DNA EXTRACTION AND PCR OPTIMIZATION OF MITOCHONDRIAL COl GENE IN THE GENUS BETTA

- .

Aisyah Binti Shamsuddin

o QH 603 M5 Bachelor of Science with Honours Al99 (Animal Resource Science and Management)18n 2012

---- ---Pusat Khidmat Maklumat Akademik U1\1VERSm MALAY lA SARAWAK

DNA EXTRACTION AND PCR OPTIMIZATION OF MITOCHONDRIAL

COl GENE IN THE GENUS BETTA

P.KHIDMAT MAKLUMAT AKADIMIK

11111111 100012351736

AISYAH BINTI SHAMSUDDIN

This project is submitted in partial fulfillment of the requirements for the degree of Bachelor

of Science with Honours (Animal Resource Science and Management)

FACULTY OF RESOURCE SCIENCE AND TECHNOLOGY

UNIVERSITY MALAYSIA SARA W AK

2012

DECLARATION

I hereby declare that the thesis is based on my original work except for citation which has

been duly acknowledgement. I also declare that it has not been previously or concurrently

submitted for any other degree at UNIMAS or any other institutions of higher learning.

Aisyah binti Shamsuddin

Animal Science Resource and Management Programme

Department of Zoology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ACKNOWLEDGEMENTS

First and foremost, 'A lhamdulillah , and praise be to Allah. The Almighty, thank you for

giving me the strength and wisdom to complete this final year project.

I would like to record my thanks and appreciation to my supervisor Dr. Yuzine bin Esa for all

the help and guided me in ensuring this project is complete as required by the Faculty.

Thanks you again for your precious and valuable advices.

Special thanks to my thanks and strongest admiration to my mother, Azizah binti Abas, my

father, Shamsuddin bin Othman and my siblings Norazlee Rizal, Mohd Amin, Muhammad

Aliff and Muhammad Ashraf for being emotionally and financially supportive, not only in

helping me completing this final year project, but also throughout my three years of studying.

I would like to record too. To all postgraduate especially Muhammad Fadzil bin Amram and

Elvy Quatrin Deka Duncan and Ho Licia for their kindness in assisting and giving me

comments on my project.

Last but not least, to all my family, friends and acquaintances, who are involved directly or

indirectly in helping me to complete this study, especially Nurul Fadilah binti Norzelan,

Sharizzaty binti Mat Rais, Khatijah Binti Ismail, Melynda Cheok and Nurul Alwanie. I give

all of you my deepest gratitude.

- - -- -p"aat kkldmat MakJum,t Akadtmik VNlVERSm MALAVSIA ~ARAWAK

TABLE OF CONTENTS

Acknowledgements I

Table of contents II

List of Abbreviations IV

List of Tables V

List of Figures VI

Abstract

1.0 Introduction 2

2.0 Literature Reviews 4

2.1 Opshronemidae 4

2 .2 Taxonomy of Genus Betta 5

2.3 Distribution and Life History 6

2.4 Mitochondrial DNA (mtDNA) 7

2.5 Cyctochrome Oxidase I (COl) 8

2.6 Polymerase Chain Reaction (PCR) 9

3.0 Material and Methods to

3.1 Study site 10

3.2 Identification of Betta species 10

3.2.1 Processing and Morphological technique 10

3.3Laboratory work 11

3.3.1 DNA Extraction II

3.3.2 Visualization of DNA Product 12

3.3.3 DNA Amplification by PCR 13

3.3.4 Purification of PCR Product 15

II

4.0 Result 16

4. I DNA Extraction of Sample 16

4.2 Polymerase Chain Reaction 18

4.3 Purification 20

5.0 Discussion 22

5. I DNA Extraction 22

5.2 Polymerase Chain Reaction (PCR) 23

5.3 Electrophoresis 25

5.4 Purification 26

6.0 Conclusion 27

REFERENCES 28

APPENDIX 31

III

I

LIST OF ABBREAVIATIONS

COl Cytochrome Oxidase I

CTAB Cetytrimethyl Ammonium Bromide

ddH20 Deionizer distilled water

DNTP Deoxyribonucleotide triphosphates

EDTA Ethylenediaminetetraacetic acid

PCR Polymerase Chain Reaction

mtDNA Mitochondrial DNA

MgCh Magnesiun Chloride

NAOAc Sodium Acetate

NaCI Sodium Chloride

Rpm Rotation per Minute

TAE Tris-acetate-EDT A

V Volt

Kb Kilobase

DC Degree Celcius

IV

i

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LIST OF TABLES

Table 2.1 Scientific classification of genus Betta 6

Table 3.1 Location and number ofBetta sample 10

Table 3.2 The master mix for PCR reaction mixture 13

Table 3.3 Amplification cycle for PCR process 14

Table 3.4 Primer for partial cytochrome oxidase I (COl) gene sequence 15

v

I

LIST OF FIGURES

Figure I: Picture of Betta unimaculata 5

Figure 2: Picture of Betta splendens 5

Figure 3: Picture of Betta splendens 6

Figure 4: Diagram of amplification cycle in peR 14

Figure 5: Gel electrophoresis of DNA extraction products for 15 samples 16

Figure 6: Gel electrophoresis of DNA extraction for II samples 17

Figure 7: Gel electrophoresis of DNA extraction for 15 samples 17

Figure 8: Gel electrophoresis of gradient peR 18

Figure 9: Gel electrophoresis of peR product 19

Figure 10: Gel electrophoresis of peR product 19

Figure 11: Gel electrophoresis of peR product 20

Figure 12: Gel electrophoresis of purification product 21

Figure 13: Gel electrophoresis of purification product 21

VI

DNA Extraction and peR Optimization of Mitochondrial COl gene in the genus Beua

Aisyah Binti Shamsuddin

Animal Resource Science and Management Programme

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Studies were conducted to study the optimization of the extraction of DNA and mitochondrial COl gene in two species of the genus Bella; Bella splendens and Betta unimaculata. A total of 15 individuals from Betta fish have been taken from Temerloh, UNIMAS campus, and several locations in Kuching as well as from local aquarium shops and wet markets. From optimization, eight individuals from 15 samples of Bella fish has been successfuny showed a single band. Various measures have been changed in the form of PCR temperature and time for each steps of PCR to identify the most effective parameters for the PCR product. Initial denaturation of the PCR parameters: 94°C for 3 minutes; annealing: 48.3°C for 1 minute; extension: 72 o C for 1.30 minutes; extension ends: 72 ° C for 5 minutes. 100bp DNA ladder was used to estimate the size of about 520 bp.

Key words: COl mtDNA gene, PCR, B. splendens, B. unimaculata.

ABSTRAK

Kajian telah dijalankan IIntuk mengkaji pengekstrakan DNA dan pengoptimuman gen mitokondria cythocrome oxidase I (COl) dua spesies dalam genus Betta iaitu !l/ll1!I splendens dan Betta unimaculata. · , Sejumlah 15 individu dari ikan Bella telah diambil dari Temerloh,-kampusUNlMAS,dan beberapa lokasi di Kuching serta dari kedai akuarium tempatan dan pasar basah. Dar! pengoptimuman, 8 individu dari 15 sampel ikan Betta telah Berjaya menunjukkan jalur tunggal. Pelbagai langkah PCR telah diubah dalam bentuk suhu dan tempoh masa bagi setiap langkah PCR untuk mengena pasti parameter yang paling berkesan untuk mendapatkan produk PCR. Parameter PCR penyahaslian awal: 9.f'C selama 3 minit; penyepuhlindapan: 48.3°C selama I minit; lanjutan: 72°C untuk 1,30minit; lanjutan akhir: 72°C selama 5 minit. 100 bp tangga DNA telah digunakan untuk menganggarkan saiz kira-kira 520 bp.

Kata kunci: COl mtDNA gene, PCR, fl.. splendens. fl.. unimaculata.

1

1.0 INTRODUCTION

The island of Borneo has one of the highest diversity of freshwater fishes in the world with

more than 350 species have been recorded (Inger and Chin, 1962). Fish is an aquatic

vertebrate with an internal skeleton made of bone. Fish are separated from other vertebrate

(amphibians, reptiles, and mammals) by lacking of limbs (Fong, 2010). However, they also

share a number of unique characteristic.

Fish has been focused as food because of their overwhelming economic importance, but

fish not only freshwater resources and also been used for fertilizer, medicine, the pet trade

and integral part of a dynamic food chain (Lim and Chou, 1990). Besides being used as

food, some species of fish are collected for the aquarium fish trade. For example the main

species fish trade in Indonesia is the clown loach Bolia macracanthus (Rainboth, 1996).

This species is the most important wild caught pet fish of the world.

Genus Betta is one of the most known freshwater fish around the world mainly due to the

popularity among the aquarium hobbyist of it beautiful appearance and easy to breed

(Yeong, 2009). According Monvises et at. (2009), Bettasjs.preferable not only for ethical

reasons but also for commercial reasons in that the export market for the latter is larger and

has higher profit margins. Although the wild types are eagerly sought and command high

prices as breeding stocks. It is the one of fish that bred in captivity for the commercial with

has a variety of colours such as red, blue, green, purple and albino (Monvises et at., 2009).

According to Rainboth (1996), Betta species is complicated of their systematic status and

still unclear due to the great diversity of this genus. It is difficult to distinguish their species

because most of them are hybrid. It can see this species has been subject to many years of

artificial selection that particularly the males for showing various mutant colour

morphology and long fins and tails. Addition, the local species are difficult to find

2

..

according the destruction on their habitat area by water pollution, urbanization,

agricultural, and logging.

Molecular technique based on polymerase chain reaction (PCR) has proven to be more

credible for species identification (Comi et at., 2005). In addition, study of molecular data

can help to strengthen the taxonomy of Betta fish based on morphology or can assist in

resolving taxonomic status among some of the problematic species. Mitochondrial DNA is

a suitable target DNA, due to the faster evolutionary rate of mitochondrial DNA than

nuclear DNA and thus contains more sequence variation thereby facility identifications of

closed related species (Donalson and Wilson, 1999).

In this study, the PCR method was used to examine the mtDNA profiling of Betta species

using COl gene. The objectives are:

i) To extract DNA from selected of several Betta species among Family

Osphronemidae.

ii) To optimise the Polymerase Chain Reaction (PCR) amplification ofBetta fish.

3

2.0 LITERATURE REVIEW

2.1 Ospbronemidae

Betta fish is one of several genera in the Family Osphronemidae. The monophyletic genus

of Betta consists of 55 nominal species with several species groups (Tan and Ng 2005a,

2005b). According to their brood care behaviour, the species were divided into bubble nest

builders and mouthbrooders (Schindler and Schmidt, 2006). Each one of Betta was also

displays one of two different types of egg brooding care either nest building or mouth

(Ruber et al., 2004). Earlier, the genus Betta was considered a part of the Anabantidae

family. This is because all Betta species have labyrinth organs just like the Anabantidae

species. This special organ is located just above the gill arches (Ruber et al., 2006) It was

enables them to breathe atmospheric air which they obtain from the water surface. They

can also thrive comfortably in low oxygen habitat because of this respiratory structure.

According to Witte and Schmidt (1992), this genus is characterized by having a low count

of the dorsal fin rays and unserrated lachrymal and predorsal. Besides, their caudal fin is

rounded or pointed and their pelvic fin consists of a single spine and five branched rays .. ~ .

(Rainboth, 1996). It also has long flowing fins with various types of tail shape including

pointed, split, round, crown, comb and fantail which adds further splendour to its

morphology (Schindler and Schmidt, 2004). Usually, the size for males Betta can be reach

a total length of about 7cm and females are slightly smaller. Females are look difference

with the male by their less of brightly coloured and shorter fins than male (Ng, 1993b).

Siamese fighting fish is one of famous Betta fish in world (Figure 2 and Figure 3). In

Thailand, the fighting fish is known as PIa-Kat which means biting and tearing fish. They

were used for contests such as games and betting medium (Kotteiat, 1994). The male of

this Betta has an aggressive behaviour toward other males (Lim and Chou, 1990). The two

4

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· - -- ......... Pusat Khid ••t Maklu.mat Aka4emlk UNlVERSm MALAYSIA SARAWAK

of male Betta can fight to death when they encounter each other. In aquarium, the only one

Betta can be occupied at that time. It is because the males are extremely aggressive

towards males of their own species and will readily fight to complete exhaustion (Witte

and Schmidt, 1992). The male may fight to claim territory or to protect their eggs or

offspring from rival males. They were spread his fins, extend his gill opercula and

membranes, and generally appear much larger than his resting size.

2.2 Taxonomy of the species from genus Betta is shown below:

Figure 1: Picture of Betta unimaculata species

Figure 2: Picture ofBella splendens species

5

Figure 3: Picture of Red Betta splendens species

Table 2.1: Scientific classification of genus Betta

Common name Siamese fighting fish

Kingdom Animalia

Phylum Chordata

Class Actinopterygii

Order Perciformes

Family Osphronemidae

Genus Betta

Species B. unimaculata (most common at Sarawak)

2.3 Distribution and Life History

Betta species originates in the shallow water in Thailand (Siam), Indonesia, Malaysia,

Vietnam, and parts of China (Witte and Schmidt, 1992). Usually, their home countries of

the Betta are tropical climate. This is because of Southeast Asia can provides a stable

environment with various types of niches and habitat. Usually, they can be found in paddy

fields, ponds, lagoons and marshes (Monvises et al., 2009). The presence of freshwater

environment such as rivers, lakes, forest stream, open country stream, hill stream,

freshwater and peat swamp can also support a great diversity of this genus (Ng and

Kottelat, 1992). The species also had been survived at extreme environment condition for

6

adaptation such as in peat swamp with high acidity. Betta species also tend to be restricted

to one or few drainage system by various natural obstacles like mountain ranges and large

rivers (Kottelat, 1994).

According to Monvises et al. (2009), in 1934 the first published of classification genetic

basis for the pigment of Betta splendens. Based on the statistical analysis of Betta scale

pigments, the scientist was found that the body, fin, and scale colour of this Betta were

regulated by genes for melanin and iridescence pigment as well as the genes for pigment

density. Recently, the work has begun on the taxonomy and population structure of Bettas

using genetic markers of mitochondrial and nuclear DNA sequences (Monvises et al.,

2009). The study was showed that Betta to be in the Osphronemidae family. It was also

found that the B. splendens nesters are genetically more closely related to B. imbellis than

the others whereas the B. pi brooders are genetically close to B. simplex as supported by an

RAPD study.

2.4 Mitochondrial DNA (mtDNA)

Mitochondrial DNA variation is relatively well-characterized for many fish speCIes.

Mitochondrial DNA was occur as a circular loop of 16, 000 to 20, 000 base pairs (bp) in

fish (Kapuscinski and Miller, 2007). It is use in combination with nuclear DNA markers

that can tell about sex-specific differences in behaviours such as migration. Each cell

contains multiple copies of the mtDNA genome. So that DNA amplification via the

polymerase chain reaction (PCR) of small and degraded samples might be more feasible

with mtDNA primers than with some nuclear DNA primers (Kapuscinski and Miller,

2007). Universal primers for PCR developed for use among diverse taxa have effectively

amplified fish mtDNA.

7

I.'

Mitochondria DNA are suitable target DNA because it has faster evolutionary rate of

mitochondrial DNA than nuclear DNA and also contain more sequence variation thereby

facility identification of closed related species (Donaldson and Wilson, 1999).

Furthermore, mitochondrial DNA has many advantages as the best molecular marker used

in studying genetic variation (Esa et at., 2008). For example, the mtDNA genetic markers

have been widely used as a tool to distinguish and relationship among individuals,

populations and species (Patemello et at., 1994). Moreover, mtDNA has a number of

specific biological properties which make it as a marker of molecular biodiversity (Shen et

at., 1997).

2.5 Cytochrome Oxidase I (COl)

COl is a catalytic enzyme Cytchrome Oxidase C that located in mtDNA genome. It was

subunit of the cytochrome oxidase complex that a part of the electron transport chain

(ETC) and also one of the superfamily of protein which function as the terminal enzymes

of respiratory chains (Hebert et at., 2003). Moreover, their amino acid sequences is highly . . conserved across phyla and making it easy to align sequences to one another and possible

to design useful universal primers (Hillis et at., 1996). The size of COl gene generated

about approximately 550 bp using the same primer, COl-forward and COl-reverse

designed (Palumbi, 1996).

Besides, COl gene in mitochondrial DNA is a good genetic marker for both intraspecific

and population genetics studies due to high variation (ranged from 7% to 9.5%) inferred in

Paramecium species (Nielsen et at., 1998). COl gene also contained a significant amount

of information to detect variation among species. Moreover, COl gene could be fast and

8

accurate marker to the resolution of the diversity of animals and has been used successfully

for species level identification in several animal groups (Hebert et al., 2004).

2.6 Polymerase Chain Reaction (PCR)

peR is a common and often indispensable technique used in medical and biological

research laboratory for variety of applications developed. These include DNA cloning and

sequencing, DNA-based phylogeny, or functional analysis of gene (Erlich and Arnheim,

1992). This method can helps to resolve the problem of low or incomplete amplification in

cross-species amplification (Smith et al., 2000).

PCR is a rapid, inexpensive and simple way of copying specific DNA fragment from

minute quantities of source DNA material (Erlich, 1989). It does not necessarily require the

use of radioisotopes or toxic chemicals. It was involves preparing the sample DNA and a

master mix with primers and followed by detecting reaction products. PCR also gives more

advantages in molecular study that are efficiently an amplifying the targets. A specific

DNA segments either mtDNA or nDNA can be amplified until millions of copies are

obtained (Nielsen et al., 1998). This amplification of DNA isallowed genetic analyses from ~ .

non-lethally collected, very small and even degraded tissues (Yeong, 2009).

PCR requires a repetitive senes cycle of heating and cooling which involved three

fundamental steps that defines as for one PCR cycle. The steps are involving in PCR

process are denaturation, annealing and extension (Carvalho et al., 2004). Through the

successive cycles of heating and cooling, double-stranded DNA is separated and replicated.

Then, it will leading to an exponential increase in copy number. Primers may need to be

developed for each species or closely related species because they are sequence-specific

and the homologous sequence in the organism's genome may differ across species (Saiki et

al., 1988).

9

3.0 MATERIALS AND METHODS

3.1 Study site

Fish sampling was collected from several locations in Temerloh, UNIMAS museum, Satok

wet market and from local aquarium in Kuching. Mostly, the samples were bought from

wet market, Kuching. All the 15 individuals of Betta fish were been collected and

identified their morphological. Two species were involved in this project which is Betta

splendens and Betta unimaculata.

Table 3.1: Location and number ofBetta samples

Location No. of individuals Temerloh 4

UNIMAS museum 1 Wet market 5 Aquarium 5 Total 15

3.2 Identification of Betta species

3.2.1 Processing and Morphological Technique

The specimens collecting were transferred to 70% ethan~l s.olution and stored in freezer at

-20°C for long tenn storage. Ethanol is suitable to storage sample and has been used

successfully in DNA hybridization and sequencing (Dessaur et ai., 1996). All the

specimens has been tagged before setting and label containing the name of captured, name

of species, the place that captured, and date.

The morphological technique an important technique uses to differentiate among species,

population or group within species. It involves identification an anatomical structure like

the shape of fins, tails and colours (Ng and Kottelat, 1992). The colourations of Betta

species are useful in distinguishing live specimen (Yeong, 2009). The colour may vary

10

depending on maturity, sex, reproductive condition and geographic variation. The large

and old species tend to be darker. Thus, the way of identification is difficult to use if it is

unfamiliar with the intraspecific variation (Ng, 1993b).

3.3 Laboratory work

3.3.1 DNA Extraction

Total genomic DNA extraction has been done using modified CT AB (Cetyl-trimethy

Ammonium Bromide) method (Grewal, 2000). All the 15 individuals of Betta fish were

extracted using several of body parts to get DNA. Firstly, a total of 1-2 cubic milimetres of

tissue from sample Betta fish was grinded and transferred into 1.5 eppendorf tube which

contains 700 J.1l of CTAB buffer. Then, 15 III of proteinase K was added into the eppendorf

tube. After that, the tube was incubated in the water bath at 65°C for 2-3 hours until tissue

are completely dissolved. Every 30 minutes, the tube should be monitored and shakes to

make sure the sample are not over lyses.

After two hours, a total amount 600 III of Chloroforrn-isoatPyl alcohol or CIA was added

the tube that containing lysed sample. The content of each tube were shakes for about two

to three minutes. Then, tube was centrifuge at 13 000 rounds per minute (rpm) for 20

minutes. As a result of this process, three layers of mixture will be visible. The only upper

layer of the supernatant was pipette for about 450 III and transferred to a newly label tube.

An equal volume cold absolute ethanol (99%) as a pipette product was added into the same

tube. The tube was sat on the bench for a few minutes before proceed centrifuge for 20

minutes at 13 000 round per minute (rpm).

11

After centrifuge, the supernatant was discarded carefully by using pipette and ensure DNA

pellet is still intact at the bottom of the tube. The pellet has been observed whether visible

or not by yellow or white colour pellet. Then, an equal volume (450 ~l) or more of cold

70% ethanol, and 25).11 of 3M NAOAc/NaCI were added into the tube and mixed by slowly

inversing it. The tube was then centrifuge at 13 000 round per minute (rpm) for 15 minutes.

Again, the supernatant from the tube was discarded and checked for the presence DNA

pellet. The tube was left at room temperature until there is no more liquid in the tube. The

pellet was dissolved by adding 50-100 ).11 of distilled water or ddH20 before storing it into

the freezer (-20°C).

3.3.2 Visualization of DNA Products

The DNA yield from the extraction was visualized using agarose gel electrophoresis

method (Palumbi et al., 2002). One percent of agarose gel will be prepared and stained

with two ).11 of ethidium bromide (EtBr). The ethidium bromide is often used to stain the

DNA molecules for subsequent visualization under UV Jrght. The gel was then immersed

in Ix TAE buffer. TAE buffer is a common buffer used for prepared agarose gel and for

agarose gel electrophoresis in the analysis of DNA product from PCR amplification

experiment. This solution also contains a minimal amount of ionic strength to prevent

denaturation of DNA.

Then, for loading the gel, 2).11 of loading dye and 2 ).11 of DNA sample will be pipette and

mixed. One kb DNA ladder (Fennentas GeneRuler™) with loading dye was also loaded

into wells as an indicator of the products' size. The agarose gel electrophoresis or AGE

will be run at 85volts for 40 minutes. After the electrophoresis complete, the gel was

visualized using transiluminator. The photographs of the gel were taken for documentation.

12

3.3.3 DNA Amplification by Polymerase Chain Reaction

All the success extraction DNA was subjected to amplification by using Polymerase Chain

Reaction (PCR). Before the PCR process took place, the master mix was prepared based on

pre-configuration of the reagents which consists of 5 x reaction buffer, dNTP mix,

magnesium chlorides (MgC b), 10 mM of COl universal forward primer, 10 mM of COl

universal reverse primer, I U Taq DNA polymerase and deionized distilled water (ddH20).

The chemical reagents that were obtained from Prom ega kits were mixed briefly and quick

spin for one minute to bring all reaction components to the bottom of tube.

After that, 23 JlI of master mix was transferred into each PCR tubes. Then, 2 JlI of template

DNA was also added into the PCR tube to make the volume into 25 Ill. Amplification

process was carried out in PCR machine for 30 cycles about three hours. This process

involves repetitive series of cycles consists of three major stages, denaturation, annealing

and extension. The components needed for the PCR mixture is shown table 3.1 table 3.2

shows the steps of PCR cycles.

Table 3.2: The Master Mix for PCR Reaction Mixture

Component Stock solution I x reaction (Ill)

13.3

5 x reaction buffer 5.00

0.2 mMdNTPs 2mM 0.50

1.5 mMMgCh 25mM 1.50

10 mM COI-f IOmM 1.25

10 mM COI-e 10mM 1.25

80 ng DNA template 40 ng! III 2.00

I U Taq DNA polymerase 5 VIlli 0.20

Total 25 .0

13

Table 3.3: The amplification cycle for PCR process

Steps Pre-denaturation

Temperature (0C)

94 Time (min)

3 Number of Cycles

Denaturation 94

Annealing

Extension

48 .3

72 1.3

30

Final Extension 72 5

Soak 4 00

Pre-denaturation

94 C 94 C

Optimum temperature

Final

3mins 1 min 72 C

Extension

72 C

A A Denaturation

Annealing

48.3°C

1.3mins

C 5 mins

1 min Extension C

B

30 cycles

Figure 4: Diagram of amplification cycle in Polymerase Chain Reaction

14

Table 3.4: Primers for partial cytochrome oxidase I (COl) gene and sequences (Palumbi et al., 2002)

Primer Sequence Direction

COl-f 5' -CCTGCAGGAGGAGA YCC- 3' Forward

COl-e 5'-CCAGAGATTAGAGGAATCAGTG-3 ' Reverse

3.3.4 Purification of peR Product

The purification of PCR was carried out using PROM EGA Purification Kit following the

protocols provided by the manufacturer. Separation and purification of the desired PCR

products is necessary before sequencing is possible (Natalia et at., 2007), in order to avoid

contaminants such as reagents and primer dimer in the PCR product. Purification process

involved three parts, which are removal of the protein, RNA and fragmented DNA. Then,

the purified products were sent to a private company (First Base Laboratories Sdn. Bhd)

with the 25 JlI of forward primer or COI-f(Table 3.3).

15