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Name : Joyleen Mutubuki

Reg no : H200638G

Program : Biotechnology

Course : Bioseperation Engineering

Code : SBT 123

Lecturer: Mrs Sengudzwa

Year : 2021

Level: 1.2

Title : Practical write up

Title:

DNA extraction

Abstract :
An experiment was carried out in which deoxyribonucleic acid (DNA) was extracted
from an onion. The onion was cut up into small pieces and crushed with a mortar and
pestle in a salt solution in order to release the contents of the cells. The mixture was
drained and a detergent (sodium dodecyl sulfate) was added to the solution to help
break down the nuclei membrane and release the DNA. 95% alcohol was added to
this mixture to help precipitate the DNA. The DNA was found to be a clear, mucus
like substance floating in the alcohol layer of the resultant mixture.

Introduction:

In 1953 Waston and Crick discovered the structure of DNA.Deoxyribonucleic

acid(DNA) is a nucleic acid polymer ,consisting of the monomers called nucleotides
(Rettner 2017).Each nucleotide consists of a phosphate group ,a deoxyribose pentose
sugar and a nitrogenous base pairs ( adenine ,guanine ,cytosine and thymine)DNA
found in the nucleus of cells contains a set of code dinstructions made up of a specific
order of nucleotides particularly the nitrogen bases (Robert 2016). According to “life
Sciences Cyberbridge ’’,adenine always pair with thymine and guanine pairs with
cytosine .DNA molecule is made up of two nucleotide chains that are binded by
hydrogen bonds between the complementary base pairs and arranged in a spiral
shaped ,known as doube helix structure.

There are various methods for DNA extractions, each having its advantages and
disadvantages. One method is the enzymatic extraction method. It involves the usage
of the enzyme, protease which digests the protein content of the sample that is heated
to suitable enzymatic action temperature. Ice-cold ethanol is then used to precipitate
and store the broken down. The DNA will be left between the mixture and the ethanol
(Genetic Education, 2018). The advantage of this method is that it is fast and produces
high yield of DNA. The disadvantage of this method however, is that protease is an
enzyme, short in shelf life time, needs to be preserved and can only be effective at
specific temperature. Another method is the phenol-chloroform extraction method. It
uses chloroform, phenol to remove protein and isoamyl alcohol to separate the DNA
content (Genetic Education, 2018). The advantage of this method is that the yield of
DNA is very high while the disadvantage is that a great quantity of sample is required
for the extraction. The next method is the CsCl density gradient extraction method.
By centrifugation, the DNA is separated by its density, which appears when its
density is the same with the CsCl gradient (Genetic Education, 2018). The advantage
of such method is that the purity of the DNA content is high but the it is very time-
consuming.

The experiment conducted used a mechanical method to disrupt the cells. A mortar
and pestle can be used to crush the cells and release the cellar contents. The onion
cells are crushed in a warm salt solution. The warmth softens the onion tissue making
it easier to disrupt the cells. The warmth also denatures some enzymes and proteins
including DNAase which would break down DNA. The positive sodium ions from the
salt solution stabilise the DNA by forming a shell around the negatively charged
phosphate groups in DNA. This occurs due to electrostatic attraction (Hadke et al,
2018).

Sodium dodecyl sulphate (SDS) is an anionic detergent that is used to degrade the cell
membranes of the nuclei of cells (Ghosh, 2006). It also degrades the histones
associated with DNA by destroying their secondary and tertiary structures. When 95%
alcohol is added to the mixture two separate layers form. The alcohol disrupts the
electrostatic interactions between the water molecules in the solution and the DNA
causing them to separate and the DNA is precipitated into the alcohol layer (Madden,
2015).
DNA extraction experiment is very important to be conducted because it helps
scientist to study and understand more of life and its connection to various species
here on earth at the level of genetics. By successfully extracting DNA, it can be
preserved and duplicate the genetic codes of life. Medical research can be lengthen so
as to detect the faulty codes responsible for many genetic diseases. Through genetic
engineering with the knowledge of DNA extraction, scientist bring an end to such
diseases that have affected mankind. DNA knowledge can be used as forensic
evidence to be used for crime case investigations (What Is Biotechnology?, n.d.).
Hence, further research and development on DNA extraction will greatly benefit the
world.

Aims and objectives:

Aim: to extract DNA from onion cells and to see how it looks like.
Objectives: to learn how to effectively disrupt cells

Methodology:
Materials:
Onion
Blender
Salt
Toothpicks
Rubbing alcohol
Strainer
Warm water
Beaker
Clear liquid dish soap

Method:
1. Small pieces of onion were cut.
2. The pieces were added into the blender and filled it with enough salty water so as to
cover it (10% NaCl solution).
3.The small pieces of onion were then blended for 5-10 minutes ,but they were not
liquefied
4.The blended mixture was poured through a strainer into a clear glass ,filled the glass
about half .
5. 2-5 teaspoons of soap were added and gently stirred the mixture ,tried not to make
bubbles.
6. Alcohol(Chilled 95% alcohol) was then slowly poured into the glass,poured the
alcohol down the side of the glass so that it formed a separate layer on top of the
soapy cell mixture.Filled near to the top.
7.The mixture was then allowed to stand for 3-5minutes and observed what happened.
The DNA slowly raised from the watery lower layer up into the alcohol layer above it.
The DNA looked stringy and had small bubbles attached to it. It was clear, “snotty”
substance and was hard to see.
8. The substance was then slowly twisted onto a toothpick . (Do not pick up cell scum
from the lower layer).

Results:
There was a clear mucus like substance which was observed rising from the aqueous
solution.Bubbles were observed around the DNA strands.

Fig 1: Precipitated DNA from the alcohol layer

Questions:
1. What does the salt do?
The salt stabilises the DNA by providing positive sodium ions that neutralise the
negative charge on DNA.
2. What does the blender do?
The blender crushes the cells, thereby allowing their contents to be released into the
solution.
3. When you mix the blended cell source with the soap, what is happening?
The soap is further degrading the components of the solution by breaking down the
cell membranes of the nuclei present in the solution.
4. What does the alcohol do?
The alcohol breaks the electrostatic attraction of water and DNA and allows the DNA
to be precipitated.
5. Why does the DNA rise to the top after adding alcohol?
The alcohol is also less dense than water and so sits above the water. The DNA is also
less dense and insoluble in alcohol.

6. If you try a seed food such as peas, there will be more protein residue in
the liquid. Why?
Because seeds stored proteins in them for the nutrition of the new plant.
7. Why can’t you see the double helix?
The double helix is too small to be seen with the naked eye. What is seen is many,
many strands of DNA
8. What part of the cell does the DNA come from?
The nucleus.

Discussion:

The experiment called for a blender but a mortar and pestle was used. A blender could
have been a better way to disrupt the cells as the blade of the blender would cut right
through the cell whereas with a mortar and pestle some of the cells may not have been
sufficiently crushed.
The DNA molecule was hardly to be seen and the molecules were in fewer
numbers .This result might be caused by ,during the course of the experiment alcohol
was supposed to be added to water but the researcher did not do like wise. ,
According to Piškur and Rupprecht (1995) the addition of ethanol to water lowers the
dielectric charge of the solution. This prevents the nucleic acid from dissolving into
the water and causes it to precipitate out of the solution. In addition to that, ethanol
also induces conformational changes in the nucleic acid structure to allow the
repulsive forces between nucleic acids to diminish, allowing the nucleic acids to
aggregate.

Since the procedure called for using 2-5 teaspoons of detergent but the researcher
used a spatula for the measurement of detergent ,since a teaspoon and a spatula are
not equal in their graduation so the detergent which was used was not enough for the
mixture thus few amounts of DNA was liberated .Detergents are amphipathic
molecules and they also contain both non-polar tails and polar heads (Khan Academy,
n.d.b). When the cell membranes of the onions are mixed with detergent solution,
detergent molecules form micelles around the hydrophobic portions of the lipids and
proteins and causes the cell membranes and nuclear membranes to disperse, degrade
and dissolve into the solution (Thermo Fisher Scientific, n.d.). Thus, liberating the
DNA material from the cell.

The experimental procedure called for a chilled 95% alcohol but the researcher used
70 % alcohol and also the alcohol was removed from the chiller whilst the researcher
was still busy crashing the onions ,so the alcohol started to take the room temperature
thus why the extracted DNA was hardly to be seen .If chilled 95% alcohol was used
the result were possibly to be seen clearly with a naked eye .According to Maniatis,
Fritsch, and Sambrook (1982),ethanol is most commonly used alcohol in alcohol
precipitations of nucleic acid. This is because the structural differences of ethanol
allows the dielectric constant of water to be lowered. Low-permittivity in solvents
allow ions of opposite charges to form ion pairs (Izutsu, 2011). According to Zumbo
(2012), he suggests that the attraction between two charges over a distance can be
described using Coulomb’s law, and while Coulomb’s law is only valid for point
charges, it can still predict the trends of how ions interact with each other within a
solvent.Eickbush and Moudrianakis (1978), (as cited in Zumbo, 2012), the
Coulomb force of attraction between cations and the negative backbones of the
nucleic acid is increased when the dielectric constant is lowered, which allows
molecules to interact, neutralise, and aggregate.

The extraction of DNA can also be improved by substituting the use of ethanol with
isopropanol. This is because isopropanol is less polar than ethanol and has a lower
dielectric constant than ethanol (Zumbo, 2012). The lower dielectric constant of
isopropanol will help the nucleic acids form bonds easier, effectively increasing the
yield of DNA samples. In addition to that, isopropanol is also less volatile than
ethanol and less volume of isopropanol is required to precipitate nucleic acid at the
same efficiency at a given volume of ethanol. DNA is also less soluble and can
precipitate faster in isopropanol, even at a lower concentrations, such that DNAwill
precipitate at 35% isopropanol and 0.5M salt, while only precipitating in 75% ethanol
with 0.5M. According to New England Biolabs (2017), the typical precipitation
protocol involves using 0.7-1 volumes of sample while ethanol is required at 2-2.5
volumes, this makes isopropanol more efficient, and especially useful when dealing
with large numbers of samples.

Conclusion:
The experiment was carried out to extract the DNA from an onion. After each step of
the extraction process was carried out in order, a mixture of 2 layers were produced.
The topmost layer being ethanol, followed by the DNA of the onion . The method of
DNA extraction, called the enzymatic extraction method is effective in obtaining
higher yield of samples, however, it might not be the quickest, or most cost-efficient
method of DNA extraction. Improvements can be made to the method to allow higher
quality yield to be made, and alternative methods could also be used to extract DNA
for different uses. Since the aims and objectives of the experiment were achieved so
the practical was successful.
Reference:
1. Eickbush, T., & Moudrianakis, E. (1978). The compaction of DNA helices into
either continuous supercoils or folded-fiber rods and toroids.

2. Genetic Education. (2018). Different Types of DNA Extraction Methods. Genetic

EducationRetrieved from http://geneticeducation.co.in/different-types-of-dna-
extraction-methods/

3. Izutsu, K. (2011). Electrochemistry in nonaqueous solutions. Weinheim: Wiley-

VCH Verlag GmbH & Co.

4.Khan Academy. (n.d.a). DNA structure and function. Retrieved from

https://www.khanacademy.org/test-prep/mcat/biomolecules/dna/a/dna-structure-and-
function .

5. Khan Academy. (n.d.b). Structure of the plasma membrane. Retrieved from

https://www.khanacademy.org/science/high-school-biology/hs-cells/hs-the-cell-
membran e/a/structure-of-the-plasma-membrane .

6. Life Sciences Cyberbridge. Retrieved from

http://cyberbridge.mcb.harvard.edu/dna_1.html

7.Maniatis, T., Fritsch, E.F., Sambrook, J. (1982). Molecular cloning

. A Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
Press.

8.Piškur, J., & Rupprecht, A. (1995). Aggregated DNA in ethanol solution. FEBS
Letters, 375(3), 174-178. doi: 10.1016/0014-5793(95)01206-t .

9.R. Ghosh, 2006, Principles of Bioseparation Engineering, World Scientific Pte. Ltd.
ISBN 981- 256-892-1
10.Rettner, R. (2017). DNA: Definition, Structure and Discovery. LiveScience.
Retrieved from https://www.livescience.com/37247-dna.html

11.Roberts, R. J. (2016). Nucleic Acid. Encyclopedia Britannica. Retrieved from

https://www.britannica.com/science/nucleic-acid#ref594016

12.Thermo Fisher Scientific. (n.d.). Detergents for Cell Lysis and Protein Extraction.
Retrieved from Thermo
https://www.thermofisher.com/my/en/home/life-science/protein-biology/protein-
biologylearning-center/protein-biology-resource-library/pierce-protein-methods/
detergents-cell-l

13.Zumbo, P. (2019). Ethanol Precipitation [PDF]. Retrieved from

http://physiology.med.cornell.edu/faculty/mason/lab/zumbo/files/
ETHANOL_PRECIPITATION.pdf