of PCR:

Cut with same REs

Bacteria Recombinant plasmid DNA

Desired gene DNA Plasmid DNA

Cut with same REs

Bacteria Recombinant plasmid DNA

Testing for GM foods by PCR