LP 10 FST 6 2002 Abstract

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Pefpusfakaon
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1100024579
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1100024579
DNA
using
fingerprinting of marble goby, Oxyeleotris marrnoratus
RAPD-PCR technique / Leong Lok Ping.
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DNA FINGERPRINTING OF MA
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GOBY,
marmoratus
USING RAPD-PCR
TECHNIQUE
By
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PU
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LEONG LOK PING
This
in
submitted
is
report
the requirements for the
proj ect
Bachelor of Educational
Faculty
KOLE]
fulfilment of
partial
Degree of
Science (Biology)
of Science and
Technology
VERSITI SAINS DAN TEKNOLOGI
VERSITI PUT
AYSIA
A YSIA
2002
00024579
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"
This report should be cited as:
Leong, L.P. 2002. DNA Fingerprinting of Marble
"
-
Goby, Oxyeleotris marmoratus Using
Technique. Undergraduate thesis, Bachelor of Educational Science
(Biology), Faculty of Science and Technology, Kolej Universiti Sains Dan Teknologi
Malaysia, Universiti Putra Malaysia, Terengganu. 54p.
D-PCR
or
this
be
mechanical,
reproduced by any
of
project may
photographic,
electronic process, or in the form ofphonographic recording, nor may it be stored in
No part
retrieval system, transmitted, or otherwise copiedfor public or private use, without
written permission from the author and the supervisor ofthe project.
a
OWLEDGEMENTS
I would like to express my
deepest gratitude
to my
supervisor,
Miss Noraznawati Ismail.
Thank you for your caring, supportive and patience supervision.
Thank you for all your contribution and advices throughout this project.
I
grateful to meet Andi, Kak Wan and Awie,
for they are always standing by to offer their valuable guidance
Thanks for sharing your knowledge with me.
I'll remember what you all taught me.
am
To the
Dean, Prof. Azmi,
thank you for
kindly provided me
the fish
samples.
Johny,
helping
up with
samples collection.
HI
thank you for
RA
H
To
and support.
and Kak Ani.
TA
GI
DI
AN
I would like to thank my family­
Dad, Mom and little sister
LS
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TA
NA
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NU
R
ZA
My sincere appreciation to Kak Faridah, Azrnan
Without their patience and consideration,
I won't have been finished with this project.
EL
AJ
AR
for their Love, Courage and Support.
That's the strength that cheers me up
this
study.
SA
T
PE
MB
throughout
PU
To my beloved friends and 382 housemates
know who you are),
thank you for being with me,
(you
sharing all the good and bad times together.
Your loyalty made me stronger to face challenges.
Though it's really frustrating sometimes,
BUT,
we
all make it
through finally!
might be neglected their names
make this project possible.
Thanks to all that I
that
helped
to
•
•
11
APPROVAL AND CERTIFICATION FO
I
certify
that the report of this fmal year
Goby, Oxyeleotris
marmoratus
Using
project
entitled 'DNA
RAPD-PCR
Fingerprinting
Technique' by
of Marble
LEONG LOK PING,
•
532 have been read and all the alteration and correction recommended
metnc no.
Exarniners have been done. This thesis submitted to
Department
have been
for
Science
accepted
as
in
(Biology)
Faculty
requirement
of Science and
Universiti Putra
Biological Science,
degree of Bachelor Educational
Technology, Kolej
Universiti Sains Dan
Malaysia.
H
Miss Noraznawati Ismail
LS
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NA
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Supervisor
Faculty of Science and Technology
Kolej Universiti Sains Dan Teknologi Malaysia
Universiti Putra Malaysia Terengganu
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Date:...
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Teknologi Malaysia,
fulfilment of the
of
by
Madya Dr. Sayed Zain S. Hassan
Head of Department Of Biological Science
Faculty of Science and Technology
Kolej Universiti Sains Dan Teknologi Malaysia
Universiti Putra Malaysia Terengganu
Prof.
•••
III
ABST
Marble
fish
important
species
DNA based
on
study owing
to its
study
from the
goby (Oxyeleofris marmoratusi
of the
effectiveness that
to establish the DNA
were
Gobiodei. Random
large suborder,
Chain Reaction
Polymerase
applied
Eleotridae is
family
to a
fingerprinting
commercially
Amplified Polymorphic
D-PCR) technique
(
a
chosen in this
was
wide range of organisms. The aim of this
of O. marmoratus, to
the
verify
,
genetic
\
and to
variability
analyze
the
Genomic DNA of 20
species.
Phenol-Chloroform Protocol
of
degree
samples
polymorphism
extracted from the muscular tissues
was
The
technique.
DNA level marker of this
on
of DNA
purity
using
from 1.301-
samples ranged
1.807, quantitatively estimated from the ratio between the reading of absorbence
from
270ng/JlL
685ng/JlL.
RA
H
HI
band formed
ethidium bromide.
A total of 19
primers
Quantity
of DNA also
0.80/0
on a
of DNA
(w/v)
samples ranged
with 60-700/0 GC content
were
tested
from each DNA
sample separately.
The PCR
stained with
0.5Jlg/mL
TA
then
LS
amplify fragments
in 1.70/0
GI
were
to
electrophoresed
(w/v)
agarose
gel
OPA07, OPA09 and OPA20
AJ
primers
-
were
chosen for further
primers generated
a
total of 28 loci
PE
The
(fragments)
with 91.70/0
polymorphic
loci
The
was
similarity
0.400
±
marmoratus.
RAPD
banding ranged
PU
(25 fragments) and the
SA
T
study.
MB
EL
ethidium bromide. Three
AR
AN
products
ability
DI
for their
UL
TA
-
0.5Jlg/mL
ZA
stained with
single
R
gel
the appearance of
through
purity
NU
agarose
observed
in UV -Spectrophotometer. The
H
qualitatively
(OD26oI0D28o)
NA
260nm and 280nm
at
index among individuals
0.167.
550bp
Further studies
needs of conservation and
and
on
this
0.442
was
400bp
were
species
propagation
from 1-10
of O.
±
fragments
0.123 whereas
found to be
with
350-1750bp.
genetic distance value
diagnostic
markers of O.
should be carried out in order to fulfill the
marmoratus
•
IV
.
,.
,
....
,
.
-
.
.,
ABSTRAK
Ikan Ketutu
ikan
(Oxyeleotris marmoratus)
dalam
suborder
Pemilihan teknik
Amplifikasi
Rantai Polimerase
telah
Gobiodei
yang berasal dari famili Eleotridae adalah
nilai
mempunyai
yang
spesis
kepentingan pcrdagangan.
Rawak Polimorfik DNA yang berdasarkan Tindak Balas
(RAPD-PCR) dalarn kajian ini disebabkan oleh keberkesanannya
ditunjukkan pada banyak organisma. Tujuan kajian
ini adalah untuk
yang
r
memaparkan
\
pencapjarian
DNA ikan
ketutu, O. marmoratus, memeriksa kepelbagaian genetik
ini. DNA
menganalisa darjah polimorfik pad a tahap penanda DNA spesis
20 contoh ikan diekstrak dari tisu otot
ikan
didapati
berada dalam
julat
1.301-1.807. Ia
penganggaran nisbah antara bacaan penyerapan
kuantitatif
260nm dan 280nm
pad a
RA
H
diperhatikan
melalui
kewujudan jalur-jalur
agaros yang telah diwamakan
(w/v) gel
dengan
(OD26010D28o)
HI
0.80/0
secara
dengan
UL
LS
GI
secara
DI
AR
60-700/0
scbanyak
(w/v) gel
diuji
berasingan.
untuk kebolehan
Basil peR dilarikan
agaros yang diwamakan
PE
MB
etidium bromida. OPA07, OPA09 dan OPA20 telah
dipilih
untuk
dengan
kajian
T
0.5�glmL
1.7%
pada
julat 270ngl�L-685ng/�L.
EL
melalui proses elektroforesis
GC
DNA ikan
mengamplifikasikan fragmen
AJ
mereka
primer dengan kandungan
TA
Sembilan belas
TA
NA
H
etidium bromida. Kuantiti DNA ikan berada dalarn
AN
0.5�glmL
NU
R
pada
diperolehi
ZA
yang terbentuk
tunggal
dalam
teknik Fenol-Kloroform. Ketulenan DNA
dengan
dalam UV -Spektrofotometer. Ketulenan DNA
genomik
serta
PU
SA
seterusnya. Ketiga-tiga primer menghasilkan jumlah 28 loci dengan 91.70/0 (25 fragmen)
daripada
IO
loci-loci tersebut adalah
polimorfik. Jalur-jalur RAPD didapati berjulat
fragmen dengan 350-1750bp. Penunjuk
adalah 0.442
Diagnostik
±
pembiakan
O.
(Similarity Index)
0.123, manakala nilai Jarak Genetik adalah 0.400
untuk O. marmoratus yang
terhadap spesis
Kesamaan
ini
perlu dijalankan
diperolehi
adalah
550bp
untuk memenuhi
marmoratus.
v
dan
±
antara I­
antara individu
0.167. Penanda
400bp. Kajian lanjut
keperluan pemuliharaan
dan