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RAPD MARKER VARIATION AMONG SELECTED SPECIES OF
ACANTHACEAE
ALIFF OMAR BIN DAUD
BACHELOR OF SCIENCE (Hons.) BIOMOLECULAR SCIENCE
FACULTY OF APPLIED SCIENCES UNIVERSITI TEKNOLOGI MARA
NOVEMBER 2010
RAPD MARKER VARIATION AMONG SELECTED SPECIES OF
ACANTHACEAE
ALIFF OMAR BIN DAUD
Final Year Project Report Submitted in Partial Fulfillment of the Requirements for the
Degree in Bachelor of Biomolecular (Hons.) Science in the Faculty of Applied Sciences
University Teknologi MARA
NOVEMBER 2010
ii
This Final Year Project entitled “RAPD Marker Variation Among Selected Species of Acanthaceae” was submitted by Aliff Omar bin Daud, in partial fulfilment of the requirements for the Degree of Bachelor of Science (Hons) Biomolecular Science in the Faculty of Applied Science and was approved by:
________________________________
Associate Professor Zainon A. Rahman Supervisor
Degree of Bachelor of Science (Hons) Biomolecular Science Faculty of Applied Science
University Teknologi MARA 40450 Shah Alam Selangor
___________________________ _________________________________ Prof. Madya. Dr. Tengku Elida Tengku Mulok Prof. Madya Dr. Faiz Foong Abdullah Project Coordinator Head of Programme B. Sc. (Hons.) Molecular Biology B. Sc. (Hons.) Molecular Biology Faculty of Applied Sciences Faculty of Applied Sciences Universiti Teknologi MARA Universiti Teknologi MARA 40450 Shah Alam 40450 Shah Alam Selangor Selangor
Date: ________________
iii
ACKNOWLEDMENT
Upon completion of this project, I would like to express my gratitude to many parties for their help and advice throughout the course of this study. First of all, I would like to thank my supervisor, Associate Professor Zainon bte A. Rahman for her fully support, advice and guidance throughout this project. I would also like to thank my parents, Daud bin Sawabi and Rusminah bte Buang for their support throughout my study period. Finally, I would like to thank all my course-mates, friends and Microbiology laboratory assistants for their support and ideas in completing this project. Aliff Omar bin Daud
iv
TABLE OF CONTENTS
Page ACKNOWLADGEMENT iii TABLE OF CONTENT iv LIST OF TABLE vi LIST OF FIGURES vii LIST OF ABBREVIATION viii ABSTRACT ix ABSTRAK x CHAPTER 1: INTRODUCTION 1.1 Background 1 1.2 Significant of study 2 1.3 Objectives of study 2 CHAPTER 2: LITERATURE REVIEW 2.1 Introduction 2.1.1 Acanthaceae family 3 2.1.2 Andrographis paniculata 4 2.1.3 Acanthus ilicifolius 6 2.1.4 Asystasia gangetica 7 2.1.5 Justicia gendarussa 8 2.2 Molecular genetics 2.2.1 Deoxyribonucleic acid 10 2.2.2 DNA Polymorphism 11 2.2.3 Uses of Polymorphism 11 2.3 Random Amplified Polymorphic DNA (RAPD) 12 2.3.1 PCR principle and procedures 13 CHAPTER 3: METHODOLOGY 3.1 Source of samples 15 3.2 General descriptions of Acanthus species 3.2.1 Andrographis paniculata 15 3.2.2 Acanthus ilicifolius 16 3.2.3 Asystasia gangetica 16 3.2.4 Justicia gendarussa 17 3.3 DNA isolation 3.3.1 Lysate Preparation for Plants Genomic DNA 20 3.3.2 Binding to column 20
v
3.3.3 Washing bound DNA 21 3.3.4 Elution of clear DNA 21 3.4 Determination of DNA 3.4.1 Genomic DNA using 1% of Agarose gel 22 3.4.2 Loading DNA into the gel 22 3.5 Oligonucleotide primers 22 3.6 PCR amplification of primer used / RAPD 23 CHAPTER 4: RESULTS AND DISCUSSIONS 4.1 Genomic DNA for the plant samples 27 4.2 PCR-RAPD 28 CHAPTER 5: CONCLUSION AND RECOMMENDATIONS 5.1 Conclusion 35 5.2 Recommendation 35 CITED REFERENCE 36 CURRICULUM VITAE 39
vi
LIST OF TABLE
Table Caption Page
2.1 The Traditional Uses of Andrographis paniculata 5
2.2 Administration and application of medicinal plants species 8
2.3 Administration and application of medicinal plants species 9
3.1 List of primers used in PCR 23
3.2 PCR-RAPD first attempt 24 3.3 PCR-RAPD second attempt 25 3.4 PCR-RAPD third attempt 25 3.5 PCR-RAPD fourth attempt 26
vii
LIST OF FIGURES
Figure Caption Page
4.1 Genomic DNA for the plant samples 28
4.2 PCR products with the name of primers used (first attempt) 29 (Primers used: OPX-3, OPX4, OPX-6, OPX-12, OPX-19,
OPB-07)
4.3 PCR products with the name of primers used (second attempt) 31 (Primers used: OPX-3, OPX4, OPX-6, OPX-12, OPX-19,
OPB-07)
4.4 PCR products with the name of primers used (third attempt) 32 (Primers used: OPX-3, OPX4, OPX-6, OPX-12, OPX-19,
OPB-07)
4.5 PCR products with the name of primers used (third attempt) 33 (Primers used: OPX-3, OPX4, OPX-6, OPX-12, OPX-19,
OPB-07)
Plate Caption Page
1 Andrographis paniculata 18
2 Acanthus ilicifolius 18
3 Asystasia gangetica 19
4 Justicia gendarussa 19
viii
LIST OF ABBREVIATION
AE : Elution buffer
AW : Wash buffer
DNA : Deoxyribonucleic acid
dNTP : Deoxyribonucleoside Triphosphates
EDTA : Ethylenediaminetetraacetis acid
MgCl : Magnesium chloride
PCR : Polymerase chain reaction
RAPD : Random Amplified Polymorphic DNA
TBE : Tris/Borate/EDTA
ix
ABSTRACT
RAPD MARKER VARIATION AMONG SELECTED SPECIES OF
ACANTHACEAE
The aim of this study was to determine the genetic diversity of the genus Acanthaceae. The genetic diversity of Acanthaceae can be identified by using random amplified polymorphic DNA (RAPD) technique. Six primers were used to study the genetic polymorphisms of the selected Acanthaceae. The selected Acanthaceae were Andrographis paniculata, Acanthus ilicifolius, Asystasia gangetica, and Justicia gendarussa. The six primers used for RAPD were known to have high scorable numbers of markers and consistent in PCR amplification. However, the RAPD of genetic variation of the genus Acanthaceae in this study was not accomplished.
x
ABSTRAK
VARIASI ANTARA SPESIES ACANTHACEAE YANG TERPILIH
DENGAN MENGGUNAKAN TEKNIK RAPD
Kajian ini adalah bertujuan untuk mengkaji variasi genetik dari spesies Acanthaceae. Variasi genetik dari spesies Acanthacea ini boleh dipastikan dan dikaji menggunakan teknik Random Amplified Polymorphic DNA (RAPD). Enam primer telah digunakan demi mengkaji kepelbagaian genetik yang hadir untuk kesemua spesies Acanthacea. Spesies Acanthaceae yang telah dipilih adalah Andrographis paniculata, Acanthus ilicifolius, Asystasia gangetica, dan Justicia gendarussa. Enam primer yang digunakan dalam teknik RAPD mempunyai nombor petanda yang tinggi dan konsisten dalam teknik PCR. Namun begitu, variasi genetik yang terdapat di dalam spesies berkenaan tidak dapat diperolehi di dalam kajian ini.
1
CHAPTER 1
INTRODUCTION
1.1 Background and problem statement
Plants have been used for many thousands of years to treat human pains and
disorders. To the people who live in villages, plants that live in their
surroundings are not only important for the food and material for shelter but it
also an important source in the medicinal fields (Faridah, et al., 1999).
Malaysia has about 7000 species of angiosperms and 600 species of ferns.
From those number, there are about 1150 species have been reported to have
medicinal properties (Latif, 1985). One of the plant families that have known
to have medicinal properties is Acanthaceae.
Many of Acanthaceae members are known as medicinal plants because they
have biologically active phytochemicals. Although these plants family are
important components of Malaysia habitat, little is known about the molecular
relatedness of the genus Acanthaceae particularly at the level of genetic
diversity among the genus or species. Morphological identification of the
species may not reliable because it does not reflect the real genetic variation
due to the genotype-environment interaction.
2
Given the economic important value of the Acanthaceae, it would be very
useful to have a reliable identification tool that can distinguish species from
rare species or those of economic importance.
This study is to characterize genetic diversity of Malaysian Acanthaceae on
the basis of Polymerase Chain Reaction - Random Amplified Polymorphic
DNA (PCR-RAPD) analysis.
1.2 Significance of study
This study will investigate and characterize the selected genus of Acanthaceae
on the basis of PCR-RAPD marker. This study may provide useful
information in the identification of Acanthaceae family using molecular
technique.
Objectives of study
The objectives of this project are:-
1. To collect genomic DNA from the selected species of Acanthaceae.
2. To amplify DNA sequence by using PCR technique.
3. To characterize the genetic variety of the species studied using
PCR - RAPD technique.
3
CHAPTER 2
LITERATURE REVIEW
2.1 Introduction
2.1.1 Acanthaceae
The Acanthaceae family (or Acanthus family) is a taxon of dicotyledonous
flowering plants containing almost 250 genera and there are about 2500
species of it. Most of it is tropical herbs, shrubs, or twining vine and some are
epiphytes. Only a few species are distributed in temperate regions. The four
main centers of distribution are Indonesia and Malaysia, Africa, Brazil, and
Central America. The representatives of the family can be found in nearly
every habitat, including dense or open forests, in scrublands, on wet fields and
valleys, at the sea cost and in marine areas, and in swamps and as an elements
of mangroves woods. The leaves are simple, opposite and decussate; stipules
are lacking. The flowers are bisexual, zygomorphic, and usually are associated
with conspicuous, often brightly colored bracts. The calyx is usually deeply 4-
5 lobed or sometimes is highly reduced with more numerous minute teeth.
The corolla is sympetalous, usually 5-merous, mostly zygomorphic, and
commonly two lipped. The androecium usually consists of four didynamous
4
stamens or only two stamens adnate to the corolla tube or epigynous zone,
alternate with the lobes. The gynoecium consists of a single compound pistil
of two carpels, a single style, and a superior ovary with two locules, each with
usually 2-10 axile ovules in one or two collateral vertical tiers. An annular
nectary disk is usually found around the base of the ovary. The fruit is
commonly an elastically dehiscent loculicidal capsule. The seed stalk or
funiculus of each seed is modified into a hook shaped jaculator or retinaculum
that functions in flinging out the seeds during dehiscence.
2.1.2 Andrographis paniculata
Andrographis paniculata is an herbaceous plant and is commonly known as
“King of Bitters” in the family Acanthaceae (Kanokwan et al., 2008). Bitter
herbs generally have an affinity with the heart, liver and gall bladder and most
have a cooling effect on the body and can bring down a temperature. This
plant is widely cultivated in southern Asia due to its benefits towards human.
The parts of the plants that mostly being used are the leaves and roots and
have been traditionally used over the centuries for many different medicinal
purpose as a folklore remedy or as an herbal supplement for health promotion.
In traditional Chinese medicine, it is important “cold property” herb used to
rid the body of heat, as in fevers, and even to dispel from the body (Deng et
al., 1978). While in the Scandinavian countries, it is commonly being used to
prevent and treat the common cold (Caceres et al,. 1997). Andrographis
5
paniculata is also a powerful immune system enhancer and may also be useful
in cancer therapy while it also have the potent as antiviral herb and helps the
growth of viruses (Barilla et al., 1999).
Andrographis paniculata is an annual plant with characteristic white-purple or
spotted purple flowers that flourishes in South-East Asia, China and India. It
has been valued for centuries by herbalists as a treatment for upper respiratory
infections, fever, sore throat and herpes while other reported applications
include its use in cases of malaria, dysentery and even snakebites.
Table 2.1: The Traditional Uses of Andrographis paniculata (Mishra et al., 2007, Caceres et al., 1997)
Natives names Traditional uses
Traditional Indian
Medicine
Kalmegh Diabetes, dysentery,
enteritis, peptic ulcer
Malaysia Hempedu Bumi, sambiloto Diabetes, hypertension
Traditional Chinese
Medicine
Chuan-Xin-Lian,
Chunlianqialio
Fever, common cold,
pneumonia
6
2.1.3 Acanthus ilicifolius
Acanthus ilicifolius, popularly known as “Harkach Kanta” belong to family
Acanthaceae while the common name of this plant is Holy Leaved Acanthus
(Tridib et al., 2007). It have stout, erect or reclining shrub, up to 1.5 m tall,
which is scarcely branched, smooth and with adventitious aerial roots. The
leaves are oblong measuring 6.5-11 cm x 4-6 cm. The spike is up to 16.5 cm
long, dense or interrupted, and with lance-shaped bracts which are 10 mm
long while the bracteoles are in two pairs, oblong-Iance-shaped and up to 1.5
cm long. The sepal lobes are obovate-oblong and fringed with small hairs
while the petal lobe is obovate, measures 3 cm x 2.5 cm and pale to bright
blue. The petal tube is white. The leaves of Acanthus ilicifolius are used to
treat rheumatism, neuralgia and poison arrow wounds (Malaysia). It is widely
believed among mangrove dwellers that chewing the leaves will protect
against snake bite (Amritpal et al., 2009).
The pounded seeds of Acanthus ilicifolius are used to treat boils, the juice of
leaves to prevent hair loss and it is also used to treat kidney stones. The whole
plant is boiled in fresh water, and the patient drinks the solution instead of
water, half a glass at a time, until the signs and symptoms disappear
(Thailand). Water extracted from the bark is used to treat colds and skin
allergies. Ground fresh bark is used as an antiseptic while the tea brewed from
the leaves relieves pain and purifies the blood (Amritpal et al., 2009).
7
2.1.4 Asystasia gangetica
Asystasia gangetica is an attractive, fast-growing, spreading, herbaceous
groundcover that grows rapidly, up to 0.5 m high alone but to 3 m high on
supporting vegetation. It forms roots when the nodes (the joins between
segments on the stem) make contact with moist soil, ultimately forming mats
or a sprawling mass of stems.
Both of the leaves and the stems have scattered hairs. It has green, oval shaped
leaves sometimes with nearly triangular shape, paler on the underside, and
may be up to 25-165 mm long and 5-55 mm wide with rounded base
occurring in opposite pairs. The flower is white-cream coloured with purple
marking and the fruit is a club shaped capsule (the neck is attached to the
stem) and contain four flattened seeds held in place by conspicuous hooks.,
splitting from tip to base (Ezike et al., 2008). It is widely distributed from
tropical Asia too Africa.
Pharmacological studies have shown that the leaves of Asystasia gangetica
posses bronchosplasmolytic and anti inflammatory properties (Ezike et al.,
2008).
8
Table 2.2: Administration and application of medicinal plants species (Faridah et al., 1999)
Scientific names Uses and parts used Method of application
Aquilaria malaccensis Bark and root decoction as tonic preparation during pregnancy.
Drink
Asystasia gangetica Juice from leaves for eye treatment Leaves chewed raw and applied externally to wound
Bath Rub Poultice
Barringtonia racemosa Leaves of roots and bark for itch and chicken pox
Rub
2.1.5 Justicia gendarussa
Justicia gendarussa belong to the family Acanthaceae and is well-known for
many of its medicinal properties. Justicia gendarussa is a shade-loving, quick-
growing, evergreen plant mostly found in most areas. It is believed to be
native to China and is distributed widely across India, Sri Lanka, and
Malaysia. In Indian and Chinese traditional medicine, the leaf of the plant is
recommended to treat ailments such as fever, hemiplegia, rheumatism,
arthritis, headache, earache, muscle pain, respiratory disorders, and digestive
trouble (Jaijesh et al., 2009). Justicia gendarussa is a deciduous shrub plants
that growing from 0.8-1.5 m. The flowers are hermaphrodite (have both male
and female organs) and the plant prefers light sandy, medium loamy and
9
heavy clay soils. The plant prefers acid, neutral and basic soils. It can grow in
semi-shade (light woodland) or no shade and most important it requires moist
soil.
The plant might succeed outdoors in the mildest areas. It should be grown in a
warm greenhouse. Seed - sow spring in a greenhouse. Prick out the seedlings
into individual pots when large enough to handle and grow on in the
greenhouse for at least the first winter. Plant out in late spring or early
summer after the last expected frosts and give some protection over the
winter.
Table 2.3: Administration and application of medicinal plants species (Source: Faridah H., et al., 1999)
Scientific names Uses and parts used Method of application
Ixora concinna Flower decoction to treat dysentery and stimulate gastric secretions
Drink
Justicia gendarussa Leaves pounded with lemon for deworming and stomach ache Pounded roots for mouth during fits
Poultice Rub
Kyllinga Brevifolia Pounded roots applied externally for skin complaints
Poultice
10
2.2 Molecular genetics
2.2.1 Deoxyribonucleic acids
DNA (deoxyribonucleic acid) is a nucleic acid that contains genetic
information and instruction that being used in the development and
functioning of all known living organism and also in some viruses. The DNA
chain is 22 to 26 Ångstroms wide (2.2 to 2.6 nanometers), and one nucleotide
unit is 3.3 Å (0.33 nm) long. The main role for DNA is to store genetic
information and it’s often compared to a set of blueprints or a code since it
contains the instruction needed to construct other components of cells, such as
proteins and RNA molecules. The DNA segments that carry this genetics
information are called genes, but other DNA sequences have structural
purpose, or are involved in regulating the used of this genetic information.
Chemically, DNA consist of double strand of long polymers of simple unit
called nucleotides, each nucleotides has three parts: a sugar molecule, a
phosphate molecule, and a structure called a nitrogenous base.
The nitrogenous base is the part of the nucleotide that carries genetic
information with the backbone was made by sugars and phosphate group
joined by ester bonds. These two strands actually run in opposite direction to
each other and are therefore anti-parallel. Bases molecules are attached to
each sugar and there are four types of bases (adenine, thymine, guanine, and
cytosine) along the backbone that encode information and this information is
read using the genetic code, which specifies the sequence of amino acids
11
within proteins. The code from DNA was copied into the related nucleic acid
RNAs, in the process called transcription.
2.2.2 DNA Polymorphism
In biology, polymorphism means when two or more clearly different
phenotypes exist in the same population of a species or in the other words, the
occurrence of more than one form or morph. Polymorphism is common in
nature; it is related to biodiversity, genetic variation and adaptation; the
function of it is usually to retain variety of form in a population living in the
varied environment. The most common example is sexual dimorphism, which
occurs in many organisms, and for other examples are mimetic forms of
butterflies and human hemoglobin and blood types. Polymorphisms are
actually results from evolutionary process, as does any aspects of a species. It
is heritable, and is modified by natural selection, and this natural selection
means that the species must have the properties or advantages phenotypes so
that the species can endure to live and survive on the certain condition or an
environment.
2.2.3 Uses of Polymorphism
There are various DNA-based technique and technologies that are useful in
genotyping and quick identification of botanical especially according to DNA
polymorphism between plant species or family. DNA-based techniques have
been widely used for authentication of plant species of medical importance
(Dageri et al., 2008). This is especially useful in case of those that are
12
frequently substituted or adulterated with other species or varieties that are
morphologically and/or phytochemically indistinguishable and various types
of DNA-based molecular techniques are utilized to evaluate DNA
polymorphism (Dageri et al., 2008).
2.3 Random Amplified Polymorphic DNA (RAPD)
In the last decade, the Random Amplified Polymorphic DNA (RAPD)
(pronounced as “rapid”) technique based on the polymerase chain reaction
(PCR) has been one of the most commonly used molecular techniques to
develop DNA markers (Fevzi et al., 2001). This method employs short single
primers of arbitrary nucleotide sequence with 8-12 nucleotides to amplify
anonymous PCR fragments from genomics template DNA. DNA
fingerprinting techniques such as RAPD (Williams et al., 1990) permit the
identification of taxa and the determination of phylogenetic relationship and
intraspecific diversity at a molecular genetics level. RADP markers were
found to be easy to perform by different laboratories, but reproducibility was
not achieved to a satisfactory level (Jones et al., 1997) and therefore, the
method was utilized less for routine identification. The RAPD technique has
been successfully used in a variety of taxonomic and genetic diversity studies
in plants (Jain et al., 1994). Besides that, RAPDs have the advantages where
the materials are processed by an efficient and inexpensive technique that
suitable especially for students to do their project and it also does not require
prior knowledge of the genome (Elisabetta et al., 2001). Due to these
13
advantages and perhaps the main reasons for this success of RAPD analysis is
the gain of a large numbers of genetic markers that require only small amount
of DNA samples or DNA template without the requirement for cloning,
sequencing or any other form of molecular characterization of the genome of
the species in question (Fevzi et al., 2001)
2.3.1 PCR principle and procedures
PCR is a technique used to amplify or make many thousands of copies of a
piece of particular DNA (deoxyribonucleic acid) sequence by the
simultaneously primer extension of complementary strands of DNA.
PCR can be extensively modified to perform a wide array of genetic
manipulation. In PCR, there are three essential steps that are required during
incubation in different temperatures. These three steps make up a PCR
“cycle”, the steps are double-stranded DNA separation or denaturation, primer
annealing to the template DNA, and the extension of the new DNA strands. In
the first cycle that is during denaturation, the temperature will go above 90ºC
for 20-30 seconds and the double-strands DNA will denature. This means the
weak hydrogen bond that usually hold the two complementary strands
together at normal temperatures are disrupted and resulting in two single
stranded DNA strands. During the annealing step, the temperature is lowered
to 50-65ºC for 20-40 seconds so that the primers can collide with their
complementary sequence on the DNA single strand template and hybridized
to it.
14
Stable DNA-DNA hydrogen bonds will form when the primer sequences are
very closely matches with the template sequence. Then the DNA polymerase
will bind to the primer-template hybrid and begins DNA synthesis. The last
step for this cycle is DNA extension. At the extension temperature, that is
around 75-80ºC, the DNA polymerase will bind to the hybridized primer and
begins to add complementary nucleotides. The DNA polymerase will
synthesis a new DNA strand that is complementary to the DNA template
strand (old strand) by adding dNTPs that are complementary to the DNA
template in 5´ to 3´ direction, condensing the 5´-phosphate group of the
dNTPs with the 3´-hydroxyl group at the end of the extending DNA strand.
The process is than repeated by cycling through the temperatures over and
over again (25 times). Each cycle results in a new DNA duplex, each strand
acting as a potential template for one or other primer.
15
CHAPTER 3
METHODOLOGY
3.1 Source of samples
All the plant samples were taken from several places in Selangor.
Acanthaceae were found mostly in tropical and subtropical regions and most
of it was herbaceous plants or shrubs that grow in tropical rainforests.
3.2 General Description on Acanthus species
3.2.1 Andrographis paniculata
Family : Acanthaceae
Genus : Andrographis
Species : Andrographis paniculata ( Plate 1)
Characteristic : Its height is from 30-110 cm mostly in moist in
shady places with leaves and white flowers with the
rose-purple spots on its petals. It have dark green
stems with 0.3-1.0 m in height, 2–6 mm in diameter
and quadrangular with longitudinal furrows while it
have wings on the angles of the younger parts.
16
Uses : Anti-inflammatory / antibacterial / antiviral
3.2.2 Acanthus ilicifolius
Family : Acanthaceae
Genus : Acanthus
Species : Acanthus ilicifolius (Plate 2)
Characteristics : Erect herbs, up to 2.5 m tall, with spiny, often
yellowish stem; leaves like those of holly, leaf blade
dark green. Flowers in neatly organized spikes at
branch tips; petals large, showy and light violet;
capsules squarish and slightly flattened, exploding
when ripe to send its whitish
Uses : Treatment for rheumatism, neuralgia and wounds of
poison arrows.
3.2.3 Asystasia gangetica
Family : Acanthaceae
Genus : Asystasia
Species : Asystasia gangetica (Plate 3)
Characteristic : It is herb or groundcover and can reach 600 mm in
height and if supported it can reach until one m. The
17
leaves are opposite and simple while the fruit has
explosive capsules which start out green in color but
dries to brown after opening.
Uses : Anthelminthic / anti-inflammatory (Ezike et al.,
2008).
3.2.4 Justicia gendarussa
Family : Acanthaceae
Genus : Justicia
Species : Justicia gendarussa (Plate 4)
Characteristic : It has erect branched with smooth undershrub about
0.8-1.5 m tall. The leaves are lance-shaped about 7-14
cm long and 1-2.5 cm wide with pointed at the ends.
The flowers are about 1.5 cm long either white or pink
in colour with purple spots while the capsule has club-
shaped, about 12 mm long and smooth.
Uses : Treatment for cough, cold, and throat infection.
18
Plate 1: Andrographis paniculata
Plate 2: Acanthus ilicifolius
19
Plate 3: Asystasia gangetica
Plate 4: Justicia gendarussa
20
3.3 DNA Isolation
The plant samples were ground to lyse the cell, and then alcohol was added to
the sample lysate so that all the unwanted molecules such as proteins and
lipids were being precipitated. Then the DNeasy Plant Mini Kit was used to
isolate the plants DNA. All of the buffers such as AP1, AP3/E, AW and AE,
except AP2 provided in this mini kit were diluted with proportional amount of
alcohol for the working solution.
3.3.1 Lysate Preparation for Plant Genomic DNA
One gram of each young fresh leaves was ground in liquid nitrogen to a fine
powder using a mortar and pestle. The tissue powder and liquid nitrogen were
transferred to an appropriate appendorf tube and was allowed to evaporate.
Four hundred microlitre of Buffer AP1 and four µl of RNase a stock solution
were added to one gram of tissue powder and was vortexed vigorously. Any
clump tissue was removed by further vortex or pipetting. The mixture was
incubated for 10 minutes at 65°C and mixed to 2 or 3 times by inverting tube
to lyse the cells. One hundred and thirty microlitre of Buffer AP2 was added
to the lysate, mixed and incubated for 5 minutes on ice. Then the lysate was
centrifuged for 5 minutes at 14,000 rpm.
3.3.2 Binding to Column
The supernatant was applied into a QIA shredder Mini Spin Column (lilac)
and was placed in a 2ml collection tube then being centrifuged for 2 minutes
21
at 2,000 x g (14,000 rpm). Then the flow through of the fraction from step 1
was transferred into a new tube without disturbing the cell-debris pallet. 1.5ml
buffer APE3/E was added to the cleared lysate and was mixed by pipetting.
Next, 650µl of the mixture was applied, including any precipitate that have
formed into the DNeasy Mini Spin Column sitting in the 2ml collection tube.
After that the sample was centrifuged for 1 minute ≥ 6,000 x g and the flow
through was discarded then the remaining sample was repeated.
3.3.3 Washing Bound DNA
The DNeasy Mini Spin Column was placed in a new 2ml collection tube and
500µl of buffer AW was added to the DNeasy Mini Spin Column and being
centrifuged for 1 minute at ≥ 6,000 x g and the flow through was discarded
while the collection tube was being reused. Then 500µl buffer AW was added
to the DNeasy Mini Spin Column and being centrifuge for 2 minutes at 20,000
x g (14,000 rpm) to dry the membrane.
3.3.4 Elution of Clear DNA
The DNeasy Mini Spin Column was transferred to a 1.5ml or 2ml
microcentrifuge tube and 100µl of preheated (65°) buffer AE was pipetted
directly onto the DNeasy membrane. The sample was then incubated at the
room temperature (15-25°C) for 5 minutes then was centrifuged for 1 minute
at ≥ 6,000 x g (≥ 8,000 rpm) to elute the DNA sample.
22
3.4 Determination of DNA
3.4.1 Genomic DNA using 1% of Agarose gel
For this study, 1% concentration of agarose gel was used. To prepare this gel,
four gram of agarose gel was dissolved with 50ml of diluted tris-borate-EDTA
(TBE) buffer in a beaker. This solution was swirled with a glass rod and then
was heated in the oven until it was completely melted. Then, gel red was
added into the gel solution to facilitate visualisation of the DNA after
electrophoresis. Next, the gel was poured into a casting tray with its comb
which formed the wells on the gel. The gel was left to solidify and then TBE
buffer solution was poured into the tank until 2-5mm depth. This buffer was
used as the running buffer.
3.4.2 Loading DNA into the gel
The third well was loaded with 5µl of 1 kb DNA ladder while the other well
was loaded with the plant DNA samples. For the DNA ladder, 2µl of loading
dye was mixed with 4µl of DNA sample. Finally, the lid and the power leads
were placed on the apparatus and the current was supplied.
3.5 Oligonucleotide primers
In this study, six different types of primers were used. Each of the arbitrary
primers are expected to anneal to the sites to which to which they are matched
or partially matched. The sequences of PCR primers used in this study as
follows:
23
Figure 3.1: List of primers
3.6 PCR amplification of primer used / RAPD
DNA amplification was performed in a thermocycler by using oligonucleotide
primers listed RAPD markers. The PCR processes were done in which, one
type of primer was mixed with individual genomic DNA samples of four
different species. The master mix was prepared according to Table 3.2.
Number Primer Name Primer sequences (5’ to 3’)
1 OPX-3 TGGCGCAGTG
2 OPX-4 CCGCTACCGA
3 OPX-6 ACGCCAGAGG
4 OPX-12 TCGCCAGCCA
5 OPX-19 TGGCAAGGCA
6 OPB-07 GGTGACGCAG
24
Table 3.2: PCR-RAPD 1st attempt
Initial Final Volume
PCR Buffer 10X 1X 2.5 µl
MgCl 25 mM 1.5 mM 1.5 µl
dNTP mix 10 mM 0.2 mM 0.5 µl
Primer 100 µM 0.2 µM 0.5 µl
DNA sample 2.5 µl
Taq Polymerase 5U/ µl 1U/ µl 0.5 µl
Sterile dH2O 17 µl
TOTAL 25 µl
RAPD reactions were performed in 25 µl total reaction volume. The
thermocycler was programmed for an initial denaturation of 3 minutes at 94°C
followed by 30 cycles of 45 s at 94°C, 1 minute at 37°C and 1 minute at 72°C
and finally for 7 minutes extension at 72°C and a hold temperature at 4°C.
Amplified DNA fragments was separated by electrophoresis at 60 V in 1X
TBE buffer for 3 hour on 2% agarose gels stained with loading dye. Gels with
amplifications fragments were visualized and photographed under UV light.
25
Table 3.3: PCR-RAPD 2nd attempt
Initial Final Volume
PCR Buffer 10X 1X 2.5 µl
MgCl 25 mM 1.5 mM 1.5 µl
dNTP mix 10 mM 0.2 mM 0.5 µl
Primer 100 µM 0.5 µM 1.25 µl
DNA sample 2.5 µl
Taq Polymerase 5U/ µl 1U/ µl 0.5 µl
Sterile dH2O 16.25 µl
TOTAL 25 µl
Table 3.4: PCR-RAPD 3rd attempt Initial Final Volume
PCR Buffer 10X 1X 2.5 µl MgCl 25 mM 1.5 mM 1.5 µl dNTP mix 10 mM 0.2 mM 0.5 µl Primer 100 µM 0.5 µM 1.25 µl DNA sample 5 µl Taq Polymerase 5U/ µl 1U/ µl 1 µl Sterile dH2O 13.25 µl TOTAL 25 µl
26
Table 3.5: PCR-RAPD 4th attempt
Initial Final Volume
PCR Buffer 10X 1X 2.5 µl MgCl 25 mM 2.5 mM 2.5 µl dNTP mix 10 mM 0.2 mM 0.5 µl Primer 100 µM 0.5 µM 1.25 µl DNA sample 5 µl Taq Polymerase 5U/ µl 1U/ µl 1 µl Sterile dH2O 13.25 µl TOTAL 25 µl
27
CHAPTER 4
RESULTS AND DISCUSSION
4.1 Genomic DNA for the plant samples
Genetic analysis of plants relies on high yields and pure DNA samples. In this
study, Plant DNA Isolation Kit was used to isolate DNA from the four plant
samples. DNeasy Plant Mini Kit Plant was used because it provides a rapid
method for the isolation and purification of total DNA from a wide range of
plant species. The kit also provides convenient method for the detection of
pathogens which may be infecting a plant, as it allows for purification of any
pathogen DNA along with the purification of the total DNA. The kit allows
the total DNA to be purified from fresh or frozen plant tissues. In this study,
fresh plant tissues were used since fresh tissues give more reproducible result
with high yield and pure total DNA. The procedure was rapid and convenient
as it does not rely on the use of liquid nitrogen in order to homogenize the
samples. However, this seem does not work efficiently and therefore in this
study, liquid nitrogen was used to homogenize the samples. As shown in
Figure 4.1, although the genomic DNA is slightly contaminated with RNA it
shows clear band of high molecular weight DNA for all plant samples. The
28
genomic DNA sized obtained were approximately around 12,000 bp for all
plant samples studied.
Figure 4.1: Genomic DNA bands. Lane (i) Andrographis paniculata (ii) Asystasia gangetica (iii) 1 kb DNA ladder (iv) Acanthus ilicifolius (v) Justicia gendarussa
4.2 PCR-RAPD
The PCR-RAPD of the samples running on the electrophoresis gel was
observed under UV transilluminator. There was no DNA bands observed for
all plant samples under study except the DNA marker. The presence of primer
dimmers was also not observed. The gels were clearly blank. This shows that
there was no DNA sample amplified by the PCR machine. The PCR-RAPD
results with the primers used of the first attempts are shown in Figure 4.2.
i ii iii iv v
10,000 bp
1,500 bp
250 bp
29
Figure 4.2: PCR products with the name of primers used. Lane (i) 1 kb ladder, Lane (ii) Andrographis paniculata (iii) Asystasia gangetica (iv) Acanthus ilicifolius (v) Justicia gendarussa
OPX-3 OPX-4 OPX-6
OPX-12 OPX-19 OPB-07
i ii iii iv v i ii iii iv v i ii iii iv v
i ii iii iv v i ii iii iv v i ii iii iv v
30
Due to negative results obtained, there were adjustments made for the amount
of the component used for the PCR reaction. The adjustments were made
which are written in bold in methodology section for the amount of primers
(Table 3.3), DNA and enzymes (Table 3.4) and MgCl2 (Table 3.5). The
changes of the amount of distilled water were also made due to the changes of
the concentration of the PCR components and mixture. Despite PCR-RAPD
experiments were repeated, there was still none of the plant genomic DNA
being amplified. The results are shown in Figure 4.2, Figure 4.3, Figure 4.4
and Figure 4.5. The absence of the amplified PCR-RAPD results may be due
to DNA samples had been degraded during storage. The increasing amount of
primer or high concentration of primer may lead to the production of primer
dimmer.
31
Figure 4.3: PCR products with the name of primers used of the second attempt. Lane (i) 1 kb ladder, Lane (ii) Andrographis paniculata (iii) Asystasia gangetica (iv) Acanthus ilicifolius (v) Justicia gendarussa
OPX-3 OPX-4 OPX-6
OPX-12 OPX-19 OPB-07
i ii iii iv v i ii iii iv v i ii iii iv v
i ii iii iv v i ii iii iv v i ii iii iv v
32
Figure 4.4: PCR products with the name of primers used of the third attempt. Lane (i) 1 kb ladder, Lane (ii) Andrographis paniculata (iii) Asystasia gangetica (iv) Acanthus ilicifolius (v) Justicia gendarussa
OPX-3 OPX-4 OPX-6
OPX-12 OPX-19 OPB-07
i ii iii iv v i ii iii iv v i ii iii iv v
i ii iii iv v i ii iii iv v i ii iii iv v
33
Figure 4.5: PCR products with the name of primers used of the fourth attempt. Lane (i) 1 kb ladder, Lane (ii) Andrographis paniculata (iii) Asystasia gangetica (iv) Acanthus ilicifolius (v) Justicia gendarussa
OPX-6 OPX-4 OPX-3
OPX-12 OPB-07 OPX-19
i ii iii iv v i ii iii iv v i ii iii iv v
i ii iii iv v i ii iii iv v i ii iii iv v
34
Most of medicinal and aromatic plant contain high amount of
polysaccharides, polyphenols, tannins and other secondary metabolites such
as alkaloids, flavanoids, terpenes, and quinines. These compounds can
interfere in DNA isolation procedures (Padmalatha et al., 2005). The presence
of polyphenols, which are powerful oxidizing agents present in many plants,
can reduce the yield and purity of the extracted DNA. The polyphenols will
bind covalently to the extracted DNA which will make it useless for most
research application (Katterman et al., 1983; Porebski et al., 1997).
Certain polysaccharides can inhibit RAPD reaction (Padmalatha et al., 2005).
Most of DNA isolation procedures yield large amount of DNA, such as 18S
and 25S rRNA (Doyle and Doyle, 1987). The large amount of RNA being
yield can chelate the Mg2+ and reduce the yield of the PCR. There were also
the lost of DNA sample (degraded) during the storage.
Optimization is the process, where PCR contents are increasing in
concentration of the content, in order to obtain better result. Optimization
were done step by step in which the concentration of contents and
temperatures were adjusted to suit the condition. (Padmaltha et al., 2005).
35
CHAPTER 5
CONCLUSION AND RECOMMANDATION
5.1 Conclusion
In the conclusion of this study, the genomic DNA of selected species of
Acanthaceae, Andrographis paniculata, Asystasia gangetica, Acanthus
ilicifolius, and Justicia gendarussa were successfully extracted but the
amplification of these targeted DNA by using PCR RAPD was unsuccessful
and the genetic variation between these species cannot be characterized during
this study.
5.2 Recommendations
If the present study would be continued, it would important to study and test
the optimum temperature of the PCR for each sample because each of the
primers has its own Tm point so it has different temperature of annealing.
Besides that increase the number and types of primers for RAPD purposes
with some adjustments to the mixture of the PCR component used.
36
REFERENCES
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Barilla MS, J. “ Andrographis paniculata.” Better Nutrition; June 1999.
Caceres D. D., Hancke J L., Burgos R. A., and Wikman G. K., (1997). Prevevntion of common colds with Andrographis paniculata dried extract: A pilot double-blind trial. Phytomedicine, 4, 101-104
Dageri A., Hasibe C. V. 2008 Optimization of DNA isolation for RAPD-PCR analysis of selected species (Enhinaceae purpurea L. Moench) medicinal plants of conservation concern from Turkey.
Deng W. L., (1978). Preliminary studies on pharmacology of the Andrographis product dihydroandrographolide sodium succinate. Newslatter Chinease Herbal Medicine, 8, 26-28
Elisabetta S., Stefano P., Assunta B., (2001). Use of Random Amplified Polymorphic DNA (RAPD) to Detect Genetic Variation in Pyrus Soecies. Plants Molecular Biology Reporter. 19: 271
Ezike A. C., Akah P. A., Okoli C. O., (2008). Bronchospasmolytic activity of the extract and fraction of Asystasia gangetica leaves. International Journal of Applied Research in Natural Product. 1(3), 8-12
Faridah H., Nurulhuda H., (1999). The use of Medicinal Plant Species by the Temuan Tribe of Ayer Hitam Forest, Selangor, Peninsular Malaysia.Pertanika J. Trop. Agric. Sci. 22(2): 85-94
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Fevzi B., (2001). Random Amplified Polymorphic DNA (RAPD) Markers. Turk J. Biol. 185-196
Jain A., S. Bhatia, S.S. Banga, S. Prakash, and M. Lakshmikumaran. 1994. Potential use of random amplified polymorphic DNA (RAPD) technique to study the genetic diversity in Indian mustard (Brassica juncea) and its relationship to heterosis. Theor. Appl. Genet. 8:116-112
Jaijesh P., Srinivasan K. K., Bhagath K.P., Sreejith G., Raju S. K., Sareesh N. N., and Sudheer M., (2008). Anti-Arthritic Potential of the Plant Justicia GendarussaBurm F. 64(4): 357–362.
Jones C. J., Edward K. J., Castaglione S., Winfield M. O., Sala F., Wiel C. van de, Bredemeijer G., Vosman B., Matthes M., Daly A., Brettschneider R., Bettini P., Buiatti M., Maestri E., Malcevschi A., Marmiroli N., Aert R., Volckaert G., Rudea J., Linacero R., Vazquez A., Karp A. (1997): Reproducibility testing of RAPD, AFLP, and SSR markers in plants by a network of European laboratories. Mol. Breed., 3:381-390
Kanokwan J.,Nobuo N.,(2008). Pharmacological Aspects of Andrographis paniculata on Health and Its Major Diterpenoid Constituent Andrographolide. Journal of Health Science., 54(4) 370-381
Katterman, F.R.H. and V.I. Shattuck, (1983). An effective method of DNA isolation from the mature leaves of Gossypium species that contain large amounts of phenolic terpenoids and tannins. Preparative Biochem., 13: 347-359
Latiff A.,(1985). Ethnobotanical and Phytochemical Studies on Malaysian Menispermaceae. Malays. Appl. Biol. 14(2): 68-75
Mishra > K., Sangwan N. S. and Sangwan R. S., (2007). Andrographis paniculata (Kalmegh): A review. Pharmacog. Rev. 1: 283-298
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Padmalatha K, Prasad M.N.V., (2005). Optimization of DNA isolation and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India. African Journal of Biotechnology Vol. 5 (3), pp. 230-234
Porebski, S., L. Grant Bailely and B.R. Boum, (1991). Modification of a CTAB DNA extraction protocol from plants containing high polysaccharide and polyphenol components. Plant Mol. Biol. Rep., 15: 8-15.
Tridib C., Dipak B., Mary C., Mosiur R., Dipak S., Baidya N. C., Subrata D., Ajay R., Kartick S., Sunil S., Sankar K M., Malay C., (2007). Acanthus ilicifolius plant extract prevents DNA alterations in a transplantable Ehrlich ascites carcinoma-bearing murine model. 28;13(48): 6538-6548
Williams, J.G.K., A.R. Kubalik, K.J. Livak, J.A. Rafalski, and S.V Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic-markers. Nucleic aciss Res. 18:6531-6535
39
CURRICULUM VITAE
A. Personal profile
B. Hobbies and interests
I enjoy reading and listening to music. I love to read books that can inspire and also that give additional of general knowledge. Listening to music sometimes can make me calm from stress and also can give me passion to do something. I am very passion in doing something important in order to get it perfectly done.
I am fluent in written and spoken Malay, and also English.
Full name Aliff Omar bin Daud
National IC no 890314-23-5433
Birth date 14th March 1989
Citizenship MALAYSIA
Place of birth Johor, MALAYSIA
Gender Male
Correspondence address
285, Jalan Paya, Rumah Awam, Bakri Batu 6,
84200, Muar,
Johor Darul Ta’zim
Telephone no.(H) 06-9860693
Telephone no. (HP) 017-6870693
Email address aliffomar_daud@yahoo.com
40
C. Academic qualifications
Degree Area Institution Year awarded
B.Sc. (Hons.) Biomolecular science
Universiti Teknologi MARA, Malaysia.
2012
Matriculation Life Science Johore Matriculation College
2008
S.P.M Science MARA Junior Science College, Muar, Johor.
2006
D. Related experience
Post Place Year
Committee member
BIOVILLE Family Day at Janda Baik 2011
AJK BIOCARE Family Day at Port Dickson
2010
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