GENETIC ENGINEERING

Genetic engineering refers to the direct manipulation of DNA to alter an organism’s

characteristics (phenotype) in a particular way. Genetic engineering, sometimes called genetic
modification is the process of altering the DNA in an organism’s genome.

This may mean changing one base pair (A-T or C-G), deleting a whole region of DNA, or
introducing an additional copy of a gene. It may also mean extracting DNA from another organism’s
genome and combining it with the DNA of that individual.

Genetic engineering is used to enhance or modify the characteristics of an individual organism.

Genetic engineering can be applied to any organism, from a virus to a sheep. For example, genetic
engineering can be used to produce plants that have a higher nutritional value or can tolerate exposure
to herbicides.

HOW DOES GENETIC ENGINEERING WORK?

An example of the process of genetic engineering is as follows – Insulin is a protein that helps
regulate the sugar levels in our blood. Insulin is normally produced in the pancreas, but people with
type1 diabetes have a problem with insulin production. For this reason, people with diabetes therefore
have to inject insulin to control their blood sugar levels.

Genetic engineering has been used to produce a type of insulin, very similar to our own, from
yeast and bacteria like E. coli. This genetically modified insulin, Humulin was licensed for human use in
1982.

THE GENETIC ENGINEERING PROCESS

STEPS

A small piece of circular DNA called a plasmid is extracted from the bacteria or yeast cell. A small
section is then cut out of the circular plasmid by restriction enzymes, molecular scissors.

The gene for human insulin is inserted into the gap in the plasmid, this plasmid is now
genetically modified. The genetically modified plasmid is introduced into a new bacteria or yeast cell.
This cell then divides rapidly and starts making insulin

To create large amounts of the cells, the genetically modified bacteria or yeast are grown in

large fermentation vessels that contains all the nutrients they need. The more the cells divide, the more
the insulin is produced. When fermentation is complete, the mixture is filtered to releases the insulin

The insulin is then purified and package into bottles and insulin pens for distribution to patients
with diabetes.
USES OF GENETIC ENGINEERING

The first genetically modified organism to be created was a bacterium, in1973. in 1974, the
same techniques were applied to mice. In 1994 the first genetically modified foods were made available.

Genetic engineering has a number of useful applications, including scientific research,

agriculture and technology. In plants, genetic engineering has been applied to improve the resilience,
nutritional value and growth rate of crops such as potatoes, tomatoes and rice.

In animals it has been used to develop sheep that produce a therapeutic protein in their milk
that can be used to treat cystic fibrosis, or worms that glow in the dark to allow researchers, scientists to
learn more about diseases such as Alzheimer’s.

STAGES OF GENETIC ENGINEERING

DNA cleavage (stage 1) restriction endo nuclease cleaves DNA into fragments.

Recombinant DNA production (stage 2) DNA fragments inserted into vectors.

Cloning (stage 3) more recombinant DNA created

Screening (stage 4) most challenging part of any genetics experiments

GENE CONCEPT

The concept of the gene is and has always been a continuously evolving one. In order to provide
a structure for understanding the concept, its history is divided into classical, new classical and modern
periods. The classical views conceived the gene as an indivisible unit of genetic transmission,
recombination, mutation, and function. The discovery of intragenic recombination in early 1940s and
the establishment of DNA as the physical basis of inheritance led to the neoclassical concept of the gene
which prevailed until 1970s. In this view the gene (or citron, as it was called then) was subdivided into its
constituent parts, muttons and recons, identified as nucleotides. Each citron was believed to be
responsible for the synthesis of a single mRNA and hence for one polypeptide. This collinearity
hypothesis prevailed from 1955 to the 1970s. Starting from the early in 1970s, DNA technologies have
led to the modern period of gene conceptualization, wherein none of the classical or neoclassical criteria
are sufficient to define a gene. Modern discoveries include those of repeated genes, split genes and
overlapping genes, transposable genes, complex promoters, multiple polyadenylation sites, polyprotein
genes, editing of the primary transcript, and nested genes. Currently we are left with a rather abstract,
open, and generalized concept of the gene even though our comprehension of the structure and
organization of the genetic material has greatly increased.

MODERN CONCEPT OF GENE - A gene is a region of DNA that encodes function. A chromosome consists
of a long strand of DNA containing many genes. A gene is a locus (or region) of DNA which is made up of
nucleotides and is the molecular unit of heredity. A gene is a sequence of nucleotides in DNA or RNA
that encodes the synthesis of a gene product, either RNA or protein.

During gene expression, the DNA is first copied into RNA. The RNA can be directly functional or
be the intermediate template for a protein that performs a function. The transmission of genes to an
organism’s offspring is the basis of the inheritance of phenotypic trait. These genes make up different
DNA sequence called genotypes. Genotypes along with environmental and developmental factors
determine what the phenotype will be. Most biological traits are under the influence of polygenes
(many different genes) as well as gene-environmental interactions. Some genetic traits are instantly
visible, such as eye color, or the number of limbs, and some are not, such as blood type, risk for specific
diseases, or the thousands of basic biochemical processes that constitute life.

The broad definition of a gene is any discrete locus of heritable, genomic sequence which affect
an organism’s trait by being expressed as a functional product or by regulation of gene expression.

HOW GENES WORK

A gene is a stretch of DNA that carriers a set of instructions on how a protein should be made.
These proteins carryout the functions of the body. There are about 25,000 different genes in humans for
example, there are genes that control eye color, genes that encode enzyme that regulate how cells grow
etc.

CHROMOSOME STRUCTURE AND BEHAVIOUR

The DNA molecule is package into the thread like structures called chromosomes in the nucleus of each
cell. Each chromosome is made up of DNA tightly coiled many times around proteins called histones that
support its structure. Chromosomes are not visible in the cell nucleus – not even under microscope,
when the cell is not dividing. However, the DNA that make up chromosomes becomes more tightly
packed during cell division and is then visible under a microscope. Each chromosome has a constriction
point called the centromere, which divides the chromosome into two sections or “arms”. The short arm
of the chromosomes is labeled the “p arm”. The long arm of the chromosome is labelled the“q arm”.
The location of the centromere on each chromosome gives the chromosome its characteristics shape
and can be used to help describe the location of specific genes.

Most cells of higher plants and animals are diploid i.e. they contain two copies of each
chromosome. Formation of the germ cells (the sperm and egg), involves a unique type of cell division
(meiosis) in which only one member of each chromosome pair is transmitted each progeny cell see fig1

.
Consequently, the sperm and egg are haploid, containing only one copy of each chromosome. The
union of these two haploid cells at fertilization creates a new haploid organism, now containing one
member of each chromosome pair derived from the male and one from the female parent see fig2.

The behavior of chromosome pairs thus parallels that of genes leading to the conclusion that genes are
carried on chromosomes. The genomes of prokaryotes are contained in single chromosomes, in
contrast, the genomes of eukaryotes are composed of multiple chromosomes, each containing a linear
molecule of DNA. Although the numbers and sizes of chromosomes vary considerably between different
species (Table1) their basic structure is the same in all eukaryotes.
CHROMATIN

The complexes between eukaryotic DNA and proteins are called chromatin, which typically
contains about twice as much protein as DNA. The major protein of chromatin are the histones (small
proteins containing a high proportion of basic amino acids – arginine and lysine) that facilitate binding to
the negatively charged DNA molecule. There are five major types of histones called H1, H2A, H2B, H3
and H4 which are very similar among different species of eukaryotes. The histones are extremely
abundant proteins in eukaryotic cells. In addition, chromatin contains an approximately equal mass of a
wide variety of no histone chromosomal proteins. Histones are not found in eubacteria (e.g., E. coli).
Archaebacterial, however, do contain histones that package their DNAs in structures similar to
eukaryotic chromatin. The basic structural unit is the nucleosome which is composed of repeating 200-
base pair units. See fig3

CHROMOSOME BEHAVIOUR DURING CELL DIVISION

OUTLINE THE BEHAVIOUR OF CHROMOSOME DURING MITOSIS

Prophase

1. Chromatin shorten and thicken/condense and become distinct structure chromosome.

Metaphase

2. Spindle fibers from each pole of the cell are attached to one of the two chromatids of each
chromosome at the centromere region.
3. Spindle fibers pull on the centromere, arranging the chromosomes in a single row on the
metaphase plate (equator of the spindle).

Anaphase

4. Centromere divides and sister chromatids separate at centromere, forming daughter

chromosome.

5. Shortening of microtubules occurs and spindle fibers attached to the centromeres pull the
daughter chromosome to the opposite poles of the cell.

6. With centromere leading the way.

Telophase

7. Daughter chromosome arrive at the opposite poles.

8. Chromosomes uncoil, lengthen and become indistinct to form chromatin again.

The digestion of chromatin with micrococci nuclease yields particles which contains 146 base pairs of
DNA wrapped 1.65 times around a histone core consisting of two molecules each of H2A, H2B, H3 and
H4 (the core histones), one molecule of the fifth histone, H1, is bound to the DNA as it enters each
nucleosome core particle. This forms a chromatin sub unit known as a chromatosome, which consists of
166 base pairs of DNA wrapped around the histone core and held in place by H1 (a linker histone) see
fig4.
INTERPHASE

In the interphase (non-diving) cells, of the chromatin (called euchromatin) relatively

decondensed throughout the nucleus. During this period of the cell cycle, genes are transcribed and the
DNA is replicated in preparation for cell division. In contrast to euchromatin, about 10% of interphase
chromatin (called heterochromatin) is in very highly condensed state that resembles the chromatin of
cells undergoing mitosis. Heterochromatin is transcriptionally inactive and contains highly repeated DNA
sequences, such as those present at centromeres and telomeres.

As cells enter mitosis, their chromosomes become highly condensed so that they can be
distributed to daughter cells. The loops of 30-nm chromatin fibers fold upon themselves further to form
the compact metaphase chromosomes of mitotic cells, in which the DNA has been condensed nearly
10,000-fold. Such condensed chromatin can no longer be used as a template for RNA synthesis, so
transcription ceases during mitosis (fig5).

CENTROMERER

The centromere is a specialized region of the chromosome that plays a critical role in ensuring
the correct distribution of duplicated chromosomes to daughter cells during mitosis.
 The cellular DNA is replicated during interphase, resulting in the formation of two copies of each
chromosome prior to the beginning of mitosis. As the cell enters mitosis, chromatin condensation leads
to the formation of metaphase chromosomes consisting of two identical sister chromatids. These sister
chromatids are held together at the centromere, which is seen as a constricted chromosomal region. As
mitosis proceeds, microtubules of the mitotic spindle attach to the centromere, and the two sister
chromatids separate and move to opposite poles of the spindle. At the end of mitosis, nuclear
membrane reform and the chromosome decondense, resulting in the formation of daughter nuclei
containing one copy of each parental chromosome.

The centromeres thus serve both as the sites of association of sister chromatids and as
attachment sites for microtubules of mitotic spindle. They consist of specific DNA sequence to which a
number of centromere- associated proteins bind, forming a specialized structure called the kinetochore.
The binding of microtubules to kinetochore proteins mediates the attachment of chromosomes to the
mitotic spindle. Proteins associated with the kinetochore then act as “molecular motors” that drive the
movement of chromosomes along the spindle fibers, segregating the chromosome to daughter nuclei.

TELOMERES

The sequences at the ends of eukaryotic chromosomes called telomeres, play critical roles in
chromosome replication and maintenance. The telomere DNA sequence of a variety of eukaryotes are
similar, consisting of repeats of a simple-sequence DNA containing clusters of G residues on one strand
(Table 2). For example, the sequence of telomere repeats in humans and other mammals is AGGGTT.
These sequences are repeated hundreds or thousands of times, thus spanning up to several kilobases,
and terminate with an overhang of single-stranded DNA. The repeated sequence of telomere DNA form
loops at ends of chromosomes thereby protecting the chromosome termini from degradation. (fig7).

GENE
A gene is a sequence of nucleotides in DNA or RNA that encodes the synthesis of a gene product, either
RNA or protein. A gene can also be referred to as the basic physical and functional unit of heredity.
Some genes act as instruction to make molecules called proteins. However, many genes do not code for
proteins. In humans, genes vary in size from a few hundred DNA bases to more than 2 million bases.

GENE ACTION

Gene action refers to the way in which certain genes exert their effects on the body. They could be
dominant, or recessive, or they could be sex-linked or be involved in chromosomal aberrations. A
combination of such gene action results in the observable phenotype of an organism.

There are three broad types of gene actions:

I. Additive gene action- the action of a gene is said to be additive where one allele of a gene is
substituted by another allele and it produces the same effect, a positive effect or a negative
effect. This can happen only if the alleles or not dominant (or recessive). In the table below,
addition or removal of A adds or subtracts 2 units of action, respectively, and is not affected by
the presence of gene B. The same is true for gene B too.

Additive gene action model

AA Aa Aa Mean

BB 10 8 6 8

Bb 8 6 4 6

Bb 6 4 2 4

Mean 8 ■4

II. Dominance gene action

When a dominant gene (A) is substituted by a recessive gene(a) and there is no effect, either
positive or negative, then it is called a dominance gene action. In the table below there is no
effect when the recessive gene in Aa is replaced by a dominant gene. In this model, AA exerts
the same effect as Aa.

This illustration shows that Aa=AA +a9/2= 8, implying absence of dominance. This time at Bb and bb
levels also. Replacement of a by A produces a plus effect of 2 units disregarding presence of B gene and
genetic phase at B locus showing no interaction between A and B. further, total of any 2 diagonal values
are equal showing no interaction
Dominance gene action

AA Aa Aa

BB 9 9 7

Bb 9 9 7

bb 7 7 5
 Mean 8.3 8.3 6.3

III. Epistatic gene action

In this type of gene action, the presence or absence of an allele in one locus affect the expression of
another allele in a different locus. Epistasis can either exert additive effect or dominance. in other
words, Epistatic action of one gene can completely mask the effect of an another gene or it can also in
completely unmasking the effect of a gene that remains dominant by default.

The interaction here could be additive X additive, additive X dominance or dominance type. An additive
X additive type interactions shown in the table below.

Additive X Additive epistatic gene action model

AA Aa As Mean

BB 9 6 3 6

Bb 6 4 2 4

bb 3 2 1 2

Mean 6 4 2

This model shows that Aa = AA disregarding whether at other locus there is BB or Bb or bb likewise
Bb=BB. Thus, there is complete dominance for both the genes and interaction is absent. No interaction
between A and B genes is further reflected by the fact that total of any 2 diagonal values is the same.

In this given model, Aa = mean of AA and aa and similarly, Bb = (Bb + bb)/2 showing no
dominance. Further, substituting a by A has equal effect from aa and Aa and from Aa to AA (3units with
BB, 2 units with Bb and 1 unit with bb). Same is time for B gene. This shows additive gene action.
Epistalsis could be additive X dominance (AXD) or dominance X dominance (DXD) type.

The overall consideration leads to:

P=G+E=A+D+I+E=A+D+I+E=A+D+AA+AD+DD+E.

INTERACTION IS OF 2 TYPES

1. Complementary type:

This involves interaction between 2 non-alleles complementing each other to produce a new
phenotype which is not ascribable to them individually. This is created by the interaction of 2
homozygotes, each acting additively (additive X additive i.e. A X A). Complementary (9:7), recessive
epitasis (9:3:4) and polymerism (9:6:1) are example of this type.

2. Duplicate type
 This involves 2 non-allelic genes which tend to cancel or weaken the effect of each other in
hybrid combination. Such a variation arises from homozygote-heterozygote (additive X dominance) or
heterozygote-heterozygote (dominance X dominance) combination. Inhibitory genes (13:3), duplicate
genes (15:1) and dominant epistasis (12:3:1) fall in this category of interaction.

When dominance (D) and DXD components have same sign (+or-) epitasis is complementary type,
otherwise duplicate type.

GENE MUTATION

A gene mutation is a permanent alteration in the DNA sequence that makes up a gene, such that
the sequence differs from what is found in most people. Mutation range in size, they can affect
anywhere from a single DNA building block (base pair) to a large segment of a chromosome that
includes multiple genes.

Gene mutation can be classified in two major ways:

Hereditary mutations are inherited from a parent and are present throughout a person’s life in
virtually every cell in the body. This mutation is also called germline mutations because they are present
in the parent’s egg or sperm cells, which are also called germ cells. When an egg and a sperm cell unite,
the resulting fertilized egg cell receives DNA from both parents. If this DNA has a mutation, the child that
grow from the fertilized egg will have the mutation in each of his or her cells.

Acquired (or somatic) mutations occur at some time during a person’s life and are present only
in certain cells, not in every cell in the body. These changes can be caused by environmental factors such
as ultraviolet radiation from the sun, or can occur if an error is made as DNA copies itself during cell
division. Acquired mutation in somatic cells (cells other than sperm and egg cells) cannot be passed to
the next generation.

Genetic changes that are described as de novo (new) mutations can be either hereditary or
somatic. In some cases, the mutations occur in a person’s egg or sperm cell but is not present in any of
the person’s other cells. In other cases, the mutation occurs in the fertilized egg shortly after the egg and
sperm cells unite. (It is often impossible to tell exactly when a de novo mutation happened). As the
fertilized egg divides, each resulting cell in the growing embryo will have the mutation. De novo
mutations may explain genetic disorders in which an affected child has a mutation in every cell in the
body but the parent does not and there is no family history of the disorder.

Somatic mutation that happens in a single cell early in embryotic development can lead to
situation called mosaicism. These genetic changes are not present in a parent’s egg or sperm cells, or in
the fertilized egg but happen a bit later when the embryo includes several cells. As all the cells divide
during growth and development cells that arise from the cell with the altered gene will have the
mutation, while other cells will not. Depending on the mutation and how many cells are affected,
mosaicism may or may not cause health problems.

Most disease causing gene mutation are uncommon in the general population. However, other
genetic changes occur more frequently. Genetic alterations that occur in more than 1% of the
population are called polymorphisms. They are common enough to be considered a normal variation in
the DNA.
 Polymorphisms are responsible for many of the normal differences between people such as eye
color, hair color, and blood type. Although many polymorphisms have no negative effects on a person’s
health, some of these variations may influence the risk of developing certain disorders.

Few mutations are bad for you. In fact, some mutations can be beneficial over time. Genetic
mutation creates genetic diversity, which keeps population healthy. Many mutations have no effect at
all. These are called silent mutation. But the mutations we hear about most often are the ones that
cause disease. Some well-known inherited genetic disorders include cystic fibrosis, sickle cell anemia,
Tay-sachs disease, phenylketonuria and colour-blindness, among many others. All of these disorders are
caused by the mutation of a single gene.

Most inherited genetic diseases are recessive which means that a person must inherit two
copies of the mutated gene to inherit a disorder. This is one reason that marriage between close
relatives is discouraged, two genetically similar adults are more likely to give a child two copies of a
defective gene. Diseases caused by just one copy of a defective gene, such as Huntington's disease are
rare. These dominant genetic diseases tend to get weeded out of populations over time, because
afflicted carriers are more likely to die before reproducing.

SICKLE CELL

These are the sickle- shaped blood cells of someone with sickle cell anemia, a genetic disease
common among Africans. Sickle cell anemia is the result of a point mutation, a change in nucleotide in
the gene for hemoglobin. This mutation causes the hemoglobin in red blood cells to distort to a sickle
shape when deoxygenated. The sickle shaped blood cells clog in the capillaries, cutting off circulation.
Having two copies of the mutated genes cause sickle cell anemia but having just one copy does not, and
can actually protect against malaria: an example of how mutations are sometimes beneficial.

Cancer usually results from a series of mutations within a single cell often, a faulty, damaged or
missing p53 gene is to blame. The p53 gene makes a protein that stops mutated cells from diving.
Without this protein cells divide unchecked and become tumors.

CHROMOSOMAL MUTATIONS/ABBRATIONS

A chromosomal mutation is a mutation that changes the structure of an individual chromosome,

leading to imbalance involving only a part of a chromosome. Such as duplication, deletion or
translocation.

A chromosomal mutation is alteration or errors that occur within a chromosome. Such errors
can be attributed to any mistakes or problem that occur during cell processes like mitosis and meiosis.
Unlike gene mutations that involve the alteration of a gene or a segment of DNA in the
chromosome, chromosomal mutation occurs and change the entirety of the chromosome itself.

There are three types of chromosomal mutations-

These are structural chromosomal mutations, chromosomal number mutation and sex chromosomes
mutation.

STUCTURAL CHROMOSOMAL MUTATION

 This kind chromosomal mutation usually occurs during any errors in cell division. This happens
when homologous chromosomes paired up, genes in chromosomes broke apart, genes inserted in the
wrong chromosome, or genes or set of genes are completely lost in the chromosome.

Basically, structural chromosomal mutation is classified into four: deletion, duplication, inversion
and translocation (or shift places). They are illustrated below:

Deletion

Deletion this type of mutation occurs when a part of the DNA is not duplicated or is lost during DNA
replication. The size of this region can either be a mere nucleotide or can be large as an entire
chromosome.

Disorders due to deletion

Common disorders due to deletion mutation in humans are Cridu chat, Duchene muscular dystrophy, Di
George’s syndrome.

Duplication

This type of mutation occurs when an extra copy of a region (or regions) in the DNA is produced.
This duplicated region can either be located in its normal location in the chromosome or sometimes be
located in other parts of the chromosome or even in another chromosome.

This duplication can now supply additional material that has the ability to evolve new functions.

Disorders due to duplication

Common disorders due to duplication mutation in humans is Charcot Marie-tooth disease type1.

Inversion
 During inversion, a portion in the chromosome is reversed and gets inserted back into the
chromosome. Two types of inversion exist:

Pericentric and paracentric.

During a pericentric inversion, the inversion encompasses the centromere of the chromosome.

During a paracentric inversion, it only involves either the short or long arm of the chromosome
and the inversion point does not include the centromere.

Disorders due to inversion

Common disorders due to duplication mutation in humans is Amniocentesis during pregnancy.

Translocation

Translocation happens when a fragmented chromosome tends to join with a non-homologous

chromosome. This newly formed segment then detaches from the chromosome and moves to a new
position on another chromosome

Disorders due to translocation

Common disorders due to duplication mutation in humans are XX male syndrome, Down syndrome,
Infertility and cancer

CHROMOSOMAL NUMBER MUTATIONS

1. Aneuploidy

Aneuploidy is a type of mutation in chromosome number where in the ploidy (chromosome

number) of the new individual is different from its wild type. This is typically a result of the non-
disjunction of chromosomes during mitosis or meiosis, hence producing offspring with either extra or
lost chromosome, instead of having two chromosomes.

The naming of aneuploid conditions is generally based on the number of chromosome added or deleted.
For instance, a monosomic (2n-1) individual bears only one copy of a chromosome, instead of having
two.

Other variations of aneuploidy are trisomic (2n+1), nullisomic (2n-2), and disomic (n+1).

2. Polyploidy

Polyploidy is a type of mutation that occurs when an individual bear more than one haploid set of
chromosomes. If the individual with polyploidy bear three sets of haploid chromosomes, the condition is
said to be triploidy whereas if it has four haploid sets, the condition is said to be tetra ploidy.

Interestingly, polyploidy is a common phenomenon among plants as well as certain groups of

fish, salamanders, frogs and leeches.

THE IMPORTANCE OF CHROMOSOMAL MUTATION

 The random errors that occur during cell division can be beneficial for organisms. Below are
some examples.

1. Survival

Mutation are very essential for populations because they help some individuals of the
population to adopt to their environment while they are also an important force in evolution because
they balance out the frequency of alleles present in the population.

In humans some successful mutations include malaria resistance, lactose tolerance and
atherosclerosis tolerance.

2. Diversity

Mutation in the chromosomes is highly connected to diversity (not only genetically but also
physically) of living organisms. Ultimately the close interactions between inherited mutations and
environmental pressures generate diversity among species.

Without genetic diversity, some of the fundamental mechanisms of evolutionary change cannot
(and continue to) operate.

Examples of some chromosomal mutations that are harmless include different eye colour such
as black, brown, grey, green or blue.

THE DISADVANTAGES OF CHROMOSOMAL MUTATIONS

Some chromosomal mutations can be quite detrimental for example.

1. Genetic disorder

Mutations in the chromosome can cause a wide variety of genetic disorders. The severity of the error in
even a small portion of the chromosome can be highly devastating

2. Other diseases

Aside from inheritable disorder certain mutations in the chromosomes can also bring about the onset of
other disease like cancer. (i.e. lung, breast and bladder).

No matter what we do, the random changes in our genome are highly inevitable, and in this case
does the saying “the only constant is change proves true”.

Most chromosome anomalies occur as an accident in the egg or sperm, and are therefore not
inherited. The anomaly is present in every cell of the body. Some anomalies, however, can happen after
conception, resulting in mosaicism (where some cells have anomaly and some do not). Chromosome
anomalies can be inherited from a parent or be “de novo”. This is why chromosome studies are often
performed on parents when a child is found to have an anomaly.

CHROMOSOMAL MUTATIONS IN MICROORGANISMS

A chromosome is the large circular piece of DNA in each bacterium that includes all of its
essential genetic material.
 Mutations in chromosomal DNA occur at a low level every time the DNA is copied during
growth. This variation causes differences in how the organisms interacts with the environment, which
leads to evolution by natural selection when the population (each containing different mutants) is faced
with a selective pressure. Antibiotics are a strong selective pressure for bacteria populations. They
remove bacterial that do not have the relevant resistance causing chromosomal mutations, and select
for bacteria that have the mutation.

BUILDING BLOCK OF DNA AND RNA

Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA) are nucleic acids which are the principal informational
molecules of the cell. DNA has a unique role as the genetic material which in eukaryotic cells is located in the
nucleus.
The DNA and RNA are a long thin organic polymer made of chemical building blocks called nucleotides. These
building blocks are made of three parts, a phosphate group, a sugar group and one of four types of nitrogen bases.
To form a strand of DNA, nucleotides are linked into chains, with the phosphate and sugar groups alternating. The
nitrogen bases are derivatives of two parent compounds, pyrimidine and purine.
Both DNA and RNA contain two major purine bases, adenine (A) and guanine (G) and two major pyrimidines. In
both DNA and RNA one of the pyrimidines is cytosine (C) but the second common pyrimidine is not the same in
both. It is thymine (T) in DNA and Uracil (U) in RNA. Only occasionally does thymine occur in RNA or Uracil in DNA.
Nucleic acids have two kinds of pentoses. The recurring deoxyribonucleotide units of DNA contain 2’-deoxy-D-
ribose, and the ribonucleotide units of RNA contain D-ribose. Both types of pentoses are in their β-furanose (closed
five-membered ring) form. The pentose ring is not planar but occurs in one of a variety of conformations generally
described as “puckered”.
Although DNA and RNA seem to have two distinctions – different pentoses and the presence of uracil in RNA and
thymine in DNA – it is the pentoses that define their identity. If the nucleic acid contains 2’-deoxy-D-ribose, it is
DNA by definition even though it may contain uracil. Similarly, if the nucleic acid contains D-ribose it is RNA
regardless of its base composition.
Both DNA and RNA also contain some minor bases. In DNA the most common of these are methylated forms of the
major bases, in some viral DNAs certain bases may be hydroxymethylated or glucosylated. Altered or unusual bases
in DNA molecules often have roles in regulating or protecting the genetic information. Minor bases of many types
are also found in RNAs. The successive nucleotides of both DNA and RNA are covalently linked through phosphate
group “bridges” in which the 5’-phosphate group of one nucleotide joined to the 3’-hydroxyl group of the next
nucleotide creating a phosphodiester linkage. The backbones of both DNA and RNA are hydrophilic. The hydroxyl
groups of the sugar residues form hydrogen bonds with water. The phosphate groups, with a pka near 0, are
completely ionized and negatively charged at pH 7 and the negative charges are generally neutralized by ionic
interactions with positive charges on proteins, metal ions and polyamines. All the phosphodiester linkages in DNA
and RNA have the same orientation along the chain giving each linear nucleic acid strand a specific polarity and
distinct 5’ and 3’ends. The 5’ end lacks a nucleotide at the 5’ position and the 3’ end lacks a nucleotide at the 3’
position.

It is important to note that

● The base composition of DNA generally varies from one species to another.

● DNA specimens isolated from different tissues of the same species have the same base composition.
● The base composition of DNA in a given species does not change with an organism’s age, nutritional state or
changing environment.

In all cellular DNA, regardless of the species, the number of adenosine residues is equal to the number of thymine
residues (that is A=T), and the number of guanosine residue is equal to the number of cytosine residue (G=C). From
these relationships it follows that the sum of the purine residues equals the sum of the pyrimidine residues, that is
A+G = T+C. These quantitative relationship is sometimes called the “Chargaff’s rules”.

WORKED EXAMPLES ON BASE PAIRING IN DNA

In samples of DNA isolated from two unidentified species of bacteria, X and Y, adenine makes up 32% and 17%
respectively of the total bases. What relative proportions of adenine, guanine, thymine and cytosine would you
expect to find in the two DNA samples?
What assumptions have you made?
One of these species was isolated from a hot spring (64 oC). Which species is most likely the thermophilic bacterium
and why?

SOLUTION
For any double helical DNA, A=T and G=C. The DNA from species X has 32% A and therefore must contain 32% T.
This accounts for 64% of the bases and leaves 36% as G=C pairs: 18% G and 18% C. The sample from species Y, with
17% A must contain 17% T, accounting for 34% of the base pairs. The remaining 66% of the bases are thus equally
distributed as 33% G and 33% C. This calculation is based on the assumption that both DNA molecules are double
stranded.
The higher the G+C content of a DNA molecule, the higher the melting temperature. Species Y having the DNA with
the higher G+C content (66%), most likely is the thermophilic bacterium; its DNA has a higher melting temperature
and thus is more stable at the temperature of the hot spring.

The three-dimensional model of DNA structure proposed by Watson and Crick in 1953 consist of two antiparallel
chains in a right-handed double-helical arrangement. Complementary base pairs, A=T and G=C are formed by
hydrogen bonding within the helix. The base pairs are stacked perpendicular to the long axis of the double helix,
3.4A apart with 10.5 base pairs per turn.
THE GENETIC CODE
The genetic code is the relationship between the nucleotide sequence in nucleic acids and the amino acid sequence
in proteins. As a result of this relationship, the information for the structure and function of all living things is
passed from one generation to the next.
The genetic code translates the language of DNA which contains four bases, into the language of the 20 common
amino acids that are formed in proteins. The genetic code is a set of rules used by living cells to translate
information encoded within genetic material (DNA or RNA sequence of nucleotide triplets or codons) into proteins.
A triplet code known as a codon refers to a sequence of three bases needed to specify one amino acid.
Translation is accomplished by the ribosome, which links amino acids in an order specified by messenger RNA
(mRNA) using transfer RNA (tRNA) molecules to carry amino acids and to read the mRNA three nucleotides at a
time. With three bases, there are 4 3 possibilities or 64 possible codons which is more than enough to encode the 20
amino acids. The genetic code is highly similar among all organisms. No codon can encode more than one amino
acid. All 64 codons are assigned meanings, with 61 of them coding for amino acids and the remaining 3 serving as
the termination signals.
Two amino acids, tryptophan and methionine have only one codon each but the rest have more than one. A single
amino acid can have as many as six codons, as in the case with leucine and arginine. Multiple codons have one or
two bases in common. The bases that are common to several codons are usually the first and second bases, with
more room for variation in the third base which is called the wobble base. The degeneracy of the code acts as a
buffer against deleterious mutations. For example, for eight of the amino acids (L, V, S, P, T, A, G and R), the third
base is completely irrelevant. Thus, any mutation in the third base in the codon would not change the amino acid at
that location. A mutation in the DNA that does not lead to a change in the amino acid translated is called a silent
mutation. The second base of the codon is very important for determining the type of amino acid. For example,
when the second base is U, all the amino acids generated from the codon possibilities are hydrophobic. Thus, if the
first or third base were mutated, the mutation would not be silent (although we say silent) but the damage would
not be as great because one hydrophobic amino acid would be replaced with another. Codons sharing the same
first letter often code for amino acids that are products of one another. The genetic code is one of the best ways to
protect an organism from DNA mutations.

HOW WAS THE GENETIC CODE DETERMINED

The assignment of triplets in the genetic code was based on several types of experiments. One of the most
significant experiments involved the use of synthetic polyribonucleotides as messengers. When
homopolynucleotides (polyribonucleotides that contain only one type of base) are used as a synthetic mRNA for
polypeptide synthesis in laboratory systems, homopolypeptide (polypeptide that contain only one kind of amino
acid) are produced. When poly U is the messenger, the product is polyphenylalanine. With poly A as the
messenger, poly lysine is formed. The product for poly C is polyproline, and the product for poly V, G is polyglycine.
This procedure was used to establish the code for the four possible homopolymers. When an alternating copolymer
(a polymer with an alternating sequence of two bases) is the messenger, the product is an alternating polypeptide
(a polypeptide with an alternating sequence of two amino acids). For example, when the sequence of the
polynucleotide is -ACACACACACACACACACACACAC- the polypeptide produced has alternating threonines and
histidines. There are two types of coding triplets in this polynucleotide, ACA and CAC, but this experiment cannot
establish which one codes for threonine and which one codes for histidine. It is interesting to note that this result
proves that the code is a triplet code. If it were a doublet code, the product would be a mixture of two
homopolymers, one specified by the codon AC and the other by the codon CA. the terminology for the different
ways of reading this message as a doublet is to say that they have different reading frames, /AC/AC/ and /CA/CA/.
In a triplet code, only one reading frame is possible, namely /ACA/CAC/ACA/CAC/, which give rise to an alternating
polypeptide. Another method on codon assignment is the filter binding assay.
It is important to note that the genetic code is based on a series of three bases for an amino. The code is nearly
universal in all organisms from viruses through humans. The code has no punctuation, meaning the mRNA is read
three bases at a time with no spaces in between. The code is non-overlapping as well, meaning that each base is
part of only one codon. The genetic code was determined by a variety of techniques, such as using synthetic mRNA
with known sequence to see what proteins would be translated from them. Although there are 64 combinations of
three bases leading to 64 codons, there are fewer types of tRNA anticodons. This means that standard Watson-
Crick base pairing must be broken on occasions. The Wobble model of codon-anticodon base pairing shows that
some bases at the 5’ end of the anti-codon of the tRNA can base-pair with multiple bases on the codon.

Replication is the copying of parental DNA to for daughter DNA molecules with identical nucleotide sequences. The
DNA acts as a template for the replication and transmission of genetic information.

DNA REPLICATION

DNA replication occurs before the cell divides, the two DNA strands separate and each serves as a template (a
template is a structure that would allow molecules to be lined up in a specific order and joined to create a
macromolecule with a unique sequence and function or a macromolecule mold, or a pattern for the synthesis of an
informational macromolecule) for the synthesis of a new, complementary strand, generating two identical double-
helical molecules, one for each daughter cell. If either strand is damaged at any time, continuity of information is
assured by the information present in the other strand, which can act as a template for repair of the damage.

DNA replication is semiconservative, that is each DNA strand serves as a template for the synthesis of a new strand,
producing two new DNA molecules, each with one new strand and one old strand. The hypothesis of
semiconservative replication of DNA was proposed by Watson and Crick in 1953 but later proved by Meselson-Stahl
who grew E coli cells for many generations in a medium in which the sole nitrogen source (NH 4Cl) contained 15N the
heavy isotope of nitrogen, instead of the normal more abundant light isotope 14N.

DNA replication begins at an origin (a unique point in which the replication loops always initiates) and usually
proceeds bi-directionally in most organisms, with the exception of a few viruses and plasmids. DNA synthesis
proceeds in a 5’→ 3’ direction and is semi-discontinuous, that is a new strand of DNA is always synthesized in the
5’→ 3’ direction, with the free 3’OH as the point at which the DNA is elongated. Because the two DNA strands are
antiparallel, the strand serving as the template is read from the 3’ end towards its 5’ end. The work of Reijl Okazaki
and colleagues in the 1960’s led to the conclusion that one strand of the DNA is synthesized continuously and the
other discontinuously. The continuous strand, or leading strand is the one in which 5’→3’ synthesis proceeds in the
same direction as replication fork movement. The discontinuous strand or lagging strand, is the one in which 5’→3’
synthesis proceeds in the direction opposite to the direction of fork movement.

NOTE
When a molecule of DNA is replicated each of the two strands is used as a template to create a complementary
strand. When a cell divides into two, each of the two cells has one of the original template strands and one of the
new strands. This process is called the semiconservative replication. When DNA molecules are replicated, the
strands are separated at origin of replication. Synthesis occurs in both directions from the origin along replication
forks.
One origin of replication exists in the circular DNA of prokaryotes. In eukaryotes, several origins of replication and
thus several bubbles exist. The bubbles grow larger and eventually merge, giving rise to two complete daughter
DNAs.

ENZYMOLOGY OF DNA REPLICAION / DNA POLYMERASES

DNA is synthesized by DNA polymerases. It catalyzes the successive addition of each new nucleotide to the growing
chain. The first DNA polymerase discovered was found in E. coli. At least five DNA polymerase are present in E. coli
known as DNA polymerase I, II, III, IV and V. Polymerase II is not required for replication, rather, it is strictly a repair
enzyme. Two important things to note about the polymerases are the speed of the synthesis reaction (turnover
number) and the processivity, which is the number of nucleotides joined before the enzyme dissociates from the
template. DNA polymerase III has the highest turnover number and a high processivity compared to polymerase I
and II. DNA polymerases must have a nucleotide with a free 3-hydroxyl already in place so that they can add the
first nucleotide as part of the growing chain. In natural replication, this primer is RNA.

DNA polymerase reaction requires all four deoxyribonucleoside triphosphates – dTTP, dATP, dGTP and dCTP, Mg 2+
and a DNA template. Because of the requirement for a primer, all four ribonucleoside triphosphates – ATP, UTP,
GTP and CTP – are needed as well. They are incorporated into the primer. The primer (RNA) is hydrogen bonded to
the template (DNA); the primer provides a stable framework on which the nascent chain can start to grow. The
newly synthesized DNA strand begins to grow by forming a covalent linkage to the free 3’-hydroxyl group of the
primer. DNA polymerase I has a specialized function in replication- repairing and ‘patching’ DNA – and DNA
polymerase III is the enzyme primarily responsible for the polymerization of the newly formed DNA strand. The
major function of DNA polymerase II IV and V is as repair enzymes. The exo-nuclease activities are part of the
proofreading and repair functions of DNA polymerase, a process by which incorrect nucleotides are removed from
the polynucleotide so that the correct nucleotides can be incorporated. The 3’→5’ exonuclease activity which all
three polymerases possess is part of the proofreading function. Incorrect nucleotides are removed in the course of
replication and are replaced by the correct ones. Proofreading is done one nucleotide at a time. The 5’→3’
exonuclease activity clears away short stretches of nucleotides during repair, usually involved several nucleotides at
a time. This is also how the RNA primers are removed.

IMPORTANT TO NOTE

● To achieve 5’→3’ synthesis of DNA on two strands that are antiparallel, DNA polymerase synthesizes one
strand continuously and the other discontinuously.
● The strand synthesized continuously is called the leading strand and the one synthesized discontinuously is
called the lagging strand.
● The pieces of DNA formed discontinuously are called Okazaki fragments and they are later joined together
by DNA ligase.
● The reaction of DNA synthesis involves the nucleophilic attack of the 3’-hydroxyl of one nucleotide on the ɣ-
phosphate of the incoming nucleoside triphosphate.
● At least five DNA polymerases exist in E. coli, called polymerase I through polymerase V. polymerase III is
the primary enzyme responsible for synthesis of new DNA and it is a multiple-subunit enzyme.
● DNA polymerases I and II are involved in proofreading and repair processes.

● All DNA synthesis requires an RNA primer.

POPULATION GENETICS

Population genetics is the study of genetic variation within populations, and involves the
examination and modelling of changes in the frequencies of genes and alleles in populations over space
and time. Many of the genes found within a population will be polymorphic – I.e., they will occur in a
number of different forms (or alleles).

Mathematical models are used to investigate and predict the occurrence of specific alleles or
combination of alleles in populations, based on developments in the molecular understanding of
genetics. Mendel’s laws of inheritance and modern evolutionary theory.

The focus is the population or the species not the individual.

The collection of all the alleles of all of the genes found within a freely interbreeding population

is known as the gene pool of the population. Each member of the population receives its alleles from
other members of the gene pool (its parents) and passes them on to other members of the gene pool
(its offspring).

Population genetics is the study of the variation in alleles and genotypes within the gene pool
and how this variation changes from one generation to the next.

Factors influencing the genetic diversity within a gene pool include population size, mutation,
genetic drift, natural selection, environmental diversity, migration and non-random mutating patterns.
The Hardy-Weinberg model describes and predicts a balanced equilibrium in the frequencies of alleles
and genotypes within a freely inter breeding population, assuming a large population size, no mutation,
no genetic drift, no natural selection, no gene flow between populations and random mating patterns.

In natural populations, however, the genetic composition of a population’s gene pool may
change over time. Mutation is the primary source of new alleles in a gene pool, but the other factors act
to increase or decrease the occurrence of alleles. Genetic drift occurs as the result of random in
fluctuations in the transfer of alleles from one generation to the next, especially in small populations
formed, say, as the result adverse environmental conditions (the bottle neck effect) or the geographical
separation of a subset of the population (the founder effect). The result of genetic drift tends to be a
reduction in the variation within the population, and an increase in the divergence between
populations. If two populations of a given species become genetically distinct enough that they can no
longer interbreed, they are regarded as new species (a process called specification).

In many cases, the effects of natural selection on a given allele are directional. The allele either
confers a selective advantage, or spreads throughout the gene pool, or it confers a selective
disadvantage, and disappears from it.

In other cases, however, selection acts to preserve multiple alleles within the gene pool and a balanced
equilibrium is observed. This situation, labelled balanced polymorphism, can arise because of a selective
advantage for individuals heterozygous for a given allele. For example, the disease sickle cell anemia is
caused by a mutation in one of the genes responsible for the production of hemoglobin. Individuals with

two copies of the mutant gene for sickle hemoglobin (Hbs/Hbs) develop the disease. Individuals that are
heterozygous – one copy of the sickle gene and one copy (Hbs/HbA) are carriers of the condition. It is
believed that these heterozygous individuals are more resistant to malaria then individuals homozygous
for normal gene (HbA/HbA) and that this selective advantage maintains the presence of the Hbs gene in
the population. As a result of balanced polymorphism, the gene pools of most populations contain a
number of deleterious alleles that reduces the overall fitness of the population (known as the genetic
load).

Genetic variation within populations and species can now be analyzed at the level of nucleotide
sequences in DNA (genome analysis) and the amino acids sequences of proteins (proteome analysis).
The genetic differences between species can be used to infer evolutionary history on the basis that the
closet relatives will have gene pools that are most similar. Recent advances in the sequencing of
genomes, allied to computer-based techniques for storing and comparing this information, have led to
the construction of detailed evolutionary trees. The use of molecular clocks nucleotide sequences (or
amino acid sequences) in which evolutionary change accumulates at a constant rate allows dates to be
attached to the points at which populations start to diverge to form new species. These approaches are
also proving useful in other areas (for example, in tracing the transmission routes of infectious diseases).

HUMAN GENETICS

Human genetics is the study of inheritance as it occurs in human’s beings.

Human genetics encompasses the following fields – classical genetics, cytogenetics, molecular
genetics, biochemical genetics, genomics, population genetics, developmental genetics, chemical
genetics and genetic counselling.

Genes are the common factor of the qualities of most human inherited traits.

BASIC CONCEPT OF HUMAN GENETICS

The genetic information of an individual is contained in 23 pairs of chromosome. Every human

cell contains the 23 pair of chromosomes.

One pair of the 23 pairs is called sex chromosome

Male XY

Female XX

Other 22 pairs of homologous chromosomes are called autosomes. The autosome chromosome
pairs are called homologous pair. Two chromosome in the same pair are called homologous
chromosomes.

One member of each chromosome pair is from mother, the other is from father or mother
transmit each of the two chromosomes with equal probability.

There are two DNA strands/chains in one chromosome. The DNA has four bases A, G, T and C. A
combine with T and G combined with C.

The unit bp (base pair) is the length unit of chromosome or DNA sequence. The DNA sequence has
directions there are two ends (side) called 5’ end and 3’ end. The homologous chromosomes
(chromosome 1 for example) have exactly same length for every individual.
MITOSIS – NORMAL CELL DIVISION

Normally old cells die and new cells grow. The new cells grow through normal cell division. The
process for the normal cell division (mitosis) includes

1. The double DNA strands in each of the chromosomes split into two single strands.
2. DNA replication. After step (2) each chromosome produces another identical one.

All the 23 pairs of the chromosomes undergo this process of replication, producing two identical
set of 23 chromosomes pairs. The two sets of chromosomes are separated and distributed into two
daughter cells.

Chromosomes are inherited through meiosis.

1. The 23 pair of chromosomes in the cell are duplicated every time a cell division occurs.
2. The only exceptions to this rule are gametes (ovum and sperm), which are produced by sex
organ.
3. Gametes are produced by a special cell division called meiosis.
4. Meiosis gives rise to daughter cells (ovum or sperm) which contain only a haploid (single
chromosome, not pair) set of 22 autosomes and a sex chromosome.

Inheritance of traits for humans are based upon Gregor Mendel’s model of inheritance. Mendel
deduced that inheritance depends upon discrete units of inheritance, called factors or genes.

AUTOSOMAL DOMINANT INHERITANCE

Autosomal traits are associated with a single gene on an autosome (non-sex chromosome) – they are
called “dominant” because a single copy – inherited from either parent is enough to cause this trait to
appear. This often means that one of the parents must also have the same trait, unless it has risen due
to an unlikely new mutation.

Examples of autosomal dominant traits and disorders are Huntington’s disease and
achondroplasia.

Autosomal recessive traits are one pattern of inheritance for trait, disease, or disorder to be
passed on through families. For a recessive trait or disease to be displayed two copies of trait or disorder
needs to be presented. The trait or gene will be located on a non – sex chromosome. Because it takes
two copies of a trait to display a trait, many people can unknowingly be carriers of a disease. From an
evolutionary perspective, a recessive disease or trait can remain hidden for several generations before
displaying the phenotype. Examples of autosomal recessive disorders are albinism, cystic fibrosis.

X – LINKED AND Y – LINKED INHERITANCE

X – linked genes are found on the sex X chromosome. X – linked genes just like autosomal genes
have both dominant and recessive types. Recessive X – linked disorders are rarely seen in females and
usually only affect males. This is because males inherit their X chromosome X – linked genes will be
inherited from the maternal side. Fathers only pass on their Y chromosome to their sons, so no X-linked
traits will be inherited from father to son. Men cannot be carriers of recessive X-linked traits, as they
only have one X chromosome, so any X-linked trait inherited from the mother will show up.
 Females express X – linked disorders when they are homozygous for the disorder and become
carriers when they are heterozygous. X-linked dominant inheritance will show the same phenotype as a
heterozygote and homozygote. Just like X-linked inheritance, there will be lack of male to male
inheritance which makes it distinguishable from autosomal traits. One example of an X – linked trait is
coffin – Lowry syndrome, which caused by a mutation in ribosomal protein gene. This mutation results in
skeletal, craniofacial abnormalities, mental retardation, and short stature.

X chromosome in female undergo a process known as X activation. X activation is when one of

the two X chromosomes in females almost completely inactivated. It is important that this process
occurs otherwise a woman would produce twice the amount of normal X chromosome proteins. The
mechanism for X inactivation will occur during the embryonic stage. For people with disorders like
trisomy X, where the genotype has three X chromosomes, X-inactivation will inactivate all X
chromosome until there is only one X chromosome active. Males with Klinefeifer Syndrome, who have
an extra X chromosome, will also undergo X inactivation to have only one completely active X
chromosome.

Y – linked inheritance occurs when a gene trait, or disorder is transferred through the Y
chromosome. Since Y chromosomes can only be found in males, Y linked are only passed on from father
to son.

The testis

determining factor, which is located on the Y chromosome, determines the maleness of individuals.
Besides the maleness inherited in the Y-chromosome there are no other found Y-linked characteristics.

An example of a family pedigree displaying an autosomal recessive trait

A pedigree is a diagram showing the ancestral relationships and transmissions of genetic traits
over several generations in a family. Square symbols are almost always used to represent males, whilst
circles are used for females. Pedigrees are used to help detect many different genetic diseases. A
pedigree can also be used to help determine the chances for a parent to produce an offspring with a
specific trait.

Four different traits can be identified by pedigree chart analysis: autosomal dominant,
autosomal recessive, X – linked or Y – linked. Partial penetrance can be shown and calculated from
pedigrees. Penetrance is the percentage expressed frequency with which individuals of a given genotype
manifest at least some degree of a specific mutant phenotype associated with a trait.

Inbreeding, or mating between closely related organisms, can clearly be seen on pedigree
charts. Pedigree charts of royal families often have a high degree of inbreeding because it was
customary and preferable for royalty to marry another member of royalty. Genetic counselors
commonly use pedigrees to help couples determine if the parents will be able to produce healthy
children.

KARYOTYPE

A karyotype of a human male showing 46 chromosome including XY sex chromosomes.

 A karyotype is a very useful in cytogenetics. A karyotype is picture of all the chromosomes in the
metaphase stage arranged to length and centromere position. A karyotype can also be useful in clinical
genetics, due to its ability to diagnose genetic disorders. On a normal karyotype, aneuploidy can be
detected by clearly being able to observe any missing or extra chromosomes.

Giemsa banding, g-banding of the karyotype can be used to detect deletions, insertions,
duplications, inversions and translocations. G-banding will stain the chromosomes with light and dark
bands unique to each chromosome. A FISH, fluorescent in situ hybridization, can be used to observe
deletions, insertion and translocations. FISH uses fluorescent probes to bind to specific sequences of the
chromosomes that will cause the chromosomes to fluoresce a unique color.