DEPARTMENT OF BIOCHEMISTRY, MICROBIOLOGY AND BIOTECHNOLOGY

MODULE : GENE STRUCTURE, FUNCTION, MOLECULAR BIOLOGY

MODULE CODE : SMIB 032

SURNAMES & INITIALS : MAYIKO RR

STUDENT NUMBER : 201727601

DATE : 29 JULY 2022

ACTIVITY 1 : POST PRACTICAL ASSIGNMENT

1. DNA extraction is the process of isolating Deoxyribonucleic Acid (DNA) from a
sample containing DNA by breaking and separating the DNA from the cell and
purify it from proteins and any other cellular components, the first DNA
extraction was perfomed by Friedrich Miescher around late 1860s.[1] The
process of DNA extraction includes a variety of steps such as DNA analysis
where the quality and quantity of the DNA extracted is being analysed for further
application depending on where the extracted DNA will be applied, for instance
the extracted DNA can be applied at forensic labs or for paternity tests. There
many techniques used for DNA extraction from the sample, some of the
techniques include organic extraction technique (Phenol – Chloroform),
inorganic extraction technique (Salting out and proteinase K treatment),
adsorption technique (Silica gel membrane) these techniques are chemical
techniques.[2]
The purpose of DNA extraction includes the use of DNA in many places for
many various reasons such as extracting DNA for forensics, where it will be
used as evidence to prove crime cases and also the extracted DNA can be
used for medical studies, where biologists will study the genetic sequence of it
by identifying certain genetic diseases and attempt to dig up more details on
those diseases, how are they caused, how they can be cured, and how life
threatening are they.[3] Laboratories study DNA with the purpose of developing
medicine for genetic diseases, for instance Down syndrome is a genetic
chromosome 21 disorder which causes development delays and intellectual
delay. Another example is cancer which is a genetic disease caused by gene
mutation. These are kinds of disease that are being studied with the aim of
having a better understanding on why and how these diseases are developing,
and all these diseases explains the purpose of DNA extraction.
The principle of DNA extraction for extracting good quality of DNA involves
numerous steps from disrupting the cell wall, cell membrane and nuclear
membrane to release the DNA into a solution, where the solution will be
precipitated and DNA within the solution will precipitate leaving all the unwanted
molecules such as proteins in the solution unbound, then alcohol will be used
to purify the precipitated DNA removing all cell debris.[4] After removing the
alcohol, the DNA will be dissolved in water for analysis and quantification by
checking if the extracted DNA is pure or contaminated.
2. Forensics
Forensic is one of the applications of isolated DNA, as broad as forensic is,
most of the forensic departments depend on DNA extraction to do their work,
to name few of the departments, forensic anthropology, forensic pathology,
forensic entomology etc.[3] These departments use DNA extraction on daily
basis in solving cases may it be criminal cases or identifying death causes of
individual as far as proving the innocence or guilty of individuals that are being
suspects. The extracted DNA plays a crucial role in forensics by identifying
individuals that are involved in crimes since, every person has a unique DNA
profile, DNA can be extracted from many cells or sample, any material
containing DNA in the form of hair, blood, skin, semen, saliva etc.()
Relationships
DNA extraction is also applied in relationships also known as paternity tests
where it is used to match the DNA pattern of parents (mother/father) with the
DNA pattern of the offspring, with the aim of proving who is the biological parent
of a child and who is not. The medical practitioners use the DNA extracted from
a sample, it can be from a hair sample or blood, purify the DNA run it if gel
electrophoresis, then a pattern will be issued showing similarities or differences
between a parent and a child.[5]
Medical diagnostics
For some medical conditions, DNA extraction is very important to officially
diagnose it, especially if the medical condition is genetic, it is carried within the
genes of an individual. Most examples include cancer, Down syndrome, cystic
fibrosis, sickle cell disease, Huntington’s disease, Turner syndrome, Triple X
syndrome etc. DNA extraction is used in identifying if the person is having or
carrying that kind of disease within their genetic information.[6]
3. Deoxyribonucleic acid (DNA) is the hereditary material found in almost all living
organisms where it is found in prokaryotic and eukaryotic cell. The DNA which
is found in prokaryotes is in a circular DNA shape while the DNA which is found
in eukaryotes is a linear DNA shape.[7] In a prokaryotic cell the DNA is located
in the central area of a cell called nucleoid since the prokaryotic cell doesn’t
have a nucleus, the DNA molecule in a circular shape is called plasmid DNA.
 The eukaryotic cell carries the DNA molecule within the nucleus, where DNA is
organised in long molecules called chromosomes. The extraction procedures
between the plasmid and genomic DNA may differ that might be because the
DNA molecules are being extracted from different environments. Both DNA
procedures they involve cracking the cell open and releasing the DNA.
4. The method used to extract the bacterial plasmid DNA from E.coli was alkaline
lysis method. The first step is to crack and open the bacterial cell extract the
plasmid DNA, then the process involves 3 solutions of lysis buffer (lysis buffer
I, lysis buffer II, lysis buffer III). Lysis buffer I which contains EDTA at a pH of 8,
which will disrupt the cell because of its pH. Lysis buffer II was added and it
contains SDS and NaOH where NaOH and SDS will break the disrupt the
hydrogen bonding between DNA stand and high pH will cause the proteins to
denature. Then centrifugation will help isolating the DNA from all the unwanted
molecules.(SMIB032, 2022 practical Guide)
5. Advantages of alkaline lysis method includes:
➢ Alkaline lysis method is the best method for plasmid DNA extraction
because it is fast, less time is consumed no need to spend weeks like
some other methods
➢ Alkaline lysis method is a reliable technique for extracting plasmid DNA
➢ It is relatively one of the cleanest methods to obtain good quality DNA
from the cell
6. Disadvantages of alkaline lysis method is that even though it is rated as one of
the best extraction techniques for good quality DNA, it also has some
challenges such as entrapment of plasmid DNA cell debris which may cause
lower quantity of plasmid DNA recovery and another challenge is that they
might be an increase of volume due to the buffers used which might cause the
increase cost of production.[8]
7. The expected results in terms of size, concentration and quality of DNA, a good
quality of DNA is expected which will be free from contaminants and lower
concentration to show that it is not contaminated with other nucleic acids. The
experiment yielded very poor results and because the concentration of the
plasmid DNA was high and it showed that the DNA was contaminated. The
cause of this error I think its because of the centrifugation step, these shows
that the sample was supposed to be given time for centrifugation. To improve
 the experiment I would repeat the experiment and increase the time for
centrifugation and the DNA drying step.

References

[1]. Dahm R (January 2008). "Discovering DNA: Friedrich Miescher and the early years
of nucleic acid research". Human Genetics. 122 (6): 565–81

[2]. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from
micro-organisms. Journal of Molecular Biology 3(2)

[3]. Elkins KM (2013). "DNA Extraction". Forensic DNA Biology. pp. 39–52

[4]. J. Sambrook and D. Russel, Molecular Cloning: A Laboratory Manual, vol. 3, Cold
Spring Harbor Laboratory Press, New York, NY, USA, 3rd edition, 2001

[6]. Boycott, K., Vanstone, M., Bulman, D. et al. Rare-disease genetics in the era of
next-generation sequencing: discovery to translation

[7]. Hearn, R. P., & Arblaster, K. E. (2010). DNA Extraction Techniques for Use in
Education. Biochemistry and Molecular Biology Education, 3, 161-166

[8]. H. C. Birnboim and J. Doly, “A rapid alkaline extraction procedure for screening
recombinant plasmid DNA,” Nucleic Acids Research, vol. 7, no. 6, pp. 1513–1523,
1979