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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
------.
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
j
· - -- ......... 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