BCH 405 Genetic Engineering and Biotechnology

Credit Units:2 Course Status Compulsory Session:2023/2024

• Gene mutation, mutagenic agents and their applications to gene-

transfer

• Bacterial Artificial Chromosomes and Yeast Artificial Chromosomes

• Protein expression and Electrophoretic technique in molecular

biology
 GENE MUTATION, MUTAGENIC AGENTS AND THEIR
APPLICATIONS TO GENE-TRANSFER
Terminologies
• Genes: The gene is referred to as the basic unit of inheritance. Genes are generally
passed from parents to offspring and they contain the information needed to indicate
physical and biological traits. Most genes code for specific proteins, or segments of
proteins, which have differing functions within the body. They are segments of
DNA located on chromosomes .

• DNA: This is generally referred to as Deoxyribonucleic acid. It is the molecule that

carries genetic information for the development and functioning of an organism.
DNA is made of two linked strands that wind around each other to resemble a
twisted ladder — a shape known as a double helix.

• Chromosomes: Chromosomes are threadlike structures made of protein and a

single molecule of DNA that serve to carry the genomic information from cell to
cell. Scientists have numbered the chromosome pairs from 1 to 22, with the 23rd
pair labeled as X or Y, depending on the structure. The first 22 pairs of
chromosomes are called autosomes. The 23rd pair of chromosomes is known as
the sex chromosomes, because they decide if you will be born male or female.
• Nucleotides: A nucleotide is the basic building block of nucleic acids (RNA and
DNA). A nucleotide consists of a sugar molecule (either ribose in RNA or
deoxyribose in DNA) attached to a phosphate group and a nitrogen-containing base.
The bases used in DNA are adenine (A), cytosine (C), guanine (G) and thymine (T).
Altering nucleotide sequences most often results in non-functioning proteins.
• Mutation: A mutation is a change in the DNA sequence of
an organism. Mutation can result from errors in DNA
replication during cell division, exposure to mutagens or a
viral infection. Mutation cause changes in the genetic
code that lead to genetic variation and a variety of effects.
• Gene mutations: These are changes in single DNA bases,
or small intragenic deletions and rearrangements that can
results into disruption of normal gene function. In other
words, a gene mutation is defined as an alteration in the
sequence of nucleotides in DNA .
• This change can affect a single nucleotide pair or larger
gene segments of a chromosome.
• Gene mutations can be generally categorized into two types:
• Point mutations and
• Base-pair insertions or deletions.
• POINT MUTATIONS

A point mutation is a type of mutation in DNA or RNA, the cell’s

genetic material, in which one single nucleotide base is added, deleted
or changed. DNA and RNA are made up of many nucleotides. There
are five different molecules that can make up nitrogenous bases on
nucleotides: cytosine, guanine, adenine, thymine (in DNA) and uracil
(in RNA), abbreviated C, G, A, T, and U.

Point mutations are sometimes caused by mutations that

spontaneously occur during DNA replication. The rate of mutations
may also increase when a cell is exposed to mutagens, which are
environmental factors that can change an organism’s DNA. Some
mutagens are X-rays, UV rays, extreme heat, or certain chemicals like
 FROM DNA TO PROTEIN

• DNA and RNA have a double helix structure.

• Phosphate groups and 5-carbon sugars make up the backbone, while the middle
of the double helix is formed by pairs of nitrogenous bases.
• Each type of nitrogenous bases pairs with another specific base.
• Cytosine pairs with guanine, while
• Adenine pairs with thymine in DNA and Uracil in RNA, and vice versa.
• In order for DNA to make proteins, it must be transcribed by messenger RNA
(mRNA).
• The mRNA “reads” the DNA three bases at a time, matching its
complementary bases to it.
• These groups of three bases are called codons, and each codon codes for a
different amino acid.
• Chains of amino acids make up proteins. Therefore, it is vitally important that
the DNA has the correct sequence of base pairs in order to make proteins
correctly.
• A single point mutation could have no effect, or it could alter the protein that is
 TYPES OF POINT MUTATIONS

Substitution
• A substitution mutation occurs when one base pair is
substituted for another. For example, this would occur when
one nucleotide containing cytosine is accidentally
substituted for one containing guanine. There are three types
of substitution mutations:
• Nonsense

• Missense

• Silent
 NONSENSE MUTATION

• A nonsense mutation occurs when one nucleotide is

substituted and this leads to the formation of a stop
codon instead of a codon that codes for an amino acid.
A stop codon is a certain sequence of bases (TAG,
TAA, or TGA in DNA, and UAG, UAA, or UGA in
RNA) that stops the production of the amino acid
chain. It is always found at the end of the mRNA
sequence when a protein is being produced, but if a
substitution causes it to appear in another place, it will
prematurely terminate the amino acid sequence and
prevent the correct protein from being produced.
 MISSENSE MUTATION

• A missense mutation occurs when one nucleotide is

substituted and a different codon is formed; but this time,
the codon that forms is not a stop codon. Instead, the
codon produces a different amino acid in the sequence of
amino acids. For example, if a missense substitution
changes a codon from AAG to AGG, the amino acid
arginine will be produced instead of lysine. A missense
mutation is considered conservative if the amino acid
formed via the mutation has similar properties to the one
that was supposed to be formed instead. It is called non-
conservative if the amino acid has different properties
that structure and function of a protein.
 SILENT MUTATION

• In silent mutation, a nucleotide is substituted but the

same amino acid is produced anyway.
• This can occur because multiple codons can code
for the same amino acid.
• For example, AAG and AAA both code for lysine,
so if the G is changed to an A, the same amino acid
will form and the protein will not be affected
 BASE-PAIR INSERTIONS OR DELETIONS

• An insertion changes the DNA sequence by adding one or

more nucleotides to the gene. As a result, the protein made
from the gene may not function properly.
 DELETION
• A deletion changes the DNA sequence by removing at least
one nucleotide in a gene. Small deletions remove one or a
few nucleotides within a gene, while larger deletions can
remove an entire gene or several neighboring genes. The
deleted DNA may alter the function of the affected protein
or proteins.
 DUPLICATION
NOTE: With one or two bases added or deleted, all of the three-base codons change. This is called a frameshift mutation.

• Duplication occurs when a stretch of one or more

nucleotides in a gene is copied and repeated next to the
original DNA sequence. This type of variant may alter the
function of the protein made from the gene.
• NOTE: With one or two bases added or deleted, all of the
three-base codons change. This is called a frameshift
mutation.
 Examples of Diseases Caused by Point Mutations

• CYSTIC FIBROSIS
• Cystic Fibrosis (CF) is a recessive inherited disorder most common
among people of European descent. There are many different
mutations that can cause CF, but the most common one is a deletion
of three nucleotides in the cystic fibrosis transmembrane conductance
regulator (CFTR) gene that results in the loss of the amino acid
phenylalanine and causes an incorrectly folded protein.

• (Note that this deletion is not a frameshift mutation because three

bases next to each other are deleted, and all the other amino acids in
the chain remain the same.) CF is associated with thick, sticky mucus
in the lungs and trouble breathing, salty sweat, infertility in certain
individuals, and a shortened life expectancy (about 42-50 years in
developed countries).
• SICKLE-CELL ANEMIA
• Sickle-cell anemia is a recessive disorder caused by a single
substitution in the gene that creates hemoglobin, which
carries oxygen in the blood. Normally, glutamic acid is
produced in the chain, but the substitution causes valine to
be produced at that spot instead. When people have two
copies of this mutation, it results in thin sickle-shaped blood
cells that sometimes cannot carry oxygen properly
• About 80% of people with sickle-cell disease are in sub-
Saharan Africa, where being a carrier for sickle-cell anemia
(having only one copy of the gene, not two) actually helps
protect against malaria.. Symptoms include anemia,
obstruction of blood vessels, and chest pain, and it is treated
with folic acid, blood transfusions, bone marrow
transplants, and certain prescription drugs
TAY-SACHS
• Tay-Sachs disease is another recessive disorder
caused by point mutations. Different mutations can
cause this disorder, but they are all found on
the HEXA gene on chromosome 15.

• Tay-Sachs causes nerve cells to deteriorate over

time, which in turn results in the decline of physical
and mental functioning. Both child and adult-onset
forms of the disease occur, and children with the
disease usually die before the age of four.
 MUTAGENIC AGENT

• Mutagens are chemical compounds or forms of radiation (such

as ultraviolet (UV) light or X-rays) that cause irreversible and
heritable changes (mutations) in the cellular genetic material,
deoxyribonucleic acid (DNA). Mutagenic lesions persist when
they escape detection by protective cellular DNA repair
mechanisms, when mistakes occur in the repair process, or when
repair mechanisms are overwhelmed by extensive damage.

• In other word, a mutagenic agent is a substance or factor that

can induce genetic mutations. Mutations are changes in the
DNA sequence of an organism, and they can occur
spontaneously or be triggered by external factors, including
mutagenic agents. These agents can lead to alterations in the
genetic code, which may have various effects on an organism's
 Mutagenic agents can be classified into different
categories
• Chemical Mutagens: These include various chemicals that
can interact with DNA and cause changes in its structure.
Examples of chemical mutagens include certain pesticides,
industrial chemicals, and certain components of tobacco
smoke.
• Physical Mutagens: These agents cause mutations by
directly damaging the DNA structure. Examples of physical
mutagens include ionizing radiation (such as X-rays and
gamma rays) and ultraviolet (UV) radiation from the sun.
• Biological Mutagens: Certain biological agents, such as
some viruses and transposons (mobile genetic elements),
can also induce mutations by interacting with the host
organism's DNA.
 APPLICATIONS OF GENE MUTATION AND
MUTAGENIC AGENTS TO GENE-TRANSFER

• Gene Editing and CRISPR-Cas9 Technology:

Gene mutations can be deliberately induced using gene-editing
technologies like CRISPR-Cas9. Researchers can use this tool to
specifically modify definite genes, introducing targeted mutations
or correcting existing ones. This is important for understanding
gene function and developing new therapeutic approaches.
Functional Genomics:
• By inducing mutations in specific genes, scientists can study
the effects of those mutations on the organism. This helps in
elucidating the function of genes and their role in biological
processes. Understanding gene function is critical for
designing effective gene therapies or manipulating traits in
Creation of Transgenic Organisms:
• Mutagenic agents can be used to induce mutations in
particular genes, and this information can be applied to
create transgenic organisms. Transgenic organisms carry
foreign genes that have been purposely introduced,
leading to the expression of desired traits. This is
commonly done in agriculture to create crops with
improved characteristics such as resistance to pests or
tolerance to environmental conditions.
• NOTE: Transgenic refers to an organism or cell whose
genome has been altered by the introduction of one or
more foreign DNA sequences from another species by
artificial means. Transgenic organisms are generated in
the laboratory for research purposes
• Drug Development and Biotechnology:
Understanding the impact of mutations on disease susceptibility can
aid in drug development. By studying the mutations associated with
certain diseases, researchers can identify potential targets for
therapeutic intervention. Furthermore, mutagenesis can be used to
improve the production of proteins in biotechnological processes.
• Evolutionary Studies:
Mutations are a natural source of genetic variation, and understanding
the role of mutations in evolution is important. By studying
mutagenic agents and their effects on genes, scientists can gain
insights into how genetic diversity arises and contributes to the
adaptation and evolution of species.
• Model Organisms for Research:
Mutagenesis can be used to create model organisms with specific
genetic changes that mimic human diseases. These models are very
useful for studying disease mechanisms, testing potential therapies,
and gaining a deeper understanding of the genetic basis of various