DNA ISOLATION

Genetics Class D
Study Program of Biology Education, Faculty of Teacher Training and Education, Jember University
Kalimantan Streat number 37, Kampus Tegalboto, Jember 68121

ABSTRACT
One of the topics of discussion in the genetics practicum is DNA isolation, this practicum aims to
determine simple methods of isolating fruit DNA and to determine the effectiveness of detergents / soap used
for DNA isolation. The DNA isolation process carried out is simple DNA isolation, where the materials and
tools needed are easily available in every area during this pandemic. However, simple DNA isolation has a
lower yield quality than other methods, such as PCR and other methods which must be performed in a sterile
laboratory without contamination of other particles that can affect the DNA results. In this practicum, each child
does a practicum individually in their respective areas. So, we can obtain and collect data on the results of class
practicum and conclude which materials can have the greatest influence, as well as what factors can affect the
results of the practicum in this DNA isolation process.

Keyword : Isolation DNA, DNA, Genetics

INTRODUCTION DNA isolation from bacteria (Maneeruttanarungroj

A molecule of deoxyribonucleic acid (DNA)
consists of two long polynucleotide chains. Each
chain, or strand, is composed of four types of
nucleotide subunits, and the two strands are held
together by hydrogen bonds between the base
portions of the nucleotides (Alberts et al., 2014:
173).
DNA isolation is one of the basic techniques
in molecular biology that can be developed into
complex studies regarding the genetic information
possessed by an organism. Information on DNA is
stored in the form of nucleotide bases, namely
adenine (A), guanine (G), cytosine (C), and
thymine (T). Each nucleotide base sequence
contains genetic information that plays a role in the
development and regulation of organisms. A cell
has DNA which is genetic material and is
hereditary in all living systems. A complete cell of
genetic material (DNA) possessed by organisms
and organized into chromosomes is called a
genome. DNA can be isolated, whether in humans,
plants, animals, or microorganisms (Widyastuti,
2017: 311).
DNA-based molecular biology will help
explain and enable researchers to fully understand
the system in vivo in more detail. Many standard
protocols available for DNA extraction, to varying
degrees success, such as organic extraction, sodium
dodecyl sulfate (SDS) method,
cetyltrimethylammonium bromide (CTAB)
method, high salt low pH method, salt extraction
method, and the NaOH method. However, some of
these standard protocols may be suitable for certain
organisms, for example, CTAB is suitable for DNA
isolation from plants, whereas SDS is preferred for
& Incharoensakdi, 2016: 98).
An ideal extraction protocol should optimize
DNA yield, minimize DNA degradation, and be
efficient in terms of cost, time, labour and supplies.
Most commonly used methods from diverse
organisms are sodium dodecyl sulphate (SDS) and
cetyltrimethyl ammonium bromide (CTAB).
Absolute ethanol or isopropanol are routinely used
for DNA precipitation in the presence of sodium
ions. However, there are several modifications
regarding the volume of ethanol or isopropanol,
incubation temperature and time used for DNA
precipitation (Haxhijaha et al., 2020: 62).
DNA extraction is a series of processes to
separate DNA from other cell components. DNA or
deoxyribose acid is a storage place for genetic
information. A cell has DNA which is genetic
material that is hereditary in all living systems.
DNA in plants and animals can be isolated easily.
There are two ways of DNA isolation, namely the
simple / conventional method and the modern way.
But basically, the two ways go through the same
stage. Broadly speaking, DNA isolation goes
through the stages of tissue isolation; lyse cell walls
and membranes to excrete cell contents; lyses
membrane proteins, cytoplasmic proteins, and
nucleolar proteins; DNA leaching and finally the
precipitation of funds for DNA binding. Simple
methods will produce "dirty" DNA and can be
easily seen with the naked eye, whereas advanced
methods will produce pure DNA dissolved in the
solvent (Nugroho & Rahayu, 2016: 22-23).
Molecular identification requires an initial
stage, namely the isolation of genomic DNA. 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
CTAB (Cetyltrimethylammonium bromide) and
SDS (Sodium Dedocyl Sulfate) (Murtiyaningsih,
2017: 85).
DNA isolation is an important step in
modern molecular research. The choice of isolation
method depends on the type of DNA source sample
and the type of test. DNA isolation is often a
limiting factor for the success of molecular analysis
because this process requires intensive work which
is influenced by many variables and targets very
few extraction products. The working principle of
DNA isolation, which is to lyse proteins then filter
out debris and DNA precipitation (Daryono &
Perdamaian, 2019: 93).
DNA isolation is needed to obtain the pure
DNA for genetic analysis in plant breeding. DNA
isolation technique is needed to obtain DNA from
organism with high purity and concentration. DNA
isolation can conduct using SDS method, CTAB,
phenol chloroform method and detergent method.
DNA isolation technique consists of three main
procedures, they are cell lysis, removing the
contaminant and purification of DNA. DNA
isolation method was chose based on some
consideration, such as amount and the molecular
weight, purity, time, species and the cost. General
method of DNA isolation is CTAB methods, a
DNA isolation method which can produce high
concentration and purity of DNA, but the cost is
expensive. DNA isolation technique using
detergent method, which is relative easy and cheap.
The method can be used to get DNA isolation
which is effective, easy, cheap and simple (Arfa et
al., 2018: 16)
High DNA yields are often required for
studies requiring multiple analyzes, and the
purified DNA must be free of contaminants that
interfere with the digestion of restriction
endonucleases or the electrophoretic separation of
DNA fragments. contaminants, such as
polysaccharides, can cause shifts in mobility during
electrophoresis resulting in misinterpretation of
fragment differences between genotypes
(Beckmann & Osborn, 2012: 1)
High quality DNA isolation is essential in
molecular research and genetic diversity.
Polysaccharide contamination is one of the
problems in DNA isolation activities in woody
plants. DNA samples from woody plants are often
contaminated by polysaccharides, phenols, and
their derivatives which greatly disrupt the quality
of the genomic DNA produced. The quality of
genomic DNA produced during isolation will affect
DNA storage and the appearance of enzymes and
inhibitors during the DNA amplification and
sequencing stages (Sundari, 2017: 31).
Despite the development and use of several
standard protocols for reproducibly extracting high
quality DNA, it is quite impossible to develop a
single isolation protocol which suits every plant
species (Riahi et al., 2019: 1231)
RESEARCH METHODS
This research was conducted on November
12, 2020, through Zoom media with laboratory
assistants and lecturers from the Study Program of
Biology Education, Faculty of Teacher Training
and Education, Jember University. This practicum
aims to determine the simple method of isolating
fruit DNA and to find out the effectiveness of
detergents / soap used for DNA isolation.
The tools and materials used include glass
beaker or aqua glass, knife, stirrer, filter (tissue /
cotton), blender machine, distilled water, 96% cold
ethanol (ethanol and ice), table salt, dab soap
(wing, bukrim), detergent (surf, rinso), tomatoes,
papaya, pears, reaction tubes and tube racks, and
spatulas.
Meanwhile, our group used pears and
detergent for this DNA isolation practice. The
procedure works by dissolving detergent in 60 ml
of distilled water, stirring slowly for 15 minutes.
Then, take 100 grams of fruit pulp plus 100 ml of
distilled water put in a blender, then blend for 50
seconds. After that, mix 100 ml each of the soap
solution mixed with 100 ml each of fruit juice. add
1 spatula of table salt then stir for 10 minutes until
a homogeneous mixture is obtained. After
obtaining a homogeneous solution, filter the
previously produced mixture twice. After being
filtered, 6 ml of the filtered results are then put into
a test tube and add 5 ml of cold 96% ethanol.
Finally, observing the process of DNA emergence,
including the time required, color, and how much
DNA is formed.
RESULT AND DISCUSSION
DNA isolation is a step in studying DNA.
DNA isolation is the process of removing DNA
from where it is (extraction or lysis), usually
carried out by homogeneation and adding an
extraction buffer or lysis buffer to prevent DNA
damage. In eukaryotic cells, including plants and
animals, the largest part of the DNA is in the
nucleus, which is the organelle which is separated
from the cytoplasm by a membrane. The nucleus
comprises 90% of all cellular DNA. The rest of the
DNA is other organelles such as mitochondria and
chloroplasts. Since DNA is present in the nucleus,
it is necessary to have a cell coating method to
harvest cells. Where this method is part of the DNA
isolation method.
Cell genetic material is contained in nucleic
acids. In organisms, there are two types of nucleic
acids, namely deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA). In the body, DNA and
RNA together form an interdependent control unit.
DNA is an inherited molecule. DNA is also
included in the basic molecule that determines all
the characteristics of each organism. DNA also has
a long deoxyribonucleotide polymer which is the
combination of many nucleotides from the ends
that form one long DNA chain. The length of DNA
can be measured by the number of nucleotides
incorporated into it. DNA is shaped like a long
chain twisted together with a double helix structure.
DNA is the carrier of genetic information and is
involved in all hereditary and biosynthetic
processes in organisms. Apart from that, DNA also
synthesizes RNA and indirectly, controls protein
synthesis. Whereas RNA, RNA is usually found in
the nucleolus and cytoplasm. In RNA, ribose acts
as a pentose sugar, while urasi acts as a substitute
for thymine. The form of RNA is single-stranded or
single-stranded. RNA also has a role in protein
synthesis. Some of the characteristics that
differentiate between DNA and RNA. From the
name alone, DNA is a deoxyribose sugar, while
RNA is a ribose sugar. The structure of DNA is in
the form of two complementary antiparallel strands
to each other, while RNA has only one strand. The
nitrogenous bases found in DNA are adenine,
guanine, cytosine, and thymine. In RNA, the
nitrogenous bases are adenine, guanine, cytosine,
and uracil. DNA does not have a secondary
structure, while RNA can form a secondary
structure. DNA is also non-catalytic and stable. In
contrast, RNA is catalic and very reactive.
In this DNA isolation practicum, there are
several materials used, including glass beaker or
aqua glass, used as a container for the solution
used. A knife, to cut the fruit to be isolated in
DNA. Stirrer, to stir the solution to make it
homogeneous. Filter (tissue / cotton), to filter out a
homogeneous solution so that no other particles
enter the experimental solution. Blender machine,
to crush fruit. Spatukla, as a stirrer and to take the
ingredients to be mixed. The reaction tube and tube
rack function to carry out reactions and
observations, while the tube rack is to place the test
tube so that the solution does not spill. Tomato /
papaya / pear, in this practicum we in group 3 use
pears as an ingredient for DNA isolation. Detergent
(surf, rinso), functions to dissolve fat in the cell
membrane, so that cells can lysis. In addition, this
detergent can inhibit DNase which can damage
Fruit Group NIM Soap
Concentration
DNA and can denature proteins, so that protein can
be removed from the solution. Table salt (NaCl), to
remove protein and carbohydrates because salt can
cause both of them to precipitate, and together with
detergents, they both function like a lysing buffer.
Cold 96% ethanol (ethanol and ice), precipitates
plasmid DNA and is also used to rinse and wash
plasmids. The addition of acetate made the salt
concentration high and single stranded rna
insoluble in this situation. Aquades, as a solvent or
a mixture of the materials used.
DNA isolation can be carried out through
several steps including: cell extract preparation,
DNA purification from cell extracts and DNA
precipitation. Although DNA isolation can be
carried out in various ways, each type or part of the
plant can give different results, this is because there
are high concentrations of polyphenols and
polysaccharides which inhibit DNA purification. If
DNA isolation is carried out with fruit samples, the
water content in each fruit is different, which can
give different results. Fruit with high water content
will produce different isolates when compared to
fruit with low water content. The higher the water
content of cells dissolved in the extract, the less, so
that the DNA that is precipitated will also be less.
The DNA isolation process begins with the
DNA extraction process. This aims to separate
DNA from other unwanted particles. This process
must be done carefully, so as not to cause damage
to the DNA. DNA isolation usually begins with
lysis or destruction of tissue or cells. This process
is essential for the breakdown of protein structures
and allows for the release of nucleic acids from the
nucleus. Lysis is carried out in saline solutions,
detergents that contain denatured proteins or
proteases (enzymes that digest protein), such as
proteinase K, this is a chemical method. In
addition, it can also be done by mechanical means,
namely by blending or grinding using a mortal and
pistil.
The addition of detergent in DNA isolation
can be done because detergents can cause damage
to cell membranes, through the bonds formed
through the hydrophobic side of the detergent with
proteins and fats on the membrane to form "lipid
protein-detergent complex" compounds. These
compounds can form protein and lipid compounds
that have hydtophilic ends, as well as detetrgen, so
they can form a chemical bond.
In this DNA isolation practicum we
obtained class data, which included the identity of
the practitioner, the type of fruit used, the brand of
soap / detergent, the concentration of alcohol, the
length of time it took for DNA to appear, and the
amount of DNA that appeared as follows:
Alcohol Estimated Amount of DNA
Time Of + = very less
Appearance ++ = less
Of DNA +++ = much
 ++++ = very much
13 70% ± 3 minutes ++
1 90 Bukrim 95% ± 2 minutes ++
133 70% ± 3 minutes +++
Papaya 01 70% 1 minutes +++
Bukrim
20 70% 6 minutes +++
2
28 70% 3 minutes +++
Wing Ekonomi
111 70% 2 minutes +++
11 Rinso 70% ± 2 minutes ++
67 Soklin 70% ± 3 minutes ++
3 84 Daia 70% ± 2 minutes +++
18 Rinso 70% 5 minutes +++
Pear 136 Soklin 70% 4 minutes +++
33 Rinso 70% 4 minutes ++
53 Rinso 70% 18 minutes +++
4
94 Soklin 70% 10 minutes +
128 Rinso 98% ± 2 minutes +++
30 95% ±5 minutes ++
129 70% ±7 minutes ++
5 73 70% ±6 minutes ++
12 95% ±5 minutes ++
Tomato 02 Mama lime 95% ±30 minutes ++
77 70% ±7 minutes ++
86 95% ±5 minutes ++
6
98 70% ±5 minutes ++
26 70% ±3 minutes ++

From the table the results of this observation
can be seen the various kinds of experimental
results that have been carried out by each child
from our class. The first experiment was by groups
1 and 2 who carried out DNA isolation experiments
with papaya fruit, various soap used, 5 children
using the Bukrim brand and 2 children using the
Wing Ekonomi brand. Almost all of them used a
70% alcohol concentration and only 1 child used a
95% alcohol concentration. The longest time
needed until the appearance of various DNAs is 6
minutes and the fastest is 1 minute. The results of
the large number of papaya DNA that appear
mostly get a lot of results (+++).
Then, the second experiment was carried out
by groups 3 and 4 who carried out DNA isolation
experiments with pears, the detergents used came
from different brands which contained different
contents such as Rinso, So Klin, and Daia. Like the
previous experiment, most of them used an alcohol
concentration of 70% and 1 child used an alcohol
concentration of 98%. The longest time needed to
emerge DNA was 18 minutes and the fastest was 2
minutes, including the experiment using 98%
alcohol concentration, it took 2 minutes to appear
DNA. The results of the large number of pear DNA
that appeared 5 children got a lot of results (+++), 3
children got less results (++), and 1 child got very
less results (+).
The third experiment was group 5 and 6
who carried out DNA isolation experiments with
tomatoes, all of whom used mama lime branded
dish soap. In this DNA isolation experiment on
tomatoes 4 children conducted an experiment with
an alcohol concentration of 95% and 5 children
using an alcohol concentration of 70%. The
estimated time needed for DNA to appear is at
most 30 minutes and the fastest is 3 minutes.
Meanwhile, the amount of DNA that appears all
shows less (++).
Based on the results of this class of data, in
my opinion detergent is more effective for use in
DNA isolation, because detergent functions to
dissolve fat in the cell membrane, so that cells can
lysis. In addition, this detergent can inhibit DNase
which can damage DNA and can denature proteins,
so that protein can be removed from the solution.
When using soap mostly shows less DNA yield
than using detergent.
However, this cannot be the only
determinant in the DNA isolation process. Because
there are many things that can affect the success of
DNA isolation. Among them are: The first is the
water content of the fruit used, the more water
content, the less DNA will be precipitated. The
water content in each fruit is different, it can give
different results. Fruit with high water content will
produce different isolates when compared to fruit
with low water content. The higher the water
content of the cells dissolved in the extract, the less
it will be. So that the precipitated DNA will also be
small. The next factor is soap or detergent used,
different levels of soap composition such as SDS
and SLS, soap or detergent which has a greater
concentration will also have a greater influence on
the results of DNA isolation. The third is the tool
used in the isolation practicum, the sterilization of
the tool is very important in this case, because
contaminated tools and materials can cause DNA
from other organisms to enter the experimental
solution.
The next factor is filtering, filtering
including an important step so that no other
particles are involved in the solution, thus affecting
the results of the observation. The filtering tool that
should be used is filter paper which has very small
pores, however, due to the unfavorable conditions,
tissue or cotton wool is used for filtering. This
certainly greatly affects the results of the DNA
isolation. Failure may occur due to inadequate
filtering so that many other particles are included in
the solution. Then the level of alcohol
concentration can also affect the success in the
DNA isolation process. So that in this DNA
isolation practicum we must be very careful to
minimize the possibility of failure and to determine
the results of DNA isolation experiments we need
to consider many things as a reference.
CONCLUSSION
The conclusion is that we can perform
simple DNA isolation that is easy for anyone to do
with affordable tools and materials. However, there
are still some things that must be considered in the
simple DNA isolation process, such as the moisture
content in the fruit used, the alcohol concentration,
the type and content of SDS (Sodium Dedocyl
Sulfate) from the soap and detergent used which
can affect the DNA results, cleanliness. the tools
used, and the filtering process, which can affect the
results or the success of the DNA isolation process
itself.
Based on the data obtained, in my opinion
detergent is more effective for use in DNA
isolation, because detergent functions to dissolve
fat in the cell membrane, so that cells can lysis. In
addition, this detergent can inhibit DNase which
can damage DNA and can denature proteins, so
that protein can be removed from the solution.
When using soap mostly shows less DNA yield
than using detergent.
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