ISOLATION OF DNA

Yolanda Anggraeni
190210103128
Genetics of Class D
Biology Education Study Program, Faculty of Teacher Training and Education, University of Jember
Kalimantan Street Number 37 Kampus Bumi Tegalboto Jember 68121
ohyoland31@gmail.com

ABSTRACT
This practicum discusses about DNA isolation. DNA isolation (Deoxyribose Nucleid Acis)
was carried out with the aim of separating DNA from other materials such as proteins, fats and carbohydrates.
There are three main principles in DNA isolation, namely destruction (lysis), extraction or separation of DNA
from solid materials such as cellulose and protein. The aim of this practicum is to determine the simple method
of isolating fruit DNA and the effectiveness of detergent / soap used for DNA isolation. The materials used in
this practicum include papaya, tomato, pear, and detergent (surf, rinso), dab soap (wing, bukrim) as well as
mamalime and alcohol with a concentration of 70%, while certain groups use alcohol with a concentration of
70%. concentration 95%. The results of this experiment show that the addition of liquid soap / mamalime can
show quite a lot of white fibers / DNA and is faster than others.

Key Words: DNA, RNA, DNA Isolation, alchohol

The principle of DNA isolation is to
obtain pure DNA which is not mixed with other
cell components such as proteins and
carbohydrates. Isolation of genomic DNA can be
carried out by physical and chemical cell lysis
methods. Physically, the cell is broken down by
mechanical force, namely by freeze thaw, bead mill
homogenization and resonance, for example by
sonication. Meanwhile, cells are chemically
damaged with lysis buffers containing chemical
compounds that can damage the integrity of the cell
wall barrier, for example SDS (Sodium Dedocyl
Sulfate) and CTAB (Cetyltrimethylammonium
bromide). Good quality genomic DNA is an
important thing needed in molecular biology
applications. These applications include PCR
(Polymerase Chain Reaction), RFLP (Restriction
Fragment Length Polymorphism), RAPD (Random
Amplified Polymorphic DNA), and other
molecular analyzes (Murtiyaningsih, H. 2017: 2).
Cytochrome b (cyt b) is one of the genes
present in mitochondrial DNA and is often used in
DNA analysis to identify species of raw materials
in fishery products. Species identification using
mitochondrial DNA has the advantage of being
smaller in size, with a large number of copies,
complete available DNA sequence information. for
aquatic organisms, and no non-coding ranges.
DNA barcoding is a method often used in
taxonomic forensics because it is effective in
identifying under a variety of conditions and does
not generate ambiguous data. Using cyt b to
identify adulterated fillet pufferfish. The use of the
cyt b coding gene for species identification has
been carried out by Wulansari et al. (2015) to
identify counterfeit products using tuna as raw
material (Maulid, D. Y. 2016: 9-10).
 DNA is a polymer consisting of a
phosphate group, deoxyribose sugar and a joined to each other to form polynucleotides like
nitrogenous base. The shape of DNA is two strands
twisted (double helix), these two strands are held
together by hydrogen bonds between the bases
contained in each strand. The four bases present in
DNA are adenine (A), cytosine (C), guanine (G)
and thymine (T). DNA matching is a process that is
almost always used in a variety of ways to
manipulate DNA, the aim of which is to get the
characteristics of a DNA. Therefore an algorithm is
needed that can help the DNA matching process so
that the process can be carried out efficiently in
terms of time (Prabowo, Y. D. 2016: 197).
The term genome refers to the entire DNA
contained within the cell of an organism. DNA is
contained within the nucleus of the cell, as discrete
bodies called chromosomes, or within the intra
cellular bodies of mitochondria and chloroplasts.
The DNA can be classified within a genome based
on its function, lack of function, or structure. This
chapter leads the reader through the basics of DNA,
from the structure of the molecule to how variation
creates different species as we see them today.
DNA is arelatively simple molecule. DNA is a
polynucleotide first discovered in 1868; however,
scientists were slow in understanding its role in
inheritance. DNA is a polymer composed of
nucleotides which contain a sugar, a base and a
phosphate group. The sugar group is a five-carbon
molecule (a pentose). The bases consist of a
structural derivative of either a purine or a
pyrimidine of which adenine (A) and guanine (G)
are purines, and cytosine (C) and thymine (T) are
pyrimidines. In the late 1940s Erwin Chargaff
discovered that DNA has equal numbers of adenine
and thymine (A = T) residues and equal numbers of
guanine and cytosine (G = C) residues. These
relationships were the first reliable methods for the
quantitative compositional analysis of DNA and
are known as Chargaffs Rules. Nucleotides can be
DNA. The phosphates bridge the 3' and 5' positions
of neighbouring ribose units, and each nucleotide
in the linkage is known as a nucleotide residue. The
linkage between each nucleotide is known as a
phosphodiester bond and the unlinked fifth carbon
(C5‘) residue is called the 5' end, while the third
carbon (C3‘) residue is called the 3' end. The
polymer of non-identical residues has the ability to
contain information in the form of its sequences
(Linacre, Adrian M. T., and Shanan S. T. 2013:2).
There are many published protocols for
isolation and purification of plasmid DNA. The
theoretical basis for these procedures is described.
In most instances, plasmid DNA, isolated by these
procedures, is of sufficient quality and quantity for
cloning and PCR analysis. However, the plasmid
DNA obtained by many of these methods is a poor
template for double-stranded dideoxy DNA
sequencing, largely due to inconsistency of
template purity and yield. To obtain plasmid DNA
of good quality for DNA sequencing, many
sequencing protocols recommend the use of
plasmid templates prepared by cesium chloride
centrifugation or column chromatography.
Although these methods give satisfactory results,
they are time consuming, expensive and not
suitable for preparation of templates from multiple
samples. This chapter describes two protocols for
large-scale plasmid isolation, and two protocols for
minipreps of plasmid DNA. All four methods yield
DNA that is an excellent template for dideoxy
dsDNA automated sequencing, as well as for
cloning and PCR. The procedures are a
modification of the alkaline lysis procedure devised
by Brinboim and Doly. The alkaline lysate is
neutralized with ammonium acetate rather than
potassium or sodium salts, and if necessary, the
remaining proteins can be removed by precipitation
with 1.87 M ammonium acetate. This allows
purification of plasmid DNA without the use of
toxic organic solvents, CsCl centrifugation or
column chromatography. The procedures are quick
and yield plasmid DNA of purity comparable to the
CsCl prepared DNA. Long procedures are suitable
for DNA isolation from both large und small
plasmids (Surzycki, Stefan. 2010: 101).
DNA (Deoxyribo Nucleic Acid) is a
nucleic acid genetic messengers in life. The genetic
information lies in the cell nucleus and neatly
arranged to form chromosomes. DNA pattern this
chromosome constituent determines the
characteristics of the trait / type hair, skin color and
special properties that differ from one individual to
other. DNA was first discovered in 1869. Through
technology X-rays show that DNA has a neatly
arranged structure and has a DNA double chain
model published by Watson and Crick in the
journal Nature in 1953. Since then, engineering
DNA purification is experiencing rapid
development and becoming routine procedures
performed in molecular research in various fields.
DNA is located inside the cell. Hence to get DNA
required a special stage which is usually carried in
specific laboratory. To remove DNA from cells,
then a technique biochemical purification of DNA
is carried out in a destructive manner cell walls
using a specific buffer solution and mixture various
types of detergents. With the opening of the cell
membrane layer then DNA can be removed and
precipitated by addition alcohol. DNA isolation is a
series of processes carried out to separate DNA
from cell components such as lipids
(Puspitaningrum, Rini, et al. 2018: 30).
The first stage of DNA isolation is the
destruction of the cell wall by mechanical means
by then filtering the results. Fruit is used because
the destruction of its cell walls is easier because it
does not contain much cellulose, lignin, pectin and
other cell wall hardening agents. The second stage
of DNA isolation is to lyse the cell membrane by
adding detergent and salt to the mixture. Detergent
application functions to open or break down cell
membranes (both cytoplasmic membranes and
membranes nucleus). The third stage is DNA
purification. This method is done by adding 96%
cold alcohol. Alcohol serves to separate DNA with
other molecules, such as proteins. Alcohol has
lighter molecules than water so it will be on the
surface of the mixture. Protein and fat will be in the
bottom and other parts of the cell. DNA structure
that has been isolated is a lump of white, thread-
like fibers that lie between the aliquot and the
alcohol (Safilu, Amiruddin, Ahdiat A., et all. 2019:
33).
Extractionof high-quality DNAin
sufficient quantity is importantfor studying the
molecular genetics of cotton. However, high
endogenous levels of polysaccharides and
polyphenols interfere with the isolation of good
quality DNA,thereby rendering it unsuitable for
down stream analyses. During cell disruption,
phenolic compounds come outofthe vacuoles,
become readily oxidized an dirreversibly bind with
nucleic acids and proteins, thus resulting in a dark
DNA pellet unsuitable for most enzymatic
manipulations. On the other hand, the viscous
nature of polysaccharides makes extracted DNA
fractious to pipetting; and, it also interferes with
various biological enzymes, and especially hinders
the PCR reaction by inhibiting the Thermus
aquaticus DNA polymerase. Previously reported
extraction protocols for cotton are comparatively
expensive, time-consuming, require liquid nitrogen
or lyophilization and ultracentrifugation in CsCl
gradients. Although, these methods may yield good
quality DNA; however, they are not suitable for the
local Pakistani varieties and high-throughput
applications, such as restriction fragment length
polymorphism (RFLP) and screening of
transformants, that require inexpensive and
reproducible DNA extractions. Another major
problem for most laboratories in developing
countries is the continuous procurement and
storage of liquid nitrogen. Over the years to
overcome these problems, numerous modifications
have been introduced into the original CTAB
method to reduce the cost and time of routine
DNA isolation; however, none of the modifications
have been found to be universally applicable for
every plant species due to their chemotypic
heterogeneity. Most recent CTAB methods,
including this protocol, omit the CsCl gradient
ultracentrifugation, selective precipitation steps,
use of liquid nitrogen and toxic phenol in favour of
a simple, rapid and safe procedure. In this regard,
the described procedure was modified from
Paterson et al. To reduce time, cost, and resolve the
problems associated with high endogenous levels
of secondary metabolites, especially
polysaccharides and polyphenols. This method
consistently yields high-quality DNA in sufficient
quantity suitable for most projects, such as cloning,
mapping and marker-assisted plant breeding. An
individual can routinely process 24–48 samples and
isolate 10–15mg of high-quality DNA in about 3 h.
In addition, this procedure may also be adapted to a
96-well microplate format for high through put
DNA extraction (Ali, Q., Salisu, I., et al. 2019:
260).
DNA molecules carried in the Poiseuille
stream are subjected to resist electrophoresis,
resulting in a transverse force oriented towards the
capillary walls. Simple DNA isolation can be done
by breaking the cell wall, plasma membrane and
nuclear membrane either mechanically or
chemically. DNA isolation is a technique used to
obtain pure DNA, that is, without protein and RNA
from a cell in the tissue (Milon, et al. 2019: 8051).
It has been estimated that about 13 billion
tons of detergents are using about 1000 tons of
lipase enzymes annually. The practice of using
lipases as detergent additives decrease the need of
using synthetic detergents and increases the
capability of household detergents to remove tough
oily or greasy stains from clothes. Lipases can be
utilized efficiently as detergent additives due to
their property of hydrolyzing lipids. Lipases are
particularly applied in detergents because of their
possession of following qualities: (1) Ability to
catalyze fats in different combinations (2)
capability to survive in comparatively harsh
washing environment (pH 10–11, 30–60ºC); (3)
being able to retain activity in presence of other
destructive enzymes, surfactants and inhibitors,
which are significant components of many
detergent preparations ( Faiz, et al. 2020: 210).
For the ethanol extraction method, the
gDNA released was in large quantities. One litre of
broth culture was used for mycobactin S extraction.
The weight of the cell pellet obtained was 400 mg
wet weight. The yield of gDNA was
approximately 2.88μg DNA/mg cells.
Determination of the optimum concentration of
ethanol for DNA isolation from M. smegmatis was
carried out using 3 mL of spent broth. The
effective concentration of ethanol was found to be
60%, where the A260 nm was maximumat 60%
ethanol concentration used. Figure 1 shows the
bands of DNA isolated by ethanol extraction
method from three different strains of (Gokarn, et
al. 2016: 39014).
METHODOLOGY
The first step is to prepare the necessary
tools and materials, namely beaker glass, knife,
stirrer, filter, (tissue / cotton), blender, spatula, test
tube and tube rack, pears, rinso brand detergent,
table salt, cold ethanol 96% and distilled water.
After all the tools and ingredients are ready, the
steps are taken, namely dissolving one spoon of
detergent by adding 60 ml of water, stirring gently
so that no foam appears until about 10 minutes.
After the detergent is dissolved, proceed by
crushing the pears using a blender by adding 100
ml of water, for about 50 seconds. After that, mix
the detergent solution with pear juice, stirring until
everything is well mixed. Then add 1 tablespoon of
salt and stir until all the ingredients are mixed.
After everything is mixed, filter the solution using
a filter that has been given a tissue, after getting a
clear solution (there are no coarse parts of the
undissolved solution) measure the solution as much
as 10 ml and measure the alcohol with the same
volume, which is 10 ml, then enter the alcohol into
the solution beforehand. Stir until DNA fibers
form, for about 2-3 minutes. The final step is to
observe the process of DNA generation, including
the time required, color, and how much DNA is
formed
DISCUSSION
DNA isolation is a mechanical and
chemical process to obtain DNA from an organism,
this process includes mechanical destruction of the
cell wall (destroyed), the next process is to lyse the
cell membrane by adding detergent and salt to the
juice obtained during the process. previous. The
function of detergents here is to open or break
down the cell membrane. And the third process is
by adding alcohol with a concentration of 96%, the
function of alcohol is to bind DNA and separate
DNA from other molecules, such as proteins.
Perbedaan of DNA and RNA, first, DNA
is composed of sugar deoxyribose, while RNA is
composed of ribose sugar. The second is that
Thymine belongs to DNA and Uracyl is owned by
RNA. The nitrogenous bases contained in DNA are
composed of purines from the Guanine (G) and
Adenine (A) composition and pyrimidines from the
Cytocine (C) and Thymine (T) composition. While
the nitrogen base RNA is composed of purines
Guanine (G) and Adenine (A), as well as Uracyl
Pyrimidines (U) and Cytocine (C). The third
difference is that DNA has long and double helix
chains, while RNA has single and short chains. The
fourth difference is that DNA can be found in
chloroplasts, mitochondria, and nucleus, while
RNA can be found in ribosomes (r-RNA),
cytoplasm. (t-RNA), and in the nucleus (m-RNA).
Fifth, DNA has a role to inherit and synthesize
protein, while RNA has a role only to synthesize
protein. And finally, DNA cells have fixed cells,
while RNA levels vary. This is adjusted to the
amount of protein synthesis required.
From the practicum that has been carried
out, there must be materials used, including: Pears,
their function as an object whose DNA will be
isolated; Rinso detergent, its function is to lyse
DNA chemically; Kitchen salt, its function is to
insulate DNA because it has Na⁺ ion which is able
to form negative bonds at the DNA phosphate pole;
96% cold ethanol, its function is to bind DNA to
separate it from other molecules, such as proteins
etc .; The last ingredient is Aquades, its function is
as a solvent so that all the ingredients used can
dissolve and mix well.
In the DNA isolation process, there are
mechanical and chemical steps, the mechanical step
is a work step that requires a tool or machine to
help a process carried out, in DNA isolation, the
mechanical work step is carried out when crushing
pears using a blender. While the chemical work
step is a work step that requires chemical
substances to help the process carried out, in the
DNA isolation that I have done, the chemical work
step occurs when adding detergent, salt, and cold
alcohol to the pear juice solution. , which will make
the DNA split with other molecules.
 Based on the class data that we obtained,
it was obtained from 25 students who used the
following brands of soap and detergent, 5 people
for Bukrim, 2 people in Economic Wing, 5 people
for Rinso, 3 people from Soklin, 1 person, Mama
lime 9. DNA that varies, ranging from very little to
very much, this occurs due to several factors, such
as the amount of SDS and EDTA content in the
soap and detergent used, according to the theory of
soap has a greater number of SDS than detergent,
but the results of practicum were found. some data
use soap, but the amount of DNA that is formed is
small, this can also be because the type of fruit
used contains a lot of water, so that the DNA
precipitation is small or little.
In my opinion, what is more effective to
use for DNA isolation is soap, because soap has
higher SDS and EDTA than detergents, which
causes the cell membrane of the material to be
isolated by DNA to undergo lysis faster. However,
the choice of fruit type also affects, because the
fruit with high water content will cause the DNA
precipitation to be small or a little.
CONCLUSION
The conclusion that we get after doing a
practicum regarding DNA isolation in fruit, namely
the success of the DNA isolation process is
influenced by many factors, the first is the fruit
factor, fruit that contains a lot of water or has high
ar levels, it will cause the amount of DNA that can
be isolated to be small, this is because the high
water content causes a little DNA precipitation, the
second factor is the use of this type of soap as a
chemical used to mechanize the fruit cell
membrane, when the soap used has a high SDS or
EDTA content, the process of coating the fruit cell
membrane will be more effective, the third factor is
the use of alcohol, in DNA isolation, the
recommended alcohol content for DNA isolation is
alcohol with levels above 96%, because the higher
the alcohol content, the faster it will be to bind
DNA to separate it from other molecules.
REFERENCES
Ali, Q., Salisu, I. B., Raza, A., Shahid, A. A., Rao,
A. Q., & Husnain, T. (2019). A modified
protocol for rapid DNA isolation from
cotton (Gossypium spp.). MethodsX, 6,
259-264.
Faiz, S., Nasreen, Z., Sha, A., & Naz, S. (2020). 18.
Isolation, screening and characterization
of lipase from bacterial isolates and its
application in detergents and oily waste
water degradation. Pure and Applied
Biology (PAB), 10(1), 209-224.
Gokarn. K, V.Sarangdhar and Ramprasad B. Pal,
2016. “Ethanol Extraction Method for
DNA Isolation from International. Journal
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39015.
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M. A., Cauet, S., Moreau, S., ... &
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assisted targeting for sequencing and
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SUPPLEMENTARY
 ATTACHMENT

Class Data:

Identity Material estimated Amount of DNA

Group NIM Fruit Soap alcohol time of + = very less
concentration appearance ++ = less
 of DNA +++ = much
++++ = very
13 Bukrim 70% ± 3 minutes ++
1 90 Papaya Bukrim 95% ± 2 minutes ++
133 Bukrim 70% ± 3 minutes +++
01 Bukrim 70% 1 minutes +++
20 Bukrim 70% 6 minutes +++
2 Papaya
28 Wing Ekonomi 70% 3 minutes +++
111 Wing Ekonomi 70% 2 minutes +++
11 Pear Rinso 70% ± 2 minutes ++
67 Pear Soklin 70% ± 3 minutes ++
3 84 Pear Daia 70% ± 2 minutes +++
18 Pear Rinso 70% 5 minutes +++
136 Pear Soklin 70% 4 minutes +++
33 Pear Rinso 70% 4 minutes ++
53 Pear Rinso 70% 18 minutes +++
4 94 10 minutes
Soklin 70% +
Pear ± 2 minutes
Rinso 98% +++
128
30 95% ±5 minutes ++
129 70% ±7 minutes ++
73 70% ±6 minutes ++
5 Tomato Mama lime
12 95% ±5 minutes ++
±30
02 95% ++
minutes
77 Tomato Mama lime 70% ±7 menit ++
86 Tomato Mama lime 95% ±5 minutes ++
6
98 Tomato Mama Lime 70% ±5 minutes ++
26 Tomato Mama lime 70% ±3 minutes ++

Result of group 4:
Febiola Ery Freitas (33)

Alifa Khairatun Nisa’ (53)

Rizki Ilma Silsilia Putri Aghni (94)

Yolanda Anggraeni (128)