MAKERERE UNIVERSITY

COLLEGE OF EDUCATION AND EXTERNAL STUDIES

SCHOOL OF EDUCATION

REPORT : NAMULONGE FIELD REPORT

BIIRA RONET YUNASI 23/U/0372

AFOYORWOTH ANNA PATIENCE 23/U/0104

PAATA JOHN 23/U/1347

MUKISA ELIAS NSEREKO 23/U/0844

MUTUMBA ROBERT 23/U/2062

AYEBAZIBWE EDWIN 23/U/07156/PS

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LECTURER : DR REYMICK OKETCH.
 GENETIC MATERIAL ANALYSIS DURING GENETIC MODIFICATION.
Obtaining samples (sampling)
The samples of tissues from which the genetic material is going to be obtained can be collected
at either cold temperature or normal temperature depending on the state of the source of the
genetic material i.e whether it degrades easily or can stay for long periods of time when it's
intact.

Cold temperature methods.

These methods are important when using swmples that degenerate easily due to action of
autolysis and nuclease enzymes so that cold temperatures are required to inactivate the enzymes.
1. Use of a cooling flask
In this, the sample is placed in a microfuge tube and placed in a cooling flask containing ice-salt
mixture at around -35.0C. This method is most useful when DNA is to be analysed. If RNA is of
interest then colder temperatures should be used because unlike DNA, most RNA doesn't have a
double stranded structure and associated molecules to protect from action of nuclease enzymes.
2. Use of liquid nitrogen
In this case the sample is wrapped in aluminium foil and dropped into liquid nitrogen and kept at
around-700C. This is most useful if RNA is of interest.

Room temperature methods.

These are used if the specimen can be extracted in an undamaged form thus doesn't degenerate
over a short period of time. They are very reliable when the specimen is to be got from a far
away place.
1. Use of a news print.
In this,the sample e.g leaves are detached carefully, and attached the onto a news print using
tape. The news print is then placed between 2 card boards that are ridged and then numerous
cardboards are wrapped together.
2. Use of silica gel.
The sample is put into a bottle containing silica gel to keep the bottle free of moisture to prevent
degeneration of the specimen.

TOTAL NUCLEIC ACID EXTRACTION.

During extraction of nucleic acids different protocols are followed depending on the type of
reagents to be used. Common examples include the CTAB protocol and the DELLAPORTA
protocol.
However all extraction procedures follow the following steps.
1. Grinding of the tissue.
This ensures that the tissue is broken into smaller pieces so that cell contents could be
released. While doing this the tissue has to be placed in a homogeniser solution to ensure
that cell contents do not degenerate. The tissue can be grinded using;
a. Pestle and motor to form a paste.
 b. Using a grinder: The small pieces of the tissue are placed in small tubes and then
beads are inserted and put in a tissue grinder machine. The machine makes the
beads to move up and down grinding the tissue.
c. Using a tissueliser: this is the fastest.

2. Incubation.
The grinded tissues are incubated in a suitable reagent at a suitable temperature (65 0C) in a water
bath. The reagent used is hypotonic to the cells and thus causes further lysis of the cells releasing
cell contents including the genetic material in solution.

3. Centrifugation.
The formed suspensions are then put in centrifuge tubes and spin at very high speeds so that the
contents can separate out according to their densities. The top most supernatant containing the
least dense genetic material can then be pipetted off.

4. Precipitation.
The pipetted supernatant is then put in isopropanol or 70% ethanol to precipitate out the genetic
material based on the fact that it's non polar. The set up is left at freezing temperatures overnight
to precipitate more genetic material and the it is pelleted. The pellets are washed with 70%
ethanol and then dissolved in water to which selection of the genetic material of interest is done
by addition of a nuclease enzymes that digests the the other genetic material of no interest.
The remaining genetic material can the proceed to amplification by PCR.

GENE AMPLIFICATION BY POLYMERASE CHAIN REACTION (PCR).

Polymerase chain reaction (PCR) is a texhnique used to produce very many copies of sections
of DNA in a short period of time. It involves use of an enzyme called TAQ polymerase extracted
from a thermo stable bacteria called Thermus aquaticus. An enzyme stable at high temperatures
is required because there's use of high temperatures at which animal enzymes or E. Coli enzyme
would degenerate. TAQ polymerase is very fast, but however lacks proof reading mechanisms
which makes the PCR process prone to error.
PCR also requires primers to be added and this provides the basis for the selectivity of gene of
interest because since DNA polymerase enzymes require primers to start synthesizing
complementary strands of DNA, only the required section of the DNA will be amplified.
PCR reactions proceed via series of temperature changes in a cycle called the thermionic cycle.
For the process of PCR to be successful a pH buffer e.g magnesium chloride, some water,
Deoxyribose nucleotides, TAQ polymerase and the DNA containing the gene of interest have to
be added to PCR tubes and then the tubes are placed in PCR tubes.
The steps involved include the following.

1. Unwinding the DNA helix.

This is done by heating the DNA helix to high denaturing temperatures of about 980C to denature
break the hydrogen bonds and open the DNA helix.
2. Annealing.
During annealing the temperature is lowered and DNA primers align along the DNA strand
according to rules of complementary base pairing. The annealing temperatures are about 40 0C to
68 0C.
3. Extension.
In this TAQ polymerase starts extending the DNA strand till the section of the gene of interest is
covered and the cycle repeated. The extension temperature is at about 720C which is around the
Optum temperature for TAQ polymerase.
The above cycle is a 3 step cycle.
The systhesises multiple sDNA strands in a an exponential pattern making it very first.
If RNA is to be analysed, it first has to be converted into a complementary DNA strand using
reverse transcriptase enzyme and then the DNA is amplified.
Due to errors involved in PCR, gene modification can not totally rely on PCR and thus use of
cloning vectors cannot be done away with.

The tubes containing the amplified copies of a gene (amplicon) can then be anlaysed using GEL
ELECTROPHORESIS to determine whether the gene of interest is available in the sample.

GEL ELECTROPHORESIS.
Electrophoresis is a method of separating charged substances based on their difference in
mobility in an electric field as a result of difference in mass. If DNA is to be analysed, the
difference in mass is a result of difference in number of base pairs.
A reagent called agrose gel is heated with a buffer and ethilium bromide to melt and then
placed on an electrophoresis plate. Combs are inserted to create holes before the gel solidifies.
The amplicon is then pored into the holes that have been oriented so that they are on the negative
side of the tray. Current is switched on to make the DNA move towards the positive electtrode
since it is negatively charged.
The gel is then illuminated with UV light and the different thick lines that indicate the position of
the DNA strands according to number of base pairs are noted. These are then compared with a
standard gel diagram that has the number of base pairs of the gene under investigation. If they
match then the gene of interest is present.
Alternatively the gel can be put in a machine having a gel documentation system for analysis.
This machine takes a picture of the gel and displays it on the screen.
TISSUE CULTURE.
Tissue culture is a method of growing tissues or cells in an artificial environment away from the
parent organisms.
Requirement
● Basic laboratory setup
● Nutrient medium
● Sterile medium
● Sample tissue.
The culture medium is prepared by mixing a liquid medium containing adequate quantities of
mineral salts, organic nutrients like glucose and plant hormones with a gelling agent e g agar that
forms a solid substrate.
A sample tissue is then sterilised by passing it through sodium chloride or calcium hypochlorite
solutions and then placed on the solid growth medium or it can be directly iserted into the liquid
nutrient medium.
Organisms grown via tissue culture are grown in sterile environments and before transferred to
the natural environment they have to go through a series of stages involving gradual exposure to
the natural conditions.
Tissues culture provides analytical services in the field of risk assessment of genetically
modified organisms.
Applications of tissue culture
Rapid clonal propagation: Soma-clonal variation, induction and selection of mutations, resistance
to weedicides, hasgenuc plants, mechanism for propagating and genetically improving
commercially valuable plants to study characteristics of the growth, metabolism, reproduction
and physiology and nutritional necessities of the plants under controlled circumstances mutagens
are added to single cell liquid cultures for initiation of mutation, intraspecific hybridization, large
scale fabrication of artificial seeds through somatic embryogenesis the production of multiple of
plants in the lack of seeds or essential pollinators to produce seeds.
Micropropagation is widely used to produce clones of a plant and this offers certain advantages
over traditional methods of propagation. They include;
The production of exact copies of plants that have good flowers, fruits or having more desirable
traits, to quickly produce mature plants, it allows regeneration of whole plants from plant cells
that have been genetically modified, it allows the production of multiples of plants in absence of
seeds or necessary pollinators to produce seeds, plants that are grown are free from pathogen,
diseases and pests.

CLONING

Cloning is the process of generating a genetically identical copy of a genetic molecule, cell or an
organism. Cloning happens often in nature; for example, when a cell replicates itself asexually
without any genetic alteration or recombination. Prokaryotic organisms such as bacteria create
genetically identical duplicates of themselves using binary fission or budding. In eukaryotic
organisms such as humans, all the cells that undergo mitosis, such as skin cells and cells lining
the gastrointestinal tract, are clones; the only exceptions are gametes (eggs and sperm), which
undergo meiosis and genetic recombination.

In biomedical research, cloning is broadly defined to mean the duplication of any kind of
biological material for scientific study, such as a piece of DNA or an individual cell. For
example, segments of DNA are replicated exponentially by a process known as polymerase
chain reaction, or PCR, a technique that is used widely in basic biological research. Cloning
can be classified into;

1. Gene cloning or DNA segment cloning

2. Cell cloning: the division of an asexually reproducing cell such as a bacterium into a
group of genetically identical cells
3. Organism cloning; cloning produces one or more organisms that are genetically identical
to the “parent”.
The type of cloning that is the focus of much ethical controversy involves the generation of
cloned embryos, particularly those of humans, which are genetically identical to the organisms
from which they are derived, and the subsequent use of these embryos for research, therapeutic,
or reproductive purposes.

The current interest in organismal cloning is primarily due to its ability to generate stem cells. A
stem cell is a relatively unspecialized cell that can both reproduce itself indefinitely and, under
appropriate conditions, differentiate into specialized cells of one or more types. Stem cells have
great potential for regenerating damaged tissues.

Cloning plants: single cell culture

Differentiated cells taken from the root (e.g. carrot) and incubated in culture medium could grow
into normal adult plants, each genetically identical to the parent plant. These results showed that
differentiation does not necessarily involve irreversible changes in the DNA.

In plants, mature cells can “dedifferentiate” and then give rise to all the specialized cell types of
the organism; any cell with this potential is said to be totipotent.

Plant cloning is used extensively in agriculture. For plants such as orchids, cloning is the only
commercially practical means of producing new plants. In other cases, cloning has been used to
reproduce a plant with valuable characteristics, such as resistance to plant pathogens.

Cloning Animals: Nuclear Transplantation

Differentiated cells from animals while in culture, generally do not divide or develop into the
multiple cell types of a new organism. Therefore, early researchers had to use a different
approach to answer the question of whether differentiated animal cells are totipotent.
This approach involved removal of the nucleus of an egg (thereby creating an enucleated egg)
and replace it with the nucleus of a differentiated cell, a procedure called nuclear
transplantation or somatic cell nuclear transfer. If the nucleus from the differentiated donor
cell retains its full genetic potential, then it should be able to direct development of the recipient
cell into all the tissues and organs of an organism. Such experiments were conducted on one
species of frog (Rana pipiens) and on another frog species (Xenopus laevis). These researchers
transplanted a nucleus from an embryonic or tadpole cell into an enucleated egg of the same
species. The transplanted nucleus was often able to support normal development of the egg into a
tadpole.

However, it was found that the potential of a transplanted nucleus to direct normal development
was inversely related to the age of the donor: The older the donor nucleus, the lower the
percentage of normal tadpoles.

In frogs and most other animals, nuclear potential tends to be restricted more and more as
embryonic development and cell differentiation progress. These were foundational experiments
that ultimately led to stem cell technology.

Reproductive Cloning of Mammals

Researchers were able to clone mammals using early embryonic cells as a source of donor
nuclei. Later researchers implanted early embryos into surrogate mothers. Out of several hundred
embryos, one successfully completed normal development. This is because a certain step of
reprogramming in the process involves epigenetic changes that lead to changes in chromatin
structure.

A number of other animals were cloned by Somatic Cell Nuclear Transfer (SCNT), including
pigs, goats, rats, mice, dogs, horses, and mules. SCNT is a technique in which the nucleus of a
somatic cell is transferred into an enucleated metaphase-II oocyte for the generation of a new
individual generally identical to the somatic cell donor. In 2001 a team of scientists cloned a
rhesus monkey through a process called embryonic cell nuclear transfer, which is similar to
SCNT except that it uses DNA from an undifferentiated embryo.

Progress in cloning mammalian embryos, including primates, has heightened speculation about
the cloning of humans, which has not yet been achieved past very early embryonic stages. The
main reason researchers have been trying to clone human embryos is not for reproduction, but
for the production of stem cells to treat human diseases.

Many early animal embryos contain stem cells capable of giving rise to differentiated cells of
any type. Stem cells can be isolated from early embryos at a stage called the blastula stage or its
human equivalent, the blastocyst stage.

Epigenic differences in cloned animals

Many cloned animals exhibit defects with only a small percentage develop normally to birth.
Many are prone to obesity, Pneumonia, liver failure and premature death. A small subset of
genes is turned on and expressed and the rest of the genes are repressed. This is a result of
epigenic changes in chromatin; like acylation of histone or methylation of DNA. Such changes
must be reversed in later stage nucleus from donor animals for genes to be expressed or
repressed in earlier stages of development.

Success in cloning attempts may depend on whether or not the chromatin in the donor nucleus
can be artificially modified to resemble that of a newly fertilized egg.

Stem cells in Animals

Human embryo cloning is mainly not for reproduction but for production of stem cells to treat
human diseases. This is called therapeutic cloning. Many early animal embryos contain stem
which are capable of giving rise to differentiated cells of any type. Stem cells can be isolated
from embryo at blastula stage (blastocyst stage in humans). The embryonic stem cells can
reproduce indefinitely in suitable culture conditions. They can be made to divide into a variety of
specialized cells including reproductive cells.

Adult body contains stem cells which serve to replace non-reproducing specialized cells as
needed. However, adult stem cells are not able to give rise to all cell types in the organism
though they can generate several cell types. For example

Region of origin of stem cell Cells that can be generated

Bone marrow All blood cells

Another bone marrow type Bone, cartilage, fat, muscle, lining of blood
vessels

Brain Nerve cells

Stem cells can maintain their own population and generate differentiated cells. A stem cell can
divide into another stem cell(s) and a progenitor cell(s). the progenitor cell can differentiate into
several cell types.

However, none of the adult stem cells are as versatile as embryonic cells (ES). The ultimate aim
of studying, isolating of adult stem cells and reproducing differentiated cells is to supply cells fo
repair of damaged or diseased organs; for example

● insulin-producing pancreatic cells for people with type 1 diabetes

● certain kinds of brain cells for people with Parkinson’s disease or Huntington’s disease.
 ● Adult stem cells from bone marrow have long been used in bone marrow transplants as a
source of immune system cells in patients whose own immune systems are nonfunctional
because of genetic disorders or radiation treatments for cancer

Embryonic stem (ES) cells are better than adult stem cells because ES cells are pluripotent
meaning; they are capable of differentiating into many different types of cell.

However, fully differentiated cells can be programmed to act like embryonic stem cells. Such
kind of cells are called “Induced Pluripotent Stem” cells (iPS cells). Differentiated stem cells
can transformed into a type of embryonic stem cells by using a modified retrovirus to introduce
into them extra cloned copies of four stem cell master regulatory genes. The cells are
deprogrammed to restore their undifferentiated state; that is to restore pluripotency, thus the
name Induced pluripotent stem cells.

Induced pluripotent stem cells have some differences compared to embryonic stem cells for
example in gene expression and other cellular functions. These differences are still under study
to improve stem cell therapies. The major importance of iPS cells include;

1. To act as a model for studying and treatment of some diseases as cells of patients have
been reprogrammed to become iPS cells.

2. In regeneration medicine whereby a patient’s cells could be reprogrammed into iPS cells
and then be used to replace nonfunctional tissues.

How a fully differentiated human cell can become a stem cell

A retroviral vector can be used to induce four genes into fully differentiated human skin
fibroblast cells. The cells are then cultured in a medium that would support growth of stem cells.
In two weeks, cells that resemble embryonic stem cells in appearance appear actively dividing.
Their gene expression patterns, gene methylation patterns, etc are consistent with those of
embryonic stem cells.

This shows that the four genes induced the differentiated skin cells to become pluripotent stem
cells with characteristics of embryonic stem cells.