Biotechnology283

284 Getting 5** in 9 Weeks: Biology

Pagier Major steps in recombinant DNA technology:

1. A suitable restriction enzyme is used to cut the DNA and the gene is removed.

Biotechnology 2. A plasmid is taken froma bacterium and cut with the same restriction enzyme.
3. The target gene is inserted into the plasmid. It is fixed onto the open plasmid by
another enzyme known as a DNA ligase.
4. Genetically engineered plasmids are then introduced into the host bacteria cell.
5. The bacteria which have been taken up the recombinant DNA are selected.
6. Transgenic bacteria are cultured in huge industrial fermenters. They secrete their
products which can be collected and purified.

DNA
plasmids containing
antibiotic resistance gene
cut by restriction
Biotechnology enzyme
CC©
• It covers various techniques for using organisms, biological systems or biological
processes in producing goods or providing services. target gene

Recombinant DNA technology +

• Important terms: recombinant DNA bacteria
plasmids uptake
Terms Meaning
recombinant
Restriction enzyme An enzyme that cuts DNA at a specific base sequence. each colony is a clone of A bacterialcells
the offspring from a single cell grownin
An enzyme that joins DNA sections. O culture medium
Ligase
containing
antibiotic
Vector A DNA molecule used as a vehicle to carry foreign genetic material
into another cell, where it can be replicated and expressed.
selected transformed bacteria
Plasmid A small circular piece of extrachromosomal DNA in bacterial cells. It are cultured in a large scale

can be taken up by bacterial cells easily because of its small size.

Marker gene A gene that is added to a plasmid at the same time as the target
gene. It allows checking whether the bacteria have taken up the (plasmid) DNA # FE (vector)
# A:
plasmids or not, e.g. an antibiotics resistance gene. 1. A

Recombinant The plasmid with the target gene inserted.

2. (marker) TÊ#NWILT#A
plasmid
 286 Getting 5** in 9 Weeks: Biology
Biotechnology 285

• Amount of DNA is double in each cycle. The total amount of DNA obtained after PCR is
Polymerase chain reaction (PCR)
2"xC.
• A polymerase chain reaction is used to amplify minute quantities of DNA. (where n is the number of PCR cycles; C is the initial number of copies of the DNA

• It starts with a reaction tube containing: template)

- templateDNA The application of PCR:
primers (i.e. short pieces of single-stranded DNA that are complementary to the two This is useful when only a small amount of DNA is available but a large amount is
ends of the DNA sequences to be amplified) required to undergo testing (e.g. crime scene samples of blood, semen, tissue, hair).
DNA polymerase
nucleotides
DNA fingerprinting
DNA fingerprinting is a technique by which individuals are identified on the basis of their
• PCR comprises three principal steps in each cycle:
respective DNA profiles.

Steps Temp (°C) Events • Within the non-coding region of an individual's genome, there exist short lengths of
highly repetitive bases called short tandem repeats (STRS).
1. Denaturation A DNA sample is heated to separate into
95
two single strands. • These repeating sequences can be cut by certain restriction enzymes to form fragments.

2. Primer annealing Primers bind to the single stranded DNA • Gel electrophoresis is used to separate these fragments according to their length.
55 templates by using complementary base
• As these repeated sequences vary in length from person to person, individuals all generate
pairing.
unique fragment profiles.
3. DNA synthesis A heat-tolerant DNA polymerase uses the
72
nucleotides to make new DNA strands.

double stranded target DNA

6
length 896 550 240 100 48
ofSTR 1100

Step 1: denaturation restriction

two new DNA targets available for PCR
sites
90% T DNA
PCR 8
amplification
cycle

9 direction of
movement DNA
fingerprint
Step 2: primer annealing Step 3: DNA synthesis

55°C primers bind to target DNA 72°C double stranded DNA duplicated
 Biotechnology 287
288 Getting 5** in 9 Weeks: Biology

• Applications of DNA fingerprinting:

Benefits and hazards of genetic engineering
Underlying principles
1. Benefits:
Parental testing Children inherit half of their DNA from each parent and thus their
To develop crop plants that are resistant to pests and diseases
DNA is a shared mix of only the mother's and father's DNA.
• To develop crop plants with higher yield
Forensic A suspect's DNA should be a complete match with the crime- • To improve the nutritional content and value of the food source
investigation scene DNA. • To develop transgenic animals to produce medically important proteins
• To develop transgenic animals as models for studying human diseases
Genetically modified (GM) organisms
2. Potential hazards
• A new gene can be added to give the organism a new characteristic.
• The GM organisms may cause diseases to animals, plants and man.
• It involves the artificial introduction of the desired gene from a donor species to the • The GM organisms could have currently unknown effects (e.g. cause a allergy in a
recipient organism using physical, chemical or biological means. percentage of the population).
• The GM microorganisms may escape to the environment and become 'super bugs
• The recipient organism with the new characteristic is calleda 'transgenic organism'.
that threaten public health.
• The basic steps used in the production of transgenic plants: 4 • The GM organisms may produce toxic compounds in the environment.
donor plant cell • The GM organisms may grow too fast and become out of control
agrobacterium
with useful gene • The GM organisms may be accidentally released into the environment, and this may
5
result in competition with native plant species.

Cloning
6
• Cloning is the artificial production of genetically identical individuals by using asexual
plasmid is cut open useful gene
methods.
by restriction enzyme
recombinant DNA inserted 1. Plant cloning
in agrobacterium •
recipient plant A small amount of meristematic tissues from the plant parts are cultured in a medium
containing essential nutrients and growth hormones under aseptic conditions.
• The cultured tissues will eventually develop into new whole plants which are identical
crop plant infected transplant
with GM bacterium to the parent.
callus

-cropplant
agar with new gene
cells removed
I ABËAth(GM
food)aj#R#)F
from recipient plant (a transgenic plant) AH3IRHASB AE Â
 290 Getting 5** in 9 Weeks: Biology
Biotechnology 289

Biotechnology in medicine
2. Animal cloning (The cloning of Dolly the sheep)
1. The production of pharmaceutical products
black-faced
sheep) white-facedsheep () • For example: insulin, growth hormones, vaccines and monoclonal antibodies

2. Gene therapy
• The treatment of genetic diseases by introducing the normal copy of a gene into the
genome is called gene therapy. Severe combined immunodeficiency (SCID) is treated
in this way:
an unfertilized egg a cell was taken - A healthy gene is cut from the donor DNA using a restriction enzyme.
collected and its from the The gene is then incorporated into a virus.
nucleus was removed mammany gland
The virus infects cells isolated from the patient, thus introducing the normal gene

O- fused cell and enucleated egg

into the cells.
The GM cells are selected and expanded and re-introduced into the patient.
Possible hazards of gene therapy:

Problems Possible hazards

fused cell
Immune response The immune system's secondary response to invaders makes it
difficult for gene therapy to be repeated in patients.

Problem of gene The therapeutic genes may be integrated into a wrong position
addition and this may cause cancer.
embryo
Problem with viral 1. The viruses used in gene therapy present a variety
vectors of potential problems to the patient, e.g. toxicity and
inflammation.
implanted embryo to
2. A viral vector may recover its ability to cause disease inside
the patient's body.

black-faced sheep (Z) 3. Stem cell therapy

gave birth to
(a) The nature of stem cells
white-faced baby • Stem cells are unspecialized cells with two key qualities:
sheep (Dolly)
- Self renewal: cary out unlimited mitotic cell division
• Basic steps: - Potency: capable of differentiation
1. Unfertilized egg is collected from a black-faced sheep (sheep X). • There are two broad types of stem cells:

2. The nucleus of the unfertilized egg is removed. Embryonic stem cells (obtained from an early embryo) which can differentiate
into almost all cell types
3. A mammary gland cell is collected from a white-faced sheep (sheep Y).
Adult stem cells (obtained from specialized tissues, e.g. bone marrow) which
4. The two cells are induced to fused together.
can differentiate into a limited range of cell types
5. The fused cell is incubated in a culture medium for 6 days to develop into an embryo.
6. The embryo is then implanted into the uterus of another black-faced sheep(sheep Z,ie.
the surrogate mother).
I. Sheep Z finally gives birth to a white-faced baby sheep (i.e. Dolly), which has genes
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292 Getting 5** in 9 Weeks: Biology

(6) Applications of stem cells in medical treatments

Stem cells can be used to repair damaged or diseased tissue. Bioethics
The treatment requires:
Stem celIl
- certain chemicals to trigger ifferentiation of thestemcells. GM food Cloning HGP Gene therapy
therapy
- transplantation of the differentiated cells into the patient's body.
suppression of the recipient's immune system to prevent rejection. Ethical issues |Maycontain 1. Thevalue of The diagnosis There are risks |Human embryos
- monitoring the new cells to ensure they don't becomecancerous. genesfrom ife is lower. of anincurable ofmisuse are killed to

Examples of therapeutic uses of stem cells:

organisms that 2. There are genetic defect ofgenetic obtainhuman
some people risks of abuse causes anxiety. improvement embryonic stem
Grafting new skin cells in burn victims
would not eat for of human cells.
- Transplanting bone marrow into patients after chemotherapy religious reasons cloning.
Transplanting stem cells into a patient's brain to treat Parkinson's disease 3. Mostembryos
die in the
Transplanting stemn cells into an injured spinal cord to cure spinal cord injury
process.
4. Biotechnology in agriculture
Safety issues May cause Viral vectors may
(a) Transgenic plants in agriculture and the food industry allergies causes diseases
Transgenic plants usually carry some beneficial features, such as:
Legal issues |1. Who owns How toprevent How toensure
- Herbicideresistance genetic misuse? the stem cells
Pest resistance information? are only used in
Disease resistance |2. Who has the the treatment of
Tolerance to cold, drought or high salinity of soil right to access disease?
personal
Delay softening or ripening
genetic
Improved nutritional value information?

(b) Transgenic animals in agriculture and the food industry

Social and May affect |High cost The public |It may widen the
• Transgenic animals usually carry some beneficial features, such as: economic the livelihood perception of social gap.
Higher growth rate issues of farmersin anindividual
developing may be affected
- Resistanceto cold
COuntries |by hisgenetic
- Improvedmeatcomposition information.
- Improved milkcompositionandproduction
Environmental 1. May reduce May reduce
- Improved wool qualityandproduction
issues biodiversity biodiversity
2. May
produce
Super weeds