RAPD Marker Variation Among Selected Species of Acanthaceae

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


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: ________________

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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

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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

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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

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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

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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


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

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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).

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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).

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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

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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


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


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

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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


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.

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Plate 1: Andrographis paniculata

Plate 2: Acanthus ilicifolius

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Plate 3: Asystasia gangetica

Plate 4: Justicia gendarussa

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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


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


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


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




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




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



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 [email protected]

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

Documents

RAPD Marker Variation Among Selected Species of Acanthaceae