CAPE Biology Unit 1 Notes

Genetic Engineering
Contents
Principles of biotechnology..................................................................................................................... 3
Isolating and cloning genes ..................................................................................................................... 5
Cloning a Gene .................................................................................................................................... 5
Steps in cloning a gene ....................................................................................................................... 5
Applications and Techniques of Genetic Engineering in Plants .............................................................. 6
Human Gene Therapy (HGT) ................................................................................................................... 7
In vivo and Ex vivo Methods of Gene Therapy ....................................................................................... 8
Principles of biotechnology

Biotechnology includes a wide range of applications ranging from the traditional production of
beers, wine, rum and cheeses, to the use of recombinant DNA technology to produce
pharmaceutical drugs, and transgenic crops and animals.

 Transgenic crops can contain genes from the original plant plus genes from microorganisms
or other species of plant.

 Biotechnology has applications in many industrial sectors. It is routinely used in the

detection, diagnosis and treatment of diseases in both plants and animals and the cleaning
up of toxic waste.

 It provides opportunities for investigating and enhancing agricultural production. Since

somatic plant cells can often produce new plants, they are readily manipulated by
biotechnology. Therefore if the genetic constitution of such plant cells is altered, the change
would be present in any offspring of the individual plant.

Genetic engineering is the formation of new combinations of heritable material in an organism by

the insertion of nucleic acid molecules into the organism’s genome.

 A vector such as a virus or a bacterial plasmid is used to insert the new DNA into the
genome.
 A plasmid is a circular strand of DNA found in the cytoplasm of a bacterial cell. It carries one
or several genes and can replicate itself independently of the bacterial host chromosome.
 A vector allows DNA sequences to be incorporated into the DNA of a host organism in which
they do not naturally occur.
 The incorporated sequences are propagated or multiplied when the host organism DNA
replicates.
 Recombinant DNA techniques allow the creation of new combinations of genes, which do
not exist under natural conditions.

Genetic engineering is the modification of the genetic composition of an organism by the use of
recombinant DNA technology.

 Foreign DNA is introduced into the genome of the recipient organism in such a way that the
genome of the host is unchanged except for the incorporation of the foreign gene.
 DNA can be isolated from cells of plants, animals or microbes and cut into groups of one or
more genes using restriction enzymes.
 The DNA fragments can be joined to vector DNA and transferred to the host organism cell
where they become part of the genetic makeup of the host.
 When the recipient host cells are propagated, they display the new genetic properties
encoded in the new DNA.
 Traditional plant and animal breeding techniques change the genetic code in a gradual and
indirect manner, while genetic engineering introduces genetic changes that would not be
possible in conventional ways of breeding.
Isolating and Cloning Genes

 Cloning is the production of many identical copies of a gene or an organism.

 Recombinant DNA is DNA which contains genes from more than one species or organism.
The production of recombinant DNA begins with the cloning of the gene of interest. The
function of a gene can be determined by comparing its sequence to genes of known function
from other organisms. If the sequence is similar the function may be similar. Gene function
can also be determined by mutating the genes inside the organism and observing the effect
of the mutant gene.

Cloning a Gene
 This requires isolation of the gene and its insertion into a plasmid vector.
 When the vector is inserted into the host cell, multiple copies of the gene of interest are
produced when the recombinant DNA multiplies within the host cell.
Note that the gene of interest incorporated into the plasmid is the recombinant DNA and the
gene-cloning occurs when the plasmid multiplies.
 A restriction enzyme is an enzyme that cuts DNA at specific sites that are characterised by a
particular sequence of nucleotides.
 The nucleotide sequences where restriction enzymes cleave DNA molecules are called
restriction enzymes.
 Many different microorganisms produce restriction enzymes and hundreds have been
isolated and purified.
 Restriction enzymes cut DNA at unique sites and these enzymes are essential tools in
recombinant DNA technology.

Watch video on restriction enzymes.

https://www.youtube.com/watch?v=5hgbcdQPISI

 Restriction enzymes catalyse the cleavage of DNA with the particular sequences of
nucleotides regardless of the type of organism from which the DNA came.
 All DNA consists of the same nucleotides and the restriction enzyme produces the same
complementary single-stranded ends.
 This means that complementary single-stranded ends from DNA of different organisms can
be joined thereby creating recombinant DNA molecules.

Steps in cloning a gene

Watch both videos
Steps in cloning a gene

https://www.youtube.com/watch?v=DiUH4nCUSV0

Key steps of Molecular CLoning

https://www.youtube.com/watch?v=sjwNtQYLKeU
Applications and Techniques of Genetic Engineering in Plants

Genetic engineering allows the breeder to select a particular gene required for a desired
characteristic and modify only that gene. Examples include:

 Conferring resistance of specific herbicides

 Conferring resistance to insect pests and microbial diseases (e.g. Caterpillars, virus-resistant
papaya)
 Improvements for marketing (e.g. delayed ripening in tomato)

The enzyme polygalacturonase breaks down cell walls during tomato ripening. If polygalacturonase
is inhibited, tomatoes can be harvested when partially ripe, transported to market in a very firm
state then ripened for sale.

The bacterium Agrobacterium tumnifaciens is one of the systems used in the genetic engineering of
plants. It can transfer part of its Ti (T-DNA) into the host plant genome. Foreign genes can be
inserted into the Ti plasmid DNA. The foreign genes are integrated into the plant genome when the
plasmid enters plant cells. Plant cells within the foreign gene can be identified. Adult plants are
reconstituted from the plant cells and the new genetic material is transmitted to offspring as a
Mendelian trait.
Human Gene Therapy (HGT)

HGT is defined as the transfer of DNA encoding a therapeutic gene to the somatic cells of a
patient in order to treat a disease.

The therapy works either by correcting genetic defects or by expressing proteins that are
therapeutically useful. HGT can also be described as the treatment or prevention of disease by gene
transfer.

HGT is regarded as a potential revolution in medicine. This is because gene therapies target
the causes of disease, whereas most drugs treat the symptoms. Gene therapy has found therapeutic
applications in cancer, infectious diseases and degenerative disorders. Although gene transfer into
humans has been demonstrated in several clinical trials, there is still no single study showing a cure
or consistent benefit for the patient.

Effective gene therapy requires the target transfer if DNA into the human cells and the
subsequent regulated expression of the corresponding gene product. Additional research is needed
to determine the future role of gene therapy.

HGT is a complex process, involving multiple steps in the human body e.g. delivery to organs,
tissue targeting, regulation of gene expression level, regulation of the biological activity of
therapeutic protein), as well as issues involving safety of the vector and the gene product. Not all of
the processes and issues are completely understood.

The prerequisites of successful HGT include therapeutically suitable genes with a proven role in the
development of the disease and appropriate gene delivery systems. The most promising areas for
the use of gene therapy today are the treatment of haemophilia and cardiovascular diseases.

Diseases such as cancer require further development in gene delivery vectors and gene
expression systems. It is important to note that there is no universal vector – each therapy may
require a specific individual vector and therefore a specific set of problems to be overcome.
In vivo and Ex vivo Methods of Gene Therapy

 In ex vivo gene therapy cells are withdrawn from the patient and DNA is transferred into
them before the cells are returned to the patient. In in vivo gene therapy the changes to the
cells of the patient are done by means of vectors inside the patient’s body.

 Experimental in vivo gene therapy has been applied to patients with lung cancer. A
retrovirus carrying a gene that causes cells to die was injected directly into tumours. This
therapy resulted in shrinking of the tumours or tumours not growing larger.