Jilid 15 No. 1 / ISSN 1394-5750 Jun 2009

Jilid 15 No. 1 / ISSN 1394-5750
Jun 2009
Content
Message from the Editor
€€ Executive Council 2007/2009
2
€€ Molecular markers for Eurycoma longifolia
and Orthosiphon stamineus
4
€€ Potential refugia and post-glacial
recolonization routes of Neobalanocarpus
heimii (Dipterocarpaceae) in Peninsular
Malaysia inferred by chloroplast DNA
9
€€ GENETIC RELATIONSHIPS AMONG MALAYSIAN
BANANA CULTIVARS REVEALED BY CHLOROPLAST
SSR
10
€€ Genetic Diversity of Purebred Boer Goats of
South African and Australian Origin
10
€€ GENETIC POLYMORPHISMS OF GLUTATHIONE
S-TRANSFERASE PI, M1 AND T1 GENOTYPES IN
MALAYSIAN POPULATION AND INFLUENCE ON
SUSCEPTIBILITY TO COLORECTAL CANCER: A
PRILIMINARY REPORT
11
€€ G6PD MUTATIONS SCREENING IN MALAYSIA ORANG
ASLI
14
€€ THE IDENTIFICATION OF HUMAN MISMATCH REPAIR
GENE, MLH1 -93G>A VARIANT IN MALAYSIAN
HEREDITARY NONPOLYPOSIS COLORECTAL CANCER
SYNDROME PATIENTS: A PRELIMINARY REPORT
14
€€ MOLECULAR CLONING OF FULL-LENGTH
HAEMAGGLUTININ (H5HA) GENE OF HIGHLY
PATHOGENIC AVIAN INFLUENZA VIRUS (HPAI) H5N1 15
€€ SCREENING ROSELLE (HIBISCUS SABDARIFFA L.)
ACCESSIONS FOR HIGH HYDROXYCITRIC ACID (HCA)
CONTENTS USING HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
16
€€ ISOLATION AND IN SILICO CHARACTERIZATION OF
CINNAMOYL-CoA REDUCTASE (CCR) FULL LENGTH
GENE IN ACACIA HYBRID
16
€€ GENETIC RELATEDNESS AMONG CYPRINIDS
INFERRED BY CYTOCHROME b
wo important events were held by the society
this year. The first was the society’s AGM which
was held on 19th March 2009 at Universiti Putra
Malaysia, Serdang. A new president was elected to the
committee which largely remains the same, with the
exception of minor changes in the EXCO members
line-up.
The second important event was the 8th Malaysia
Genetic Congress which was held at Awana Resort
Genting Highlands from 4th to 6th August 2009. This
year’s congress attracted a record number of 242
participants. Fifteen keynote papers, 41 oral papers and
94 posters were presented during the meeting.
A message from our new president, Professor Mohamad
Osman is a key feature of this issue. Professor Mohamad
is no stranger to us; he has been a member of the society
for more than 10 years and has served the society in
various capacities. This issue also features a report on
the 8th Genetic Congress (including photographs) and
the abstract of the winners of the free paper sessions.
Zilfalil Bin Alwi, MBBS, PhD
Editor-in Chief
17
€€ DIFFERENTIAL DISTRIBUTION OF MICROSATELLITE
ALLELE FREQUENCIES IN KOOMPASSIA
MALACCENSIS OF CONTRASTING HABITAT
PREFERENCES 18
€€ ISOLATION AND IDENTIFICATION OF
MICROSATELLITE FROM EPINEPHELUS
FUSCOGUTTATUS (TIGER GROUPER) AS BROODSTOCK
SELECTION
18
€€ GENOME-WIDE DETECTION OF DIFFERENTIALLY
METHYLATED DNA IN CHILDHOOD ACUTE
LYMPHOBLASTIC LEU KEMIA
19
PGM EXECUTIVE COUNCIL LINE-UP
President
Prof Mohamad Osman
Vice President
Assoc Prof Maude Elvira Phipps
HON-Secretary
Dr Abd Rahman Milan
€€ HISTOLOGICAL ANALYSIS OF Jag2 MUTANT MICE
REVEALED THE FORMATION OF SECONDARY CLEFT
PALATE; SUGGESTING THE IMPORTANCE OF Jag2
DURING PALATOGENESIS
20
ASSISTANT Treasurer
Dr Norshariza Nordin
€€ PRELIMINARY STUDY OF GAINS AND LOSSES IN
PROSTATE CANCER CASES BY ARRAY COMPARATIVE
GENOMIC HYBRIDIZATION.
20
HON-Treasurer
Assoc Prof Jothi Malar Panandam
€€ Isolation of CONSTANS- like gene from teak
(Tectona grandis)
21
EXCO Members
Prof Wickneswari Ratnam
Assoc Prof Zilfalil Alwi
Assoc Prof Rozita Rosli
Assoc Prof Zarina Abdul Latiff
Dr Norwati Muhammad
Dr Halimi Mohd Saud
Dr Mohd Rafii Yusop
€€ MONOCHLOROACETIC ACID BIODEGRADATION BY
LOCALLY ISOLATED PRESUMPTIVE BACILLUS SP
TW1
21
€€ TRANSFORMATION OF LETTUCE WITH A CHITINASE
GENE, DERIVED FROM RICE (ORYZA SATIVA) TO
CONTROL BOTRYTIS CINEREA PER FR.
22
Editorial Board
Printer
Assoc Prof Zilfalil Bin Alwi (Editor-in-Chief)
Prof Tan Soon Guan
PST Enterprise Sdn. Bhd.
33, Jalan 9/2, Taman IKS, Seksyen 9,
43650 Bandar Baru Bangi,
Selangor Darul Ehsan.
Bulletin Frequency and Distribution
Bulletin PGM is issued half yearly. It is circulated free to all
members. If you wish to be included in the mailing list, please
write to:
The Honorary Secretary,
Dr. Abd. Rahman Milan
Pusat Penyelidikan Hortikultur, MARDI,
Peti Surat 12301, Pejabat Besar Pos,
50774 Kuala Lumpur.
E-mail :armilan@mardi.gov.my
The view and opinions in all the acticles are entirely those
of the authors unless otherwise specified
Short articles, services, news, etc. on genetics and allied
disciplines are welcome. Comments on published articles in this
bulletin are also welcome.
GENETIK June 2009 page 3
SHORT COMMUNICATION
Molecular markers for Eurycoma longifolia and Orthosiphon
stamineus
K. F. Rodrigues1 and H. K. Tam
Biotechnology Research Institute
Universiti Malaysia Sabah
Locked Bag 2073
Kota Kinabalu 88999 Sabah Malaysia
E-mail: kennethr@ums.edu.my
1
ABSTRACT
This paper describes the first reported attempt to isolate DNA sequences containing repeat motifs in Eurycoma longifolia
and Orthosiphon stamineus. A library enriched for genomic repeat motifs was developed using novel oligonucleotides designed
with inosine residues incorporated at predetermined positions. A total of eight and twelve specific molecular markers were
developed for O. stamineus and E. longifolia respectively. These markers have a potential application in estimating population
diversity levels and QTL mapping in these two medicinal plants, which are widely used in the Malaysian herbal industry.
Keywords: Eurycoma longifolia, Orthosiphon stamineus, inosine, microsatellite
INTRODUCTION
The Malaysian herbal industry utilizes extracts derived from and as active ingredients in a range of medicinal products. The
first reported population genetic diversity study (Osman ., 2003) utilized SNPs to characterize the population genetic structure of
varieties in Malaysia. In the case of , no prior genomic characterization has been reported in published literature. The objective of
this study was to isolate sequences containing repeat motifs and to design specific primers pairs which could be utilized as potential
molecular markers in population genetic studies and varietal identification of these two species. Direct PCR amplification of genomic
DNA, using oligonucleotides containing degenerate bases, for the isolation of repeat units, has been reported earlier (Fisher ., 1996),
however the inclusion of degenerate bases can result in a tendency to self-anneal resulting in a significant increase in the amount of
PCR dimer formation and the generation of non-specific PCR products. The incorporation of inosine residues at specific positions in an
oligonucleotide has been reported to improve specificity (Fujiwara ., 1995). The primers utilized for the isolation of repeat units were
designed as follows: the 5’ end consisted of the motifs ‘GIVRI’, the sixth position comprised the repeat unit being targeted followed
by a single Inosine residue, a series of repeat motifs were included from positions 9 to 24 and a single inosine residue was included
at the 3’ end in order to facilitate the extension by DNA polymerases in case of the mismatch of a single base pair. For instance, the
primer BRION3, [5’-GIVRI(CT)I(CT)7(CT)I-3’] was designed to isolate sequences containing (CT) repeat motifs. Similarly, an array
of oligonucleotides was designed to isolate DNA sequences containing (ATG), (GA), (GT) and (AC) repeat motifs. The purpose of
incorporating a single ‘G’ residue at the extreme 5’ end of the oligonucleotides was to ensure 5’ stability.
MATERIALS AND METHODS
Fully expanded leaf samples were obtained from one
specimen of (Accession number: EL128B) and one specimen of
(Accession number: OS142). DNA was extracted according to
the method described by Doyle and Doyle (1987) with a minor
modification in the extraction buffer, which was supplemented
with 2% LMW Polyethylene glycol (PEG) (Sigma). Amplicons
containing repeat units were isolated using primers incorporating
inosine Three degenerate primers with a range of repeat motifs,
BRION3
[5’-GIVRI(CT)I(CT)7(CT)I-3’],
BRION7
[5’GIVRI(ATG)I(ATG)5(ATG)I-3’], BRION6 [ 5’- GIVRI(AC)
I(AC)8(AC)I- 3’ ], BRION1 [ 5’- GIVRI(GT)I(GT)8(GT)I- 3’ ]
and BRION9[ 5’- GIVRI(GA)I(GA)8(GA)I- 3’ ] (where V = G /
C / A, R = G / A and I = inosine ) were used in PCR amplification
to isolate a range of repeat motifs. PCR amplification was carried
out in a total volume of 20 µL containing 50 ng of template DNA,
2.5 mM MgCl2,1X PCR buffer containing 10 mM Tris-HCl (pH =
8.0), 50 mM KCl, 1 U Taq DNA polymerase (Qiagen), 10 pmol of
degenerate primer (BRION3, BRION7, BRION6, BRION1 AND
BRION9 ), 0.2 mM of dATP, dGTP, dCTP and dTTP. Amplification
GENETIK June 2009 page 4
was carried out in a thermocycler (MJ Research thermal cycler)
with an initial denaturation at 96 oC for 5 minutes followed by
30 cycles of 30 s at 94 oC, 40 s at 58 oC, 90 s at 72 oC and a final
extension step of 10 min at 72 oC. The amplification products were
separated by electrophoresis on a 2% TBE Agarose gel with 100
bp and 1 Kb DNA ladders (Promega) as size standards. The gel
was stained with Ethidium Bromide (5 µg / ml) and visualized
using an ALPHAIMAGER 2000 (Alpha Innotech Corp. USA ).
PCR products containing distinct amplicons with size in excess
of 300 bp were purified using a PCR purification kit (Qiagen)
and 2 µL of the purified PCR products were blunted with 1.0 U
T4 DNA Polymerase (Fermentas) ligated onto a pJET1.2 blunt
cloning vector and transformed into chemically competent TOP
10 cells according to the instructions from the manufacturer
(Fermentas). A total of 50 recombinant clones derived from
each of the two species were selected randomly from LuriaBertani plates containing Ampicillin (100 mg / liter). Plasmids
were extracted and purified using the alkaline lysis miniprep
protocol (Sambrook 1989). Plasmids containing inserts were
identified by PCR amplification of each of the purified plasmids.
PCR amplification was carried out in a total volume of 20 µL
containing 50 ng of template plasmid DNA, 2.0 mM MgCl2, 1X
PCR buffer containing 10 mM Tris-HCl (pH = 8.0), 50 mM KCl,
0.5 U Taq DNA polymerase (Qiagen), 10 pmol of specific primers
pJET1.2F (5’-CGACTCACTATAGGGAGAGCGGC-3’) and
pJET1.2R (5’-AAGAACATCGATTTTCCATGGCAG-3’), 0.2
mM of dATP, dGTP, dCTP and dTTP. Amplification was carried
out in a thermocycler (MJ Research thermal cycler) with an initial
denaturation at 96 oC for 5 minutes followed by 30 cycles of 30 s
at 94 oC, 30 s at 58 oC, 60 s at 72 oC and a final extension step of 10
min at 72 oC. PCR products were electrophoretically resolved on
a 2% TBE Agarose gel. Plasmids with an insert size in excess of
300 bp were sequenced with the primers pJET1.2F and pJET1.2R
using the BigDye Terminator v3.0 Cycle Sequencing Ready
Reaction Kit (Applied Biosystems) on an ABI Prism 377 DNA
sequencer. All sequences containing repeat motifs were deposited
at the GenBank (NCBI). Similarity searches were done using the
NCBI blastn algorithm (Stephen ., 1997). Specific primer pairs
were designed to flank repeat motifs using the online software
PRIMER 3 ( Rozen and Skaletsky 2000). PCR amplification
of a specific locus was carried out in a total volume of 20 µL
containing 50 ng of template DNA, 2.0 mM MgCl2,1X PCR
buffer containing 10 mM Tris-HCl (pH = 8.0), 50 mM KCl, 0.5 U
Taq DNA polymerase (Qiagen), 10 pmol each of a specific primer
pair, 0.2 mM of dATP, dGTP, dCTP and dTTP. Amplification
was carried out in a thermocycler (MJ Research thermal cycler)
with an initial denaturation at 96 oC for 5 minutes followed by
30 cycles of 30 s at 94 oC, 30 s at 58 oC, 30 s at 72 oC and a final
extension step of 5 min at 72 oC. The amplification products were
separated by electrophoresis on a 10% Poly Acrylamide Gel with
a 25 bp DNA ladder (Promega) as a size standard. The gel was
stained with Ethidium Bromide (5 µg / ml) and visualized using
an ALPHAIMAGER 2000.
RESULTS AND DISCUSSION
The DNA extraction procedure was modified by including
2% LMW Poly ethylene glycol (Sigma) in the extraction buffer,
this was done in order to sequester phenolic components associated
with DNA, which have been demonstrated to be PCR inhibitors
(Pandey ., 2002). The specificities of the primers and their abilities
to target the designated repeat motifs were demonstrated by the
sequencing results, which indicated that all the 5’ terminal ends
consisted of sequences containing the repeat motifs being targeted.
The degenerate primers have a potential for application in the
isolation of repeat motifs in organisms whose genomes have not
been characterized. A total of 11 sequences from and 9 sequences
from were deposited at the NCBI GenBank. Similarity searches of
the NCBI GenBank with the blastn algorithm indicated that three
of the sequences derived from exhibited significant similarities
to sequences available in the GenBank. EU009928 had a high
similarity (E = 2.6e-23) to chromosome 8 (AP010798), EU009930
was similar (E = 0.64) to sequence AM445465, EU009935 was
similar (E = 1.1e-35) to sequence AM449306. In the case of ,
sequence EU036683 had a high similarity (E = 5e-33) with sequence
AM486505, EU0036676 demonstrated a high similarity (E = 7.3
e-44) with sequence AM426783, The 3’ terminal end of sequence
EU036674 was similar to the gene encoding Aminoimidazole
ribonucleotide carboxylase in (AAR06291) implying that the 5’
terminal end is located upstream of the gene coding region. These
comparisons provide the first evidence of genomic similarity
of and with plants whose genomes are in the process of being
characterized. A total of 12 and 8 specific molecular markers were
designed for single locus amplifications. The primer designs and
expected sizes of the PCR products are shown in Tables 1 and 2.
The molecular markers developed can be applied to characterize
the population genetic diversity of naturally occurring as well
as cultivated varieties of these two important medicinal plants.
Another potential application is in DNA fingerprinting and QTL
mapping of varieties with desirable traits.
REFERENCES
Doyle, J. J., Doyle, J. L. 1987. A rapid DNA isolation procedure
for small quantities of fresh leaf material. Phytochem Bull
19: 11–15
Fisher PJ, Gardener RC, Richardson TE .1996. Single locus
microsatellites isolated using 5’-anchored PCR. Nucleic
Acids Research24: 4369-437
Fujiwara, H., K. Fujiwara, and K. Hashimoto. 1995. PCR with
Deoxyinosine containing primers using DNA polymerases
with proofreading activity. PCR Methods and Applications
4:239-40.
Osman, A., Jordan, B., Lessard, P., Muhamad, N., Haron,
M.R.,Riffin, N.M., Sinskey, A. J., Rha, C. and Housman, E.
2003. Genetic Diversity of Inferred from Single Nucleotide
Polymorphisms. Plant Physiology. 131. 1294–1301
Pandey, R. N., Adams, R. P. and Flournoy, L. E. 1996. Inhibition
of random amplified polymorphic DNAs (RAPDs) by plant
polysaccharides. Plant Molecular Biology Reporter. 1: 17 22
Rozen, S. and Skaletsky, H. J. 2000. PRIMER3 on the WWW
for general users and for biologist programmers. In: (eds
Krawetz S, Misener S), pp. 365-386. Humana Press, Totowa,
New Jersey.
Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schäffer,
Jinghui Zhang, Zheng Zhang, Webb Miller, and David J.
Lipman . 1997. “Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs”. Nucleic
Acids Research. 25:3389-3402.
Sambrook, J., Fritsch, E. F., Maniatis, T. 1989. Cold Spring Harbor
Laboratory PressCold Spring Harbor. New York.
GENETIK June 2009 page 5
Table 1: Sequences isolated from Eurycoma longifolia indicating clone, repeat motif(s), specific primer pairs, expected size of
PCR amplicon and GenBank Accession number.
Sr.
No.
Clone
1
EL11B
2
EL04B
Repeat
motif(s)
targeted
(CT)20
(CT)17
3
EL182B
(TA)5
(CT)15
4
EL181B
(CT)18
5
EL14B
(TG)10
6
EL03B
(TTCA)4
(CT)15
7
EL01B
(GA)15
8
EL05B
(AG)4
(GT)10
9
EL06B
(CT)18
10
EL08B
(CT)15
11
EL10B
(CT)15
12
EL09B
(GT)11
Primers
5'- 3'
F: GTAGCACCTCTCTCTCTCTCTCTCT
R: AATGGCCAGAAAAGATGGTT
F: GGGGACCTCTCTCTCTCTCTCT
R: TACGGGGGTTAGTTCCAACA
F: GGCGGAGCTCTCTCTCTCTCT
R: TTAGAAGGTAAAATGGGGAAAAA
F: GGGGAACCTCTCTCTCTCTCT
R: CAAGGAATCCAGAAGGGAAAA
F: GGAAGAATGTGTGTGTGTGTGT
R:GCATCAGCATCATCAGGAGA
F: GCGCGCCTCTCTCTCTCTCT
R:TTTGTTGTTGGACGATTGGA
F: TGGAGTGTACAGTGCGATTTTC
R: GAGGGGCTCTCTCTCTCTCTCT
F: GCGCGATGTGTGTGTGTGT
R: CAGCATCAGCTCTTCCCATA
F: GGGGAAGCTCTCTCTCTCTCTCT
R:AACCTGATACCTGCGTGACTC
F: GAAGAACTCTCTCTCTCTCTCTCTCTC
R: CAATGGCCAGAAAAGATGGT
F: GGGACACCTCTCTCTCTCTCTCT
R: TGCAGAAGCAATAAGCTTGG
F: GGGGTGTGTGTGTGTGTGTGT
R: GATGAATCGCCCTTCCTACA
Expected size of
PCR amplicon
(bp)
GenBank
Accession
Number
203
EU036683
221
EU036682
188
EU036681
233
EU036680
216
EU036678
226
EU036679
226
EU036677
212
EU036676
211
EU036675
242
EU036673
210
EU036674
221
EU036684
Table 2: Sequences isolated from Orthosiphon stamineus indicating clone, repeat motif(s), specific primer pairs, expected size of
PCR amplicon and GenBank Accesion number.
Repeat
motif(s)
targeted
Sr.
No.
Clone
1
OS056
2
OS040
(AC)10
3
OS036
(ATG)9
4
OS038
(AG)15
5
OS0035
(AC)10
6
OS037
(AC)10
7
OS004
(CAT)9
8
OS001
(CT)10
(CA)10
GENETIK June 2009 page 6
Primers
5'- 3'
F: GCAAGGGCACACACACACA
R: GTTGGAGGAAGACAGGGTGA
F: GAAGGAACACACACACACACA
R: GGTGTGGCAATCTTGGTTTT
F: GGTGGCCATGATGATGATGAT
R: GCTTAGCAACCTTTTAAACTGGA
F: GCTCAGCTGGCCATTTTTAT
R: GGGAAAGCTCTCTCTCTCTCTCTC
F: GTGGCGAACACACACACACA
R: AAGAGGAAGAGCAGGAGCGTA
F: GGGACAGACACACACACACACA
R: TCAAGTTTGATGCATGGTTTG
F: CATGAGGTTGACCTTGTGGTA
R: GTTGGGAATGATGATGATGATGA
F: GGGGCTCTCTCTCTCTCTCTCT
R: ATCAGCCATTGGGAAATCAG
Expected size of
PCR amplicon
(bp)
GenBank
Accession
Number
212
EU009934
205
EU009932
204
EU009931
150
EU009930
173
EU009929
240
EU009928
174
EU009927
169
EU009935
8th MALAYSIAN GENETIC CONGRESS
The 8th Malaysian Genetics Congress was held at the Awana Resort, Genting Highlands, Pahang, from 4th to 6th August 2009.
The theme of the Congress was “Role of Genetics in Wealth Creation and Quality of Life”. Various issues were discussed
during the congress; from food supplies to health care, energy and breeding. We strongly believe that genetics will continue
to contribute towards scientific and economic development, both locally and globally.
The congress was supported by Universiti Kebangsaan Malaysia, Universiti Putra Malaysia and the Ministry of Science,
Technology and Innovation, Malaysia (MOSTI). The Honorable Datuk Dr. Maximus Johnity Ongkili, the Minister of
Science, Technology and Innovation, graciously officiated at the Opening Ceremony.
The First National Congress on Genetics was held 15 years ago, and since then, there has been considerable and remarkable
progress in many areas of genetics. The Congress was held over two and a half days and the papers presented covered
medical and human, plant, animal, microbial and molecular genetics, biodiversity, biosafety and policy issues.
This Congress attracted 242 participants and received 15 keynote papers, 41 oral papers and 94 poster papers. At this
congress, the 8th Mendel Lecture was delivered by Datin Paduka Prof. Dr. Khatijah Mohd. Yusoff, Deputy Secretary General
(Science) of MOSTI. Others keynote speakers included Emeritus Professor Dato’ Dr. Zakri A. Hamid, the Tuanku Chancellor
Chair of the Centre for Policy Research and International Studies at Universiti Sains Malaysia, and Prof. Michael Patton,
Head of the Department of Medical Genetics at St. George Medical School, London.
We would like to thank all speakers, chairpersons, members of the panel discussion and forum, exhibitors, sponsors,
participants and individuals for their valuable contributions and support to make this Congress a success.
Prepared by:
Dr Abdul Rahman Milan
Prof Mohamad Osman
GENETIK June 2009 page 7
PGM SEMINAR
&
th
14 PGM Annual General Meeting
19 March 2009
Bilik Pertanian, Fakulti Pertanian, UPM, Serdang
The 14th PGM Annual General Meeting (AGM) was held at Bilik Pertanian, Fakulti Pertanian, UPM, Serdang
on 19th March 2009. The AGM was preceded by a half day seminar. Four lectures were delivered during the
seminar which was attended by more than 50 members and non members of PGM.
The following lectures were delivered:
Lecture 1: Rice biotechnology using molecular markers (Tentative)
by Dr. Leocadio Sebastian
Regional Director, Bioversity International-Malaysia, Serdang
Lecture 2: Genetics in Malaysia: Bridging the Present and the Future in Medicine by Assoc. Prof. Dr.
Zilfalil Alwi
Director, Human Genome Center, School of Medical Sciences
Universiti Sains Malaysia, Kubang Kerian
Lecture 3: Determinants of the Bacterial Diversity in Soil
by Dr. Delita Zul
FMIPA, Universitas Riau, Pekanbaru, Indonesia
Lecture 4: Induced Breeding,Triploidy Induction and Sperm Cryopreservation
of Catfish
by Assoc. Prof. Dr. Anuar Hassan
Deputy Director, Institute of Tropical Aquaculture (AKUATROP)
Universiti Malaysia Terengganu
During the AGM, the following new EXCO members were elected:
President: Prof Mohamad Osman
Vice President: Assoc Prof Maude Elvira Phipps
Hon-Secretary: Dr Abd Rahman Milan
Assistant Secretary: Dr Norshariza Nordin
Hon-Treasurer: Assoc Prof Jothi Malar Panandam
EXCO members:
Prof Wickneswari Ratnam
Assoc Prof Zilfalil Alwi
Assoc Prof Rozita Rosli
Assoc Prof Zarina Abdul Latiff
Dr Norwati Muhammad
Dr Halimi Mohd Saud
Dr Mohd Rafii Yusop
Auditors:
Dr Ho Boon Peng & Dr Abhimanyu Veerakumara-Sivam
The meeting ended at 230pm.
GENETIK June 2009 page 8
WINNERS OF THE FREE PAPER
ORAL PRESENTATION COMPETITION
Theme A: GENETIC RESOURCES AND BREEDING
1st Place
Potential refugia and post-glacial recolonization routes
of Neobalanocarpus heimii (Dipterocarpaceae) in Peninsular
Malaysia inferred by chloroplast DNA
Lee Hong Tnah#, Soon Leong Lee#, 1, Kevin Kit Siong Ng*, Subha Bhassu†, Rofina Yasmin
Othman†
Forest Research Institute Malaysia, 52109 Kepong, Selangor Darul Ehsan, Malaysia
†
Universiti Malaya, 50603 Kuala Lumpur, Malaysia
1
Corresponding author: leesl@frim.gov.my
#
Abstract
During Pleistocene climatic oscillations, the tropical rain forests are thought to have contracted into a few glacial refugia. In
Peninsular Malaysia, the extents of rain forest refugia and recolonization events have never been fully investigated. In the present
study, the parameters of genetic diversity and geographical distribution of chloroplast DNA (cpDNA) haplotypes in 32 populations
of Neobalanocarpus heimii were used to infer the evolutionary history of the species, including the potential refugia sites and their
post-glacial recolonization routes. Fifteen haplotypes were defined from ten substitutions of the five noncoding cpDNA regions: trnL
intron, trnS-trnG spacer, trnG intron, trnK intron and psbK-trnS spacer. The geographical distribution of haplotypes elucidated two
major genealogical cpDNA lineages of N. heimii: a widespread central-southern lineage and a northern lineage. Based on the haplotype
network and the pattern of genetic diversity, three potential refugia sites were inferred: the Perak and Kedah region (R1), the northern
region (R2), and the eastern and southeastern region (R3). Recolonization of refugia R1 and R2 could have first expanded into the
northern region and migrated both northeastward and northwestwardly during the interglacial retreat, and stopped at the central of
Peninsular Malaysia. At the meantime, refugia R3 could have commenced throughout the central-southern region and followed with
postglacial invasion to the western region. In short, N. heimii is predicted to survive in the multiple refugia throughout Peninsular
Malaysia during Pleistocence oscillations.
2nd Place
GENETIC RELATIONSHIPS AMONG MALAYSIAN BANANA CULTIVARS
REVEALED BY CHLOROPLAST SSR
Tee Meng Han and Siti Khalijah Daud*
Department of Biology, Faculty of Science,
Universiti Putra Malaysia 43400 UPM Serdang, Selangor, Malaysia.
*Email: sitikd@science.upm.edu.my
Abstract
Bananas and plantains have been highly esteemed and widely used by man since ancient times. Fiber contents in bananas and
plantains act as an aid for digestion. There are also reports that ripe bananas are used in the treatment of asthma and bronchitis.
In breeding programs, the breeds valued characteristics should be maintained during its further improvement. A Chloroplast Simple
Sequence Repeat (SSR) analysis was carried out to determine variation and relationships among local banana cultivars. Eight banana
cultivars were used in this study, namely Mas (AA), Berangan (AAA), Raja (AAB), Rastali (AAB), Awak (ABB), Nipah (BBB), Kapas
(AA) and Nangka (AAB). Twenty five individuals of each cultivar were collected from Perak, Selangor, Johor, Pahang Kedah, Kelantan
and Negeri Sembilan. Sixteen Microsatellite primer pairs were selected to amplify the chloroplast DNA. The highest genetic distance
was found between Pisang Nipah and Pisang Raja (0.0654) whilst the lowest was between Pisang Berangan and Pisang Awak (0.0036).
The dendrogram showed that Pisang Raja and Pisang Rastali were grouped together and they are of similar genotypes. However, Pisang
Awak and Pisang Berangan have the lowest genetic distance and grouped together although their genotypes are different. The results of
this study suggest that the clustering of these bananas does not necessarily follow their ploidy levels.
Keywords: Banana, Musa, RAMP, microsatellite, genetic relationships
GENETIK June 2009 page 9
3rd Place
Genetic Diversity of Purebred Boer Goats of South African
and Australian Origin
Hamidah Ali Kamarulzaman,1 Jothi Malar Panandam*1,2 and Siti Khalijah Daud3
1
Institute of Tropical Agriculture, 2Department of Animal Science, Faculty of Agriculture
3
Department of Biology, Faculty of Science,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
*Email: jothi@agri.upm.edu.my
Abstract
Boer goats which originate from South Africa are popularly raised as meat goats throughout the world. They are being continuous
imported into Malaysia from South Africa and Australia in large numbers. However, no information is available on the breed’s genetic
characteristics or genetic variability. This information is vital to plan breeding programs which will ensure that the breed’s valued
characteristics are maintained during its further improvement. This project aimed to evaluate the genetic variation within and between
purebred Boer goats of South African and Australian origin. Random samples of female purebred Boer goats from South Africa (SA) and
Australia (Aus) (n = 50 each) were evaluated using 15 microsatellite loci recommended by FAO. Percent polymorphism was 67% for
both the Boer goat types. There were no differences between the two populations for observed number of alleles and allele size ranges at
all loci. Effective number of alleles (ne) was generally the same, except for loci TGLA53, ILSTS087 and OARFCB48 which displayed
slightly higher ne for Aus population. Mean observed heterozygosity too differed between the populations (0.45 for SA vs. 0.52 for Aus).
The genetic distance based on UPGMA showed that the purebred South African and Australian Boer goats were closely related (d =
0.03). The results show low genetic variability in the Boer goats, which may be due to selection and mating practices in their respective
countries of origin. The Australian Boer goats, despite having originated from the South African population some generations before,
have not changed much genetically. However, they seem to have slightly greater genetic variability than the population of origin.
Keywords: Boer goat, microsatellite, genetic variability, heterozygosity
THEME B: HUMAN AND MEDICAL GENETICS
1st Place
GENETIC POLYMORPHISMS OF GLUTATHIONE S-TRANSFERASE PI, M1
AND T1 GENOTYPES IN MALAYSIAN POPULATION AND INFLUENCE ON
SUSCEPTIBILITY TO COLORECTAL CANCER: A PRILIMINARY REPORT
Siti Nurfatimah MS1, Abdullah M F 1, Zahri M K 1, Biswal BM 2, Bhavaraju VMK 2, Ankathil
R 1.
1
Human Genome Centre, 2Department of Nuclear Medicine, Radiotherapy & Oncology, School of Medical Sciences, Universiti Sains
Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
Abstract
It is increasingly evident that genetic variance is a major determinant of differential risk for many diseases including colorectal
cancer (CRC). Interindividual variation in response to xenobiotics and their potential carcinogenic effects is mediated by genetic
polymorphisms in the genes encoding enzymes involved in xenobiotic metabolic pathways. Glutathione S Transferases (GSTs) is a
group of phase II cellular detoxification enzymes. Three members of the GST family GSTM1, GSTT1 and GSTP1 have been analyzed
in different population groups for polymorphic variants, and to elucidate their role in carcinogenesis, but nil from Malaysian population.
So a case control study was undertaken to determine the polymorphic allele frequencies of GSTM1, GSTT1 and GSTP1 in 125 Malaysian
healthy controls and 31 CRC patients and to evaluate the influential role of variant genotypes in CRC susceptibility risk. Peripheral blood
from the study subjects were collected in EDTA tubes and genomic DNA extracted using QIAGEN kit. The ile105val polymorphism
was analyzed by PCR-RFLP technique using BsmA1 restriction enzyme. The presence or absence of GSTM1 and GSTT1, genes were
determined using a multiplex PCR protocol with albumin as the housekeeping gene. The resulting PCR fragments were separated on
2.0% agarose gel for GSTP1 and 3.0% agarose gel electrophoresis for the GSTT1 and GSTM1. In the case of CRC patients 29% showed
GSTM1 null genotype, 16.1% showed GSTT1 null genotype, 19.4% showed GSTP1ile/val genotype and 12.9% showed GSTP1 val/val
genotype. Among controls, the frequencies were GSTM1 42.4%, GSTT1 15.2%, GSTP1 ile/val 38.4% and GSTP1 val/val 4.8%. When
the genotypes were analyzed singly for CRC susceptibility risk, GSTT1 null and GSTP1 val/val genotypes were positively associated,
GENETIK June 2009 page 10
but statistically not significant. When different variant genotype combinations were analyzed, the GSTM1-/GSTT1-/GSTP1 ile/ile
combination genotype emerged as significant risk genotype associated with CRC susceptibility. The smaller sample size might have
impaired the ability to recognize other association with smaller effects and so the study is being continued with larger sample size to
identify other CRC predisposition genotypes
2nd Place
G6PD MUTATIONS SCREENING IN MALAYSIA ORANG ASLI
Farahnaz Amini1, Endom Ismail1 and Bin- Alwi Zilfalil2
The School of Bioscience and Biotechnology, Faculty of Science and Technology, The University of Kebangsaan Malaysia, 43600
Bangi, Selangor, Malaysia.
2
The Human Genome Centre, The University of Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
1
Abstract
This study aims to define the molecular basis of the glucose-6-phosphate dehydrogenase (G6PD) deficiency in Malaysian Orang
Asli. Negrito, one of the Peninsula Malaysia Orang Aslis, comprise of 6 sub-ethnic groups; Kensiu, Kintak, Lanoh, Jahai, Bateq and
Mendriq. The sub-ethnic group “Mendriq” was excluded from this study due to them have practised intermarriages for over a decade
ago (as a result of infertility). Four hundred and eighty seven consented Negrito volunteers were screened for G6PD deficiency and
45 (9.2%) individuals were found to be G6PD deficient. These individuals underwent PCR-RFLP analysis using thirteen recognized
G6PD mutations known as: Viangchan, Mediterranean, Mahidol, Canton, Gaohe, Coimbra, Andalus, Orissa, Union, Chatham, Kaiping,
Vanua Lava and African A-. All of these mutations were selected based on their scoring frequency of 30% or more among the three most
prevalent races in Malaysia, namely Malay, Chinese and Indian. The remaining were selected to represent the Javanese of Indonesia,
Thailand populations, Indigenous Negrito peoples on Nicobar Island, India and Senoi (another sub-tribe of the Malaysian Orang Asli).
The African A- mutation was selected after molecular mutational screening, which used the above mutations, failed to recognize any
one of them as a common mutation for this population. Of all these, three samples were found to carry Viangchan, one Coimbra and
three Coimbra-like mutations. Study results represent the first comprehensive molecular investigation into the Malaysian Orang Asli
in relation to G6PD mutations. The unique distribution of G6PD mutant variants in this population , however, only show a trace of
Malay-Indochinese similarity as demonstrated by the presence of Viangchan and Coimbra. Thus, more novel mutations are expected to
represent this population.
Keywords: G6PD; mutation; Negrito; Orang Asli
3rd Place
THE IDENTIFICATION OF HUMAN MISMATCH REPAIR GENE, MLH1 -93G>A
VARIANT IN MALAYSIAN HEREDITARY NONPOLYPOSIS COLORECTAL
CANCER SYNDROME PATIENTS: A PRELIMINARY REPORT
Mohd Nizam Zahary1, Gurjeet Kaur2, Muhammad Radzi Abu Hassan3, Syed Hassan
Syed Abdul Aziz4, VMK Bhavaraju5, Biswa Mohan Biswal5, Lee Yeong Yeh6 , Ravindran
Ankathil1.
Human Genome Centre, 2Advanced Medical and Dental Institute, Universiti Sains Malaysia, 3Internal Medicine Department,
Hospital Alor Setar, 4Department of Surgery, 5Department of Nuclear Medicine, Radiotherapy & Oncology, 6Department of Medicine,
School of Medical Sciences, Universiti Sains Malaysia Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia.
Email: rankathil@hotmail.com
1
Abstract
Colorectal cancer is the most common gastrointestinal cancer in Malaysia and hereditary nonpolyposis colorectal cancer (HNPCC)
accounts for approximately 1-5% of all colorectal cancers. HNPCC also called as Lynch syndrome is characterized by an autosomal
dominant inheritance with multiple members affected in families, high gene penetrance (85-90%) and early onset of colorectal cancer
and extracolonic cancer such as endometrial cancer. Germline mutations in DNA mismatch repair (MMR) genes have been found to be
associated with susceptibility to HNPCC. Based on Revised Bethesda Guidelines, we identified twenty four HNPCC patients in which
multiple members are affected with HNPCC, and also with early age of disease onset. This study aims to screen the twenty four affected
HNPCC patients for germline mutations in all the 19 exons of MLH1 gene. Peripheral blood from HNPCC patients was collected and
GENETIK June 2009 page 11
VIP, Mendel Lecture Speaker
Dr Abd Rahman
Milan, Co-chairman
of the Organising
Committee
Prof Mohamad
Osman, President
of the Genetics
Society of Malaysia
and co-chairman
of the Organising
Committee
Y
O
S
&
YB Minister of Science Technology & Innovation Malaysia
The Organising Committee, 8th
YB Minister visiting one of the booths
The 8th Malaysia Genetics Cong
GENETIK June 2009 page 12
YB Dato’ Maximus
Ongkili, Minister of
Science Technology
& Innovation
h
The President presenting a token of
appreciation to the honourable Minister
The honourable Minister declaring the Congress open
Congress Dinner
Malaysia Genetics Congress
Congress Dinner; VVIP table
gress. Genting Highlands 2009
GENETIK June 2009 page 13
DNA was extracted using commercialized kit and amplified using appropriate primers. All the 19 exons of MLH1 gene were screened
by dHPLC. Mutation was confirmed by DNA sequencing. dHPLC screening revealed the heteroduplex peaks in fourteen patients.
The variant, -93G>A (rs1800734) was identified in the promoter region of MLH1 gene. To the best of our knowledge, this is the first
study reported on the DNA MMR gene mutation detection among Malaysian HNPCC patients. A study to investigate the frequency
and pattern of DNA MMR gene mutation in Malaysian HNPCC patients and establish a MMR gene mutation database of Malaysian
HNPCC kindreds is underway.
THEME C: GENOMICS AND BIOTECHNOLOGY
1st Place
MOLECULAR CLONING OF FULL-LENGTH HAEMAGGLUTININ (H5HA)
GENE OF HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS (HPAI) H5N1
1
1, 2
Mustapha Bala Abubakar , Aini Ideris
1
1, 2
, AbdulRahman Omar
1
and Mohd Hair Bejo
Biologics Laboratory, Faculty of Veterinary Medicine,
Institute Bioscience, Universiti Putra Malaysia UPM, 43400 Serdang Selangor DE, Malaysia.
e-mail:mustydvm@gmail
2
Abstract
Avian Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA
genome surrounded by a helical symmetry capsid. Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI)
such as H5 or H7 subtype are the most important pathogens for the poultry industry in recent times. The haemagglutinin protein and
neuraminidase, serves as the target for the immune response of the host. Due to recurrent genetic reassortments between avian and
human influenza viruses, global pandemics may emerge and the naive human immunity could not withstand pressure by the novel
hybrid virus. The emergence of genetic engineering technology provided the industry with new methods of manufacturing diagnostics
tools and vaccines. After extraction of RNA from the cell culture of strain influenza A/Chicken/Malaysia/2004(H5N1) of AIV, the viral
RNA was converted to cDNA by a specific primer. The cDNA was amplified by the polymerase chain reaction (PCR) and analyzed
by agarose gel electrophoresis. The intact PCR product of full length haemagglutinin gene was cloned in TOPOTM TA Cloning vector.
The full-length HA-encoding gene of H5N1 AIV was subcloned into a pPICZA vector. After successful ligation, the constructed
plasmid was transformed into E.coli.Top10, Plasmid DNA from transformed bacteria was extracted in white colony and positive clones
were confirmed by restriction digestion with Sacl and Not1 restriction enzymes, colony PCR screening and nucleotide sequencing.
Construction of a recombinant pPICZA/H5HA plasmid containing the full length haemagglutinin gene was achieved as a first step
towards the expression in Pichia pastoris. Keyword: Avian influenza virus, haemagglutinin gene (H5HA), pPICZA expression vector. 2nd Place
SCREENING ROSELLE (HIBISCUS SABDARIFFA L.) ACCESSIONS FOR HIGH
HYDROXYCITRIC ACID (HCA) CONTENTS USING HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY (HPLC)
Mohamad, O1., Mamot, S1., Ahmed Mahir, M2.,
Ahmad Bachtiar, B3., Halimaton Saadiah, O2., and Noor Baiti, A. A1. Ramadan, G1.
1
Universiti Kebangsaan Malaysia, Bangi
Universiti Sains Islam Malaysia, Nilai
3
Universiti Malaya, Kuala Lumpur
2
Abstract
Roselle (Hibiscus sabdariffa L.) from family Malvaceae is relatively a new crop in Malaysia. It was introduced into Malaysia in
early 1990s. The calyces of roselle is well-known for its content of vitamin C and anthocyanins and is mainly used to produce pro-health
drink. Recent findings have shown that roselle also contains relatively high contents of hydroxycitric acid (HCA). HCA is now been
widely used as a potent body weight controlling ingredient in many commercially available weight-loss supplemenents and aids. The
results of a local feeding trial has suggested that roselle plays an important role in the prevention of obesity. Roselle produces HCA,
and this added characteristic would promote roselle as a source of HCA. HCA is extracted from dried calyces in the form of potassium
GENETIK June 2009 page 14
hydroxycitrate, a from that is stable and biologically active. Samples from roselle accessions and their mutant lines were used to extract
HCA which were Acc. 3, Acc. 12, HS03100-29-2-1-17-15-1 (red calyx), HS03100-29-7-1-6-3-1 (white calyx), HS1250-18-18-1-11-1 (red calyx) and HS1250-1-18-1-1-1-1 (white calyx) while asam keping (Garcinia atroviridis) and Garcinia cambogia which were
known to contain HCA were used as control. Two extraction methods were employed to extract HCA which were (a) HCA extraction
in the form of potassium hydroxycitrate (according to Majeed et. al 2004) (b) modified HCA extraction method (using charcoal) to
produce potassium hydroxycitrate. Comparison of potassium hydroxycitrate contents were made between roselle accession and their
mutant lines. Apart from that, identification tests which include both physical and chemical characteristics were also done. HPLC
procedure was used to screen the presence of HCA in extracts obtained from both extraction methods. Although HPLC procedure was
sufficiently selective and accurate, not all results conclusively indicated the occurrence of high contents of HCA in the rinds of asam
keping and Garcinia cambogia and also in calyces of some roselle accessions and mutant lines. With some refinements, this procedure
could be used in screening for high contents of HCA in roselle.
3rd Place
ISOLATION AND IN SILICO CHARACTERIZATION OF CINNAMOYL-CoA
REDUCTASE (CCR) FULL LENGTH GENE IN ACACIA HYBRID
Sukganah A. 1 , Choong C.Y. 1 and Wickneswari R. 1*
School of Environmental and Natural Resource Sciences, Faculty of Science and Technology,
Universiti Kebangsaan Malaysia, 43600 Bangi,
Selangor Darul Ehsan, Malaysia
* Email: wicki@pkrisc.cc.um.my
1
Abstract
A. mangium X A. auriculiformis hybrid is highly potential as an important economic commodity especially for timber and wood
pulp. Lignin is an undesirable component in the conversion of wood into pulp and paper. Trees altered in their lignin profile, with reduced
amounts of lignin or with an increased extractable syringyl-rich composition, are desirable for pulping. Cinnamoyl-CoA reductase
(CCR) is one of the enzymes likely to regulate lignin content and composition of syringyl (S) and guaiacyl (G). In this study, full length
genomic sequence of CCR was isolated in the hybrid by using genome walking and PCR walking methods, and then characterized.
The size of the CCR full length genomic sequence was 3367 bp; containing 920 bp of promoter region, five exons and four introns.
The size of the CCR full length cDNA was 1305 bp, with an open reading frame of 960 bp. The polypeptide encoded by CCR was 319
amino acids with a predicted molecular mass of 35.1 kDa and a theoretical isoelectric point (pI) of 5.87. Adh_short domain motif I
(VTGASGAIGSWLVRLLLDRG) and motif II (YPIAK) were found in CCR of the Acacia hybrid through InterProScan and Motif Scan
analyses, indicating that this gene belongs to the short-chain dehydrogenase/reductase family. Currently, the CCR full length genomic
sequence is being used to study specific nucleotide polymorphisms (SNPs) present in this gene among Acacia individuals. The SNPs
could be further exploited through this candidate-gene-based Linkage Disequilibrium mapping for production of elite planting materials
with reduced lignin content and high S-lignin levels.
GENETIK June 2009 page 15
WINNERS OF THE FREE PAPER
POSTER PRESENTATION COMPETITION
Theme A: GENETIC RESOURCES AND BREEDING
1st Place
GENETIC RELATEDNESS AMONG CYPRINIDS INFERRED BY
CYTOCHROME b
Faezeh Yazdani Moghaddam1,4, Siti Khalijah Daud1, Siti Shapor Siraj3, Jothi Malar
Pandam4 and Mansour Aliabadian5
Department of Biology, Faculty of Science,
Department of Aquaculture, Faculty of Agriculture,
3
Department of Animal Science, Faculty of Agriculture,
Universiti Putra Malaysia 43400 UPM Serdang, Selangor, Malaysia.
4
Department of Biology, School of Science, Ferdowsi University of Mashhad, Iran.
5
Institute for Biodiversity and Ecosystem Dynamics and Zoological Museum, University of Amsterdam, Mauritskade 61, 1092 AD
Amsterdam, The Netherlands.
*Email: sitikd@science.upm.edu.my
1
2
ABSTRACT
Puntius is a genus belonging to the family Cyprinidae, the most widely distributed freshwater fish all over the world. There
are confusions in the taxonomic status of this genus whereby the classification of this genus is based solely on the morphological
characteristics. Thus, the present study highlighted the phylogenetic relationships among common cyprinids found in Peninsular
Malaysia. Fifty samples of Puntius and other genera were collected from different locations in Peninsular Malaysia, namely Sungai
Pahang, Sungai Perak, Banting (Selangor) and Langkawi. Two mitochondrial Cyt b primers, namely Cyto 1 and Cyto 2, were used in
this study. The average pairwise distance between all Puntius was 0.151 while the average value for cyprinids was 0.109. Based on
cytochrome b gene, all species of Puntius can reasonably be regarded as different species. The phylogenetic tree revealed that Puntius
is paraphyletic genus because different species of Puntius did not cluster to each other. Therefore, the revision of this genus is needed
in the near future.
Keywords: Cyprinids, Puntius, phylogenetic relationships, Cytochrome b, mitochondrial DNA
2nd Place
DIFFERENTIAL DISTRIBUTION OF MICROSATELLITE ALLELE
FREQUENCIES IN KOOMPASSIA MALACCENSIS OF CONTRASTING HABITAT
PREFERENCES
Chai-Ting Lee 1, Soon-Leong Lee 1, Faridah Qamaruz Zaman 2, Siti Shapor Siraj 3,
Kevin Kit-Siong Ng 1 and Norwati Muhammad 1
Genetic Laboratory,Forest Research Institute Malaysia (FRIM)
52109 Kepong, Selangor
2
Biodiversity Unit,Institute of Bioscience, Universiti Putra Malaysia
43400 UPM Serdang, Selangor, Malaysia
3
Department of Aquaculture,Faculty of Agriculture, Universiti Putra Malaysia
43400 UPM Serdang, Selangor, Malaysia
Email: leechait@frim.gov.my
1
ABSTRACT
Not many tropical plant species growing on dry soil can thrive in the peat swamp habitat, Koompassia malaccensis is among the
unique species. Koompassia malaccensis, or locally known as kempas, is one of the major commercial timber species in Southeast
Asia. It has been the interest of ecologists to know whether K. malaccensis found in the peat swamp and dry land are genetically
distinct or their existence in such contrasting habitats is merely due to phenotypic plasticity. Findings from a population genetic study
of K. malaccensis in 34 natural populations throughout Peninsular Malaysia revealed presence of ecotypic differentiation. A total of 20
GENETIK June 2009 page 16
microsatellite loci were analysed with an average of 29 samples per population. In the majority of the loci studied, a consistent trend
was observed in the distribution of allele frequencies among the populations, in that the peat-swamp (Kuala Langat Selatan and Pekan)
and non-peat-swamp populations shared different common alleles. In loci Kma057, Kma163 and Kma172a, the predominant alleles in
the peat-swamp populations were either absent or in low frequencies (≤ 0.115) in the non-peat-swamp populations and vice versa. The
significant divergence of allele frequencies at nuclear loci indicates that these two ecotypes are most likely of different evolutionarily
significant units (ESUs).
3rd Place
ISOLATION AND IDENTIFICATION OF MICROSATELLITE FROM
EPINEPHELUS FUSCOGUTTATUS (TIGER GROUPER) AS BROODSTOCK
SELECTION
1,2
Mohd Azinuddin Ahmad Mokhtar, 1,2Normah Mohd Noor and 1Syarul Nataqain
Baharum
Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Malaysia
Schools of Bioscience & Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, UKM Bangi,
Malaysia
1
2
ABSTRACT
Molecular DNA based marker has become important for selection of broodstock. Among various marker systems, microsatellite
is the favorable marker since it highly polymorphic, abundant throughout genome and inherited in a codominant manner. Application
of these markers in broodstock selection of Epinephelus fuscoguttatus (tiger grouper) was crucial since this species is well known as
high commercial value fishes. Besides, special ability of grouper that able to adapt to the habitat by changing its color make grouper
classification which are based on phenotype characteristic are inadequate. Isolation and identification of microsatellite sequence was
performed by enrichment method. Principle of streptavidin and biotin was employed to isolate loci with repetitive nucleotide which
later followed by cloning and sequencing. Sequences obtained were analyzed using Biology Workbench 3.2 and Microsatellite Repeat
Finder Software. A total of 205 DNA plasmids were sent for sequencing. Only 150 plasmid sequences were analyzed since 22 unaligned
between forward and reverse, 33 sequence was the same. From these 150 sequences, 252 microsatellite were identified in which 33 were
mono-, 146 were di-, 36 were tri-, 30 were tetra, 4 were penta, 3 were hexa- and 1 was heptanucleotide repeats. Out of these, 173 were
categories as perfect repeats, 53 as imperfect and 16 as compound repeats.
THEME B: HUMAN AND MEDICAL GENETICS
1st Place
GENOME-WIDE DETECTION OF DIFFERENTIALLY METHYLATED DNA IN
CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA
1
Noor Hamidah Hussin,1, Chee Wei Choo1, Maha Abdullah4, Hamidah Allias 2, Eni
Juraidah Abdul Rahman5, Mohd. Hishamshah5, Abdul Rahman Jamal3
Department of Pathology and 2Department of Paediatrics Faculty of Medicine UKM Medical Centre, 3UKM Molecular Biology
Institute, 4 Immunology Unit, Department of Pathology UPM and 5Institute of Paediatrics Kuala Lumpur.
Email: hamidah@ppukm.ukm.my
Abstract
Drug resistance is one of the major determinants in the treatment outcome of acute lymphoblastic leukaemia (ALL) patients.
Epigenetic aberration particularly DNA hypermethylation has been associated with the ‘non-sensitive effect’ of cancer cells towards
the chemotherapy. The aim of this study is to identify differentially hypermethylated genes in resistant ALL cases. DNA was
extracted from cryopreserved bone marrow specimens from five pre-B ALL cases in complete remission and five pre-B ALL cases
who were not in remission following induction therapy (resistant). Two normal peripheral blood DNA were used as control. Infinium
HumanMethylation27 BeadChip which consists of 27,000 CpG sites and encompasses more than 14,000 well-annotated human genes
was used to identify new genes for drug resistance in these cases. Our preliminary results showed that five genes were statistically
identified as differentially hypermethylated in the resistance group. These genes are involved in regulation of transcription, apoptosis,
growth rate, cell differentiation, drug resistance, cell biosynthesis, and ion transport. Methylation Specific Polymerase Chain Reaction
(MSPCR) was used to access the reliability of the methylation array result obtained. Further evaluation of the association between the
GENETIK June 2009 page 17
chemotherapeutic responses and the methylation status of these five genes in a larger cohort of ALL patients is ongoing. In conclusion,
we have identified eight hypermethylated genes that have the potential to be predictive biomarkers as well as the therapeutic targets in
ALL.
2nd Place
HISTOLOGICAL ANALYSIS OF Jag2 MUTANT MICE REVEALED THE
FORMATION OF SECONDARY CLEFT PALATE; SUGGESTING THE
IMPORTANCE OF Jag2 DURING PALATOGENESIS
Khairani Idah Mokhtar1,3, Suwanna Jitpukdeebodintra2, Michael Dixon3
School of Dental Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; 2Department
1
of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hatyai, Thailand; 3Faculty of Life Sciences,
University of Manchester, United Kingdom.
Emailr: khairani@kk.usm.my
ABSTRACT
Cleft palate; characterised by formation of a cleft at the roof of the mouth, is a common congenital anomalies observed in man.
During palatogenesis, the vertically orientated palatal shelves flanking the developing tongue subsequently elevates, adhered and fused
with each other to form an intact secondary palate. It involves multicellular processes governed by sequential activities of a variety of
genes. Disruptions that occur within these genes may lead to cleft palate. The Notch signalling pathway provides important signalling
mechanisms essential for cell fate specification and embryonic development in numerous organisms. Jagged2 (Jag2); one of the Notch
ligands, has been shown to be important during craniofacial development. Disruption of Jag2 results in craniofacial abnormalities in
the mutant mice. The aim of the study is to describe the phenotype of Jag2 mutant mice in order to compare the differences that occur
during palate development by examining the histological sections of Jag2 mutant and the wild-type littermates. Jag2 heterozygous
mice were time-mated and sacrificed at specific embryonic ages. The embryos were fixed, washed and dehydrated in graded ethanol series
and processed to wax. Serial 6 ?m sections were prepared and the sections were stained with Haematoxylin and Eosin and observed under
microscope. While normal formation of oral cavity is observed in the wild-type, fusion between the mandible and maxillary shelves
and adhesion between the palatal shelves to the lateral sides of the tongue were observed in E13.5 of the Jag2 null mice. Histological
analysis confirmed that glossopalatal fusion observed in E14.5 and E15.5 of Jag2 null mice leads to cleft palate; whereby due to this
aberrant fusion, the vertically directed palatal shelves were unable to elevate and fuse in the midline. The completely penetrant cleft
palate observed in Jag2-null mice suggested that this gene plays a highly specific role during palate morphogenesis.
3rd Place
PRELIMINARY STUDY OF GAINS AND LOSSES IN PROSTATE CANCER
CASES BY ARRAY COMPARATIVE GENOMIC HYBRIDIZATION.
Nenny NS, Siti-Aishah MA, Reena MZ, 1Zulkifli MZ, 2Zubaidah Z
Jabatan Patologi dan 1Surgeri, Pusat Perubatan Universiti Kebangsaan Malaysia (PPUKM) dan 2Institut Penyelidikan Perubatan
(IMR), Kuala Lumpur.
Abstract not submitted by the authors
GENETIK June 2009 page 18
THEME C: GENOMICS AND BIOTECHNOLOGY
1st Place
Isolation of CONSTANS- like gene from teak (Tectona grandis)
Norlia B., Norwati A., Mohd Rosli H., Norwati M. and Norihan M.S.*
Forest Biotechnology Division, Forest Research Institute Malaysia (FRIM),
52109 Kepong, Selangor, Malaysia
*Cell and Molecular Biology Department, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia (UPM),
43400 Serdang, Selangor, Malaysia
Abstract
Teak flower at the young age is one of the problems that affect the performance and management of teak plantation in Malaysia.
The flower develops at the young age of the tree lead to forking phenomenon and reduces vegetative growth. Previous research on teak
flowering genes has isolated Tectona grandis late elongated hypocotyls (Tg-LHY) gene from flowering tissues. LHY is one of genes
involved in circadian clock system of plant, which govern many process such as stomata opening, leaf movement and flowering time.
Tg-LHY gene has been observed to delay the flowering time of Arabidopsis, indicated the involvement of this gene in teak flowering
process. As LHY gene was also reported to involve in other process in plant, this gene could not be manipulated. Therefore, the genes
that integrate between the circadian clock system and flowering pathway such as CONSTANS, FT and GIGANTEA should be studied.
Screening of cDNA library through PCR approach has been applied to isolate the integrator genes. Degenerate primer were designed
and used to amplify the respective genes of interest. The amplified fragments were cloned and sequenced. The specific primers were
then designed based on the sequenced fragments and used to screen the cDNA library using PCR base method. DNA sequence analysis
carried out found that one clone was similar to CONSTANS of Arabidopsis. The finding indicates the involvement of CONSTANS gene
in flower development of teak.
2nd Place
MONOCHLOROACETIC ACID BIODEGRADATION BY LOCALLY ISOLATED
PRESUMPTIVE BACILLUS SP TW1
Nurul Asma Hasliza Zulkifly and 2Fahrul Huyop
1
Department of Biotechnology, Faculty of Agriculture and Biotechnology,
Universiti Darul Iman Malaysia, Kampus Kota,
20400 Kuala Terengganu, Terengganu-Malaysia
2
Department of Industrial Biotechnology, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia,
81310 Skudai, Johor-Malaysia
1
ABSTRACT
A total of twelve bacteria were isolated from various kinds of water samples from Terengganu. Liquid minimal media and solid
minimal media were used for enrichment and direct plating isolation methods, respectively. Bacteria strains were isolated based on their
ability to grow in liquid minimal media supplemented with monochloroacetic acid (MCA) 5 mM as sole carbon and energy source.
Bacterial cell cultures were grown in liquid minimal medium at initial pH 7.5, incubated at 30ºC on rotary shaker at 150 rpm. From 12
selected bacterial strains, only 8 cell cultures were able to degrade monochloroacetic acid. Degradation of monochloroacetic acid was
detected by measuring the amount of chloride ion released in the growth medium. The highest chloride ion released was detected in the
growth medium from strain TW1, isolated from tap water from Kuala Terengganu distribution water system. The strain was tentatively
identified as Bacillus sp. strain TW1 by basic biochemical test. Bacillus sp. strain TW1 degraded monochloroacetic acid best with
maximum chloride released of 0.104 µmolCl/mL. At 5 mM monochloroacetic acid was the best concentration to grow strain TW1 in
liquid minimal medium. Growth was not detected in liquid minimal media supplemented with monochloroacetic acid at concentration
of 30 mM possibly due to the toxicity of the chemical. The results of our current study demonstrated the potential use of Bacillus sp.
TW1 as suitable biological agent for biodegradation of MCA in contaminated drinking water distribution systems.
Keywords: Monochloroacetic acid, Bacillus sp., bacterial dehalogenation, biodegradation
GENETIK June 2009 page 19
3rd Place
TRANSFORMATION OF LETTUCE WITH A CHITINASE GENE, DERIVED
FROM RICE (ORYZA SATIVA) TO CONTROL BOTRYTIS CINEREA PER FR.
Laboh, Rozeita1, Rossall, Stephen2 and Davey, Mike Raymond2
Horticulture Research Centre, MARDI, Serdang, Selangor
Plant and Crop Sciences, University of Nottingham, Sutton Bonington Campus, Leicestershire
LE12 5RD England
Email : rozella@mardi.gov.my,
1
2
ABSTRACT
Grey mould (Botrytis cinerea Per Fr.) is a fungus that affects many plant species and causes severe yield losses world-wide,
including lettuce and many other species of commercial interest. This opportunist fungus has various modes of attack, diverse hosts and
inoculum sources, making it difficult to control. Due to this problem, growers rely heavily on fungicides to control the disease. However,
there are increasing pressures on growers to reduce pesticide inputs into horticultural products because of environmental concerns and
fungicides resistance in pathogens. Therefore, to provide a viable alternative strategy to the use of fungicides, we are developing a
transformation of plants with known antimicrobial gene which may indirectly enhance disease suppression. Lettuce (Lactuca sativa) cv.
Evola has been used as a model system. Transgenic lettuce was generated using Agrobacterium tumefaciens-mediated gene delivery. A
chitinase gene, derived from rice, was used. Transgenes were detected in transformed plants using PCR and RT-PCR analysis.
GENETIK June 2009 page 20
MOLECULAR CLONING OF FULL-LENGTH HAEMAGGLUTININ (H5HA)
GENE OF HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS (HPAI) H5N1. 1*
Mustapha Bala Abubakar , Aini Ideris
1
1, 2 **
2
, AbdulRahman Omar
1, 2
and Mohd Hair Bejo
1
Biologics Laboratory, Faculty of Veterinary Medicine, Institute Bioscience, Universiti Putra Malaysia UPM, 43400 Serdang
Selangor DE, Malaysia. *e-mail:mustydvm@gmail; **e-mail:aiini@admin.edu.my.
Abstract
Avian Influenza virus belongs to the Orthomyxoviridae family, there are enveloped viruses with segmented negative sense RNA
genome surrounded by a helical symmetry capsid. Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI) H5
or H7 subtype are the most economically important pathogens affecting the poultry industry in recent times. The haemagglutinin and
neuraminidase surface glycoprotein are the target for the host immune response. Due to recurrent genetic reassortments between avian
and human influenza viruses, global pandemics may emerge and naive human immunity could not withstand pressure by the novel hybrid
virus. The emergence of genetic engineering provides the new methods of manufacturing vaccines and diagnostics tools. In this study we
extracted viral RNA from the influenza A/Chicken/Malaysia/2004(H5N1) strain from cell culture, the viral RNA was converted to cDNA
by a specific primer. The cDNA was amplified by the polymerase chain reaction (PCR) and analyzed by agarose gel electrophoresis. The
intact PCR product of full length haemagglutinin gene was cloned in TOPOTM TA cloning vector. The full-length HA-encoding gene of
H5N1 AIV was subcloned into a pPICZA vector. After successful ligation, the constructed plasmid was transformed into E.coli.Top10’.
Plasmid DNA from transformed bacteria was extracted in white colony and positive clones were confirmed by restriction digestion
with Sacl and Not1 restriction enzymes, colony PCR screening and nucleotide sequencing. A recombinant pPICZA/H5HA plasmid
containing the full length haemagglutinin gene was constructed and sequence confirmed as a first step towards the expression in Pichia
pastoris. Keyword: Avian influenza virus, haemagglutinin gene (H5HA), pPICZA expression vector. Introduction
Influenza virus is an enveloped virus with segmented negative sense single stranded RNA genome under the family Orthomyxoviridae.
The viral genome is surrounded by a finger-like glycoprotein spike known as hemagglutinin and neuraminidase. The hemagglutinin
(HA) is the most abundant protein mediating the attachment to terminal sialic acid-containing receptors and fusion with endosomal
membrane(Carroll and Paulson 1985; Suarez and Schultz-Cherry 2000; Swayne and Suarez 2000). Three different types (A, B, &C) of
influenza viruses are classified on the basis of antigenic differences among their nucleocapsid (NP) and Matrix (M) proteins. Influenza
A viruses are further divided into subtypes based on their antigenic relationships of their hemagglutinin (HA) and neuraminidase (NA)
surface glycoproteins.
The hemagglutinin and neuraminidase genes are extremely variable in sequence, and less than 30% of their amino acids are
conserved among all the subtypes. A total of 16 different HA subtype (H1-H16) and 9 different NA subtypes (N1-N9) have been
identified. There is an expectation of more additional HA and NA subtypes to be recognized in the near future as more extensive
surveillance is being conducted on various poultry species (domestic and wild) in different geographical location (Apisarnthanarak,
Kitphati et al. 2004; Fouchier, Munster et al. 2005; Swayne and Halvorson 2008). The emergence of the first influenza pandemic of
the 21st H1N1 is an attestation to this fact. In this study, the HA gene of H5N1 subtype influenza A virus was amplified, cloned, and
sequenced as the most crucial stage in expression . This could also be a basic preparatory study for developing an effective diagnostic
kits and/or subunit vaccine.
Materials and Methods
Viruses and main reagents Influenza A/Chicken/5858/
Malaysia/2004 (H5N1) was kindly provided by Division of
Veterinary Service, DVS Ipoh Perak, Malaysia.
Total RNA extraction Total RNA was extracted using
Trizol LS reagent according to the manufacturer’s instructions.
The resultant pellets of RNA were washed once with 75% ethanol
and dissolved in RNase-free distilled water. All the RNA samples
were stored at -70°C until they were used as template in RT-PCR
RT-PCR was performed under the following conditions:
Synthesis of the first strand cDNA was carried out using reverse
transcription system (Promega, USA). The reaction mixture
contained in a final concentration of 1x of reaction buffer, 5mMof
MgCl2, 0.8μM of each primer (H5HAF & H5HAR), 0.1Mm
of dNTP, 2U of AMV reverse transcriptase, 5U of recombinant
RNasin ribonuclease inhibitor and 100ng of extracted RNA . The
mixture was initially incubated as follows:
Briefly , at 450C for 50min, Reverse transcriptase denature at
94°C for 5min, chilled at 4OC, the full denaturation at for 94°C
min, at 94°C for 1min, 1mins at 53°C, 2 min at 68°C for 40 circles
of amplification, and 10 min at 68°C for an additional extension.
DNA Sequencing The purified products obtained from
RT-PCR amplification of avian influenza virus subtype A/
Chicken/5858/Malaysian/H5N1 strain was sequence using primer
sets AIVH5F & AIVH5R respectively. The sequencing was carried
out using ABI PRISMR BigDye Terminator Cycle Sequencing
Ready Reaction Kit v2.0 (Perkin Elmer, USA) in an automated
DNA sequencer (ABI PRISMR 377 DNA Sequencer)
GENETIK June 2009 page 21
Sequencing Analysis of the one-step RT-PCR products
The 1707bp of the hemagglutinin (H5HA) gene sequences of avian
influenza virus A/Chicken/5858/Malaysian/2004(H5N1) strain
RT-PCR fragment were first identified using percentage homology
by Gene Bank using standard nucleotide BLAST Program of
National Institute of Biotechnology Information (NCBI) (http://
www.ncbi.nih.gov/Blast) under BLASTN 2.0.11 program.
the nucleotide sequence of H5N1of H5HA gene derived from the
recombinant plasmid pPICZA-H5HAand its deduced amino acid
sequence with those of H5HA of H5N1 strain, the nucleotide
sequence of the cloned H5HAgene was found to be similar
(99%homology) to that of A/Chicken/5858/Malaysian/H5N1
strain retrieved from Gene Bank AF038403, with 98% homology
of the deduced amino acids.
Construction of Plasmid DNA
TOPO Cloning Reaction: The RT-PCR products of the fulllength of avian influenza virus A/Chicken/5858/Malaysian/H5N1
strain H5HA generated was purified using GENEALLTM GEL SV
Kit (General Biosystem, Korea) and subsequently cloned into
pCRR2.1 TOPO vector from TOPO TA CloningR Kit (Invitrogen,
USA) according to manufacturer’s instructions.
Plasmid DNA Extraction and Purification Plasmid
extraction was carried out using GENEALLTM Plasmid SV Mini
Kit (General Biosystem, Korea) according to manufacturer’s
instructions. Concentration and purity of the extracted DNA were
determined by spectrophotometer (Beckman, USA),
Construction of the recombinant plasmid pPICZAH5HA The amplified products of the gene encoding H5HA
and pPICZA expression vector were digested separately with
restriction enzymes mixtures containing 2μl XhoI(10/ μl) and
1.5 μl NotI(10/ μl), 2.5 μl Orange buffer(10X) and dH20 to a
final volume of 25 μl per. The reaction were incubated at 37OC
overnight. The insert and the vector were ligated using DNA
T4 Ligase ligation system according to (Invitrogen USA). The
ligation products were transformed into Escherichia coli cells
(Invitrogen, USA) and plated on Luria broth plates containing 100
μg of ampicillin per ml. The recombinant plasmid was verified by
PCR and restriction enzyme digestion. The correct recombinant
plasmid was designated as pPICZA-H5HA.
Construction of expression vector The PCR product
was purified with a GENE ALL SV GEL purification kit and
digested with XhoI and Not I restriction sites in the expression
vector pPICZA (Invitrogen) to generate the recombinant plasmid
pPICZA-H5HA selected by Zeocin (Invitrogen). The recombinant
plasmid pPICZA-H5HA was generated and identified by restriction
enzymes digestion PCR colony screening and sequence analysis.
DNA sequencing The recombinant plasmid pPICZAH5HA was used to determine the sequence of H5N1 H5HA
protein gene by the dideoxy-chain-termination method [7] with
automated sequencer (ABI 377 system). The nucleotide of the
H5HA gene and its deduced amino acid sequence were compared
with the published sequences for the Influenza A virus subtype A/
Chicken/5858/Malaysian/H5N1 strain (Gene Bank)
Results and Discussion
Amplification, cloning and identification of A/Chicken/5858/
Malaysian/H5N1 full-length H5HA protein by using the RT-PCR
method described above, the full-length H5HA gene fragment about
1707bp in size was amplified, sequence and cloned into TOPO TA
cloning vector. Purified DNA clone of amplified full-length H5HA
gene fragment was inserted into the expression vector pPICZA to
generate a recombinant plasmid pPICZA-H5HA. After comparing
GENETIK June 2009 page 22
M
1
2
3
4
5
3200
1700bp
Figure 1Restriction endonuclease analysis of the recombinant
plasmid DNA extracted from PCR positive transformant colonies
(pPICZA-H5HA clones digested with XhoI and NotI). Lane M
(1kbmarker), Lane 1 (positive control), Lane 2 -5 (vector with H5
insert).
1700
Figure 2. Amplification product of full-length H5HA cDNA using
HAF and HAR primers. Lane M (1kbmarker) is a DNA marker
of Generuler Ladder mix(Fermentas), Lane 1 (positive control),
Lane 2 -5 (Amplified H5 gene ).
The 1707 bp gene encoding the full-length of H5HA A/
Chicken/5858/Malaysian/H5N1 (H5N1) virus was cloned into the
expression plasmid pPICZA to obtain pPICZA –H5HA (Fig. 1).
DNA sequence analysis showed an in-frame direct fusion of the
H5HA gene 569 encoded amino acids.
Appraisal of recombinant plasmid
Appraisal of recombinant plasmid pPICZA-H5HA: Upon
cutting the recombinant plasmid pPICZA-H5HA with XhoI I and
NotI restriction enzymes, as confirmation , there appeared two
strips of 3200 bp, 1707 bp for the parent vector and the gene
insert respectively (Fig.1).
Pichia pastoris is one of the few species of yeast expression
system that is able to metabolize methanol as it sole carbon
source. these systems employed fusion between the foreign gene
sequences and a strong methanol promoter which allows for the
metabolism of methanol (Gellissen 2000). Because of this strong
promoter, the presence of methanol will cause transcription and
translation of not only the gene encoding for the enzyme, but also
for the recombinant protein (Tschopp et al., 1987). H5HA gene
amplified by two-step RT-PCR was generated by proofreading
DNA polymerase. The H5HA gene was easily cloned into pCR2.0
TOPO TA cloning vector. This vector was designed to cloned PCR
fragment with low background of non-recombinants (Invitrogen
USA). Because a good quality and high concentration of the
insert (A/Chicken/5858/Malaysian/H5N1) was efficiently ligated
and 1.5% low melting point agarose gel was used to separate
the H5HA fragment from the excess of primers and original
template(Sambrook, Fritsch et al. 1989). Both the primers and
template can compete for the blunt end vector during ligation if
they were not removed. The formation of recombinant plasmid
from a very low amount(undetectable) of cDNA insert confirmed
further that the PCR-Blunt end vector is very efficient and highly
reliable as a cloning vector(Sambrook, Fritsch et al. 1989). In
addition, more than 90% of white colonies contained the insert
were demonstrated by digestion of the recombinant pCR-blunt.
This could be explained further by the fact that white colonies
were surviving bacteria that if they were not mutated, must
contained the insert since the insertion of the insert into pCR2.0
blunt inactivated the lethal gene ccdB allowing the transformed
E.coli Top 10 strain to grow. The amplified cloned H5HApCR2.0 recombinants plasmid were further digested with XhoI
and NotI restriction enzymes subsequently subcloning into the
corresponding sites of pPICZA expression vector in preparatory
for downstream expression studies. The constructed plasmid was
confirmed by PCR, restriction enzyme digestion and sequencing to
be in frame with the N-terminal of the pPICZA vector. However
it is easier and less costly than other method such as hybridization,
α-complementation, insertional inactivation. PCR and restriction
enzymes digestion is the method of choice for screening many
transformants (Sambrook, Fritsch et al. 1989). The H5HA gene
obtained can also be used as a marker for diagnostic probe after
Isolation from cloning vector by RE follow by its labeling with
radioisotope, enzyme or other detection systems. The use of this
probe will enhance the rapidity and the accuracy of detection
system.
CONCLUSION
DNA Cloning techniques have made invitro protein
production a reality; however, the advancement of biotechnology
in this field has been dependent upon the successful cloning and
sequencing of a putative gene prior to expression and subsequent
purification and clinical trials.
References:
Apisarnthanarak, A., R. Kitphati, et al. (2004). “Atypical avian
influenza (H5N1).” Emerging infectious diseases 10(7):
1321-1324.
Carroll, S. M. and J. C. Paulson (1985). “Differential infection of
receptor-modified host cells by receptor-specific influenza
viruses.” Virus research 3(2): 165-179.
Fouchier, R. A. M., V. Munster, et al. (2005). “Characterization
of a novel influenza A virus hemagglutinin subtype (H16)
obtained from black-headed gulls.” Journal of virology
79(5): 2814.
Gellissen, G. (2000). “Heterologous protein production in
methylotrophic yeasts.” Applied microbiology and
biotechnology 54(6): 741-750.
Sambrook, J., E. F. Fritsch, et al. (1989). Molecular cloning, Cold
Spring Harbor Laboratory Press Cold Spring Harbor, NY.
Suarez, D. L. and S. Schultz-Cherry (2000). “Immunology of avian
influenza virus: a review.” Developmental and Comparative
Immunology 24(2-3): 269-283.
Swayne, D. E. and D. A. Halvorson (2008). “Influenza.” Book
Chapter: 153-184.
Swayne, D. E. and D. L. Suarez (2000). “Highly pathogenic avian
influenza.” Revue scientifique et technique (International
Office of Epizootics) 19(2): 463.
GENETIK June 2009 page 23