GENETICS

NOTES

Compiled by TM Kathurima®

[PhD]

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TABLE OF CONTENTS
CHROMOSOMES AND GENES ....................................................................................................................... 3
MENDEL'S LAWS OF INHERITANCE ............................................................................................................... 5
TRANSMISSION GENETICS............................................................................................................................. 5
BLENDING THEORY OF INHERITANCE ........................................................................................................... 5
MENDELS EXPERIMENTS ............................................................................................................................... 5
REASONS FOR MENDEL’S SUCCESS ............................................................................................................... 6
GENE LINKAGES AND MAPPING.................................................................................................................... 9
SEX DETERMINATION .................................................................................................................................. 10
GENE INTERACTIONS................................................................................................................................... 11
BLOOD GROUPING ...................................................................................................................................... 12
TYPES OF GENETIC VARIATIONS ................................................................................................................. 12
SOURCES OF VARIATION ............................................................................................................................. 13
MUTATIONS, TYPES AND EFFECTS .............................................................................................................. 14
GENETIC ENGINEERING ............................................................................................................................... 15
GENE POOL.................................................................................................................................................. 15
APPLICATIONS OF GENE CLONING ............................................................................................... 18
TRANSGENIC ANIMALS ............................................................................................................................... 19
ECONOMIC IMPORTANCE OF TRANSGENIC ORGANISMS .......................................................................... 19
PLANT BREEDING ........................................................................................................................................ 20

Course Outline

 Chromosomes and genes

 Mendel's laws of inheritance
 Sex determination
 Gene linkage and mapping
 Gene interactions
 polygenic inheritance
 Blood grouping
 Genetic variations
 Sources of variation
 Genetic Engineering
 Transgenic Animals
 Economic Importance of Transgenic Organisms
 Plant breeding

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CHROMOSOMES AND GENES
Chromosomes are long, coiled-up strands of DNA that contain the genetic information (genes) of an
organism. DNA is a long, double-stranded molecule that carries the genetic instructions that determine
the physical and biological traits of an organism. In eukaryotic cells, such as those found in humans and
most animals, the DNA is organized into distinct structures called chromosomes.

Each chromosome is made up of genes, which are specific sequences of DNA that provide instructions for
building proteins. Proteins are the building blocks of cells and are responsible for many of the functions
in the body. Genes control the expression of proteins by providing the instructions for their synthesis.

The number of chromosomes and genes varies among different organisms. Humans have 23 pairs of
chromosomes, for a total of 46 chromosomes, and an estimated 20,000 to 25,000 genes. Other organisms
may have more or fewer chromosomes and genes, depending on their evolutionary history and the
complexity of their biological systems.

Chromosomal inheritance refers to the process by which genetic information, carried on chromosomes,
is passed from one generation to the next. In most organisms, including humans, chromosomes come in
pairs. One chromosome in each pair is inherited from the mother and the other from the father.

The specific genes carried on these chromosomes determine the physical and biological traits of the
offspring. Some traits are determined by a single gene on a single chromosome, while others are
controlled by multiple genes on different chromosomes.

In humans, certain genetic disorders are caused by abnormalities in the number or structure of
chromosomes. For example, Down syndrome is caused by an extra copy of chromosome 21. Other
chromosomal disorders, such as Turner syndrome and Klinefelter syndrome, are caused by missing or
extra sex chromosomes.

Overall, chromosomal inheritance plays a crucial role in determining the traits and characteristics of
organisms, and understanding the patterns of inheritance can provide important insights into the causes
and treatment of genetic disorders.

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DNA BASE PAIRING

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Mendel's laws of inheritance
Mendel's laws of inheritance are a set of principles that describe how genetic traits are passed from one
generation to the next. These laws were discovered by the Austrian monk Gregor Mendel, who conducted
experiments on pea plants in the mid-1800s.

TRANSMISSION GENETICS
The transmission of characters (genes) from one generation to the next that is from parents to
offspring is known as heredity.
The study of heredity is ‘Genetics’. New individual develop according to the genes inherited from
their parents.
Siblings from same parents are unique and differ from each other except the identical twins. Such
differences are termed variations.
Variation means differences between parents and offspring or between offspring of same parents
or between members of the same population.
Mendelian genetics is the term generally used in discussing the elementary aspects of inheritance
in organisms with more than one chromosome.
The term Mendelian genetics honors the Austrian monk Gregor Mendel (1822-1884) the person
who first made rigorous quantitative observations of the patterns of inheritance and proposed
mechanisms to explain them.

Mendel began his experiments at a time when various theories about heredity had been proposed.
For example the blending theory of inheritance was believed by many.

BLENDING THEORY OF INHERITANCE

This stated that parents of contrasting appearance always produce offspring of intermediate
appearance. It acknowledged that both sexes contribute equally to a new individual. Therefore
according to this theory a cross between plants with red flowers and plants with white flowers
would yield only plants with pink flowers. When red and white flowers reappeared in future
generations, the breeders attributed this to instability in the genetic material.

MENDELS EXPERIMENTS
From 1858 to 1866, he bred garden peas (Pisum sativum) in his monastery garden and analyzed
the offspring of these matings.
He chose garden pea because:
1. Many true- breeding varieties with differing traits were available
2. They were easy to cultivate
3. They have a short generation time
4. They could be cross-pollinated even though they normally self pollinate

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Many scientists before Mendel had performed similar experiments on plants but none had
produced results which had the accuracy and detail of Mendel’s nor were they able to explain their
results in terms of a mechanism of inheritance.

REASONS FOR MENDEL’S SUCCESS

1) Preliminary investigations were carefully carried out to obtain familiarity with the
experimental organism.
2) All experiments were carefully planned so attention was focused on only one variable at
any time thus simplifying the observations to be made.
3) Meticulous care was taken in carrying out the techniques, thus preventing the introduction
of other variables.
4) Accurate records were kept of all the experiments and the results obtained.
5) Sufficient data were obtained to have statistical significance.

Mendel concentrated on seven pairs of contrasting traits i.e.

o seed shape (round vs wrinkled)

o seed colour (green vs yellow)
o flower colour (white vs purple)
o stem length (tall vs dwarf)
o Flower position (axial vs terminal)
o Pod colour (green vs yellow)
o Pod shape (inflated vs constricted)

In some experiments the F1 were allowed to self-pollinate & thus produce a second filial (F2)
generation.
He performed reciprocal crosses: 1st he dusted the pollen of tall plants on the stigmas of short
plants and then he dusted the pollen of short plants on stigmas of tall plants. In both cases, all F1
offspring resembled the tall parents.

MONOHYBRID CROSS

 These consists of crosses of plants differing in one of the contrasting pair of traits.

Example of Monohybrid Cross

– Tall or dwarf, round or wrinkled etc.

 A cross was made by taking the pollen grains from one type of plant to fertilise the egg of
the other and then collecting the offspring’s in the form of seeds.

Parents Round X Wrinkled

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 AA X aa

Gametes A X a

Offsprings Aa Round

Selfing F1 Aa X Aa

AA Aa Aa aa

Offsprings F2 = 3 : 1 ratio
3 round
1 wrinkled
The ratio of dominant to recessive phenotypes of 3: 1 is called the monohybrid ratio.

 Mendel's monohybrid experiments always resulted to a 3:1 ratio in F2 generation.

 He sought an explanation for these results.
 A 3: 1 ratio among the F2 offspring was possible if:

1. The F1 generation contained 2 separate copies of each hereditary factor, one of these being
dominant and one being recessive.

2. The factors separate when the gametes were formed and each gamete carried only one copy of
each factor

3 Random fusion of all possible gametes occurred upon fertilization

In this way Mendel arrived at the first of his laws of inheritance

There are three main laws of Mendelian inheritance:

1. Law of Segregation: This law states that pairs of genes (alleles) separate during gamete formation,
such that each gamete receives only one allele from each pair. This explains why offspring inherit
one copy of each gene from each parent.

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 2. Law of Independent Assortment: This law states that the inheritance of one gene is not
dependent on the inheritance of another gene. In other words, the assortment of alleles for one
gene is independent of the assortment of alleles for other genes. This principle explains why the
traits of offspring can be unpredictable, even when considering multiple genes.

3. Law of Dominance: This law states that when two different alleles are present in an individual,
one allele may mask the expression of the other allele, which is known as the dominant allele. The
other allele, which is not expressed, is known as the recessive allele.

Mendel's laws of inheritance provide the basis for our understanding of genetic inheritance and are still
used today in the study of genetics.

DIHYBRID CROSS

 Mendel extended his work by including 2 pairs of contrasting characters in his crosses which were
called Dihybrid.

 The process of crossing remained the same as above.

 The first generation (F1) were selfed or self- fertilized to give rise to second generation (F2)
progeny.

 Mendel's dihybrid experiments showed 9:3:3:1 ratios in F2 generation. This is known as the
dihybrid ratio.

After analysing the dihybrid cross Mendel came up with the second law

Law of Independent Assortment / Inheritance Law (Second law)

Two alleles of a gene assort independently of other alleles during gamete formation.

ANALYZING A CROSS WITH A PUNNETT SQUARE

R. C. Punnett devised a method of analyzing the possible combinations of genes in zygotes which is called
a Punnett square. The gametes of one parent are written along the top line of the square & the gametes
of the parents are written on the left side of the squares. The alleles of each parental gametes are written
in the appropriate boxes which then represent the offspring genotypes.

Round Yellow X Wrinkled Green

AA BB X aa bb

AB Ab aB ab

AB AABB AABb AaBB AaBb

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 Ab AABb AAbb AaBb Aabb

aB AaBB AaBb aaBB aaBb

Ab AaBb Aabb aaBb aabb

Round Yellow = 9

Round Green = 3

Wrinkled Yellow = 3

Wrinled Green = 1

Gene linkages and mapping

Gene linkage refers to the tendency of certain genes on the same chromosome to be inherited together
because they are physically located close to each other on the chromosome. The degree of gene linkage
is measured by the frequency at which genes are inherited together.

Gene mapping is the process of determining the location of genes on a chromosome and the relative
distance between them. Gene mapping can be done using a variety of techniques, such as pedigree
analysis, genetic crosses, and molecular markers.

One of the most common methods of gene mapping is through the use of genetic crosses, such as the
dihybrid cross. By observing the inheritance patterns of two genes on the same chromosome, researchers
can determine the degree of gene linkage and the distance between the genes.

Gene mapping can also be done using molecular markers, such as restriction fragment length
polymorphisms (RFLPs) and single nucleotide polymorphisms (SNPs). These markers can be used to
identify specific regions of a chromosome and to track the inheritance of genes and traits.

Overall, gene linkage and mapping are important tools for understanding the inheritance of genetic traits
and for identifying the genes responsible for specific traits and disorders.

Gene linkage refers to the tendency of genes located close to each other on the same chromosome to be
inherited together during reproduction. It was first discovered by Thomas Hunt Morgan through his
experiments with fruit flies.

Gene Linkage and Mapping:

1. Linkage and crossing over: Gene linkage occurs because genes located close together on the same
chromosome have a lower chance of undergoing crossing over, the exchange of genetic material
between homologous chromosomes during meiosis. Crossing over can separate linked genes, but
the frequency of crossing over between linked genes is generally low.

2. Linkage mapping: Linkage mapping is a technique used to determine the relative positions of
genes on a chromosome. It is based on the principle that the closer two genes are to each other,

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 the less likely they are to undergo crossing over and thus the more likely they are to be inherited
together.

3. Genetic markers: Genetic markers, such as visible traits or DNA sequences with known locations,
are used in linkage mapping. By analyzing the patterns of inheritance of these markers in a
population, researchers can infer the positions of genes of interest.

4. Linkage maps: Linkage maps are graphical representations of the relative positions of genes on a
chromosome. They are constructed based on the frequencies of recombination events between
genes and genetic markers.

5. Centimorgan (cM): The centimorgan (cM) is a unit of measurement used in linkage mapping. One
centimorgan represents a recombination frequency of 1% between two genes or genetic markers.

6. Linkage groups: Genes located on the same chromosome form a linkage group. The number of
linkage groups in an organism is equal to the number of different chromosomes.

7. Three-point mapping: Three-point mapping is a method used to determine the order and
distances between three genes on a chromosome. It involves analyzing the recombination
frequencies between the three genes and using these data to construct a linear gene map.

8. Map units: Map units, also known as centimorgans, are used to measure the distances between
genes on a linkage map. One map unit is equal to a recombination frequency of 1% between two
genes.

9. Linkage disequilibrium: Linkage disequilibrium refers to the non-random association of alleles at

different loci. It occurs when certain combinations of alleles at different loci are more or less
frequent than would be expected by chance.

10. Applications of gene linkage and mapping: Gene linkage and mapping have important applications
in genetics and genomics, such as identifying disease-causing genes, determining the locations of
genetic markers used in genetic testing, and understanding the genetic basis of complex traits.

Sex determination
Sex determination is the process by which an organism is designated male or female. In most organisms,
including humans, sex is determined by the presence of sex chromosomes.

In humans, sex chromosomes are designated as X and Y. Females have two X chromosomes (XX), while
males have one X and one Y chromosome (XY). The Y chromosome contains a gene called SRY (sex-
determining region Y), which triggers the development of male characteristics during fetal development.

In other organisms, sex may be determined by other mechanisms, such as temperature-dependent sex
determination in some reptiles, or by the presence of certain sex-determining genes in some plants and
animals.

Sex determination can also be influenced by environmental factors, such as nutrition or social factors,
which can affect the expression of sex-related genes or hormones.

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Overall, sex determination is a complex process that involves a combination of genetic and environmental
factors and is critical for the development and survival of organisms.

Almost all animals fall under 4 systems of sex determination.

System of sex Organism Somatic cells Sex cells Heterogametic

determination gender

XY – XX Mammals, XX XY X X or Y male
drosophila

ZW – ZZ Birds, ZW ZZ Z or W Z female
butterflies,
fish

XO – XX Bedbugs, XX XO X X or O male
crickets,
grass
hoppers

ZO – ZZ moth ZO ZZ Z or O Z female

Gene interactions
Gene interactions refer to the way in which different genes can interact with each other to produce a
particular trait or phenotype. There are several types of gene interactions:

1. Complementary gene interaction: In this type of interaction, two genes work together to produce
a single trait. For example, in some plants, two different genes control the color of the flowers,
and only when both genes are present can the flowers have a particular color.

2. Epistatic gene interaction: In this type of interaction, one gene masks or suppresses the
expression of another gene. For example, in mice, a gene that controls coat color can be
suppressed by another gene that regulates the production of pigment.

3. Modifier gene interaction: In this type of interaction, one gene modifies the expression of
another gene, resulting in a different phenotype. For example, in humans, a gene that controls
the production of melanin (the pigment responsible for skin color) can be modified by other
genes, resulting in different skin tones.

4. Pleiotropic gene interaction: In this type of interaction, a single gene can affect multiple traits or
phenotypes. For example, a gene that regulates the production of insulin can also affect the
development of other organs and tissues.

Understanding gene interactions is important for predicting the inheritance of certain traits and for
identifying the genetic basis of complex diseases and disorders.

polygenic inheritance

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Polygenic inheritance refers to the inheritance of traits that are controlled by multiple genes, each
contributing a small amount to the overall phenotype. These traits are often quantitative in nature,
meaning they can be measured on a continuous scale, such as height, weight, or skin color.

In polygenic inheritance, the more genes that contribute to a trait, the greater the number of possible
phenotypes. This results in a range of phenotypes, rather than discrete categories. For example, height is
a trait that is controlled by multiple genes, and individuals can vary in height from very short to very tall.

Polygenic traits are often influenced by environmental factors, such as nutrition or exposure to certain
chemicals. The interaction between genes and the environment can have a significant impact on the
expression of these traits, making them difficult to predict.

Polygenic inheritance is common in many organisms, including humans, and is thought to be responsible
for the variation observed in many complex traits, such as intelligence, personality, and susceptibility to
diseases.

Understanding polygenic inheritance is important for predicting the inheritance of complex traits,
identifying the genetic basis of diseases and disorders, and developing new treatments and therapies for
genetic conditions.

Blood grouping
Blood grouping is a system used to classify blood into different groups based on the presence or absence
of certain proteins or antigens on the surface of red blood cells. The two most important blood grouping
systems are the ABO system and the Rh system.

The ABO blood group system is based on the presence or absence of two antigens, A and B, on the surface
of red blood cells. There are four possible blood types in the ABO system: A, B, AB, and O. Individuals with
blood type A have the A antigen on their red blood cells, those with blood type B have the B antigen, those
with blood type AB have both A and B antigens, and those with blood type O have neither A nor B antigens.
In addition, individuals with blood type A or B have antibodies to the antigens they lack (anti-B or anti-A,
respectively) in their plasma, while individuals with blood type AB have neither antibody and those with
blood type O have both antibodies.

The Rh blood group system is based on the presence or absence of the Rh antigen on the surface of red
blood cells. Individuals who have the Rh antigen are said to be Rh positive (Rh+), while those who do not
have the antigen are Rh negative (Rh-).

Blood grouping is important in medicine, particularly in blood transfusions and organ transplantation. For
example, individuals with blood type A can receive blood from individuals with blood types A and O, but
not from individuals with blood types B or AB, while individuals with blood type B can receive blood from
individuals with blood types B and O, but not from individuals with blood types A or AB. Individuals with
blood type AB can receive blood from any blood type, while individuals with blood type O can only receive
blood from individuals with blood type O. In addition, Rh- individuals must receive Rh- blood, while Rh+
individuals can receive Rh+ or Rh- blood.

Types of genetic variations

There are several types of genetic variations that can occur in an organism's DNA:

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 1. Single nucleotide polymorphisms (SNPs): These are the most common type of genetic variation
and involve a change in a single nucleotide, or DNA "letter". SNPs can occur in both coding and
non-coding regions of DNA, and they can influence an organism's susceptibility to certain diseases
or affect how they respond to medications.

2. Insertions and deletions (indels): These involve the addition or removal of one or more
nucleotides in the DNA sequence. Indels can have a significant impact on the function of genes,
particularly if they occur in coding regions.

3. Copy number variations (CNVs): These involve a change in the number of copies of a particular
DNA sequence. CNVs can have a significant impact on an organism's phenotype, particularly if
they affect genes involved in development or disease susceptibility.

4. Chromosomal rearrangements: These involve a change in the structure of chromosomes, such as

translocations, inversions, or deletions. Chromosomal rearrangements can have a significant
impact on gene expression, particularly if they affect the location or orientation of genes.

5. Mitochondrial DNA variations: Mitochondrial DNA is a separate DNA molecule that is inherited
only from the mother. Variations in mitochondrial DNA can affect an organism's metabolism and
energy production.

Understanding genetic variations is important for predicting an individual's risk of developing certain
diseases and for developing new treatments and therapies for genetic conditions. Genetic variations can
also be used to trace ancestry and to study the evolution and diversity of different species.

Sources of variation
There are several sources of genetic variation, including:

1. Mutation: This is a spontaneous change in the DNA sequence that can occur during DNA
replication, repair, or recombination. Mutations can be caused by errors in DNA replication,
exposure to mutagens (such as radiation or chemicals), or by natural processes such as
transposable elements. Mutations can result in a range of genetic variations, including single
nucleotide polymorphisms (SNPs), insertions and deletions (indels), and chromosomal
rearrangements.

2. Genetic recombination: This is the process by which genetic material is exchanged between
homologous chromosomes during meiosis. Recombination can result in the creation of new
combinations of alleles and can increase genetic diversity.

3. Gene flow: This occurs when genetic material is exchanged between different populations of the
same species. Gene flow can result in the transfer of new alleles into a population and can
increase genetic diversity.

4. Genetic drift: This is the random fluctuation of allele frequencies in a population due to chance
events. Genetic drift can result in the loss of rare alleles and can reduce genetic diversity,
particularly in small populations.

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 5. Natural selection: This is the process by which certain traits are favored or selected for in a
population, typically because they confer a selective advantage in a particular environment.
Natural selection can result in the increase or decrease of certain alleles in a population and can
lead to the evolution of new traits over time.

Understanding the sources of genetic variation is important for studying the genetics of populations and
for predicting an individual's risk of developing certain diseases or conditions. Genetic variation can also
be used to trace ancestry and to study the evolution and diversity of different species.

Mutations, types and effects

Mutations are changes in the DNA sequence that can occur spontaneously or as a result of exposure to
mutagens (such as radiation or chemicals). Mutations can be classified based on the type of change they
cause in the DNA sequence.

1. Point mutations: These are mutations that involve a change in a single nucleotide in the DNA
sequence. Point mutations can be further classified into three types:

a. Silent mutations: These are mutations that do not change the amino acid sequence of the protein
encoded by the gene, because the change in nucleotide still codes for the same amino acid.

b. Missense mutations: These are mutations that change a single nucleotide in the DNA sequence,
resulting in a change in the amino acid sequence of the protein encoded by the gene.

c. Nonsense mutations: These are mutations that change a single nucleotide in the DNA sequence,
resulting in a premature stop codon, and therefore the resulting protein is incomplete.

2. Insertions and deletions: These are mutations that involve the insertion or deletion of one or
more nucleotides in the DNA sequence. This can cause a shift in the reading frame of the gene,
leading to a completely different amino acid sequence.

3. Chromosomal mutations: These are mutations that involve changes in the structure or number
of chromosomes. These mutations can be further classified into several types, including:

a. Deletions: These are mutations that involve the loss of a segment of the chromosome.

b. Duplications: These are mutations that involve the duplication of a segment of the chromosome.

c. Inversions: These are mutations that involve the reversal of the order of a segment of the chromosome.

d. Translocations: These are mutations that involve the movement of a segment of one chromosome to
another chromosome.

The effects of mutations depend on several factors, including the type of mutation, its location in the
genome, and its impact on the function of the protein encoded by the gene. Some mutations may have
no effect on the protein's function or may result in a completely different protein, while others may
disrupt the normal functioning of the protein, leading to a variety of genetic disorders or diseases, such
as cystic fibrosis, sickle cell anemia, and cancer. However, it's important to note that not all mutations are
harmful; some may even be beneficial and lead to evolutionary adaptations.

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GENETIC ENGINEERING
Genetic engineering is the process of manipulating the genetic material of an organism to modify its
characteristics in a desired way. This is achieved by altering the DNA sequence of an organism through
the introduction, deletion, or modification of specific genes.

The basic steps involved in genetic engineering are as follows:

1. Isolation of DNA: The DNA of interest is extracted from the organism that contains the gene of
interest.

2. Gene cloning: The gene of interest is amplified by cloning it into a vector, such as a plasmid or a
virus.

3. Gene transfer: The vector carrying the cloned gene is introduced into the host organism, where
it integrates into the host genome and is expressed.

4. Expression of the gene: The cloned gene is expressed in the host organism, resulting in the
production of the desired protein or trait.

Genetic engineering has numerous applications in various fields, including medicine, agriculture, and
biotechnology. For example, genetic engineering can be used to produce medicines, such as insulin,
growth hormone, and vaccines, by introducing the genes for these proteins into bacteria or other
organisms that can be easily grown and harvested.

In agriculture, genetic engineering can be used to produce crops with desired traits, such as increased
yield, improved nutritional value, and resistance to pests and diseases. This is achieved by introducing
genes from other organisms, such as bacteria or other plants, into the crop plants.

Genetic engineering has also been used in biotechnology to produce enzymes and other proteins for
various industrial applications, such as the production of detergents, textiles, and biofuels.

While genetic engineering has many potential benefits, it also raises ethical and safety concerns,
particularly with regard to the release of genetically modified organisms into the environment and their
potential impact on ecosystems and human health. Therefore, strict regulations and guidelines are in
place to ensure the safe and responsible use of genetic engineering techniques.

GENE POOL
The gene pool refers to the complete set of genes and their different variants (alleles) that are present in
a particular population of a species. It is the sum total of all the genetic information within a population
that can be passed on to the next generation through sexual reproduction.

The gene pool is dynamic and can change over time due to various factors, including genetic drift, gene
flow, mutations, and natural selection. Genetic drift refers to the random fluctuations in allele frequencies
that occur in small populations, while gene flow refers to the movement of individuals or genes between
populations. Mutations introduce new genetic variation into the gene pool, while natural selection can
act on this variation, leading to changes in the frequency of alleles over time.

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The gene pool plays a crucial role in determining the genetic diversity and evolutionary potential of a
population. Populations with high levels of genetic diversity are more likely to be able to adapt to changing
environmental conditions and are less susceptible to the negative effects of inbreeding, such as reduced
fitness and increased risk of genetic disorders.

The study of the gene pool and the factors that influence its composition is an important area of research
in evolutionary biology and population genetics. Understanding the gene pool of a population can provide
valuable insights into its genetic health, evolutionary history, and potential for future adaptation.

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A. Isolation of the DNA fragment or gene
 The target DNA or gene to be cloned must be first isolated. A gene of interest is a fragment
of gene whose product (a protein, enzyme or a hormone) interests us. For example, gene
encoding for the hormone insulin.
 The desired gene may be isolated by using restriction endonuclease (RE) enzyme, which cut
DNA at specific recognition nucleotide sequences known as restriction sites towards the inner
region (hence endonuclease) producing blunt or sticky ends.
 Sometimes, reverse transcriptase enzyme may also be used which synthesizes complementary
DNA strand of the desired gene using its mRNA.
B. Selection of suitable cloning vector
 The vector is a carrier molecule which can carry the gene of interest (GI) into a host, replicate
there along with the GI making its multiple copies.
 The cloning vectors are limited to the size of insert that they can carry. Depending on the size
and the application of the insert the suitable vector is selected.
 The different types of vectors available for cloning are plasmids, bacteriophages, bacterial
artificial chromosomes (BACs), yeast artificial chromosomes (YACs) and mammalian
artificial chromosomes (MACs).
 However, the most commonly used cloning vectors include plasmids and bacteriophages
(phage λ) beside all the other available vectors.
C. Essential Characteristics of Cloning Vectors
All cloning vectors are carrier DNA molecules. These carrier molecules should have few common
features in general such as:
 It must be self-replicating inside host cell.
 It must possess a unique restriction site for RE enzymes.
 Introduction of donor DNA fragment must not interfere with replication property of the vector.
 It must possess some marker gene such that it can be used for later identification of
recombinant cell (usually an antibiotic resistance gene that is absent in the host cell).
 They should be easily isolated from host cell.
D. Formation of Recombinant DNA
 The plasmid vector is cut open by the same RE enzyme used for isolation of donor DNA
fragment.
 The mixture of donor DNA fragment and plasmid vector are mixed together.
 In the presence of DNA ligase, base pairing of donor DNA fragment and plasmid vector
occurs.
 The resulting DNA molecule is a hybrid of two DNA molecules – the GI and the vector. In
the terminology of genetics this intermixing of different DNA strands is called recombination.
 Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and the
technology is referred to as the recombinant DNA technology.
E. Transformation of recombinant vector into suitable host
 The recombinant vector is transformed into suitable host cell mostly, a bacterial cell.
 This is done either for one or both of the following reasons:
 To replicate the recombinant DNA molecule in order to get the multiple copies of the GI.
 To allow the expression of the GI such that it produces its needed protein product.
 Some bacteria are naturally transformable; they take up the recombinant vector automatically.
For example: Bacillus, Haemophillus, Helicobacter pylori, which are naturally competent.

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 Some other bacteria, on the other hand require the incorporation by artificial methods such as
Ca++ ion treatment, electroporation, etc.
F. Isolation of Recombinant Cells
 The transformation process generates a mixed population of transformed and non-trans-
formed host cells.
 The selection process involves filtering the transformed host cells only.
 For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector
is employed.
 For examples, PBR322 plasmid vector contains different marker gene (Ampicillin resistant
gene and Tetracycline resistant gene. When pst1 RE is used it knock out Ampicillin resistant
gene from the plasmid, so that the recombinant cell become sensitive to Ampicillin.
G. Multiplication of Selected Host Cells
 Once transformed host cells are separated by the screening process; becomes necessary to
provide them optimum parameters to grow and multiply.
 In this step the transformed host cells are introduced into fresh culture media .
 At this stage the host cells divide and re-divide along with the replication of the recombinant
DNA carried by them.
 If the aim is obtaining numerous copies of GI, then simply replication of the host cell is
allowed. But for obtaining the product of interest, favourable conditions must be provided
such that the GI in the vector expresses the product of interest.
H. Isolation and Purification of the Product
 The next step involves isolation of the multiplied GI attached with the vector or of the protein
encoded by it.
 This is followed by purification of the isolated gene copy/protein.

Applications of Gene Cloning

 A particular gene can be isolated and its nucleotide sequence determined
 Control sequences of DNA can be identified & analyzed
 Protein/enzyme/RNA function can be investigated

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 Mutations can be identified, e.g. gene defects related to specific diseases Organisms can be
‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc

TRANSGENIC ANIMALS
Transgenic animals are animals that have been genetically modified to carry one or more foreign genes
that have been introduced into their genome using genetic engineering techniques. The foreign genes are
usually derived from another species and are introduced into the animal's genome to modify its
characteristics in a desired way.

The process of creating transgenic animals typically involves the following steps:

1. Isolation of the foreign gene: The foreign gene of interest is isolated from the donor species and
cloned into a vector, such as a plasmid or a virus.

2. Introduction of the foreign gene into the animal: The vector carrying the foreign gene is
introduced into the animal's genome through various methods, such as microinjection,
electroporation, or viral transduction.

3. Integration of the foreign gene into the animal's genome: The foreign gene is integrated into the
animal's genome through homologous recombination or random integration.

4. Expression of the foreign gene: The foreign gene is expressed in the animal, resulting in the
production of the desired protein or trait.

Transgenic animals have many applications in various fields, including medicine, agriculture, and
biotechnology. For example, transgenic animals can be used to produce medicines, such as insulin and
growth hormone, by introducing the genes for these proteins into animals that can produce them in large
quantities.

In agriculture, transgenic animals can be used to produce food with desirable traits, such as improved
nutritional value or increased resistance to disease. For example, transgenic cows have been produced
that produce milk containing human proteins that can be used in medicine.

However, the use of transgenic animals also raises ethical and safety concerns, particularly with regard to
the welfare of the animals and the potential impact on ecosystems if the animals were to escape or be
released into the environment. Therefore, strict regulations and guidelines are in place to ensure the safe
and responsible use of transgenic animal technologies.

ECONOMIC IMPORTANCE OF TRANSGENIC ORGANISMS

Transgenic organisms have significant economic importance in various fields, including agriculture,
medicine, and biotechnology. Some of the economic benefits of transgenic organisms are:

1. Increased crop yields: Transgenic plants can be modified to be resistant to pests, diseases, and
environmental stresses, resulting in increased crop yields and improved food security.

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 2. Reduced use of pesticides: Transgenic crops that are resistant to pests and diseases require less
pesticide use, reducing the cost of crop production and minimizing the environmental impact of
chemical pesticides.

3. Improved nutritional value: Transgenic crops can be engineered to have improved nutritional
value, such as increased levels of vitamins and minerals, leading to improved health and reduced
healthcare costs.

4. Lower production costs: Transgenic organisms can be used to produce valuable products, such as
pharmaceuticals, at a lower cost than traditional methods, leading to reduced healthcare costs.

5. Improved animal health and productivity: Transgenic animals can be engineered to have
improved resistance to disease and increased productivity, resulting in reduced animal healthcare
costs and increased profits for farmers.

6. Reduced environmental impact: Transgenic organisms can be engineered to be more

environmentally friendly, such as crops that require less water or produce less greenhouse gases,
resulting in reduced environmental impact and lower costs.

Despite the potential economic benefits of transgenic organisms, there are also concerns regarding their
safety and environmental impact. Therefore, strict regulations and guidelines are in place to ensure their
safe and responsible use.

Plant breeding
Plant breeding involves the deliberate manipulation of plant species to develop new varieties with
desirable traits. Here are some key points to note about plant breeding:

1. Objective: The primary goal of plant breeding is to develop new plant varieties with improved
characteristics such as higher yield, disease resistance, stress tolerance, nutritional value, flavor,
or adaptability to specific environments.

2. Selection: Plant breeders use selection techniques to identify and propagate plants with desired
traits. This involves carefully choosing individual plants or lines based on their phenotypic or
genotypic characteristics and using them as parents for further breeding.

3. Genetic Variation: Genetic variation is the foundation of plant breeding. Breeders seek out and
utilize the natural genetic diversity present within plant populations to introduce new traits and
enhance overall genetic quality.

4. Crossbreeding: Crossbreeding involves mating two different plants with desirable traits to
combine their genetic traits in the offspring. This technique helps bring together different
beneficial alleles and create diverse populations.

5. Hybridization: Hybridization is a specific form of crossbreeding involving the crossing of two

genetically distinct, highly inbred parental lines. Hybrids often display improved vigor, yield, or
other desirable traits compared to their parental lines.

6. Selfing: Selfing or self-pollination is the process of allowing a plant to fertilize its own flowers. This
technique helps fix desirable traits in a population and establish true-breeding lines.

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 7. Inbreeding and Outbreeding: Inbreeding involves mating closely related plants to concentrate
desired traits and increase homozygosity, while outbreeding involves mating unrelated or
distantly related plants to introduce new genetic variation and enhance vigor.

8. Selection Methods: Plant breeders employ various selection methods, such as mass selection,
pedigree selection, and recurrent selection, to choose superior individuals with desired traits for
further breeding and development of new varieties.

9. Molecular Techniques: Advances in molecular biology and biotechnology have revolutionized

plant breeding. Techniques like marker-assisted selection (MAS) and genetic engineering allow
for more precise and efficient selection and manipulation of specific genes or traits.

10. Testing and Evaluation: New plant varieties undergo rigorous testing and evaluation in field trials,
controlled environments, and experimental plots to assess their performance, stability,
adaptability, and overall agronomic traits.

11. Intellectual Property Rights: Plant breeding can involve the development of novel plant varieties,
and breeders may seek intellectual property protection through plant patents, plant variety
protection, or other forms of intellectual property rights.

12. Importance: Plant breeding plays a crucial role in addressing global challenges such as food
security, climate change adaptation, sustainable agriculture, and the development of crops with
improved nutritional value or tolerance to biotic and abiotic stresses.

Overall, plant breeding combines scientific knowledge, genetic resources, and breeding techniques to
develop new plant varieties that meet the evolving needs of agriculture and society.

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