CHAPTER I

INTRODUCTION

A. Background
As a result, modern geneticists can discover the number and amino acid
sequences of all the polypeptides that determine phenotype. Knowledge of DNA
sequence thus opens up powerful new possibilities for understanding an
organism’s growth and development at the molecular level.In the second stage of
gene expression, the cellular machinery translates mRNA into its polypeptide
equivalent in the language of amino acids. This decoding of nucleotide
information to a sequence of amino acids is known as translation. It takes place on
molecular workbenches called ribosomes, which are composed of proteins and
ribosomal RNAs (rRNAs), and it depends on the “dictionary” known as the
genetic code, which defi nes each amino acid in terms of specifi c sequences of
three nucleotides. Translation also depends on transfer RNAs (tRNAs), small
RNA adaptor molecules that place specifi c amino acids at the correct position in
a growing polypeptide chain. The Central Dogma does not explain the behavior of
all genes. As Crick himself realized, a large subset of genes is transcribed into
RNAs that are never translated into proteins. The genes encoding rRNAs and
tRNAs belong to this group.
Basically DNA isolation can be done from various sources, including
human organs, blood,leaves, fruit flesh, insects, callus, stem roots, meat and fish
scales. In the same individual DNA obtained from various sources will have the
same type and size. DNA isolated from plants is often contaminated by
polysaccharides and secondary metabolites like tannins, pigments, alkaloids and
flavonoids. Whereas DNA from animals contains more protein. One of the
difficulties of DNA isolation from tall plants is the process of destroying cell
walls to release cell contents. This is because plants have strong and often cell
walls several types of plants, the contamination is difficult to separate from
nucleic acid extract. Presence the above contamination can inhibit enzyme
activity, for example DNA is not sensitive to restriction enzymes and interfere
with the process of DNA amplification by PCR. Likewise in animals that have a
womb chitin (like an insect), requires special techniques and methods to destroy
cells until the contents can separate or out of cell.
Basically DNA isolation can be done from various sources, including
human organs, blood,leaves, fruit flesh, insects, callus, stem roots, meat and fish
scales. In the same individual DNA obtained from various sources will have the
same type and size. DNA isolated from plants is often contaminated by
polysaccharides and secondary metabolites like tannins, pigments, alkaloids and
flavonoids. Whereas DNA from animals contains more protein. One of the
difficulties of DNA isolation from tall plants is the process of destroying cell
walls to release cell contents. This is because plants have strong and often cell
walls several types of plants, the contamination is difficult to separate from
nucleic acid extract. Presence the above contamination can inhibit enzyme
activity, for example DNA is not sensitive to restriction enzymes and interfere
with the process of DNA amplification by PCR. Likewise in animals that have a
womb chitin (like an insect), requires special techniques and methods to destroy
cells until the contents can separate or out of cell.
In addition, scientists later found that certain viruses contain an enzyme
that can reverse the DNA-to-RNA fl ow of information by copying RNA to DNA
in a process called reverse transcription. Four general themes emerge from our
discussion of gene expression. First, the pairing of complementary bases is key to
the transfer of information from DNA to RNA, and from RNA to protein. Second,
the polarities (directionality) of DNA, RNA, and polypeptides help guide the
mechanisms of gene expression. Third, like DNA replication and recombination,
gene expressionrequires an input of energy and the participation of specifi c
proteins and macromolecular assemblies, such as ribosomes. Finally, mutations
that change genetic information or obstruct the fl ow of its expression can have
dramatic effects on phenotype. Simple DNA isolation can be initiated by breaking
the cell wall, plasma membrane and core membrane, both mechanically and
chemically. Chemically can be done by giving detergent which can cause damage
to the cell membrane
B. Purpose
1. Knowing how to separate / extract DNA from plant tissue using a simple
method
2. Look directly at DNA.

C. Benefits
1. Practican can know how to separate DNA from plant tissue by a simple
method.
2. Practican can know the shape of DNA by looking at it directly.
 CHAPTER II
PREVIEW OF LITERATURE

Genetics, the science of heredity, is at its core the study of biological

information. All living organisms from single celled bacteria and protozoa to
multicellular plants and animals must store, replicate, transmit to the next
generation, and use vast quantities of information to develop, reproduce, and
survive in their environments. Geneticists examine how organisms pass biological
information on to their progeny and how they use it during their lifetime. This
book introduces you to the field of genetics as currently practiced in the early
twenty first century. Several broad themes recur throughout this presentation.
First, we know that biological information is encoded in DNA, and that the
proteins responsible for an organism’s many functions are built from this code.
These elements interact to form complex systems by which function is controlled.
We also have found that all living forms are closely related at the molecular level,
and recent technology has revealed that genomes have a modular construction that
has allowed rapid evolution of complexity. With the aid of highspeed computers
and other technologies, we can now study genomes at the level of DNA sequence.
Finally, our focus here is on human genetics and the application of genetic
discoveries to human problems. (Hartwel, 2011)
Population genetics is one branch of the science of genetics in a
population. This branch of genetic science is widely applied in various fields,
especially health, exaltation and conservation. The frequency of genes in a
population can change if there are evolutionary forces, namely factors that play a
role in changing the frequency of alleles and genotypes, including mutations,
migration, non-random marriage and natural selection. The character of the
genetic makeup and the distribution of allele alleles that are varied, are largely
determined by their parental genes. Changes in the frequency of genotypic alleles
of a population are indications of microevolution, namely evolution that occurs at
a small level, namely genes (Khoiriyah, 2014).
 Genetic diversity is a very influential factor in developing conservation
strategies. Genetic characters found in one place to grow or different provenances
can be different, this is due to genetic differences. This will show the nature and
specificity of a plant. Genetic diversity can be observed by observing genetic
characters, the properties observed are DNA that is difficult to influence the
environment (Langga et al., 2012).
DNA extraction is a series of processes to separate DNA from other cell
components. DNA or deoxyribose acid is a place for storing genetic information.
A cell has DNA which is genetic material and is hereditary in all living systems.
The genome is the full set of genetic material (total DNA) that an organism has
and organized into chromosomes. DNA in high-level organisms such as humans,
animals and plants is present in the cell nucleus, and in several organelles in cells,
such as mitochondria and chloroplasts. Core DNA (core genome) originates from
the cell nucleus, while mitochondrial DNA (mitochondrial genome) originates
from. mitochondria, and the chloroplast DNA comes from chloroplasts. At low
level organisms (Nugroho and Dwi, 2017).
There are three main steps in DNA extraction, namely cell wall destruction
(lysis), separation of DNA from solid materials such as cellulose and proteins, and
DNA purification. Through this process DNA is separated from other cellular
components such as protein, RNA (Riboxy nucleic acid), and fat. Many methods
are used to isolate DNA, depending on the specimen to be detected. The method
basically has the same principle, but there are certain things that are usually used
modifications to be able to destroy inhibitors that are in each source of specimens
(Langga et al., 2012).
DNA as a carrier of genetic information found in all living things,
contained in cells especially inside cell nucleus. DNA can be obtained from one
living things with a process extraction, making it easier for identify the DNA
called DNA isolation process. There are three main steps in DNA extraction, that
is wall destruction cell (lysis), separation of DNA from material solids such as
cellulose and protein, as well DNA purification. Through the process DNA is
separated from cellular components others such as protein, RNA (Riboksi) nucleic
acid), and fat. Many method used for isolate DNA, depending on the specimen
which will be detected. The method basically have that principle the same, but
there are certain things modification is usually used to be able to destroy
inhibitors that is in each of them source of specimens. (Langga et al, 2012).
Each type of plant has different secondary compounds which require
optimum extraction techniques. The right extraction technique determines the
quality and quantity of DNA produced. Extraction to obtain high-quality DNA is
a basic rule that must be met in molecular analysis. Problems in DNA extraction
are still important things that need to be addressed. Every plant needs an optimum
isolation procedure to obtain genomic DNA that can be used as material in
molecular analysis. Optimization of the procedure can be done on the composition
of its lysis buffer solution or physical handling techniques in the separation of
genomic DNA from other compounds. In principle, this optimization procedure
aims to protect genomic DNA from degradation due to secondary compounds
released when the cell is destroyed or damage due to physical handling
(Restu et al, 2012).
Molecular identification requires the initial stages of isolation of genomic
DNA. The principle of DNA isolation is to get pure DNA that is not mixed with
other cell components such as proteins and carbohydrates. The isolation of
genomic DNA can be done by physical and chemical cell lysis methods.
Physically the cell is broken down by mechanical strength, namely by freeze
thaw, bead mill homogenization and resonance, for example by sonication. While
chemically cells are damaged by 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). Genetic
diversity is a variation of genes in one species both among populations that are
geographically separated and among individuals in one population. The presence
of morphological diversity is closely related to genetic diversity
(Murtiyaningsih, 2017).
During DNA isolation from annual plant tissue, these inhibitors are
deposited together with DNA, thereby deteriorating the quality and results of
DNA. To overcome this problem, we tried several agents and chemicals needed
by the protocol, which were previously reported along with various modifications
of fresh and dried leaves, but none of which produced free DNA from
polysaccharides and polyphenols. This situation requires the development of new
protocols for high purity genomic DNA isolation (Deshmukh et al, 2017).
DNA, with its double-stranded spiral, is among the most elegant of all
biological molecules. But the double helix is not just a beautiful structure; it also
gives DNA incredible stability and permanence, providing geneticists with a
unique window to the past. In 1856, a group of men working a limestone quarry in
the Neander Valley of Germany discovered a small cave containing a number of
bones. The workers assumed that the bones were those of a cave bear, but a local
schoolteacher recognized them as human, although they were clearly unlike any
human bones the teacher had ever seen. The bones appeared to be those of a large
person with great muscular strength, a low forehead, a large nose with broad
nostrils, and massive protruding brows. Experts confirmed that the bones
belonged to an extinct human, who became known as Neanderthal. In the next
100 years, similar fossils were discovered in Spain, Belgium, France, Croatia, and
the Middle East. Research has now revealed that Neanderthals roamed Europe and
western Asia for at least 200,000 years, disappearing abruptly 30,000 to 40,000
years ago. During the last years of this period,Neanderthals coexisted with the
direct ancestors of modern humans, the Cro-Magnons. The fate of the
Neanderthals why they disappeared has captured the imagination of scientists and
laypersons alike. Did Cro- Magnons, migrating out of Africa with a superior
technology, cause the demise of the Neanderthal people, either through
competition or perhaps through deliberate extermination? Or did the Neanderthals
interbreed with Cro-Magnons, their genes becoming assimilated into the larger
gene pool of modern humans? Support for the latter hypothesis came from the
discovery of fossils that appeared to be transitional between Neanderthals and
Cro-Magnons. Unfortunately, the meager fossil record of Neanderthals and Cro-
Magnons did not allow a definitive resolution of these questions (Pierce, 2010).
 Nevertheless, despite its high-throughput screening potentials multiplex
real-time PCR has not yet replaced microscopy for the diagnosis of STH in large-
scale epidemiological surveys. Undoubtedly this can be explained by a range of
logistical and financial challenges, which might be faced when implementing
DNA detection-based diagnostics in a low resource laboratory setting. But an
additional explanation has been the lack of a more efficient, though simple-to-use
procedure to detect T. trichiura DNA in human stool samples within a multiplex
or multi-parallel real-time PCR context. The most likely explanation for the
relatively poor performance of the T. trichiura PCR, as seen in several studies,
seems to be the robustness of T. trichiura eggs, hampering optimal DNA isolation
Further improvement of the DNA isolation steps is therefore important, because
as long as not each of the target helminth species can be diagnosed by the highly
sensitiveDNAdetection-basedmethodology in combination with a relatively
simple-to-use uniform sample-processing procedure, complementary microscopy
examination of stool samples will remain indispensable (Maria et al, 2017).
DNA isolation is one of the important stages in molecular-based activities.
DNA of good quality is needed for various activities such as the use of molecular
markers, making genome libraries, and sequencing. The main problem that often
arises in the process of plant DNA isolation is the presence of contaminant
compounds in isolated samples such as polysaccharides, polyphenols, proteins,
RNA, and secondary metabolites found in medicinal plants. The presence of
contaminant compounds can inhibit various processes ranging from cutting DNA,
amplification, to cloning (Nugroho et al, 2015).
Mastery of basic techniques molecular biology such as DNA isolation and
protein, good electrophoresis agarose gel electrophoresis, SDS-PAGE (Sodium
Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis), and PCR (Polymerase
Chain Reaction) can be a basic capital for researchers to develop research for the
sake of improve human well-being. With mastery of basic techniques the
researcher can make new discoveries that can utilized in various fields life. DNA
isolation is one of them basic techniques in molecular biology which can be
developed into complex studies regarding genetic information that is owned by an
organism. Information on DNA is stored in an alkaline form nucleotides namely
adenine (A), guanine (G), cytosine (C), and thymine (T). Each containing
nucleotide base sequences genetic information that plays a role in development
and regulation of organisms (Dyah, 2016).
 CHAPTER III
EXPERIMENT METHODOLOGY

A. Time and Place

Day / Date : Tuesday, 16th February 2019
Time : 09.10 - 10.50 WITA
Place : Microbiology lab, 2nd floor of biology department Faculty of
Mathematics and Natural Science University of Makassar.
B. Tools and Materials
1.Tools
a. Test tube 5 pieces
b. Knife 1 piece
c. Drop pipette 1 piece
d. Mortar and pastel 1 piece
e. Measuring glass 1 piece
f. Stirrer bar 1 piece
g. Funnel 1 piece
2. Material
1. Fruits (Tomato, Kiwi, Dragon Fruit, Melon, Banana, Orange)
2. Filter paper 1 piece
3. NaCl 3 gram
4. Detergent
5. Aquadest
C. Work Procedure

1. Make a buffer solution. 45

ml of water + 5 grams of
NaCl + 5 ml of liquid
detergent
2. . Filtration. Firstly, the
fruit is peeled and put
into a plastic sugar
and crushed with
mortar.

3. . Filter the juice using filter

paper and funnel into a
measuring cup of 3 ml. The
filtrate is ready

4. Extraction. By
using different
syringes, put them in
the test tube: 3 ml of
filtrate, 2 ml of
buffer, 2 ml of
alcohol (be careful
not to mix the
solution).

5. After alcohol is
poured, attach the
stopwatch, see the time
needed when DNA is
formed between layers
1 and 2.
 CHAPTER IV
OBSERVATION RESULT AND DISCUSSION

A. Observation Result

Group Fruit Shape Time

1 Tomato Cloudy 45 second
2 Kiwi Cloudy 32 second
3 Dragon Fruit Cloudy 43 second
4 Melon Cloudy 28 second
5 Banana Cloudy 35 Second
6 Orange Ring 54 Second

B. Discussion

Based on observations it was found that melon fruit has the fastest time in
form While chemists were working out the structure of DNA, biologists were
attempting to identify the source of genetic information. Mendel identified the
basic rules of heredity. But he had no idea about the physical nature of hereditary
information. Biologists had concluded that genes reside on chromosomes, which
were known DNA. This stage is the process of destruction or destruction of the
membrane and cell walls of the fruit. The purpose of the process is to remove the
cell contents. Then the fruit is added to the detergent solution. The addition of
detergent solution serves to lyse the plant cell walls contained in the sample
solution and can also play a role in reducing the activity of the nuclease enzyme
which is a DNA degrading enzyme. damaged cell. detergent that can dissolve
lipids in cell membranes so that cell membranes destabilize.
According to Murtiyaningsih (2017), detergent solution has a strong anion
that can dissolve lipids as a constituent membrane, so that the DNA will be
exposed to the outside of the cell. This detergent acts as a destroyer of the cell
nucleus so that the DNA will be released and increase the viscosity of the
solution, so that the DNA molecule looks more real. Detergents also act as
inhibitors of the activity of all nuclease enzymes that are present during the
extraction process. The next chemical step is to add NaCl which functions to
remove the polysaccharide. Then add a protein-breaking enzyme that can obtain
only the cell nucleus and by extracting it from the cell nucleus it will obtain a
substance that is soluble in alkaline but not soluble in acid. NaCl is acidic, so the
concentration and pH of the buffer used must be in the pH range 5 to 12. The
buffer solution with a low pH will cause depurification and cause DNA to be
distributed to the phenol phase during the deproteinization process.
While the pH of the solution high above 12 will result in the separation of the
double strand of DNA. The next chemical stage is cold ethanol in the precipitation
stage. the principles of precipitation, among others, first, reduce the solubility of
nucleic acids in water. This is because water molecules that are polar surround the
DNA molecule in aquoeus solution. The positive dipole charge of water interacts
with the negative charge on the phosphodiester DNA group. This interaction
increases the solubility of DNA in water. Second, the addition of ethanol will
remove water molecules in the DNA solution so that DNA will be precipitated.
Ethanol has a lower dielectric than water, making it easier for salts that have a
positive charge (Na +) to interact with negatively charged DNA. This interaction
causes DNA to be hydrophobic and precipitate (Murtyaningsih, 2017).
From the results of observations of DNA isolation, for the kiwi fruit we
observed after being treated, then we got the form of DNA, which is cloudy.
Where according to the theory the DNA formed will float because the density of
DNA is the same as ethanol, then the DNA will move up and up to the surface of
ethanol because of the presence of NaCl which puts DNA into ethanol. The results
of the lab also show that this practicum can be claimed to be successful by
forming three layers in the test tube which are isolated DN. In ADN there are 3
types of complex macromelecules, namely gulapentosa, phosphoric acid, and
nitrogen bases. If reviewed further this molecule has two chains of
polynucleotides. The backbone of each chain of polynucleotides consists of gugu
phosphate and sugar groups alternately. The phosphate group and the sugar group
bound to the 5th carbon atom from one sugar will be bound to the 3rd carbon
atom and the next. These two chains are mutually selected as the form of the
spoiralganda ladder. This is called the double helix.
DNA isolation is one of the important stages in molecular-based activities.
DNA of good quality is needed for various activities such as the use of molecular
markers, making genome libraries, and sequencing. The main problem that often
arises in the process of plant DNA isolation is the presence of contaminant
compounds in isolated samples such as polysaccharides, polyphenols, proteins,
RNA, and secondary metabolites found in medicinal plants. The presence of
contaminant compounds can inhibit various processes ranging from cutting DNA,
amplification, to cloning. DNA binding proteins play a crucial role in
transcription, DNA replication, repair, recombination and various other cellular
activities . To completely understand these fundamental biological processes, it is
important to have knowledge about the proteins whose interplay leads to the
complex control. In the past, methods such as electrophoretic mobility shift assay
(EMSApull down assay have been reported to identify DNA binding proteins.
These methods are mainly associated with in vitro DNA-protein interactions
wherein a purified protein or a crude extract of protein is incubated with labeled
DNA. DNA binding proteins have specific or general affinity for DNA sequences.
Some proteins involved in transcriptional regulation or other varied function may
bind weakly to the DNA, hence making their isolation and identification difficult.
For example, in gel shift assay, the conditions need to be optimized to get an
optimal binding Similarly, in an in vitro pull-down assay the DNA fragment is
immobilized and the crude extract is allowed to pass through.There are only
limited reports of methods that can be used to isolate the proteins that bind in vivo
with the knowledge of short sequences of DNA. One such example of a known
short DNA fragment is the dsz promoter of desulfurizing organisms.
The genes for biodesulfurization are present in the form of an operon under
the control of this promoter. The sequence of this promoter has been identified but
the proteins that bind to this promoter have not been reported.In this study, we
describe a method for isolation and identification of transcription factorsthat bind
to DNA inside the cell.
 CHAPTER V
CLOSING

A. Conclusion
Based on the observations made, it can be concluded that:
1. There are three main steps in DNA extraction, namely cell wall destruction
(lysis) bias by using physical effects such as grinding and chemical effects
such as the addition of detergent. Then the separation of DNA from solid
materials such as cellulose and protein, as well as purification of DNA to form
fibrils.
2. The form of DNA in the form of fibrils, some of which form a ring and tone
that form fasted in melon fruit.
B. Suggestion
In the next practicum, it is expected that the practican can understands the
work procedures so that there will be no more mistakes in practicum activities.
 BIBILIOGRAPHY

Deshmukh, Vishal P., Prashant V. Thakare., Uddhav S. Chaudhari and Prashant

A. Gawande. 2017. A simple method for isolation of genomic DNA from
fresh and dry leaves of Terminaliaarjuna (Roxb.) Wight and Argot.
Electronic Journal of Biotechnology. Vol 10 (3)
Dyah, widyastuti. 2016. Isolasi Dna kromosom Salmonella sp Dan Visualisasinya
Pada Elektroforesis Gel Agarosa. Seminar Nasional Pendidikan Biologi
dan saintek.
Hartwell, Leland H. et al. 2011. Genetics. New York : McGrawHill

Khoiriyah, Yustin Nur. 2014. Karakter Genetik Populasi Bedeng 61B Desa
Wonokarto Kabupaten Lampung Timur Pasca Program Kolonisasi
Pemerintah Belanda. Jurnal Biogenesis. Vol 2(2).

Langga,Indah Fajarwati.,Muh. Restu dan Tutik Kuswinanti. 2012. Optimalisasi

Suhu dan Lama Inkubasi dalam Ekstraksi DNA Tanaman Bitti (Vitex
Cofassus Reinw)Serta Analisis Keragaman Genetik dengan Teknik RAPD-
PCR. Jurnal Sains & Teknologi. Vol 12 (3).

Maria, Kaisar. Yenny, Djuardy. Sartono, Erliyanti. 2017. Improved Diagnosis of

Trichruris trichua by using a bead-beating prosecudre on ethanol
preserved stool samples prior to DNA isolation and the performance of
multiplex rael-time PCR for intestinal parasites. Parasitologi.

Murtiyaningsih, Hidayah. 2017. Isolasi DNA Genom dan Identifikasi

Kekerabatan Genetik Nanas Menggunakan Rapd (Random Amplified
Polimorfic DNA). Agritrop. Vol 15 (1)
Pierce, Benjamin A. 2010. Genetics Essentials. New York: W.H freeman and
Company.
Nugroho, Kristianto. Rerenstradika. Puji, Lestari. 2015. Optimisasi Metode Isolasi
DNA Pada Jatropa spp. Vol 5 No 2. Jurnal Agroteknologi.
Restu., Mukrimin dan Gusmiaty. 2012. Optimalisasi Teknik Ekstraksi dan Isolasi
DNA Tanaman Suren (Toona Sureni Merr.)untuk Analisis Keragaman
Genetik berdasarkanRandom Amplified PolymorphicDNA (RAPD).
Jurnal Natur Indonesia. Vol 14 (2
 RATIFICATION PAGE

Complete Report of Biotechnology with the title “DNA (Deoxiribonucleid

Acid) Isolation” which written by:
name : Fatmah Kamaruddin
ID : 1614440002
class : Biology Education of ICP
group : I (one)
After checked and approved by assistant and assistant coordinator, this report
was accepted.

Makassar, April 2019

Assistant Coordinator, Assistant,

Suharyanti Amir , S.Pd Nyai Ratih Iha

ID.1314141017

Known by,
Responsibility Lecture

Prof. Dr. Ir. Hj. Yusminah Hala, M.S.

NIP: 19811212 198601 2 002