Biology CH.41

TE
41
Techniques in
modern
biotechnology
e-aristo.hk/r/
bioccfc41.e
Techniques in modern biotechnology allow scientists to manipulate DNA accurately and quickly.
Links to prior knowledge Chapter preview

In the Compulsory Part (Chapters 26 and 27), you
have learned the basics of molecular genetics. You 41.1 Introduction to
should have recognized how the understanding of modern biotechnology
fundamental life processes (e.g. the properties of 41.2 Genetic engineering
enzymes and life cycles of microbes) has helped
scientists to develop some techniques in
41.3 Animal and plant
biotechnology. You have also explored some of the
cloning
applications of modern biotechnology in society
(Chapter 28). In this Elective Part, you will further
study the principles, applications and ethical
implications of biotechnology.
41 Techniques in modern biotechnology
Is Supersweet corn genetically modified?
Supersweet is a variety of sweet corn that has high

sugar content and can stay sweet for a long period
of time, unlike other corns whose sugars are quickly
converted to starch after harvest.
As the public are becoming more and more

concerned about the techniques of genetic Supersweet corn
modification, some people begin to ask whether or
not Supersweet corn is genetically modified. The
answer is probably not.
Supersweet corn is traditionally developed by selective breeding. Corn plants with

high sugar content are selected by breeders for cross pollination. Breeders developed
the Supersweet variety long before the techniques of genetic modification were
invented.
However, today it is possible that a Supersweet corn plant could also have a genetically
modified trait. Nonetheless, the Supersweet trait is not created by genetic modification.
Think about …
1. The production of Supersweet corn by selective breeding is an example of traditional
biotechnology. Do you know other examples of traditional biotechnology?
2. What is genetic modification?
3. What techniques are used to produce genetically modified organisms?
4. How do we know whether a crop has been genetically modified?
Answer
(Refer to p.A1 for answers.)
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41.1 Introduction to modern

Learning objective biotechnology
• Understand what modern
biotechnology is about Biotechnology refers to the use of biological processes, biological
• Recognize that genetic
systems or organisms to produce goods or provide services to
engineering and cloning are
examples of modern humans.
biotechnology
Biotechnology has a long history. The earliest example is the
domestication of plants and animals. Humans selectively breed
organisms with desirable characteristics to produce new varieties
that are more refined and better suited to human use.
Nowadays, with increased knowledge about the structure of DNA

and its universal role in organisms, scientists have developed
various techniques to manipulate DNA. This in turn allows cells
and tissues to be genetically modified. The focus of modern
biotechnology is the applications of these techniques on organisms
to improve their products.
a b
Rice was domesticated more than 8000 years ago. Scientists modify the DNA of the rice plant to produce
Different varieties of rice were cross-bred to increase Golden Rice, which synthesizes beta-carotene. Our body
yields. uses beta-carotene to make vitamin A.
c d
×15,000
Cattle have been selectively bred to produce meat, milk, The bacteria E. coli can be genetically modified to mass
leather, and insulin, the hormone used to treat diabetes. produce human insulin.
Figure 41.1 Examples of traditional and modern biotechnology
biotechnology 生物工程
41- 3
Two rapidly expanding areas of modern biotechnology are genetic

engineering and cloning.
1. Genetic engineering
Genetic engineering, also called genetic modification, is the direct
manipulation of the genetic material of an organism, thereby
changing its genetic make-up.
For example, genes from one organism can be inserted into the
genetic material of another organism. The organism receiving the
foreign genes is a genetically modified organism (GMO) or
transgenic organism. The expression of the foreign genes gives the
GMO new characteristics.
a b
×3400
Maize plants can be genetically modified to become Yeasts can be genetically modified to make antigenic
resistant to herbicides and pests. proteins to be used in vaccines.
c d
Salmon can be genetically modified to have a higher Genetically modified microorganisms (e.g. yeasts and
growth rate. bacteria) can be used in sewage treatment.
Figure 41.2 Some applications of genetic engineering
2. Cloning
Cloning is the production of genetically identical copies of a gene,
a cell, or even a whole organism. The products are called clones.
The most famous clone is probably the first cloned mammal (using
Figure 41.3 Dolly the sheep an adult body cell), Dolly the sheep (1996–2003).
genetic engineering 遺傳工程 transgenic organism 轉基因生物

41- 4 cloning 克隆
genetically modified organism 基因改造生物
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Learning objective 41.2 Genetic engineering

• Outline the principle of
recombinant DNA technology
Scientists have discovered ‘useful tools’ from nature (e.g. restriction
• Use the production of insulin to
enzymes, DNA ligase, plasmids, etc.) and made use of them to
illustrate the process of
recombinant DNA technology develop various techniques to manipulate DNA.
• Outline the principle of the
polymerase chain reaction and
recognize its wide applications A. Recombinant DNA technology
• Outline the principle of DNA
fingerprinting and recognize its Recombinant DNA technology is a set of techniques for joining
wide applications DNA molecules from different sources to create recombinant DNA.
• Outline the principle of This technology enables scientists to transfer a gene of interest
constructing genetically
modified organisms
(target gene) from a donor organism to a host organism. The two
• Discuss the benefits and organisms can be of the same or different species.
hazards of genetic engineering
1. The principle of recombinant DNA technology

You have learned the basic steps of recombinant DNA technology
in Chapter 28. The process of making recombinant DNA involves:
 Isolation of DNA fragments that contain the gene of interest
DNA fragments that contain the gene of interest are isolated from
donor cells.
 Isolation of vectors
A vector is a DNA molecule that acts as a carrier to transfer the

gene of interest into a host cell. Inside the host cell, the vector is
replicated. This produces copies of the vector together with the
gene of interest. Bacterial plasmids and viruses are commonly
used vectors.
A plasmid is a circular double-stranded DNA (Figure 41.4),

independent of the bacterial chromosome, found in many types of
bacteria. They are usually not essential for the bacteria to survive,
bacterial
but the genes contained in plasmids may offer survival advantages
plasmids chromosome
to the bacteria (e.g. antibiotic resistance genes).
Plasmids replicate independently of the bacterial chromosome and

there are often multiple copies in a single bacterial cell. They are
naturally transferred from one bacterial cell to another. These
antibiotic resistance gene
Figure 41.4 Plasmids exist in features make plasmids useful vectors in recombinant DNA
multiple copies in a bacterium. technology.
recombinant DNA technology 重組 DNA 技術 plasmid 質粒

recombinant DNA 重組 DNA 41- 5
vector 載體
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
Restriction—cutting DNA fragments and plasmids with a
restriction enzyme
Restriction enzymes (restriction endonucleases) act like molecular

scissors. They recognize specific base sequences, called restriction
sites, on a piece of DNA and cut at those sequences.
Some restriction enzymes make a straight cut across the DNA and
produce blunt ends (Figure 41.5 a ). Other restriction enzymes cut
and produce DNA fragments with sticky ends which are single-
stranded with unpaired bases ( b ). The bases on a sticky end can
Remember this pair up with another sticky end with a complementary base
The genetic code of DNA is sequence.
universal, therefore the
complementary bases on different By cutting the DNA fragments containing the gene of interest and
DNA fragments can pair up
regardless of the source of the the plasmids with the same restriction enzyme, the sticky ends on
DNA. the cut DNA fragments and the cut plasmids are complementary
and can attach to each other easily.
a Restriction b Restriction
enzyme cuts enzyme cuts
at at
G A A T T C G A A T T C G A A T T C G A A T T C
C T T A A G C T T A A G C T T A A G C T T A A G
restriction
site
G A A T T C G A A T T C G A A T T C G A A T T C
C T T A A G C T T A A G C T T A A G C T T A A G
blunt ends blunt ends sticky ends sticky ends

Figure 41.5 Restriction enzymes cut DNA molecules at restriction sites
 Ligation—joining DNA fragments and plasmids together with

DNA ligase
The sticky ends on the cut DNA fragment (which contains the gene
of interest) and the sticky ends on the cut plasmid pair up. They can
be joined together by an enzyme called DNA ligase (Figure 41.6 ).
The process of joining the two pieces of DNA is called ligation. The
resulting DNA molecule is called a recombinant plasmid.
restriction enzyme 限制酶 ligation 連接 restriction endonuclease 限制性核酸內切酶

41- 6 sticky end 黏端 recombinant plasmid 重組質粒
DNA ligase DNA 連接酶
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The recombinant plasmids can then be transferred into host cells.

Bacteria are commonly used as host cells in recombinant DNA
technology. As plasmids are naturally transferred between bacterial
cells, it is relatively easier for bacteria to take up the recombinant
plasmids than eukaryotic cells.
2. Production of human insulin using

Bacteria can be genetically engineered with recombinant DNA
technology to synthesize human insulin:
bacterium
bacterial
plasmid
chromosome
 Obtain the DNA

fragment that contains  Isolate plasmids from
the human insulin gene bacterial cells.
through proper processes.
antibiotic
resistance
gene plasmid
sticky ends
 Cut the DNA fragment
and the plasmid using
the same restriction
sticky ends enzyme.
cut plasmid
 Insert the DNA fragment into

the cut plasmid and join
them using DNA ligase.
recombinant plasmid
 Introduce the recombinant
plasmid into a bacterium
(e.g. E. coli).
transformed bacterium
Simulation
(bacterium that has taken up
Genetic modification of
bacteria using the recombinant plasmid)
recombinant DNA
technology  Select the transformed
e-aristo.hk/r/ bacteria and culture them on
bioccsim4101.e a large scale.
Figure 41.6 Production of  Recombinant plasmids replicate inside the bacteria and gene expression is
human insulin using induced for protein synthesis. Pure and functional human insulin can be
recombinant DNA technology obtained after further processing of the gene products.
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Recombinant plasmids are produced by inserting the human insulin

gene into plasmids (Figure 41.6 –). Bacteria (e.g. Escherichia
coli) are then mixed with the recombinant plasmids. Some of the
bacteria take up the recombinant plasmids (). This process is
called transformation, and the bacteria that have taken up the
recombinant plasmids are called transformed bacteria.
Remember this
However, some bacteria may not take up any recombinant plasmids,
A selection marker can be any
genes that confer selective so they are not transformed. In order to select transformed bacteria
advantage to the transformed from the ones that have not transformed, the plasmids used carry a
cells, but not the non-transformed
selection marker, such as an antibiotic resistance gene.
cells. It allows the transformed
cells to survive in a particular
environment, while the non- When bacteria are growing in a medium containing an antibiotic,
transformed cells die. This is a only the transformed bacteria are resistant to the antibiotic, so they
type of positive selection.
survive and reproduce to form colonies (Figure 41.7).
transformed bacterium bacterium that has not taken

possesses both the human up the recombinant plasmid
insulin gene and the does not possess the
antibiotic resistance gene antibiotic resistance gene
visible colonies
incubation at 37 ºC
agar containing an antibiotic
Figure 41.7 When bacteria are cultured in an agar plate containing an antibiotic, only the transformed
bacteria can survive and reproduce.
The transformed bacteria are selected and cultured on a large scale

in industrial fermenters (Figure 41.6 ). The recombinant plasmids
replicate independently of the bacterial chromosome in each cell,
so multiple copies of the plasmids exist in each bacterium. When
the transformed bacteria reproduce, the replicated plasmids are
passed to daughter cells. As a result, numerous identical copies of
the gene of interest are produced. The process of producing
identical copies of a gene is called gene cloning.
Gene expression is induced in the transformed bacteria to synthesize

the gene products (). After further processing of the gene
products, pure and functional human insulin is obtained.
transformation 轉化
41- 8 gene cloning 基因克隆
Discovering science
Discovery of restriction enzymes and the development of
The first restriction enzyme was discovered in the bacterium Haemophylus
influenzae in the 1960s. Inside bacterial cells, restriction enzymes are
responsible for cutting the viral DNA of invading bacteriophages, thus
stopping their replication. This discovery was made by a Swiss scientist
Werner Arber (1929–2014). He was awarded the Nobel Prize in Physiology or
Medicine in 1978. Since then, hundreds of restriction enzymes from different
bacteria have been identified and studied.
In the 1970s, an American scientist Stanley Cohen
(1935–) observed the transformation of bacteria by
plasmids—bacterial cells take up plasmids and
incorporate them into their genome. Another American
scientist Herbert Boyer (1936–) studied a restriction
enzyme found in E. coli that cuts DNA and produces
sticky ends.
Stanley Cohen

Later, when Cohen and Boyer met and discussed their
work, they together hypothesized a technique making
use of restriction enzymes and plasmids to alter the
genetic material of organisms—recombinant DNA
technology. They then successfully used this technique
to transfer genes from other bacteria, amphibians and
even humans (insulin gene) into E. coli.
Herbert Boyer

Taking it further
An alternative method to obtain the gene of interest
If suitable restriction enzymes are
not available for isolating the gene
cell type actively
of interest, an alternative method is producing the target
to synthesize the DNA from its gene product
mRNA. This is particularly useful for
a gene that is actively expressed in a  Extract mRNA of
certain type of cell because a large the target gene
amount of its mRNA can be extracted
from those cells. mRNA
Using the mRNA as a template, a  Synthesize cDNA
from mRNA
complementary DNA (cDNA) strand
can be synthesized by a process mRNA
called reverse transcription. This is
catalysed by the enzyme reverse
cDNA  Remove the
mRNA with alkali
transcriptase. The cDNA produced is
single-stranded. It is then used as  Synthesize the
the template for the synthesis of the second DNA strand
other DNA strand using DNA DNA containing from the cDNA
the target gene
polymerase. The two DNA strands
 Obtaining a target gene from mRNA
make up a double-stranded DNA
molecule containing the target gene.
41- 9
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Key point
1. Recombinant DNA technology is a set of techniques for joining DNA
molecules from different sources to create recombinant DNA.
2. The basic steps of recombinant DNA technology include:
• Step 1: Isolation of DNA fragments that contain the gene of interest
• Step 2: Isolation of plasmids from bacteria to be used as vectors
• Step 3: Cutting the DNA fragments and the plasmids using the same
restriction enzyme
• Step 4: Joining the cut DNA fragments and cut plasmids together using
DNA ligase
3. One application of recombinant DNA technology is the production of
useful biological molecules (e.g. human insulin) in large quantities.
Checkpoint
Arrange the steps involved in the production of human insulin by
recombinant DNA technology in the correct order.
(1) Multiply copies of the gene coding for human insulin in the bacterium.
(2) Obtain the gene coding for human insulin.
(3) Insert the gene coding for human insulin into a vector.
(4) Screen for bacteria with the gene coding for human insulin.
(5) Introduce the vector into a bacterium.
A. (2), (3), (4), (5), (1)
B. (2), (3), (5), (4), (1)
C. (5), (2), (3), (4), (1)
D. (5), (2), (4), (3), (1)
B. Polymerase chain reaction

Remember this
The polymerase chain reaction (PCR) was invented by an American
By transferring a gene of interest biochemist Kary Mullis (1944–) in 1983. It is a technique for
into host cells with vectors, the
gene of interest can be cloned replicating specific DNA sequences outside cells. With PCR, a small
when the host cells replicate. amount of a DNA sample can be amplified to many copies quickly
Although specific DNA sequences and accurately.
can be amplified in this way, this
method is complicated and
time-consuming.
1. The principle of PCR
In a cell, DNA replication begins when the DNA double helix unwinds
Link it
and the two DNA strands separate. Each DNA strand then serves as
Refer to Chapter 26 for the a template for the synthesis of a new complementary DNA strand.
process of DNA replication.
Free nucleotides in the nucleus pair up with the exposed bases on
the template. The newly added nucleotides are then joined together
by the enzyme DNA polymerase.
polymerase chain reaction 聚合酶鏈反應
41- 10
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The principle of PCR is similar to that of DNA replication, but PCR

is performed in a reaction mixture outside cells, which consists of:

a DNA template—a sample of the target DNA sequence to be
amplified (i.e. the target sequence).
primers—a primer is a short sequence of synthetic single-

stranded DNA that is complementary to one end of the target
sequence. It marks the point where the synthesis of a new DNA
strand starts. As a DNA template consists of two strands, two
different primers are needed. Each primer binds to the starting
point on one of the DNA strands. In this way, the target sequence
to be amplified is ‘bracketed’ (Figure 41.9 on p.12).

a heat-stable DNA polymerase—an enzyme that synthesizes
new DNA strands by joining adjacent nucleotides. The most
commonly used DNA polymerase in PCR is the Taq DNA
polymerase. It can withstand the high temperature required
during PCR.

nucleotides (deoxyribonucleoside triphosphates or dNTPs)—
the building blocks of new DNA strands.
Flipped classroom The reaction mixture undergoes many cycles of polymerase chain
Polymerase chain
reaction
reaction. In each PCR cycle, there are three steps: denaturation,
e-aristo.hk/r/ primer annealing and extension.
bioccflip4101.e
 Denaturation
The reaction mixture is first heated to about 95 °C. At this

temperature, the hydrogen bonds that hold the two DNA strands
break, so that the double helix unwinds and separates into two
single strands (i.e. the DNA molecule denatures).
double-
stranded DNA
denaturation
at about 95 ºC
two pieces of
single-stranded
DNA
Figure 41.8 The double helix unwinds and separates into two single strands
at 95 °C.
primer 引物 primer annealing 引物連接
DNA polymerase DNA 聚合酶 extension 延伸 41- 11
denaturation 變性
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 Primer annealing
The reaction mixture is cooled to between 50 °C and 65 °C. This

allows primers to anneal 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 of the target sequence.
primer 1
primer 2
Figure 41.9 Two primers mark out the target sequence to be replicated.
 Extension
The temperature of this step depends on the DNA polymerase used.

For example, the optimum temperature for Taq DNA polymerase is
about 70 °C.
DNA polymerase attaches to the primers and free nucleotides pair

up with the bases on the DNA template by complementary base
pairing. The enzyme catalyses the formation of bonds between
nucleotides, joining adjacent nucleotides together. Through the
extension of primers, two new strands of DNA are synthesized. Two
copies of the target sequence are formed.
Taq DNA polymerase
primer 2
synthesis of new DNA strands
primer 1 free DNA

nucleotides
Figure 41.10 DNA polymerase catalyses the addition of nucleotides to form the new DNA strand.
41- 12
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The number of DNA strands is doubled at the end of each cycle.

Each strand acts as a template in the next cycle. With a sufficient
supply of free nucleotides and primers, as little as a single piece of
DNA segment can be amplified exponentially into billions of copies
after 30 to 40 cycles.
initial DNA
template
Number of PCR cycle at start 1st 2nd 3rd 4th

1 2 3 4
Number of DNA copy 1 2 =2 2 =4 2 =8 2 = 16
Figure 41.11 Amplification of DNA by PCR
Cycling of PCR can be performed automatically in a thermal cycler

(Figure 41.12). Programmes can be set to alter the number of PCR
cycles performed, and the temperature and duration for the different
stages of a PCR cycle. By using a thermal cycler, 30 cycles of PCR
can be completed in only a few hours.
However, there are considerable amounts of reagents (e.g. free

nucleotides and primers) mixed with the products of PCR. The
amplified DNA fragments have to be purified before they are used
in DNA sequencing, DNA fingerprinting or for other purposes.
Figure 41.12 A thermal cycler
Discovering science
Discovery of heat-stable DNA polymerase enables thermal cycling of PCR
In the early days of DNA research, DNA amplification was a time-consuming and expensive
procedure. It was because the DNA polymerase was easily denatured at high temperatures.
Therefore, it was necessary to add the enzyme to the reaction mixture after every denaturation
step. Kary Mullis invented PCR by using Taq DNA polymerase, which is extracted from the
bacterium Thermus aquaticus. This bacterium lives in very hot environments such as hot
springs, so its Taq DNA polymerase is heat-stable and can be reused for repeated cycling. For
his invention of the PCR method, Mullis was awarded the Nobel Prize for Chemistry in 1993.
Kary Mullis

thermal cycler 循環變温加熱器

41- 13
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2. Applications of PCR
PCR is fast and accurate. Specific base sequences can be targeted
by using suitable primers and amplified quickly. In addition, PCR is
highly sensitive. It can amplify even a very small amount of DNA to
a significant amount.
Scientists often use PCR to amplify DNA samples for subsequent

DNA sequencing or other analysis. Below are some examples.
a b
In police investigations, PCR can be used to amplify tiny To assist prenatal diagnosis of genetic diseases, PCR is
amounts of DNA in a single drop of blood or in a hair used to amplify the DNA extracted from foetal cells
root found at the crime scene for further analysis (e.g. found in amniotic fluid for further analysis.
DNA fingerprinting).
c d
Mycobacterium tuberculosis (x20,000) grows very slowly in

culture.
In the diagnosis of infectious diseases, even only tiny Due to its very high sensitivity, PCR can be used to
amounts of viral or bacterial DNA in a patient’s samples amplify DNA from ancient organic remains for
can be amplified and detected. This is much faster than archaeological studies. Likewise, PCR is well suited for
culturing pathogens. Sometimes prompt treatment is detecting GMO in processed food containing very little
crucial to the survival of patients. GMO content.
Figure 41.13 Some applications of PCR
Key point
1. The polymerase chain reaction (PCR) is a technique for replicating specific DNA sequences outside cells.
2. A reaction mixture containing a DNA template, primers, heat-stable DNA polymerase and nucleotides
undergoes many cycles of polymerase chain reactions. Each cycle consists of three steps: denaturation, primer
annealing and extension. At the end of each cycle, the number of DNA strands is doubled.
41- 14
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C. DNA fingerprinting
DNA fingerprinting, also called DNA profiling, is a technique for
identifying individuals using their DNA profiles. It was invented by
the English scientist Alec Jeffreys (1950–) in 1984.
1. The principle of DNA fingerprinting

The human genome consists of about three billion base pairs.
Approximately 99.9% of the base sequences in human DNA are
identical among all people. The remaining (0.1%) varies from
person to person.
Remember this
The variations are found in different loci of the non-coding DNA.
Over 95% of the human DNA is Some of these regions consist of repetitive sequences known as
non-coding DNA. Less than 5%
codes for functional proteins.
variable number tandem repeats (VNTRs).
Each VNTR contains a number of short repetitive sequences of

Test yourself nucleotides (Figure 41.14 a ). The number of repeats in a given
Why do greater variations exist in VNTR varies greatly among individuals. Therefore, a difference in
VNTRs but fewer variations exist in
the number of repeats results in VNTRs of different lengths in
the coding regions of DNA?
(Refer to p.A1 for answers.) Answer different individuals (Figure 41.14 b ). This phenomenon is called
length polymorphism.
a In individual 1, there are 24 repeats in an allele of a b In individual 2, there are 14 repeats in an allele of the
VNTR. same VNTR.
Individual 1 Individual 2
VNTR VNTR
chromosome chromosome
24 repeats 14 repeats
GGCGGTGCTGGTGTGGG GGCGGTGCTGGTGTGGG
Figure 41.14 The number of repeats in a given VNTR varies greatly among individuals.
Furthermore, each individual has two alleles of a given VNTR—one

from each parent (Figure 41.15). The chance of two individuals
having the same set of VNTRs is very low (except for identical
twins). DNA fingerprinting makes use of the uniqueness of VNTRs
to identify individuals.
maternal maternal
paternal paternal
Figure 41.15 Each individual has two alleles of a given VNTR, one from each parent.
DNA fingerprinting DNA 指紋分析 length polymorphism 長度多態性

variable number tandem repeat 可變數目銜接重複 41- 15
A VNTR is sandwiched by non-repetitive sequences (the purple

region in Figure 41.16), allowing the VNTR segment to be extracted
with restriction enzymes. The resulting DNA fragments vary in
length (according to the number of repeats in the VNTR) and can be
Link it
separated by gel electrophoresis. This method of generating a DNA
Refer to Chapter 28 for the fingerprint is called restriction fragment length polymorphism
principle of gel electrophoresis.
analysis (RFLP analysis).
alleles of
a VNTR
fragments of different lengths to be

separated by gel electrophoresis
(-) cathode
Band corresponding to the

segment of 24 repeats
Band corresponding to the
segment of 20 repeats
Band corresponding to the Band corresponding to the
segment of 16 repeats segment of 14 repeats
(+) anode
Figure 41.16 VNTR segments of different lengths can be separated with gel electrophoresis.
RFLP analysis is one of the earliest methods of DNA fingerprinting.

The method is time-consuming, requiring several weeks to
complete. It also requires relatively large amounts of DNA. The
procedure of RFLP analysis is outlined in Figure 41.17 (continued on
the next page).
 DNA extraction  Restriction fragment preparation  Gel electrophoresis

DNA is extracted from samples Restriction enzymes are used to cut the DNA fragments are then
(e.g. hair, blood, saliva or semen) DNA at specific sites, producing a large separated by gel electrophoresis
from different individuals. number of DNA fragments. Some of according to their molecular size.
these fragments contain VNTRs.
gel
DNA extracted DNA cut by restriction
samples from
enzymes
different individuals
movement of DNA fragments
Figure 41.17 DNA fingerprinting using RFLP analysis
restriction fragment length polymorphism analysis 限制性片段長度多態性分析

41- 16
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Taking it further
Short tandem repeats (STR) analysis
Nowadays, DNA fingerprinting is mostly carried out using STR analysis. STR, which stands for short tandem repeat,
is a kind of repetitive sequences of nucleotides. It is similar to VNTR in that it is located in the non-coding DNA with
the number of repeats varying among individuals. However each repetitive sequence is shorter in length. Each
repeat in VNTR may consist of 10 to 100 base pairs, while each repeat in STR may consist of 1 to 6 base pairs. In the
example below, there are five repeats of the short DNA sequence TCAT.
- - - - C C C T C AT T C AT T C AT T C AT T C AT T C A - - - -
Compared with RFLP analysis, STR analysis is a faster method. It is often coupled with PCR and requires only small
samples of DNA. The procedure of STR analysis is:
1. DNA is extracted from the sample.
2. The STR loci are targeted with specific primers labelled with fluorescent dye and amplified using PCR. Hence, the
PCR products containing STRs are fluorescent when viewed with ultraviolet light. These DNA fragments vary in
molecular size, depending on the number of repeats of the STRs.
3. The fluorescently labelled DNA fragments are then loaded into a machine that can separate and detect the
fragments according to their molecular size. The result is recorded as a DNA profile.
 A DNA profile generated with STR analysis; the X-axis represents the molecular size of the DNA fragments,
the Y-axis represents the intensity of the signal.
There are many STR loci in our genome. It is time consuming and a waste of resources if all the loci are analysed.
Forensic scientists commonly choose a few loci for analysis. For example, the Federal Bureau of Investigation (FBI) of
the United States uses 20 STR loci for criminal identification.
 Denaturation of DNA fragments  Incubation of the membrane with  Production of DNA fingerprints
and transfer to a membrane radioactive DNA probes Unbound DNA probes are washed
The gel is immersed in an The nylon membrane is incubated with off. An X-ray film is placed
alkaline solution so that the radioactive DNA probes (single- against the nylon membrane in
DNA fragments are denatured stranded DNA fragments with base the dark. The radioactive probes
and become single-stranded. The sequences complementary to VNTRs at on the DNA fragments expose
fragments are then transferred several loci). The DNA probes bind to the film, giving a pattern of dark
from the gel to a nylon the DNA fragments containing the bands. The pattern makes up the
membrane. VNTRs being examined. DNA fingerprint.
nylon membrane radioactive

X-ray film
DNA probes
gel with
DNA
nylon membrane with bound DNA fingerprint
fragments
radioactive DNA probes
Figure 41.17 DNA fingerprinting using RFLP analysis (Cont'd)
STR analysis 短銜接重複分析

short tandem repeat 短銜接重複 41- 17
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2. Applications of DNA fingerprinting

a. Criminal investigation
Remember this
Criminals may leave hairs, saliva, blood or semen in the crime
DNA samples found in a crime scene. Police collects DNA samples from these sources to create
scene may be in very small
amounts. PCR can be used to
DNA fingerprints for matching with those of victims and the suspects.
amplify the DNA samples before
analysis. If the band pattern of the crime scene sample and that of a suspect
is identical, this suggests that the crime scene DNA comes from the
suspect.
If the DNA fingerprint of a suspect does not match that of the crime
scene sample, this can also serve as evidence to suggest that the
suspect may be innocent.
hair root from blood on the suspect’s victim’s

the crime scene suspect’s shoe blood blood
Test yourself
To whom do the hair obtained
from the crime scene and the
blood on the shoe belong?
(Refer to p.A1 for answers.) Answer
Figure 41.18 DNA fingerprints from DNA samples found at a crime scene,
from a victim and from a suspect
Practical 41.1 Go to Appendix.
Crime scene investigation using DNA fingerprinting

In this practical, you will use the polymerase chain reaction (PCR) and gel
electrophoresis to create DNA fingerprints to help solve a crime.
41- 18
All answers TE
b. 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.
Each child inherits half of his/her genetic material from each parent,
so some bands on the DNA fingerprint of the child resemble some
bands of the mother and others resemble some bands of the father.
Skill-building Handling data
Activity 41.1 Using DNA fingerprints in a parentage test
The following shows the DNA fingerprints of a boy, his mother and two individuals who each claims to
be his biological father.
mother boy individual 1 individual 2
 DNA fingerprints obtained for a parentage test
1. Study the DNA fingerprints, and circle the DNA bands shared by the boy and his mother. (Use a
ruler to help you select the matching bands)
2. Compare the remaining DNA bands of the boy and the DNA bands of individuals 1 and 2. Deduce
which person is likely to be the biological father of the boy.
Test yourself
The DNA fingerprints of a pair of identical twins show the same
For many years, tests for blood
groups were done to prove band pattern. Therefore, DNA fingerprinting can also be used to
parentage. Suggest why matching determine whether new born twins are identical or fraternal.
blood groups is not as reliable as
DNA fingerprinting in determining
parentage.
41- 19
c. Victim identification
DNA fingerprinting provides a fast and accurate way for identifying
the bodies of victims, especially in disasters such as earthquakes.
This is particularly useful if the body is badly decomposed or if only
parts of the body are found.
Surf the net To determine the identity of an unclaimed body, its DNA fingerprint
Visit the following website to learn is compared with those of missing people. DNA samples recovered
more about the use of DNA from the personal items (e.g. toothbrushes) of the missing people
fingerprinting in forensic science:
can be used to produce DNA fingerprints. If these are not available,
e-aristo.hk/r/
DNA samples from close relatives of the missing people are used
bioccstn4101.e
instead.
d. Medical diagnosis of diseases

DNA fingerprinting is used in rapid tests for infectious diseases. In
such a test, the DNA fingerprint of the organism found in a patient
is compared with that of known pathogens.
DNA fingerprinting can also be used to check whether you carry a

mutant allele for a genetic disease. Considering a genetic disease
caused by a recessive mutant allele, the DNA fingerprints of a
homozygous normal person, a homozygous affected person and a
heterozygous carrier may look like those shown in Figure 41.19.
DNA homozygous homozygous heterozygous

ladder normal person affected person carrier
500 bp
400 bp
Test yourself
300 bp
Why do the DNA fingerprints of
homozygotes show only one 200 bp
band?
100 bp
(Remarks: bp = base pair)

Figure 41.19 DNA fingerprints of a homozygous normal person,
homozygous affected person and a heterozygous carrier for a certain
genetic disease
This kind of genetic tests is now used to diagnose genetic diseases

in foetuses and newborn babies. These diseases include cystic
fibrosis, haemophilia, Huntington’s disease, sickle-cell anaemia,
thalassaemia and many others.
41- 20
e. Authentication of food products and herbal medicines

Authentication of the biological ingredients used to make food
products and herbal medicines is important for ensuring food safety.
Traditionally this has been done by morphological comparison, but

now more accurate methods such as DNA fingerprinting are used.
This is done by comparing the DNA fingerprints of an uncooked
food sample with that of the species that it claims to contain. DNA
fingerprinting can also distinguish genuine herbal medicines from
substitutes.
Figure 41.20 Caviar samples can be tested with DNA Figure 41.21 Korean ginseng (Panax ginseng) and
fingerprinting to determine whether the fish eggs American ginseng (Panax quinquefolius) are widely
originate from real Sturgeon (Acipenseridae) species used medicinal plants with similar morphology but
or whether they have been replaced by less valuable different medicinal effects. It is even more difficult to
eggs. differentiate them when they are sold in powdered or
sliced forms.
Many GM foods contain foreign genes that are absent in their non-
GM counterparts. If the presence of foreign genes is detected in a
food sample, it is likely that the sample contains GM materials.
151
101
GM maize
151
Non-GM maize
Time
Figure 41.22 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.
41- 21
f. Ecological studies and conservation

When an unknown species is found, scientists can compare its DNA
fingerprint with those of the known species to identify it. If no DNA
fingerprint matches, the species is probably newly discovered.
Greater genetic variation enables a population to adapt more rapidly

to the changing environment. Species showing little genetic
variation, especially endangered species, risk extinction. Scientists
can make use of DNA fingerprinting to monitor the amount of
genetic variation within a population. Greater variation in the DNA
fingerprints within a population shows greater genetic variation.
DNA fingerprinting can also be used to identify whether goods are

made from protected species. This helps fight against the illegal
trade of endangered species and their parts, such as bear’s bile,
tiger skin, whale meat, elephant ivory and rhinoceros horn.
Figure 41.23 Rhinoceros are killed by humans for their horns, which are
traded illegally and are used by some cultures for ornaments or traditional
medicines. With DNA fingerprinting to match the DNA of rhinoceros horns
with the dead rhinoceros bodies, the origin of the horn products can be
identified. This aids in arresting the hunters and traders.
g. Evolutionary studies
DNA fingerprinting helps scientists establish 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 will share in common in their DNA
fingerprints.
41- 22
All answers
STSE connections
Species identification with DNA barcoding
DNA barcoding identifies species with a short DNA base sequence from a standard part of the genome, similar to
the way that a supermarket barcode scanner identifies products using the black stripes of the Universal Product
Code.
The region of the genome that is used as the standard barcode for almost all animals is a region in the cytochrome-c
oxidase I gene (COI). It is highly effective in identifying birds, butterflies, fish and many other animal groups. For
plants, two chloroplast genes, rbcL and matK, have been assigned as the barcode regions. Barcode sequences are
analysed and recorded for each known species and stored in species databases shared across the world. Unknown
specimens can be identified by finding the closest matching sequence in the database.
A blue jay (left), and its barcode sequence* (right)


An illustrative barcode for the blue jay*


* Source: Ratnasingham, S. & Hebert, P. D. N. (2007). BOLD: The Barcode of Life Data System (www.barcodinglife.org). Molecular
Ecology Notes 7, 355-364. DOI: 10.1111/j.1471-8286.2006.01678.x
Key point
1. DNA fingerprinting, also called DNA profiling, is a technique for
identifying individuals using their DNA profiles.
2. One method of DNA fingerprinting is to make use of variable number
tandem repeats (VNTRs) in the non-coding DNA to identify individuals.
3. DNA fingerprinting is used in forensic science, parentage testing, victim
identification, medical diagnosis of diseases, authentication of food
products and herbal medicines, ecological studies and conservation, and
evolutionary studies.
Checkpoint
Which of the following is an application of DNA fingerprinting?
A. to separate DNA fragments
B. to amplify DNA samples
C. to sequence DNA fragments
D. to identify suspects involved in a crime
DNA barcoding DNA條碼

41- 23
D. Genetically modified organisms

Genetically modified organisms (GMOs) are organisms whose
genetic compositions have been changed using genetic engineering
techniques. For example, the bacterium that has been genetically
modified to produce human insulin is a GMO. Biologists have also
created genetically modified animals and genetically modified
plants.
1. How can microorganisms be genetically

modified?
You have learned how E. coli can be genetically modified to produce
human insulin using recombinant DNA technology. The gene coding
for human insulin is transferred into the bacteria using bacterial
plasmids as vectors. Apart from plasmids, bacteriophages (viruses
that infect bacteria) can also be used as vectors to transfer a gene
of interest into bacteria.
Bacteriophages attach to the surface of bacteria and inject their

genetic materials into the bacterial cells. This property makes
bacteriophages useful as vectors.
The gene of interest is first inserted into the DNA of a bacteriophage,

so that it will be injected into the bacterium along with the
bacteriophage DNA. The bacteriophage DNA together with the
gene of interest then integrates into the bacterial chromosome.
 The DNA of a bacteriophage has bacterium

been inserted with a gene of
interest.
 The bacteriophage attaches to a
bacteriophage host cell (a bacterium) and injects
its DNA together with the gene
of interest into the host cell.
bacterial
chromosome
 The viral DNA together with the
head containing DNA gene of interest integrates into
the bacterial chromosome.
tail fibre (for attaching to

specific bacterial cell)
Simplified structure of a bacteriophage

 The transformed bacterium
reproduces. The bacterial
chromosome together with the
inserted gene is replicated and
passed to the daughter cells.
Figure 41.24 The gene of interest
can be inserted into the genome
of a bacterium using a
bacteriophage.
genetically modified animal 基因改造動物

41- 24 genetically modified plant 基因改造植物
bacteriophage 噬菌體
TE
2. How can plants be genetically modified?

To transfer a gene of interest into plants, the soil bacterium
Agrobacterium can be utilized. This bacterium contains a plasmid,
called the Ti (tumour inducing) plasmid, which can carry the gene
of interest into a plant cell so that it can integrate with the plant
DNA. The bacterium infects most dicotyledonous plants, and is
therefore widely used in modifying these plants.
Animation
Ti plasmid
The Ti plasmid
herbicide resistant gene
e-aristo.hk/r/
bioccani4101.e
Agrobacterium
 Extract Ti plasmid and cut it
using a restriction enzyme.
Cut the donor DNA with the
same restriction enzyme to
obtain the gene of interest.
cut plasmid
 Insert the gene of interest gene of interest

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

Test yourself with the transformed Agrobacterium
Agrobacterium.
How does the herbicide resistant
gene work as a selection marker
to indicate successful
transformation of plant cells?
(Refer to p.A1 for answers.) Answer  Transfer the infected plant tissues genetically modified
infected plant to an agar plate containing
plant containing the
tissues containing nutrients for growth and the
gene of interest
the gene of herbicide to which transformed
interest cells are resistant.
Figure 41.25 The procedure of creating genetically modified plants using Agrobacterium
This technique works well with tomatoes, potatoes and many other
plants. Originally it was thought that Agrobacterium could not be
used to modify monocotyledonous plants such as wheat, rice and
maize, because the bacterium does not naturally infect these plants.
However, these plants are important food crops, so scientists
conducted years of research to finally overcome this problem.
Agrobacterium 土壤桿菌
41- 25
Another method of transferring target genes into plant cells is to use

a gene gun. DNA fragments containing the gene of interest are
coated onto the surface of gold or tungsten nano-sized beads. The
beads are then physically forced into the plant cells by a gene gun.
The advantage of using a gene gun over Agrobacterium is that the

gene gun can shoot DNA into any plants with no host limitation.
nano beads coated with DNA

containing the gene of interest
gene gun
shooting out the coated beads

into plant tissues
foreign DNA
transferred into
plant cells
plant cell chromosome integrated

with foreign DNA
Figure 41.26 Plant cells can be genetically modified by shooting the gene
of interests directly into cells with a gene gun.
Sometimes, biotechnologists may wish to introduce multiple new

traits (e.g. insect resistance and herbicide resistance) into a single
plant. This can be done by genetic engineering or cross-breeding of
GM plants. Two GM plants, each expressing a different desired trait,
can be cross-pollinated in the traditional way to produce offspring
that express both traits of the parents.
gene gun 基因槍

41- 26
TE
3. How can animals be genetically modified?

A number of methods have been developed to create genetically
modified animals. If all the cells of a transgenic animal are to contain
the gene of interest, it has to be introduced into the zygote, the very
first cell of the animal. This is usually done by a method called
microinjection. The procedure of creating a genetically modified
sheep by microinjection is outlined in Figure 41.27.
female sheep
egg cell from the

 Egg cells are removed from a female sheep
female sheep. These are fertilized
by sperms in vitro.
sperm
 Foreign DNA containing the gene fertilized egg

of interest is injected into the
fertilized eggs using a very fine
needle-like pipette viewed with a DNA containing
microscope. the gene of
interest
 The fertilized eggs containing the

gene of interest are allowed to embryo
grow and develop into embryos.
 The embryos are then implanted

into the uterus of another female surrogate mother
sheep (the surrogate mother).
 The surrogate mother gives birth to

offspring containing the gene of genetically modified sheep
interest.
Figure 41.27 The procedure of creating genetically modified sheep by

microinjection
microinjection 微注射
41- 27
Animal cells can also be genetically modified by transferring target

genes into them using viruses or liposomes as vectors.
To use viruses as vectors, the genome of the virus is first modified

to remove its disease-causing genes and to insert the gene of
interest. When the modified viruses infect animal cells, the gene of
interest will be transferred into the cells and may integrate into the
host cell’s genome (Figure 41.28 a ).
Liposomes are small artificially prepared vesicles made of a lipid

bilayer. The gene to be delivered can be enclosed in these vesicles.
When a liposome fuses with the cell membrane, it transfers the gene
into the cell (Figure 41.28 b ).
There is a small chance that viral vectors may regain the ability to
cause diseases. Liposomes are non-pathogenic. Therefore,
liposomes are often regarded as a safer vector than viruses but they
are less effective.
a b
liposome
Insert the genes of

interest into the DNA fragment
genome of viruses. containing the
target gene
target cell
viruses with the genes liposome fuses

of interest with the cell
Viral vectors
introduce the
genes into cells.
Genes are transferred into the

target cells.
Figure 41.28 Genetic modification of animal cells with a viruses and

b liposomes
liposome 脂質體
41- 28
All answers TE
Extras: Do you know...

Fluorescent protein as a visual selection marker
When creating GMOs, scientists often include a gene that codes for 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
organisms have been successfully modified.
A similar technique has been applied to create genetically modified pets. In
2003, genetically modified zebrafish called ‘Glofish’ were introduced to the
market. They glow in the dark with bright red, green, orange-yellow, blue and
purple fluorescent colours.
An ordinary zebrafish
  Genetically modified zebrafish
that display fluorescent colours
Key point
1. Genetically modified organisms (GMOs) are organisms whose genetic
compositions have been changed using genetic engineering techniques.
2. Scientists use different types of vectors to create genetically modified
bacteria, genetically modified plants and genetically modified animals.
Vectors Applications
Plasmids, bacteriophages Transfer the target gene into bacteria
The soil bacterium Infects plant cells and transfers the target
Agrobacterium gene into plant cells with Ti plasmids
Gene gun Shoots the target genes coated on nano-
size beads into plant cells
Microinjection Injects the target gene into the fertilized
egg of an animal
Viruses Infect animal cells and transfer the target
gene into the cells
Liposomes Fuse with animal cells to bring the target
gene inside
Checkpoint
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
41- 29
TE
E. Benefits and potential risks of genetic

engineering
Genetic engineering offers many benefits, which include:
providing scientists with new means to study organisms at the

molecular level, e.g. a particular gene can be knocked out to
study its effect on organisms.
Link it
speeding up research in medical science and bringing advances
You will learn more about gene in medicine, e.g. new treatments could be developed with
therapy in Chapter 42.
genetic engineering techniques to correct diseases caused by
gene defects.
Link it
developing new possibilities for solving food shortages and
You will learn more about the use providing ways to improve food quality, e.g. crop plants can be
of transgenic animals and plants in
the food industry and agriculture
genetically engineered to become drought resistant so that they
in Chapter 42. can grow well in dry environments.
enabling scientists to develop new tools for cleaning up the

contaminated environment or degrading organic waste, e.g.
bacteria can be genetically engineered to break down
hydrocarbons in oil spill.
However, genetic engineering brings some potential risks:
Antibiotic resistance genes are often used as selection markers

in genetic engineering. If these genes were transferred to
pathogens, ‘superbugs’ that are resistant to many types of
antibiotics could result.
GMOs have foreign genes deliberately transferred into them.

Some people may have allergic reactions to the gene products
after having food or drugs made from GMOs.
GM food and recombinant drugs have a relatively short history,

and their long-term effects on human health are unknown.
Link it
GMOs, if they leak outside laboratories or test fields, may breed
You will learn more about the with wild types and transfer their modified genes to their
ethical, legal, social, economic and
environmental issues concerning
offspring. This may cause unknown or adverse effects on the
biotechnology in Chapter 43. non-GM species. GMOs may also out-compete the wild types.
This may disrupt the ecosystem and lead to a loss of biodiversity.
41- 30
TE
Learning objective 41.3 Animal and plant cloning

• Outline the major steps of
plant tissue culture
Cloning of organisms is the production of genetically identical
• Outline the major steps of
individuals. Some organisms such as bacteria, Amoeba and plants
cloning of mammals
• Be aware of the advantages, can naturally produce clones of themselves through asexual
disadvantages, applications and reproduction. However, asexual reproduction is less common in
limitations of cloning in animals animals. With advances in biotechnology, scientists have
and plants
successfully cloned many species of plants and animals.
A. Plant cloning by tissue culture

Link it Traditionally, plant cloning has been practised through artificial
propagation methods, such as making cuttings. Nowadays, methods
Meristematic tissues from shoot
involving tissue culture (also known as micropropagation) are
tips are preferable for tissue culture.
These consist of undifferentiated commonly used. In tissue culture, tissue samples are taken from a
cells undergoing active cell parent plant and cultured in a sterile culture medium containing
division. For revision on
essential nutrients and plant hormones. The cultured tissues will
meristems, refer to Chapter 15.
eventually develop into whole plants.
 Tissue samples are taken from a surface sterilized  Each tissue sample is placed on a sterile culture
parent plant. These samples are called explants. medium containing essential nutrients and plant
hormones. A mass of undifferentiated cells called a
callus is formed by mitotic cell division.
callus
 Small pieces of the callus are transferred to different  The plantlets formed can be planted into soil. Each
sterile culture media, which contain plant hormones will grow into a whole plant that is genetically
that promote the growth of shoots and roots. identical to the parent plant.
Figure 41.29 The main steps involved in plant tissue culture
tissue culture 組織培養 explant 外植體

micropropagation 微繁殖 callus 癒傷組織 41- 31
TE
Tissue culture provides a faster method to propagate plants than

conventional methods. It 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.
B. Animal cloning
Since the late 1970s, scientists have been trying to clone animals
Remember this
by embryo splitting. This involves isolating individual cells from an
Embryo splitting actually occurs embryo at an early stage and growing them independently. Each of
naturally when a developing
embryo splits into two masses of
the isolated cells develops into an embryo. The embryos are then
cells and continues to develop into transferred into the uteri of surrogate mothers for development.
a pair of identical twins.
embryo at an early stage
individual cells
isolated from
the embryo
embryos grown
from individual
cells in sterile
culture
surrogate
mothers
clones
Figure 41.30 Animal cloning by embryo splitting
In 1996, a sheep named Dolly was cloned by British scientist Ian

Remember this
Wilmut (1944– ) and his team using another method called somatic
Somatic cells generally refer to the cell nuclear transfer. Dolly was the first mammal cloned from an
cells forming the body of an
adult somatic cell. The main steps involved in cloning Dolly are
organism, excluding gametes and
gamete-producing cells. illustrated in Figure 41.31 on the next page.
embryo splitting 胚分割 surrogate mother 代母

41- 32 somatic cell nuclear transfer 體細胞核移植

A mammary gland cell was obtained from an adult sheep
(nucleus donor).

An egg cell was obtained from another sheep (egg donor). The
nucleus of the egg cell was removed.

The mammary gland cell and the egg cell were 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.
Simulation 
The embryo was transferred into the uterus of a surrogate
Animal cloning mother for development.
e-aristo.hk/r/
bioccsim4102.e

The surrogate mother gave birth to Dolly.
A mammary gland cell was

collected from sheep A.

The nucleus of the mammary A very small electric
gland cell fused with the shock was applied to
enucleated egg cell. stimulate cell division.
sheep A 
(nucleus donor) 
An egg cell was collected
from sheep B, whose
nucleus was then removed. fused cell embryo
 
sheep B
(egg donor)
sheep C
(surrogate mother)

Test yourself
To which sheep is Dolly genetically
identical?
Dolly
Figure 41.31 Cloning of Dolly
41- 33
TE
By comparing the genetic make-up of Dolly and the adult sheep

from which the mammary gland cell was obtained, it was confirmed
that Dolly was a clone. Since Dolly the sheep, other mammals such
as cows, cats, dogs, horses and monkeys have been cloned using
similar methods.
Clear your concepts

A clone looks the same as the nucleus donor.
Figure 41.32 Chinese
researchers announced in 2018 A clone does not necessarily look the same as the nucleus donor. Although
that a pair of cloned monkeys the genetic make-up of the clone is identical to that of the nucleus donor,
was born. They were cloned some genes may be expressed differently in the clone. Moreover, the
using somatic cell nuclear expression of a phenotype is affected by environmental factors.
transfer.
Cloning has many potential applications, but also has a number of

limitations.
Advantages and applications
Cloning in general:
• Clones are genetically identical to the parent, so desirable characteristics of the parent can be preserved.
• Organisms that are difficult or slow to breed normally can be reproduced quickly by cloning.
• Endangered species (e.g. some orchids) and organisms of considerable economic importance (e.g. a high
milk-yielding dairy cow) can be propagated quickly. It may be possible to clone extinct animals using
preserved DNA from fossils and ova from closely related species.
• GM plants or animals, with target genes inserted, can be cloned to serve as ‘biological factories’ to
produce pharmaceutical products or other useful chemicals.
Plant cloning: Animal cloning:

• Cloned plants that are free of pathogens (e.g. • Cloned animals can serve as models for studying
bacteria or fungi) can be produced as they are diseases and testing drugs.
grown under sterile conditions.
• Cloning early embryos could provide stem cells
• Plant cloning requires relatively little space for use in research or medical treatment.
compared with growing plants in the field.
Disadvantages and limitations
Plant cloning: Animal cloning:

• The cost of tissue culture may be higher than • The success rate of animal cloning using nuclear
conventional methods as it requires sterile transfer is still low and the cost is high.
conditions and is labour-intensive.
• Some cloned animals age faster and have a
• Due to a lack of genetic variations, the clone shorter lifespan, although this is not the case for
population may be less able to adapt to changes all cloned animals and the reasons are not yet
in the environment. clear.
• Not all plants can be successfully propagated as
the suitable culture media for these plants are not
yet known.
Table 41.1 Applications and limitations of cloning
41- 34
Extras: Do you know...

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 humanely put to death in 2003, at the age of six.
The normal lifespan of a sheep like Dolly is usually about 12 years.
Some people think that Dolly’s poor health was a result of being kept indoors
and overfeeding from visitors. 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.
STSE connections
Human stem cells created by somatic cell nuclear transfer
Stem cells are unspecialized cells that have the ability to differentiate into
various kinds of cells in the body. They are found in early embryos. In 2013,
Shoukhrat Mitalipov (1961‒), an American scientist, for the first time,
successfully used somatic cell nuclear transfer to clone human cells and
derived stem cells from them.
In Mitalipov's study, a skin cell from a human donor was fused with an
unfertilized egg whose nucleus had been removed. The resultant cell,
containing the donor nucleus, divided and developed into an undifferentiated
mass of cells, i.e. stem cells which were then harvested and cultured.
The stem cells obtained in this way are genetically identical to the donor.
They are therefore patient-specific. These stem cells have great potential in
medical treatment, especially for organ transplant. Organs grown from
patient-specific stem cells are genetically identical to the organ recipient, so
rejection is much less likely.
Key point
1. Cloning of organisms is the production of genetically identical individuals.
2. Plant cloning can be achieved by tissue culture. Meristematic tissues are
collected from a parent plant and grown in sterile culture medium
containing nutrients and plant hormones. The tissues will grow into whole
plants.
3. Animals can be cloned by embryo splitting or nuclear transfer. In nuclear
transfer, the nucleus of an egg cell is replaced by a nucleus from a somatic
cell of a donor. The fused cell will develop into an embryo which is then
implanted into the uterus of a surrogate mother for development.
41- 35
Worked example 41.1
The African clawed frog (Xenopus laevis) is brown in colour. There is also a mutant form that is albino.
In a cloning investigation, three adult frogs (X, Y and Z) were used to create clones. Frog X is brown,
while frogs Y and Z are albino.
Frog X Frog Y Frog Z
donates an egg donates an egg donates sperms
fertilization
fertilized egg
develops into a ball of cells

called a blastocyst
egg cell with nucleus extracted from an

nucleus removed isolated cell of the blastocyst
fused cell divides and develops into an

embryo which then grows into a tadpole
DNA samples were collected from the three adult frogs and the tadpole for analysis by DNA fingerprinting.
The DNA fingerprints obtained are as follows:
Pattern 1 Pattern 2 Frog Y Frog Z
cont'd
41- 36
All answers TE
(a) Deduce, with reasons, which DNA fingerprint belong to

(i) Frog X and (3 marks)
(ii) the tadpole. (3 marks)
(b) Describe one advantage of cloning over sexual reproduction. (2 marks)
Solution
(a) (i) ‘Pattern 2’ belongs to Frog X. .............................................................................................. (1)
Frog X is genetically unrelated to the others. ....................................................................... (1)
The majority of bands in ‘Pattern 2’ do not match with those of the other frogs. .................. (1)
(ii) ‘Pattern 1’ belongs to the tadpole. ....................................................................................... (1)
Some bands in ‘Pattern 1’ match with a few bands in the DNA fingerprint of frog Y (a parent of
the tadpole). ....................................................................................................................... (1)
Some bands in ‘Pattern 1’ that do not match with the DNA fingerprint of frog Y match with those
of frog Z. ............................................................................................................................. (1)
(b) The clones are genetically identical to the parent, ..................................................................... (1)
so any desirable characteristics of the parent will be preserved in the clones. ............................ (1)
Checkpoint
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)
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 sterile 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)
41- 37
All answers
Article reading
Hazard-indicating organisms
Humans have for many years observed animals for signs of forthcoming hazards. Some animals
are more susceptible to particular hazards than humans in the same environment, so they can
be used to detect risks for humans by providing advance warning of a danger.
For example, canaries have been used in coal mines to detect the presence of poisonous carbon
monoxide. The bird's rapid breathing rate, small size and high metabolism, compared to the
miners, cause the canaries to die in the presence of carbon monoxide before the miners, thereby
giving people time to take action.
Researchers are studying the use of genetically modified organisms as hazard-indicating
organisms. A type of transgenic fish has been developed to detect harmful chemicals such as
endocrine disruptors. When these fish are exposed to the harmful chemicals, they synthesize the
green fluorescent proteins which can be observed as green fluorescent light. This type of
transgenic fish can be used to indicate whether food and skincare products contain the harmful
chemicals.
▲ These transgenic medaka emits green fluorescent

light when exposed to endocrine disruptors.
Questions
1. Briefly outline the steps of creating genetically modified fish that carry a gene coding for
fluorescent protein from jellyfish, using microinjection. (4 marks)
2. Explain how DNA fingerprinting can be used to identify which fish have been genetically
modified to carry a foreign gene. (2 marks)
3. Give one advantage of using genetically modified organisms as hazard-indicating organisms

instead of non-GM organisms. (1 mark)
canary 金絲雀
41- 38 endocrine disruptor 內分泌干擾物
medaka 青鱂魚
e-dictionary
Key terms e-aristo.hk/r/
bioccedict.e
Agrobacterium 土壤桿菌 p.25 polymerase chain reaction 聚合酶鏈反應 p.10
bacteriophage 噬菌體 p.24 primer 引物 p.11
biotechnology 生物工程 p.3 recombinant DNA technology 重組 DNA技術 p.5
cloning 克隆 p.4, 31 restriction enzyme 限制酶 p.6
DNA fingerprinting DNA指紋分析 p.15 somatic cell nuclear transfer 體細胞核移植 p.32
DNA ligase DNA連接酶 p.6 tissue culture 組織培養 p.31
DNA polymerase DNA聚合酶 p.11 transformation 轉化 p.8
embryo splitting 胚分割 p.32 transgenic organism 轉基因生物 p.4
gene gun 基因槍 p.26 variable number tandem repeat 可變數目銜接 p.15
重複
genetic engineering 遺傳工程 p.4
vector 載體 p.5
genetically modified organism 基因改造生物 p.4, 24
microinjection 微注射 p.27
Summary
41.1 Introduction to modern biotechnology

　
1. Biotechnology refers to the use of biological processes, biological systems or organisms to produce goods
or provide services to humans. Modern biotechnology is about the manipulation of DNA, genes, cells and
tissues to improve human lives. Two rapidly expanding areas of modern biotechnology are genetic
engineering and cloning.
41.2 Genetic engineering

　
A. Recombinant DNA technology
2. Recombinant DNA technology is a set of techniques for joining DNA molecules from different sources to
create recombinant DNA. This technology enables scientists to transfer a gene of interest (target gene)
from one organism (donor) to another organism (host) of the same or a different species.
3. A vector is a DNA molecule that acts as a carrier to transfer the gene of interest into a host cell. Bacterial
plasmids and viruses are commonly used vectors.
4. Plasmids possess the following characteristics that make them useful vectors:
• Plasmids are naturally transferred between bacterial cells.

• Plasmids replicate independently of the bacterial chromosome.
• Plasmids contain genes (e.g. antibiotic resistance genes) that can serve as a marker for identifying the
host cells that contain the inserted gene of interest.
5. Restriction enzymes recognize restriction sites on DNA and cut at those sites. By cutting the DNA
fragments containing the gene of interest and the plasmids with the same restriction enzyme, the sticky
ends on the cut DNA fragment and cut plasmids are complementary.
6. The cut DNA fragments containing the gene of interest and the cut plasmid can be joined by DNA ligase.
The resulting DNA molecule is called a recombinant plasmid.
41- 39
7. The basic steps of modifying the genetic material of bacteria with recombinant DNA technology to
synthesize human insulin:
bacterium
bacterial
plasmid
chromosome
 Obtain the DNA fragment

that contains the human  Isolate plasmids from
insulin gene through bacterial cells.
proper processes.
antibiotic
resistance
gene plasmid
 Cut the DNA fragment sticky ends

and the plasmid using
the same restriction
sticky ends enzyme.
cut plasmid
 Insert the DNA fragment into

the cut plasmid and join them
using DNA ligase.
recombinant plasmid
 Introduce the recombinant
plasmid into a bacterium (e.g.
E. coli).
transformed bacterium
(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.
B. Polymerase chain reaction
8. The polymerase chain reaction (PCR) is a technique for replicating specific DNA sequences outside cells.
9. A PCR mixture containing a DNA template, primers, heat-stable DNA polymerase and nucleotides
undergoes many cycles of polymerase chain reaction. Each cycle consists of three steps:
 Denaturation  Primer annealing  Extension

Heat the reaction mixture Cool the reaction mixture Raise the temperature for
to cause DNA to unwind to allow primers to anneal DNA polymerase to
and separate into two to the single-stranded catalyse the synthesis of
single strands. DNA. new DNA strands.

10. At the end of each cycle of PCR, the number of DNA strands is doubled.
11. PCR can amplify DNA for DNA fingerprinting, detection of genetic disorders and infectious diseases,
archaeological studies, and scientific research.
41- 40
C. DNA fingerprinting
12. DNA fingerprinting, also called DNA profiling, is a technique for identifying individuals using their DNA
profiles.
13. One method of DNA fingerprinting makes use of polymorphism in variable number tandem repeats
(VNTRs) to identify people. VNTRs can be extracted with restriction enzymes. The resulting DNA fragments
vary in length according to the number of repeats in the VNTRs and can be separated by gel
electrophoresis. This method of generating a DNA fingerprint is called restriction fragment length
polymorphism analysis (RFLP analysis):
 Extract DNA from a source.

 Cut the DNA with restriction enzymes into DNA fragments.
 Perform gel electrophoresis to separate the DNA fragments according to their molecular size.
 Separate the double-stranded DNA into single strands (denaturation) and transfer the DNA fragments
to a nylon membrane.
 Incubate the nylon membrane with radioactive DNA probes.
 Place an X-ray film against the nylon membrane in the dark and develop the film to obtain the DNA
fingerprints.
14. DNA fingerprinting is used in criminal investigation, parentage testing, victim identification, medical
diagnosis of diseases, authentication of food products and herbal medicine, ecological studies,
conservation and evolutionary studies.
D. Genetically modified organisms
15. Genetically modified organisms (GMOs) are organisms whose genetic compositions have been altered
using genetic engineering techniques.
16. Plasmids and bacteriophages can be used as vectors to transfer a target gene into bacteria to create
genetically modified bacteria.
17. Genetically modified plants (transgenic plants) can be created by transferring a target gene into the plant
using the soil bacterium Agrobacterium or a gene gun.
18. Genetically modified animals (transgenic animals) can be created by introducing a target gene into a
fertilized egg by microinjection.
19. Genetic engineering offers many benefits, yet it also causes potential hazards to our health and the
environment:
Benefits Potential hazards
• Providing scientists with new tools to • The use of antibiotic resistance genes as selection
study organisms at the molecular level markers raises concerns in creating multi-antibiotic-
resistant bacteria (‘superbugs’).
• Speeding up research in medical
science and bringing advances in • GM foods and recombinant drugs may cause allergic
medicine reactions and their long-term effects on our health are
still unknown.
• Opening up new possibilities for solving
food shortages and providing ways to • GMOs may breed with wild types and transfer their
improve food quality modified genes to the offspring. GMOs may out-
compete wild types and result in a loss of biodiversity.
41- 41
41.3 Animal and plant cloning

　
20. Cloning of organisms is the production of genetically identical individuals.
21. Plant cloning can be achieved by tissue culture, which involves the following steps:
 Tissue samples are taken from a surface sterilized parent plant.

 The sample is placed in a sterile culture medium to allow the formation of callus.
 Small pieces of the callus are transferred to different culture media to promote the growth of shoots
and roots.
 The plantlets formed are planted into soil.
22. Plant cloning has many potential applications, yet there are some limitations:
Potential applications Limitations
• To propagate plants that are endangered (e.g. • The cost is higher than artificial propagation
orchids), difficult to grow by conventional methods as tissue culture requires sterile
propagation, or plants that have high economic conditions and is labour-intensive.
values.
• A lack of genetic variations in the clone
• To produce plants which are free from disease population may reduce its ability to adapt to
by growing them under sterile conditions. changes in the environment.
• To mass produce GM plants from cells with
target genes inserted.
23. Animals can be cloned by embryo splitting or nuclear transfer. Animal cloning by nuclear transfer involves
the following steps:
 A somatic cell is obtained from an animal.

 An egg cell is obtained from another animal and its nucleus is then removed.
 The somatic cell is fused with the egg cell lacking its nucleus. The fused cell develops into an embryo.
 The embryo is transferred into the uterus of a surrogate mother.
 The surrogate mother gives birth to the cloned animal.
24. Animal cloning has many potential applications, but there are some limitations:
Applications Limitations
• It is used to propagate animals of considerable • The success rate of animal cloning using
economic importance and may be used to save nuclear transfer is low and the cost is high.
endangered animals.
• Animal clones may age faster and have a
• Cloned animals are used as models for studying shorter lifespan.
diseases and testing drugs.
• Cloned GM animals 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.
41- 42
All answers
Concept map
Complete the following concept map to review the key points of this chapter.
Modern biotechnology
examples include
genetic engineering cloning
examples of techniques includes
recombinant DNA animal

technology cloning
(PCR)
enables the
production of enables is done by is done by
amplification of specific tissue

DNA sequences culture embryo
(GMOs) splitting
application
synthesis of useful
products is done by
transfer
example restriction fragment
length polymorphism
(RFLP) analysis
for treating diabetes
applications
criminal parentage victim diagnosis of authentication of

investigation testing identification diseases food products and
herbal medicine
ecological studies
and conservation
evolution studies
41- 43
All answers
Time allowed: 40 minutes

Self quiz Total score: 30 marks
Level 1: Understanding basic concepts (8 marks)
1. For each tools of genetic engineering listed in column 1, select the corresponding function listed in column 2. Put the
appropriate letter in the space provided. (4 marks)
Column 1 Column 2
(a) Vector A. Catalyses the joining of complementary DNA fragments
(b) Restriction enzyme B. Cuts double-stranded DNA at specific sites
(c) DNA ligase C. Acts as a carrier to transfer a target gene into a host cell for
(d) DNA polymerase replication and expression
D. Catalyses the synthesis of a new DNA strand from a template
2. Complete the following sentences with suitable words. (4 marks)
(a) DNA molecules contain DNA from different sources.

(b) Two vectors commonly used in genetic engineering to carry genes into bacteria are
and bacteriophages.
(c) refer to sections of DNA fragments with unpaired bases created by the action of
restriction enzymes.
(d) When bacteria are mixed with recombinant plasmids, some of them will take up the recombinant plasmids.
This process is called .
Level 2: Applying concepts (19 marks)
3. The polymerase chain reaction (PCR) is used in laboratories to replicate DNA. The diagram below summarizes the
main stages of PCR.
DNA molecule
➊ heat to 95 °C
➌ heat to 72 °C, add

free nucleotides and
DNA polymerase
➋ add molecules X
molecule Y
and Y, and slowly
cool to 55 °C
molecule X
(a) Explain why DNA is heated to 95 °C in step 1. (1 mark)Answer

(b) (i) X and Y are short lengths of single-stranded DNA. What general name is given to these molecules?
(1 mark)Answer
(ii) Explain why the reaction mixture is cooled from 95 °C to 55 °C in step 2. (1 mark)Answer
41- 44
(c) Suggest why DNA polymerase from human sources is not suitable for use in PCR. (2 marks)Answer
(d) How many DNA molecules will have been produced from one molecule of DNA after 5 complete cycles of
PCR? (1 mark)Answer
4. One application of DNA fingerprinting is to identify paternity. A puppy mother dog P dog Q
dog breeder wants to find out whether a litter of puppies were
fathered by an award-winning show-dog P or another dog Q.
DNA samples of a puppy, the mother, dog P and dog Q were
collected and analysed. The diagram on the right shows the
DNA fingerprints of these dogs.
(a) Based on the information, deduce which dog is more likely

to have been the father of the puppy. Answer (3 marks)
(b) Will all the puppies in the same litter show an identical DNA
fingerprint pattern? Explain your answer. Answer (2 marks)
5. Laboratory rats are physiologically similar to humans, so they are useful for medical research into human diseases.
The first cloned lab rat was made in 2003 by nuclear transfer as illustrated in the flow chart below.
An unfertilized egg cell (cell X) was

collected from rat A.
A somatic cell (cell Y) was taken

The nucleus of cell X was removed. from rat B.
Cell Y fused with the denucleated cell X.
The fused cell subjected to electric shock and it

began to divide.
An embryo formed and it was transferred into the

uterus of a surrogate mother for development.
The surrogate mother gave birth to rat C.
(a) State whether cells X and Y are haploid or diploid. (2 marks)Answer

(b) State and explain whether rat C is a clone to rat A, rat B or the surrogate mother. (3 marks)Answer
41- 45
(c) The following shows the DNA fingerprints of rat A, rat B and rat C. Identify the DNA fingerprint of rat C. Explain
your answer. (3 marks)Answer
pattern pattern pattern

1 2 3
Level 3: Building a better answer (3 marks)
6. Read the following question and student A’s answer. Re-write and improve the answer based on the teacher’s
comments.
Question
Outline the steps of making a recombinant plasmid that carries the human insulin gene. (3 marks) Answer
Student A’s answer

Plasmids and DNA fragments containing the human insulin gene are isolated by proper
processes. ✔
Cut the plasmids and DNA fragments containing the human insulin gene with restriction
enzymes. 
Join the cut plasmids and DNA fragments with DNA polymerase. 
Teacher's comment
 The plasmids and DNA fragments containing the human insulin gene should be cut with the same
restriction enzyme.
 DNA polymerase is not the enzyme used to join DNA fragments.
Answers are available on p.A1. If you miss any of the questions, review the relevant section(s) again.
Question 1 2 3 4 5 6
Section(s) 41.2 41.2 41.2 41.2 41.3 41.2
41- 46
All answers TE
Exam practice 3. The following shows a recombinant plasmid to be

transferred into a bacterial cell.

Multiple-choice questions gene coding for
human insulin
Section 41.2
1. The table below shows the number of fragments gene coding for
produced when a piece of DNA was digested with antibiotic resistance
different combinations of three restriction enzymes:
Sal I, EcoR I, and Hpa I. What is the use of the antibiotic resistance gene in
producing transformed bacteria?
Combination of Number of fragments
restriction enzymes produced A. To promote the uptake of the plasmid by the
bacterium
Sal I and EcoR I 4
B. To allow the bacterium to produce antibiotics
Sal I and Hpa I 3
Sal I, EcoR I and Hpa I 5 C. To indicate that the bacterium has taken up the
plasmid
Which of the following correctly shows the position D. To ensure that insulin is not affected by
of the recognition sites for all three restriction antibiotics
enzymes on this piece of DNA?
Sal I Hpa I Sal I EcoR I
A. 4. Which of the following statements about PCR is/are
correct?
EcoR I Sal I EcoR I Hpa I
B. (1) The double-stranded DNA denatures when
Sal I EcoR I Hpa I EcoR I phosphodiester bonds are broken. Hint 1
C.
(2) Primer annealing refers to the binding of DNA
Sal I Hpa I EcoR I Sal I polymerase to a DNA strand.
D.
(3) The temperature required for extension
SQA NQ Higher Biotechnology 2015 Section A Q8 depends on the DNA polymerase used.
A. (2) only
2. Which of the following enzymes is/are required in B. (3) only
the making of recombinant plasmids?
C. (1) and (2) only
(1) restriction enzyme D. (2) and (3) only
(2) DNA polymerase
(3) DNA ligase 5. Which of the following substances in a PCR
A. (1) only reaction mixture is not used up by the reaction?
B. (1) and (2) only A. DNA fragments containing the target sequence
C. (2) and (3) only B. primers
D. (1) and (3) only C. free nucleotides
D. heat-stable DNA polymerase
6. PCR can be used to
A. separate DNA fragments according to their

size.
B. insert a target gene into a plasmid.
C. cut DNA at specific sequences.
D. amplify specific DNA fragments.
41- 47
Hint 1: A phosphodiester bond is a bond between the sugar molecules of adjacent nucleotides in a DNA molecule.
All answers
7. In a case where two babies, X and Y, have 9. Arrange the steps involved in nuclear transfer
accidentally lost their identity bracelets. DNA in the correct order.
fingerprinting is used to identify the biological
parents of the children. The diagram below shows (1) Obtain a somatic cell from a nucleus
the DNA fingerprints of the alleged mother W, the donor.
alleged father M and the children X and Y. (2) The surrogate mother gives birth to the
W M X Y clone.
(3) Obtain an egg cell from an egg donor and
remove the nucleus of the egg cell.
(4) Fuse the somatic cell with the egg cell.
(5) Implant the embryo into the uterus of a
surrogate mother.
A. (1), (3), (4), (5), (2)

B. (1), (3), (5), (4), (2)
Which of the following conclusions can be drawn
C. (4), (2), (3), (5), (1)
from the above results?
D. (4), (2), (5), (3), (1)
A. W and M are the biological parents of child X.
B. W and M are the biological parents of child Y.
10. Which of the following statements concerning
C. W and M are the biological parents of child X embryo splitting and somatic cell nuclear
and Y. transfer is correct?
D. W and M are not the biological parents of child
A. Both involve the isolation of cells from an
X or Y.
embryo.
B. Only somatic cell nuclear transfer involves
Section 41.3 denucleation of egg cells.
8. Which of the following statements about plant C. Only somatic cell nuclear transfer involves
tissue culture is incorrect? implantation into the uterus of a surrogate
mother.
A. A callus is a mass of differentiated cells.
D. Both methods produce clones that are
B. Tissue culture is carried out in sterile genetically identical to the egg cell donor.
conditions.
C. The culture medium used in tissue culture
contains essential nutrients and hormones.
D. The genetic make-up of the clones is identical.
Short questions
Section 41.1
11. (a) Outline one historical use of biotechnology. (1 mark)Answer
(b) Explain why the use in (a) may be considered as traditional biotechnology. (2 marks)Answer
(c) Compared to selective breeding, state one advantage of genetic engineering as a way to improve the
characteristics of a species. (1 mark)Answer

41- 48
Section 41.2
12. In recombinant DNA technology, plasmids are commonly used as vectors to transfer a gene of interest into host
cells. The diagram below outlines how recombinant plasmids are produced.
plasmids extracted
from a bacterium (a) What are enzymes X and Y respectively?
(2 marks)Answer
(b) Describe how the DNA fragments containing the
treated with gene of interest should be treated before mixing
enzyme X DNA fragments containing a with the cut plasmids so that they can join.
gene of interest (2 marks)Answer
(c) The cut plasmids, the DNA fragments containing
the gene of interest and enzyme Y are then mixed,
and three possible combinations of DNA molecules
can be formed. Draw and label the three different
mixed and incubated
combinations. (3 marks)Answer
with enzyme Y
13. Two heat-tolerant DNA polymerases used in polymerase chain reactions (PCR) are Taq and Pfu. Pfu has ‘proof
reading’ activity. It checks that the correct nucleotides are inserted during replication of a target sequence and then
corrects any errors. The graph shows the temperatures during a single PCR cycle required to amplify a target
sequence using Taq and Pfu.
100
90 Key: Taq polymerase

Pfu polymerase
Temperature (oC)
80
70
60
50
40
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Time (minutes)
(a) (i) Calculate the time taken for 16 copies of the target sequence to be made from one DNA fragment using
Taq polymerase. (1 mark)Answer
(ii) Identify the time period during which primers bind to the original DNA fragment. (1 mark)Answer
(b) A scientist was planning to amplify DNA using PCR. State which DNA polymerase should be used and describe
the advantage of using this polymerase. (1 mark)Answer
(c) Explain the importance of using heat-tolerant DNA polymerases in PCR. (1 mark)Answer
SQA NQ Higher Biology 2017 Section 2 Q2
41- 49
14. DNA may be analysed to detect whether someone carries a particular gene. The flowchart below shows some of the
steps involved in this analysis.
Step 1: DNA is cut into fragments.
Step 2: DNA fragments are separated by gel electrophoresis.
Step 3: DNA probes are used to locate specific DNA fragments.
(a) Name the enzyme used in Step 1. (1 mark)Answer

(b) Explain how DNA fragments can be separated by gel electrophoresis in Step 2. (3 marks)Answer
(c) Explain how the DNA probes enable scientists to detect a specific gene. (3 marks)Answer
15. The harlequin ladybird (Harmonia axyridis) was first recorded in the UK in 2004 and has since spread widely over
the South East of England. It may prey on smaller ladybirds, such as the ten-spot and two-spot ladybirds, populations
of which have subsequently declined. The techniques of PCR and gel electrophoresis have been used to analyse
DNA extracted from the gut of harlequin ladybirds to determine which other ladybird species have been preyed
upon.
(a) Suggest why PCR is a necessary technique in investigations such as that outlined above. (2 marks)Answer
(b) DNA samples from each of the three ladybird species were cut using restriction endonucleases and the resulting
fragments were separated by gel electrophoresis. The diagram below shows the DNA profile of each species in
the first 3 lanes. The next 3 lanes contained DNA extracted from the guts of 8 harlequin ladybirds trapped at
each of 3 sites in SE England. The extract from the gut will contain DNA from the prey of the harlequin ladybird
along with its own DNA.
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6
Harlequin Ten-spot Two-spot Site 1 Site 2 Site 3

ladybird ladybird ladybird
(i) Researchers identified site 2 as a location where harlequin ladybirds were preying on the ten-spot ladybird,
but not the two-spot ladybird. Use the DNA profiles to outline the evidence supporting this conclusion.
(2 marks)Answer
(ii) With respect to the prey of the harlequin ladybird, suggest an interpretation for the DNA profiles from site
1 and site 3. (2 marks)Answer
CCEA GCE AS Biology Assessment Unit AS 1 Jan 2012 Q6
41- 50
Section 41.3
16. The flowchart below shows the main steps in tissue culture.
A small piece of tissue (explant) is removed from a plant (e.g. a bud).
The explant is transferred to a culture medium containing nutrients and hormones.
A mass of undifferentiated cells (a callus) develops.
The callus is transferred to a new culture medium in which shoot growth and root
growth are stimulated.
Developing plants are separated and grown under optimum conditions.
(a) Outline the purpose of tissue culture. (1 mark)Answer

(b) The entire process is carried out under sterile conditions. Explain why sterile conditions are necessary.
(1 mark)Answer
(c) State two advantages of producing plants by tissue culture rather than from seeds. (2 marks)Answer
(d) Tissue culture is increasingly used in conjunction with genetic engineering to propagate transgenic plants.
Outline three additional steps which would be required in the production of transgenic plants. (3 marks)Answer
Structured questions
Section 41.2
17. Potato blight disease is caused by a fungus that infects potato plants. In infected plants, brown spots develop on
leaves, leading to a reduction in the yield of potato tubers. Some potato plants are resistant to this disease, so
farmers can use selective breeding to develop more resistant potato plants. In addition, scientists can insert the
blight resistance gene into potato plants using modern biotechnology.
 Brown spots develop on a leave of an infected plant

(a) Give two disadvantages of developing blight-resistant potato plants with selective breeding compared with
genetic engineering. (2 marks)Answer
(b) The blight resistance gene can be transferred into the cells of a potato plant using Agrobacteria. Describe how
this can be done with recombinant DNA technology. (4 marks)Answer
(c) Following (b), how could scientists identify the plant cells that have transformed? (3 marks)Answer
41- 51
18. The diagram below shows the change in temperature during a polymerase chain reaction (PCR) cycle:
100
Stage I
90
80
Temperature (°C)
Stage III
70
60 Stage II
50
10
0
Time (min)
(a) Which stage corresponds to DNA denaturation? Explain your answer. (3 marks)
(b) Mary planned to amplify a fragment of DNA using PCR. The following diagram shows the annealing of primers
during PCR. The sequence of DNA strand X is shown below and the corresponding sequences of regions I and II
are highlighted:
region I region II
DNA strand X
DNA extension primer II
primer I DNA extension
DNA strand Y
Sequence of DNA strand X: no. of

bases
TGGCGCTGGG C G C A AT G C G C G C C A T T A C C G AGTCCGGGCT GCGCGTTGGT 50
region I
G C G G ATAT C T C G G TA G T G G G A T A C G A C G A T ACCGAAGACA G C T C AT G T TA 100
TAT C C C G C C G T TA A C C A C C A TCAAACAGGA TTTTCGCCTG CTGGGGCAAA 150
CCAGCGTGGA CCGCTTGCTG CAACTCTCTC AGGGCCAGGC GGTGAAGGGC 200
A AT C A G C T G T TGCCCGTCTC ACTGGTGAAA AGAAAACCA CCCTGGCGCC 250
C A ATA C G C A A ACCGCCTCTC CCCGCGCGTT G G C C G AT T C A T TA AT G C A G C 300
TGGCACGACA GGTTTCCCGA CTGGAAAGCG GGCAGTGAGC G C A A C G C A AT 350
TA AT G T G A G T TA G C T C A C TA AT TA G G C A C C C C A G G C T T TA C A C T T TAT G C 400
region II
TTCGGCTCG TAT G T T G T G T G G A A T T G T G A G C G G ATA A C A AT T T C A C A C A 450
41- 52
Mary designed the following primers for the PCR:

DNA extension
Primer I: CGGUAGUGGG AUACGACGAU
DNA extension
Primer II:
CCTTAACACT CGCCTATTGT
(i) There is one type of mistake in each primer. Write the correct primers to be used. (2 marks)
(ii) What is the predicted size (in terms of number of base pairs) of the PCR product? (1 mark)
(c) Mary used the following plasmid as a vector to carry the PCR product to transform bacteria. The plasmid
contained:
(I) an ampicillin resistance gene;

(II) a Z gene encoding an enzyme that converts substance X to blue compounds;
(III) some restriction sites within the Z gene.
ampicillin resistance gene restriction sites
Z gene
After the transformation of the bacteria, Mary grew the bacteria on agar plates containing both ampicillin and
substance X. Blue and white bacterial colonies were formed.
(i) What is the purpose of adding ampicillin to the agar plates? Explain your answer. (2 marks)
(ii) Explain which type of colony (blue or white) contains non-recombinant plasmids, i.e. without DNA insert.
(4 marks)
HKDSEE Biology 2018 Paper 2 Q4(b)
41- 53
19. Some populations of flies are becoming resistant to insecticides intended to kill them. Scientists developed a method
for finding out whether a fly was carrying a recessive allele, r, that gives resistance to an insecticide. The dominant
allele, R, of this gene does not give resistance. The scientists:
• crossed flies with genotype RR with flies with genotype rr
• obtained DNA samples from the parents and offspring
• used the same restriction endonuclease enzymes on each sample, to obtain DNA fragments.
(a) Explain why the scientists used the same restriction endonuclease enzymes on each DNA sample. (2 marks)Answer
The scientists added two different primers to each sample of DNA fragments for the polymerase chain reaction
(PCR).
• Primer A3 only binds to a 195 base-pair fragment from allele r.

• Primer A4 only binds to a 135 base-pair fragment from allele R.
The scientists separated the DNA fragments produced by the PCR on a gel where shorter fragments move further in
a given time. Their results are shown in the figure below.
L - DNA M - DNA N - DNA

fragments from fragments from fragments
Direction DNA one of the offspring from the other
fragments parents parent
move on gel
Bands on gel
containing DNA
fragments
(b) Explain why primer A3 and primer A4 only bind to specific DNA fragments. (2 marks)Answer
(c) Use all the information given to explain the results in the figure. (3 marks)Answer
(d) The scientists wanted to know on which chromosome the gene with alleles R and r was located. From the flies
with genotype RR, they obtained cells that were in mitosis and added a labelled DNA probe specific for allele R.
They then looked at the cells under an optical microscope. Explain why they used cells that were in mitosis.
(2 marks)Answer
AQA GCE AL Biology Unit 5 Jun 2015 Q9(a)-(d)
20. DNA fingerprinting is used in the screening of a genetic disease known as sickle-cell anaemia. The disease is a result
of a gene mutation which leads to the production of defective haemoglobin. To prepare the DNA fingerprint, copies
27 of DNA fragments containing the gene associated with sickle-cell anaemia are first produced by a polymerase chain
41 reaction (PCR). The fragments are then treated by a restriction enzyme which cuts DNA at the middle of CCTNAGG,
where N can be any nucleotide. The diagram below shows some nucleotide sequences of the DNA fragment
containing the normal allele and the mutated allele for sickle-cell anaemia:
~1.4 kb (kilo base pairs)

DNA fragment with part
of the normal allele: CCTTAGG CCTGAGGAG CCTTAGG
DNA fragment with part

of the mutated allele: CCTTAGG CCTGTGGAG CCTTAGG
Mutation
41- 54
(a) How many restriction sites are found in the DNA fragment with the normal allele and that with the mutated allele
respectively? (1 mark)
(b) Based on the principle of gel electrophoresis, explain how the cutting of the two DNA fragments shown above
would produce different DNA fingerprint patterns in a gel. (4 marks)
(c) How many DNA bands would be observed in the DNA fingerprint of a carrier of sickle-cell anaemia? Explain
your answer. (2 marks)
(d) Explain why the gene mutation will result in the production of defective haemoglobin. (3 marks)
HKDSEE Biology 2014 Paper 2 Q4(b)
Section 41.3
21. The diagram below shows how Dolly the sheep was cloned.
sheep A sheep B
A cell was taken from An egg cell was obtained

the mammary gland from sheep B and its
of sheep A. nucleus was removed.
The two cells were fused.
cell X
Cell X developed to
form an embryo.
embryo
The embryo was implanted

into the uterus of sheep C.
sheep C
Sheep C gave birth to Dolly.
Dolly
(a) Does cell X contain a haploid or diploid set of chromosomes? Explain your answer. (2 marks)Answer
(b) Name the type of cell division by which cell X underwent to form the embryo. (1 mark)Answer
(c) Dolly shows the same body characteristics as one of the three parents, sheep A, B, and C. Which parent is this?
Explain your answer. (3 marks)Answer
(d) Give two ways in which the above process is different from the natural process of reproduction in sheep.
(2 marks)Answer
(e) Give two potential applications of animal cloning. (2 marks)Answer
(f) Give two limitations of the technique involved in animal cloning. (2 marks)Answer
41- 55
22. Dairy cows are traditionally bred to improve the milk yield. Cows that produce very high yield can be cloned by
somatic cell nuclear transfer.
(a) Describe how dairy cows can be bred to improve the milk yield using selective breeding. (3 marks)Answer
(b) Outline how a high-yield cow can be cloned using somatic cell nuclear transfer. (4 marks)Answer
(c) Milk yield can also be increased by injecting cows with the hormone bovine somatotropin (BST). The use of this
hormone increases milk production by 10% to 25%. This hormone can be manufactured by genetically
engineering E. coli.
(i)
Suggest one concern raised by the use of BST to increase milk yield. (1 mark)Answer
(ii)
Describe how E. coli can be modified to synthesize BST by transferring the gene for BST into the bacteria.
(4 marks)Answer
41- 56

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Links to prior knowledge Chapter preview

Is Supersweet corn genetically modified?

Supersweet is a variety of sweet corn that has high

As the public are becoming more and more

Supersweet corn is traditionally developed by selective breeding. Corn plants with

41.1 Introduction to modern

Nowadays, with increased knowledge about the structure of DNA

Two rapidly expanding areas of modern biotechnology are genetic

genetic engineering 遺傳工程 transgenic organism 轉基因生物

Learning objective 41.2 Genetic engineering

1. The principle of recombinant DNA technology

 Isolation of DNA fragments that contain the gene of interest

A vector is a DNA molecule that acts as a carrier to transfer the

A plasmid is a circular double-stranded DNA (Figure 41.4),

Plasmids replicate independently of the bacterial chromosome and

recombinant DNA technology 重組 DNA 技術 plasmid 質粒

Restriction enzymes (restriction endonucleases) act like molecular

blunt ends blunt ends sticky ends sticky ends

 Ligation—joining DNA fragments and plasmids together with

restriction enzyme 限制酶 ligation 連接 restriction endonuclease 限制性核酸內切酶

The recombinant plasmids can then be transferred into host cells.

2. Production of human insulin using

 Obtain the DNA

 Insert the DNA fragment into

Recombinant plasmids are produced by inserting the human insulin

transformed bacterium bacterium that has not taken

agar containing an antibiotic

The transformed bacteria are selected and cultured on a large scale

Gene expression is induced in the transformed bacteria to synthesize

B. Polymerase chain reaction

The principle of PCR is similar to that of DNA replication, but PCR

primers—a primer is a short sequence of synthetic single-

The reaction mixture is first heated to about 95 °C. At this

The reaction mixture is cooled to between 50 °C and 65 °C. This

The temperature of this step depends on the DNA polymerase used.

DNA polymerase attaches to the primers and free nucleotides pair

primer 1 free DNA

The number of DNA strands is doubled at the end of each cycle.

Number of PCR cycle at start 1st 2nd 3rd 4th

Figure 41.11 Amplification of DNA by PCR

Cycling of PCR can be performed automatically in a thermal cycler

However, there are considerable amounts of reagents (e.g. free

thermal cycler 循環變温加熱器

Scientists often use PCR to amplify DNA samples for subsequent

Mycobacterium tuberculosis (x20,000) grows very slowly in

Figure 41.13 Some applications of PCR

1. The principle of DNA fingerprinting

Each VNTR contains a number of short repetitive sequences of

Furthermore, each individual has two alleles of a given VNTR—one

DNA fingerprinting DNA 指紋分析 length polymorphism 長度多態性

A VNTR is sandwiched by non-repetitive sequences (the purple

fragments of different lengths to be

Band corresponding to the

RFLP analysis is one of the earliest methods of DNA fingerprinting.

 DNA extraction  Restriction fragment preparation  Gel electrophoresis

restriction fragment length polymorphism analysis 限制性片段長度多態性分析

nylon membrane radioactive

Figure 41.17 DNA fingerprinting using RFLP analysis (Cont'd)

STR analysis 短銜接重複分析

 Isolation of DNA fragments that contain the gene of interest

 Ligation—joining DNA fragments and plasmids together with

Skill-building Handling data