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2.

0 INTRODUCTION
Deoxyribonucleic acid, or DNA, is the molecule of life. It is the chemical code specifying our
function, appearance and pedigree and is unique for all individuals except identical twins. An
individuals DNA is formed by combination of DNA from his or her parents with half coming from
the mother and half from the father. For this reason, DNA testing can be used as evidence of
paternity of a child.
DNA is found in most cells of the body, including white blood cells, semen, hair roots and
body tissue. Traces of DNA can be detected in body fluids such as saliva and perspiration.
Mitochondrial DNA, which follows the maternal line of an individual, can be extracted from hair
and bone samples. This can be used to examine relatedness and common ancestry between
individuals, and to verify the identity of buried remains.
Polymerase chain reaction (PCR) had an impact on four main areas of biotechnology:
gene mapping, cloning, DNA sequencing, and gene detection. PCR is now used as a medical
diagnostic tool to detect specific mutations that may cause genetic disease, in criminal
investigations and courts of law to identify suspects on a molecular level and also in the
sequencing of the human genome. Prior to PCR the use of molecular biology techniques for
therapeutic, forensic, pharmaceutical, or medical diagnostic purposes was not practical or costeffective. The development of PCR technology changed these aspects of molecular biology
from a difficult science to one of the most accessible and widely used tools in genetic and
medical research.
The central scientific fact that makes PCR so useful is that the genetic material of each
living organism; plant or animal, bacterium or virus possesses sequences of its nucleotide
building blocks (DNA and RNA) that are uniquely and specifically present only in its own
species. Indeed, complex organisms such as human beings possess DNA sequences that are
uniquely and specifically present only in particular individuals. These unique variations make it
possible to trace genetic material back to its origin, identifying with precision at least what
species of organism it came from.

Genes can be identified by gel electrophoresis. Gel electrophoresis is one of the staple
tools in molecular biology and is of critical value in many aspects of genetic manipulation and
study. One use is the identification of particular DNA molecules by the band patterns they yield
in gel electrophoresis after being cut with various restriction enzymes. Viral DNA, plasmid DNA,
and particular segments of chromosomal DNA can all be identified in this way. Another use is
the isolation and purification of individual fragments containing interesting genes, which can be
recovered from the gel with full biological activity.
Gel electrophoresis makes it possible to determine the genetic difference and the
evolutionary relationship among species of plants and animals. Using this technology it is
possible to separate and identify protein molecules that differ by as little as a single amino acid.
These two techniques, PCR and gel electrophoresis are very useful in DNA profiling and has
definitely helped in solving court cases and identifying target personnels.

11.0 RECOMMENDATIONS
Some of the recommendations towards the betterment of the results from this experiment
include:

The DNA sample should be placed on ice bucket as enzyme might denatured at high

temperature.
The enzyme must be kept in ice bucket to keep the temperature low as enzyme can be

denatured in high temperature.


The voltage should be set at suitable range as a high voltage would cause the gel to

melt.
Make sure that the percent of Agarose gel is not too high as higher concentrations cause

the long run times.


Page method can also be used to separate DNA fragment instead of using

electrophoresis.
The concentration of dye used during the staining should not be high as the band cannot

be seen.
Agarose gel should be handled with care as it is easily broken.

12.0 REFERENCE
Brown, T. (2010). Gene cloning and DNA analysis: an introduction: Wiley-Blackwell.
Mozayani, A. (2011). The forensic laboratory handbook procedures and practice: Springer
Science+ Business Media.
Snustad, D. P., & Simmons, M. J. (2009). Principles of genetics: Wiley.

According to Manz, Pamme and Lossifidis (2004), electrophoresis can be defined as the
movement of electrically charged particles or molecules in a conductive medium under the
influence of an applied electric field, moves towards the cathode or anode. The conductive
medium is usually an aqueous buffer, which also referred to as an electrolyte or run buffer. The
mixture of analytes is introduced into the medium containing the run buffer and an electric field
is applied. The analyte mixture contains negatively charged molecules. Upon application of the

electric field, the anions start moving towards the positive electrode (anode). Differences in
charge and size will often lead to different mobilities and thus separation of the different sample
components. Similarly, positively charged ions move towards the cathode in an applied electric
field.
Electrophoretic separations can be performed in free solution or in a solution containing
a non-conductive matrix such as an agarose or polyacrylamide gel. The separation of the
analyte ions occurs due to differences in mobility for instance, differences in the charge to size
ratio. Joule heating can interfere with the separation and thus causing band broadening. In
absence of band broadening factors, the compounds move in the form of zones or bands. Gel
has as a sieving effect which acts as heat dissipating medium which minimised the bands
broadening ability. The separation of analytes in a gel is also based on the differences in
mobility. The sieving effect in a gel causes the larger compounds to retard more that the smaller
compounds. This means that in gel electrophoresis, two compounds with same charge to size
ratio can be separated as long as they are different in size. The efficiency of an electrophoretic
separation is governed by two main factors which are electrophoretic mobility of the analytes
and the so-called electroosmotic flow (EOF) of the bulk solution (Manz, Pamme & Iossifidis,
2004).

Figure 4.1 The migration of DNA band from negaitive charge to positive charge.

The electrophoretic mobility of DNA is also affected by the composition and the ionic
strength of electrophoresis buffer. In the absence of ions, electrical conductivity is being
minimized and lead to slow migration of DNA. It is said that if the buffer is high ionic strength as
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if 10 times electrophoresis is mistakenly used, the electrical conductance is said to be very


efficienct and significant amount of heat is generated. This results in the gel melting and
denaturing the DNA molecules itself (Vennison, 2009).

As been stated by Allison (2007), since DNA is negative charged based on its phosphate
backbone, the DNA is then migrated towards the anode. Proteins and nucleic acids are being
separated by electrophoresis matrix or gel, Commonly, the gel cast in the shape of a thin slab,
with wells for loading the sample. The gel is immersed in the buffer so that it can provide ions to
carry a current and some type of buffer to maintain the pH at relatively constant value. As
agarose gel is used in this experiment, it is then submerged in a buffer solution and an electric
current is passed through the gel, with negatively charged DNA due to the phosphate in the
sugar-phosphate backbone, moving through the gel from negatively charged anode towards the
positive electrode (anode).

Figure 4.2 The separation principle of electrophoresis. Particles with different charges in this
case negative charges and different sizes migrate at different velocities in an applied electric
field (Manz, Pamme & Iossifidis, 2004).
Staining is one of the crucial part of this experiment. This is because stains are dyes
which stick to cells or parts of cell by virtue of their charge or solubility properties. Most simple
stains are dyes that have a strong positive charge; this is they are cations. Most proteins,
carbohydrates, nucleic acids have a net negative charge. By adding the stain the cells, ionic
attraction keeps the stain attached to the cells so that rinsing with water does not rinse off the
stain attached to the cells. As in this experiment, the DNA can be more visible with the helps of
staining with the attachment of stick dye on the DNA fragment itself.
DNA ladder is generally contains a set of known DNA fragments with different sizes in
base pairs. These fragments are being separated and visualized as DNA bands on agarose gel.
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The separated DNA bands together looks like a ladder and thus the DNA is then called DNA
ladder. In electrophoresis, the DNA ladder is used for determining the size and quantity of
testing DNA fragments of genomic, PCR DNA or plasmid. Basically, DNA ladder is in liquid form
where a few amount of DNA ladder is loaded in a well on agarose gel testing with DNA
fragments. Quantity of each DNA band can be determined once the loading volume is known.
The fragments determined after electrophoresis process can be compared to the DNA ladder
according to its size and quantity.

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