Techniques in modern

E4
1 biotechnology

1.1 Introduction to biotechnology

1 Biotechnology is any technological application that involves the use of organisms, biological
systems or processes to produce goods or provide services.
2 Modern biotechnology refers to a range of processes and techniques that involve the
manipulation of DNA, cells, tissues or biological processes to attain knowledge, produce
goods or provide services.

1.2 Recombinant DNA technology

1 Major steps in recombinant DNA technology (illustrated by the production of human insulin):

human pancreatic cell bacterium

ampicillin tetracycline
resistance gene resistance gene

2 Obtain a plasmid
1 Obtain DNA from a bacterium.
encoding
human insulin.

DNA encoding
plasmid
human insulin
3 Cut the DNA fragment
and the plasmid using
the same restriction
enzyme.

4 Join the DNA

open
fragment and
plasmid
the plasmid
together using
a DNA ligase.

recombinant
plasmid

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2 Plasmids are small rings of double-stranded extrachromosomal DNA in bacterial cells. They
are often used as vectors to transfer the gene of interest into host cells.
3 Restriction enzyme recognizes a specific base sequence (called restriction site (限制位點))
and cuts both strands of the DNA molecules.
- Some restriction enzymes cut the two strands of a DNA molecule at points directly opposite
each other, leaving straight cut ends called blunt ends (鈍端).
- Other restriction enzymes leave short lengths of single-stranded DNA at the cut ends called
sticky ends (黏端).
4 DNA ligase catalyses the joining of the DNA fragments and the plasmids by covalent bonds.
Recombinant plasmids are formed when the DNA fragment and the plasmid join together.
5 Production of human insulin using recombinant plasmids:

Transformation (轉化): The recombinant plasmids are introduced into bacteria (host cells).

To enable selection of bacteria carrying the DNA insert, plasmids carrying selective markers
(選擇性標記) such as antibiotic resistance genes are often used as vectors.
- The bacteria are first cultured on an agar plate containing an antibiotic to select the
bacteria that picked up a plasmid (i.e. transformed bacteria).
- A portion of bacteria from each colony is transferred to a second agar plate containing
another antibiotic to identify the transformed bacteria with a recombinant plasmid.

The bacteria carrying DNA encoding human insulin are cultured in fermenters (發酵器). Gene
cloning (基因克隆) occurs, producing many copies of the DNA encoding human insulin.

DNA encoding human insulin is expressed and polypeptides are produced. The polypeptides are
extracted from the bacteria and processed into functional human insulin.

6 Advantages of using recombinant DNA technology in human insulin production over the
extraction of insulin from animal pancreases:
- Insulin can be produced in a shorter time. The extraction cost is lower.
- The product yield is much higher because:
 many copies of the DNA encoding human insulin are present in the bacterial culture.
 the growth rate of bacteria is high.
 the bacterial culture produces insulin continuously.
- The insulin produced is structurally the same as the insulin produced by the human body.
It is not rejected by the immune system.
- The insulin produced is pure. The risk of being contaminated with pathogens and causing
infections is lower.
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1.3 Genetically modified organisms
1 A genetically modified organism (GMO) is any organism whose genetic make-up has been
changed by genetic engineering.
2 Techniques that can be used to introduce a gene into different organisms to produce GMOs:

Organisms Techniques

Bacteria - Using a plasmid

- Using a bacteriophage (噬菌體)

Plants - Using Agrobacterium (農桿菌)

- Using a gene gun (基因槍)

Animals - By microinjection (顯微注射)

- Using a virus
- Using a liposome (脂質體)

3 Production of pest-resistant GM maize plants using Agrobacterium:

Agrobacterium



toxin gene
plasmid


recombinant
plasmid

transformed
Agrobacterium

maize cell with 

toxin gene
incorporated
into its genome 


maize cells on
an agar plate

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  Obtain a DNA fragment containing the toxin gene from a toxin-producing bacterium and
cut it using a restriction enzyme.
 Obtain a plasmid from Agrobacterium and cut it using the same restriction enzyme.
 Join the DNA fragment and the plasmid together using a DNA ligase.
 Introduce the recombinant plasmid into Agrobacterium.
 Infect maize cells with the transformed Agrobacterium.
 Adult plants are developed from the infected maize cells.
 The GM maize plants produce the bacterial toxin. The plants are pest-resistant.

4 Benefits of the production of GMOs using genetic engineering:

- GMOs can act as ‘biological factories’ to produce large amounts of useful products in a
shorter time and at a lower cost.
- Growing GM crops and raising GM animals with higher productivity can increase food
supplies.
- Some GM foods have a higher nutritional value.
- Growing certain GM crops and raising certain GM animals can help reduce pollution
caused by agriculture.
5 Potential hazards of the production of GMOs using genetic engineering:
- The long-term effects of GMOs and their products on human health are still unknown.
- New genes or their products in GM food may cause allergic reactions in some people.
- Antibiotic resistance genes, which often serve as selective markers in the process, may be
accidentally transferred from GMOs to pathogens and produce ‘superbugs’ that are
resistant to multiple antibiotics.
- GMOs may transfer their genes to the wild types (i.e. causing genetic pollution). These
genes may have unexpected and dangerous effects.
- GMOs may out-compete the wild types and upset the ecological balance, resulting in a
decrease in biodiversity.
- Introducing new genes from a different species into an organism to produce a GMO adds
genes to the gene pool of a species. The long-term effects are not yet known.

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1.4 Plant and animal cloning
1 Cloning is the process of producing genetically identical organisms (i.e. clones).

Plant cloning
2 An example of cloning is producing plants by artificial vegetative propagation, which is
a method of asexual reproduction.
3 Some plants can be cloned by tissue culture (組織培養) (also called micropropagation
(微繁殖)):

Remove a piece of tissue from a plant and sterilize (消毒) it.

Put the piece of tissue into a sterile culture medium containing all the nutrients necessary for
growth. Cells divide by mitotic cell division to form a mass of undifferentiated cells called
a callus (胼胝體).

Transfer the cells of the callus to a culture medium containing hormones. The hormones promote
the growth of roots and shoots. The cells differentiate into different types of cells and plantlets are
formed.

Grow the plantlets in the soil for further development.

4 Applications of plant cloning by tissue culture:

- To produce large numbers of plants that are economically important.
- To produce disease-free plants for agricultural purposes.
- To rescue disease-infected plant populations by culturing uninfected tissues of the plants.
- To produce plants that are endangered or hard to grow from seeds or by conventional
propagation.
- To maintain a special breed of plants with desirable characteristics.
- To produce genetically identical plants for research purposes.
5 Advantages and disadvantage of cloning plants by tissue culture:

Advantages Disadvantage

- A larger number of clones can be - Plant tissues have to be transferred under

produced at the same time. sterile conditions. Highly trained staff
- Clones can be produced in a shorter time. and expensive equipment are required.

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Animal cloning
6 In embryo splitting (胚分割), individual cells from a single embryo are isolated and grown into
embryos. The embryos are then transferred into surrogate mothers for development.
7 Nuclear transfer (核移植) is another technique used for animal cloning. Dolly was the first
mammal cloned from an adult cell using this technique:

1 A mammary 2 An ovum was

gland cell was collected from
collected from another sheep
an adult sheep. and its nucleus
The nucleus was removed.
was obtained.
sheep X sheep Y

3 The nucleus of the

mammary gland cell
was transferred to
the ovum. The cells
started to develop into
an embryo in vitro.

4 The embryo was

implanted into the embryo
uterus of a surrogate
sheep to allow further
development.

sheep Z

5 The surrogate sheep

gave birth to Dolly.

Dolly
genetic make-up identical to
the nucleus donor (sheep X)

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8 Applications of animal cloning:
- To propagate farm animals or endangered animals.
- To mass-produce GM animals for the production of valuable pharmaceutical products or
other chemicals.
- To produce genetically identical animals for use in drug tests or research.
- To obtain stem cells (幹細胞) for use in research or medicine.
9 Limitations of animal cloning:
- The success rate is low.
- Animal clones may get old sooner and have shorter lifespans.
10 Advantages and disadvantage of plant and animal cloning:

Advantages Disadvantage

- Desirable characters can be preserved. - The clones produced lack genetic

- It can be used as a method to overcome variations. This reduces the adaptability
reproductive difficulties. of the population to changes in the
environment.

11 Issues related to animal cloning:

Ethical issues - Animal cloning using nuclear transfer has a low success rate. A large
number of embryos are sacrificed.
- Many cloned animals have birth defects or other health problems.

Economic issue - Animal cloning requires expensive equipment.

Environmental - Animals that are endangered due to the loss of habitats will still have
issues no place to live after they are cloned.
- The genetic variations of a population may decrease if cloning
becomes widespread.

12 Issues related to human reproductive cloning (生殖性克隆):

- Human clones may be considered as ‘unnatural’ or ‘sub-human’.
- The relationship between human clones and their nucleus donors is unclear.
- Human clones lack self-identity.
- It lowers the value of life.

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1.5 Polymerase chain reaction
1 Polymerase chain reaction (PCR) (聚合酶鏈反應) is a technique that allows scientists to make
many copies of DNA quickly without living cells.
2 PCR can be performed in a thermal cycler (循環變温加熱器). The events that happen in each
cycle of PCR:

 DNA denaturation (~95°C)

DNA
sample

 Primer annealing (~50–65°C)

primer 2
 Extension (~70°C) DNA
polymerase
primer 1

free synthesis of new

nucleotides DNA strand

 DNA denaturation: Heat the reaction mixture to about 95 oC. This separates the two
strands of the DNA sample by breaking the hydrogen bonds between them so that each
strand can be used as a template.

 Primer annealing: Cool the reaction mixture to 50–65 °C. This allows the annealing (連接)
of primers (引物) to the DNA templates by complementary base pairing.

 Extension: Raise the temperature again to about 70 °C. This allows the heat-stable DNA
polymerase to catalyse the joining of adjacent nucleotides so that free nucleotides are
added to the primers to extend the new DNA strands.

3 At the end of each cycle, the number of DNA copies doubles. This is because the newly
synthesized DNA strands act as templates in the next cycle.

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4 Applications of PCR:
- To amplify DNA for prenatal diagnosis of genetic diseases.
- To amplify DNA to produce DNA fingerprints in forensic science.
- To amplify DNA from the remains of historical figures or extinct species for studies.
- To diagnose infectious diseases.
- To identify GM foods.

1.6 DNA fingerprinting

1 About 0.1% of the base sequence in DNA represents the genetic variation between individuals.
Some of the genetic variations occur in variable number tandem repeats (VNTRs) (可變數目
串聯重複). VNTRs are present in the non-coding regions on human chromosomes.
2 Based on the variation in the lengths of VNTRs between individuals, scientists can identify
individuals by DNA fingerprinting (also called DNA profiling). One of the methods is
restriction fragment length polymorphism (RFLP) analysis (限制性片段長度多態性分析):

blood 1 DNA is extracted from blood

samples samples and cut with specific
restriction enzymes.

DNA
fragments

band containing
DNA fragments
2 DNA fragments are separated
according to their lengths by
gel electrophoresis.

gel

3 The gel with DNA fragments will be

further processed to obtain DNA
fingerprints.

DNA fingerprints

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3 Advantage and disadvantages of DNA fingerprinting using RFLP analysis:

Advantage Disadvantages

- The DNA fingerprint obtained is unique to - It is time-consuming. It may take several

each individual (except identical twins). weeks to complete.
It is a very reliable method of identifying - It requires a relatively large amount of
an individual. good quality (i.e. intact) DNA.

4 Applications of DNA fingerprinting:

- To provide evidence of the identities of individuals in forensic science.
- To establish family relationships in parentage tests.
- To identify victims in disasters.
- To authenticate (認證) foods and Chinese medicines.
- To help conserve endangered species, e.g. by tracing the origin of ivory products.
- To trace the source of infectious diseases.
- To screen for genetic diseases.

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