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SCREENING FOR FLOWER-SPECIFIC eDNA SEQUENCES IN SAGO PALM (Metroxylon sagu) VIA

A DIFFERENTIAL DISPLAY TECHNIQUE

Bong Shiaw Kong

Master of Science (Molecular Biology)

2004

Pusat Khidmat MakJumat Akademik UNlVERSITI MALAYSIA SARAWAK


A DIFFERENTIAL DISPLAY TECHNIQUE PKHIOMAT MAKLUMAT AKAOEMIK

111111111 rlI~ii 111111111 1000246514

Bong Shiaw Kong

BSc (Hons) in Biotechnology

A thesis submitted in full fulfillment of the requirement for the degree of Masters of Science

Faculty ofResource Science and Technology UNIVERSITY MALAYSIA SARA W AK

2004

DECLARATION

No portion of the work referred to in this thesis has been submitted in support of an application for anoth~r degree or qualification to this or any other university or institution of

higher learning

Bong Shiaw Kong 730302-13-5109

ii

ACKNOWLEDGEMENT

I would like to express my gratitude and sincere appreciation to my supervisor Professor Dr Mohd Azib Salleh for his guidance and inspiration throughout this work and also his help in the preparation of this thesis

My apprecation also goes to Mr Amin Manggi in the Faculty of Resource Science and Technology University Malaysia Sarawak for his technical assistance To Dr Sim Soon Liang and Dr Hairul Azman thank you for your advice and support I would also like to thank the Ministry of Science Technology and Environment (MOSTE) for giving me the Postgraduate Fellowship to support my studies

To my wife Jane my parents brother and sisters thank you so much for your understanding

and support

III

Abstract

( Differential display is rapid and economical method compared to traditional

differential screening of cDNA libraries or construction of subtracted cDNA libraries for the

identification of differentially expressed genes This technique is used in the present study to

identify genes that are specifically expressed in flower tissues of sago)

Sago palm maturation period is a major obstacle in the development of this plant as a

commercial crop Previously it has been found that the flowering of sago palm is a direct

indicator for the maximum level of starch content in the trunk This study focuses on the

isolation of specific genes that can be expressed only in the flower tissue Characterization of

these genes can lead to the discovery of the regulatory gene for maturation in sago palm A

nonradioactive differential display technique which takes advantage of chemiluminescent

technology has been adopted for this study This adaptation has proven to be successful

compared to other nonradioactive techniques The results were more convincing and

reproduceable A random primer was used to amplify the cDNA generated from mRNA of

different tissues and the differentially expressed cDNA bands were displayed in the

lwniniscent detection film Two differentially expressed bands S 1 and S2 were selected from

the cDNA fingerprints The bands were then excised from the cDNA fingerprints and

reamplified using the same random primer These differentially expressed bands were then

analyzed by blot analysis to determine their specificity They showed positive results in all

tI

blotting experiment including cDNA blotting and Northern blotting The blotting experiments

also utilized the chemiluminescent detection method The S 1 and S2 bands were then cloned

into a pPCR-Script Amp SK(+) cloning vector before it was transformed into a E coli and

stored in glycerol stock for further analysis cDNA sequencing of the S 1 bands showed high

IV

Abstrak

I-

Teknik differential display merupakan satu teknik yang cepat ekonomik dalam

mengenal pasti gen yang diekspres secara berasingan Teknik ini telah digunakan dalam

kajian ini untuk mengenalpasti gen yang diekspres secara khusus dalam tisu bunga pokok

sagu

Tempoh pematangan sagu yang tidak serentak (antara 7-12 tahun) merupakan masalali

yang paling rumit untuk menanam tumbuhan ini secara besar-besaran Peringkat awal

pembungaan telah lama dikenali sebagai peringkat yang mana kandungan kanji berada pada

tahap maksimum dalam pokok ini Projek ini menwnpukan perhatian terhadap pengasingan

gen khusus yang hanya boleh diekspres dalam tisu bunga sagu Analisis sifat-sifat gen ini

boleh menyumbang kepada pemahaman yang lebih mendalam mengenai proses permatangan

sagu Dalam kajian ini teknik differential display yang menggWlakan bahan bukan radioaktif

telah digWlakan Sebaliknya teknik ini menggunakan bahan kimla berilurnioasi Bahan kimia

berlwninasi adalah lebih berkesan berbanding dengan bahan bukan radioaktif yang lain Satu

primer rawak yang berasaskan gen yang diekspres secara khusus dalam tisu bunga telah

digunakan untuk mengampliftkasikan mRNA dari tisu-tisu yang diambil dari pokok sagu

berbunga Dua jalur amplikon yang diperolehi (dilabel sebagai S 1 and S2) dari fingerprint

cDNA yang dihasilkan Jalur-jalur tersebut kemudiannya diampliftkasi lagi dan digunakan ~

untuk analisa hibridisasi untuk memastikan kekhususannya Keputusan positif dari hibridisasi

secara eDNA blotting dan northern blotting telah menunjukkan kekhususan kedua-dua jalur

terse but sebagai mewakili gen hanya diekspres dalm tisu bunga Jalur S I dan S2 tleh

diklonkan ke dalam vektor ppeR-Script Amp SK(+) sebelum ditransformasikan ke dalam

vi

E coli Transforrnan yang diperolehi disimpan dalam stok gliserol untuk kajian yang

seterusnya Jujukan nukleotid eDNA bagi jalur S 1 telah menunjukkan homologi yang tinggi

dengan gen O-methyltransferase (OMT) yang terdapat dalam tumbuhan almond (Prunus

amygdalus) Gen tersebut adalah khusus dalam tisu bunga tumbuhan tersebuy Gen OMT

adalah terlibat dengan penghasilan lignin lni mencadangkan bahawa process pembungaan

sagu melibatkan penghasilan lignin Jujukan nukleotid jalur S2 menunjukkan homologi

dengan suatu gen yang khusus dalam bunga dalam Arabidopsis thaliana dan Zea mays bull

Fungsi gen terse but masih belum diketahui Berdasarkan keputusan analisis jujukan

nukleotida boleh disimpulkan bahawa jalur S 1 and S2 menwakili eDNA yang mengandungi

kod gen yang khusus bagi tisu bunga Kedua-dua jalur tersebut adal~ berguna untuk

dijadikan prob untuk mengenalpastfmiddot gen sebenar dalam genom pokok sagu yang mengkodkan

enzim spesifik yang terti bat dalam process pennatangan atau pembungaan

vii

Pusat Kllidraat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK

TABLE OF CONTENT

CONTENTS PAGE

TITLE PAGE DECLARATION ii ACKNOWLEDGEMENT iii ABSTRACT iv ABSTRAK vi TABLE OF CONTENT viii LIST OF TABLE xi LIST OF FIGURE xii ABBREVIAnONS USED xiii

10 LITERATURE REVIEW _

11 Introduction 1 12 Starch storage in sago and its yield at different stages of growth- 4 13 The flowering process in sago 6 14 The isolation of flower specific genes- 8

141 RAP-PCR and differential display of mRNA 9 142 RAP-PCR technique -- 9 143 Strengths and limitations of inRNA differential display and

RAP-PCR technique- 10 middot - 144 The comparison between conventional differential dispby and

RAP-PCR 14 145 Other improvement on mRNA differential display techniqueshy

----------------------------- 14 146 Ways to overcome some problems associated with

mRNA fmgerprinting- 16 147 The application of differential display and RAP-PCR--- --17 148 Identification of tissue specific genes by using other screening

~ methods----- 19

1S Aims of this project--------------------------------------20

20 MATERIALS AND METHODS

21 Preparation of media reagents and enzymes--------- -----22 22 Collection of plant materials---------------------- - - -- 22

viii

23 Extraction of total RNA------------------------------24 231 Treatment of glassware and plasticware for RNA extraction-24 232 RLIA iso la ti 0 n-------------------------------------------------- 24 233 Determination of concentration and purity of the total RNA 26 234 Gel electrophoresis------------------ 26 235 Elution of DNA from agarose gel- ----------- 27 236 Determination of the integrity of total RNA via gel

electrophoresis------------------------------------ ------ 28 24 Isolation of mRNA from total RNA 29 25 The RAP-PCR process of differential display 30

251 The synthesis of first strand cDNA from mRNA-- --------30 252 The Polymerase Chain Reaction (PCR) process --------31 253 Analysis of the RAP-PCR product 33

26 The chemiluminescent detection process ---- 33 261 Detection of DIG-IabeUed nucleic acid with chemiluminescentshy

-------- 34 27 Verification of differentially expressed bands 35

28 Hybridization----- -----36 281 Labelling of probes-- -------36 282 The prehybridization process 36 283 DNA dot blotting 37 284 cDNA blotting -------------- --- 38 285 Northern blotting - 39

29 Cloning of the differentially expressed bands 39 291 Descriptionof the cloning vector 39 292 The genotype of epicunan E coli XL-10gold ultracompetent

cells --- 40 293 Polishing of PCR product for blunt end ligation 40 294 The ligation process 41 295 The transformation process -- 41 296 Analysis of positive clonps---- ---- 42 297 Restriction enzyme analysis 44

210 DNA sequencing ---------------------------------- 44

30 REsULTS AND DISCUSSION

31 Isolation of total RNA ----------------------------------------------45 311 Qualitative analysis of total RNA -----------------------48

32 The quality and quantity of the mRNA---------middot~__48 33 Isolation of flower-specific genes cDNAs through differential displayshy

----------------------~------------------------------------51

34 Reamplification of the selected bands --------------------57

ix

35 Confirmation of the flower-specificity of the SI and S2 bands through hybridization ---------------------------------------------------63 351 The labelling of probe------------------------------------- 63 352 Dot blot hybridization ---------------------------------------------- 64 353 cDNA blotting--------------------- 66 354 The Northern blotting------------------- 66

36 Cloning of the SI and S2 bands -----------------------76 37 Verification of the insert------- --------------------------------- -- 71 38 DNA sequencing of the S1 and S2 cDNA ------------71

40 GENERAL DISCUSSIONS

41 The total RNA isolation 79 42 mRNA isolation process-- 80 43 The isolation of cDNAs containing coding sequences for flower-

specific genes through differential display ------------81 44 The RAP-PCR process 82 45 Hybridization 85 46 Nucleotide sequmiddotence of the flower-specific cDNAs -- ---- 86 47 General conclusion and future works 88

BmLIOGRAPHY------------------------~------------89

APPENDIX 1

APPENDIX II

x

LIST OF TABLES

Table No Title Page

Table 1 Nucleotide sequences of the arbitrary primers used for the 32 differential display

Table 2 RAP-peR conditions 32

Table 3 Spectrophotometer readings of total RNA isolated from different sago palm tissues 47

Table 4 The quality and quantity of the total RNA obtained from middot different sago 1alm tissues 47

Table 5 Spectrophotometric readings of mRNA preparation 50

Table 6 The quality and quantity of the mRNA yielded 50

Table 7 Recombinant plasmids carrying cDNA inserts either derived from band S 1 or-band S2 72

xi

LIST OF FIGURES

Figure No Title

Fig 1 The RAP-PCR technique

Fig 2 A diagrammatic comparison between RAP-PCR And conventional differential display

Fig 3 A flowering sago palm showing third order branching

Fig 4 Total RNA from leaf tissues

Fig S Differential display of all tissues using Cl primer showing the differentially expressed bands

Fig 6 Differential display of all the tissues using C 1 primer

Fig7a Differential display of all tissues using C2 primer

Fig7b Differential display of all tissues using C5 primer

Fig 8 Differential display of all the tissues using C3 primer

Fig 9 The reamplified S 1 and S2 bands

Fig 10 Dot blot of the differentially expressed bands

Fig 11 cDNA blotting using the same probe as in the dot blot

Fig 12 RNA hybridization

Fig 13 Electrophoresis of digested DNA of recombinant Plasmids pMASKSll pMASKSI2 pMASKS21 pMASKS22 pMASKS23

Fig 14 Nucleotide sequence of the cDNA derived from SI band

Fig IS Nucleotide sequence of the S2 band cDNA

Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin

Ammonium persurphate

Base pair

Bromo phenol blue

double-distilled water

Differential display reverse transcription

Diethyl pyrocarbonate

Digoxig~nin-ll-dUTP

Deoxyribonucleic acid

Deoxynucleotide

Ethylene diamine tetra acetate

Ethidiwn bromide

isopropylthio-j3-D-galactoside

Luria-Bertani me~ium

Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal

Moloney murine leukimia virus-reverse transcriptase

O-methyltransferase

Polyacrylamide gel electrophoresis

Polymerase chain reaction

Pound per square inch

polyvinyl-polypyroridon

Random arbitrary primer

Ribonucleic acid

Ribonuclease

rev01ution per minute

Streptavidin-paramagnetic particle

Sodium dodecyl sulfate

Tris-acetatelEDTA electrophoresis buffer

Tris-boriclEDTA electrophoresis buffer

Trist-EDTA

Tetramethy ethyl enediamine

Volt

volume over volume

Watt

5-bromo-4-chlorlt-3-indolyl-p-D-galactoside

xiv

10 LITERATURE REVIEW

11 Introduction

Metroxylon Sagu commonly known as sagu or mulung locally is one of the

earliest tropical starch plants grown by natives of South East Asia (Nakao 1985)

Bellwood (1985) noted that the sago palm was one of the most important cultivated

plants in the Indo-Malay Archipelago together with other crops such as yarn banana rice

etc Among the earliest record of sago plantation as a domestic crop was that described

in a Chinese geography text published in the late 13 th century (Takaya 1985) According

to Takaya the sago palm was grown extensively in the areas stretching from southern

Mindanao to Borneo Northern Suiawesi and the Maluk-u islands

In Sarawak sago palm has been grown for at least 400 years and is concentrated

mainly along the coastal belt and reverine areas especially in Mukah and Dalat in the

Mukah Division Matu-Daro in Binttilu Division and Kelaka and Saribas in Sri Arnan

division A detailed study on the distribution of sago palm in Sarawak has been carried

out previously (Tie et al 1989) In Sarawak sago cultivation is undertaken mainly by

smallholders and predominantly by the Melanau community as their main cash crop

(Anonymous 1986) It was the principal source of revenue during the Brooke reign in

bull the 19th century but at present it only contributes about 4 of the state revenue for

agriCUltural products (Anonymous 1998) The total acreage of sago in Sarawak is about

20000 ha and roughly 75 of these areas are located in the Mukah Igan and Oya-Dalat

Division The total annual production of sago S$ch from the state is about 55000

tonnes

The total acreage of sago plantations in South East Asia is about 375 million ha

with Indonesia claiming more then 75 followed by Papua New Guinea Malaysia and

Thailand with 102 million ha 50000ha and 3000ha respectively (Flach 1997)

Chemically sago starch is quite similar to that of com potato tapioca and wheat

starches Sago starch can be used to make biscuits bread cakes and as thickeners for

chili and tomato sauces Sago pellets and tebaloi are the two popular traditional food

made from sago flour Sago starch has also be utilized extensively in the manufacture of

high fructose syrup glucose monosodium glutamate alcohol baby foods gum candy

textile paper adhesive gum gelling agent and plastic Sago rasp is commonly used as

feed in the local pig industry The rrajor conswners of sago starch are from the Far East

such as China Japan and Korea These countries import sago starch for specialize food

and up-market food outlet There has been an increasing demand for sago starch recently

by the Japanese as it has specific properties for the manufacture of up-market products

which other starches lack (personal communication Mr J Takara [2001])

Despite recent advance in farming techniques and starch processing methods the

importance 9f sago palm as a cash crop has been decreasing This was due to several

factors Firstly the swampy natural habitat of sago palm makes it difficult to introduce

commercial plantation In addition the economic return of sago is low compared to other

crops such as pepper cocoa and oil palm However the most significant factor is the

long and non-uniform maturation period which makes harvesting difficult t~ manage if

the crop is grown in large-scale plantations

Nevertheless sago palm is said to rival the root crops as a major starch producing

crop (Flach 1973) The long duration (7-12 years) for starch accumulation to reach

maximum level is however a major disadvantage for sago palm compared to for

example 4-6 months for sweet potato Biotechnological techniques including molecular

genetics and tissue culture sago starch utilization and modification and the treatment of

waste and waste water from sago processing plants have been the topic of discussion

among researchers and major sago growers in the state Worldwide the Tsukuba Sago

Fund has been encouraging and supporting research and industrial application of sago

starch However there has been very little effort to investigate the underlying genetic and

biochemical mechanisms that control starch biosynthesis in sago

In Sarawak Sago is mainly grown under a semi-wild condition with minimal

maintenance There seems to be no definite planting spacing or pattern In older plots

this problem of spacing is further complicated by the fact that sago grows in clumps and

new suckers can creep along the ground before growing upwards at some distance from

the mother palm The ability to produce suckers also tends to vary Furthermore sago

palms have such a long maturing period that many growth st~ges can be encountered in a

single garden Thus the best planting material for sago palm will be young sucker that

were readily available in most older plots Seed gennination is not a popular choice

among small holders as it requires higher mairitenance and a well-planed cultivation

scheme The vast majority of the small holders in Sarawak still maintain the semi-wild

method of sago cultivation In the early 1990s the Sarawak Department of Agriculture

has established the Land Custody and Development Authority (LCDA) an agency tasked

3

with carrying out intensive research and development programmes for sago This has

resulted in the setting up of the first large-scale sago palm plantation in the Mukah

district

12 Starch storage in sago and its yield at different stages of growth

The biochemical pathways for starch biosynthesis in plants have been well

studied by Preiss (1988) and Okita (1992) As a predominant storage product for carbon

synthesized in the photosynthetic pathway starch is produced in the leaves of the sago

palm and then stored in the trunk However the starch content depends on the starch

density in the pith and the trunk size The starch density in the pith probably depends on

the irradiation captured by the leaves The trunk size increases quadratically with girth

The trunk height is mainly governed by light a trunk growing in the shade will try to

reach fun sunshine and thus use the limited amount of photosynthates produced in the

crown first for trunk elongation Short stout trunks of the saine palm type are thus

expected to contain more starch than tall slender trunks (Flach 1991)

Starch flour yield ofnonnal stands of sago palm varies The variation is a result of

several factors namely type of soil rain falls and most importantly stages of growth

(Flach 1971) Zwallo (1950) estimated the production of 120kg starch per palm while

Fairwhether (1937) reported the yield of crude flour varies with the size of the palm and

range between 1l4-295kg per palm Flach (1971) suggested 182kg flour could be

produced based on his researc~ at Batu Pahat Johor Whereas the sago palm in the

Singapore Botanical Garden can produce as much as 325Kg of starch (Johnson and

4

Pusat Khidmat Maklumat Akademik llNlVFISITI MALAYSIA SARAWAK

Raymond 1956) Wahby et aI (1970) estimated that the average yield of sago starch

flour in Sarawak as about 242kg per trunk Ahmad (1970) suggested another figure-

about 189kg from one matured trunk in commercially grown sago ill all these studies

time of harvesting has become the factor Sim and Ahamd (1978) had conducted an

experiment based on the stage of growth in sago palm The findings of their work showed

that at the early flowering stage (average age of about 11 years) the tree could give the

maximum yield in sago stai-ch ThllS the figure quoted previously was just an indication

of starch yield at different geographical locations and under different environmental

condition According to Sim et al (1978) in Sarawak it is a general belief that felling of

sago palm is best carried out after flowering but before the fruiting stage

Johnson and

Raymond (1965) claimed that the- maximum starch content occurs at the stage after

flowering Flach (1972) however reported that the sago trunks are best harvested during

the flower development stage (at the age of about 8-10 years) Sim and Ahmad (1978) in

their assessment agreed to this Sim and Ahmad also suggested that starch stored would

have been used for the fonnation of seed after the flowering process shy

These findings proved that the flowering stage is a vital indicator for us to identify

the maturation of the sago palm as this is the only physiological factor that can be

examined by plant breeders and plant cultivators Therefore the study of the flowering

process would provide us with a possible clue to what control the starch accumulation

and physiological development process

5

13 The flowering process in sago

The transition to flowering can be a remarkable change in the life of a plant In

many species such as in most perennials reproductive development occurs in certain

regions of the plant but vegetative growth of the plant continues The transition occurs

in shoot meristems which are reprogrammed to make inflorescence or floral organs

rather than vegetative organs on receiving appropriate environmental or development

signals From a developmental perspective therefore the floral transition is as much

about reprogramming the shoot meristems as it is about the actual production of

inflorescence flowers However it is not known whether the anatomical changes are the

cause or the results of changes in growth status of the meristem The floral transition

marks the beginning of reproductive development and in many plants such as sago

palm which bas a single bunch of flowers it also signifies the end of indeterminate

growth There are two distinct transition processes that can be distinguished genetically

Different types of inflorescence are formed in detenninate and indetenninate species

(Weberling 1989) In determinate species the inflorescence meristem forms terminal

flowers that end any fwther inflorescence growth In indetenninate species flowers are

fonned on lateral branches or inflorescence and not from terminal buds

The development of flowers is required for the alteration of the sporophytic to the

gametophytic generation the production or gametes for fertilization and seed

development These reproductive processes require the production of specialized organs

for the development of the gametophytes and to ensure fertilization The evocation

morphogenesis and function of these specialized organs is regulated through complex

6

mechanisms that have both genetic and environmental components (Greyson 1994) The

environmental component for example is the requirement that some plants have for a

specific photoperiod in order to initiate the flowering process But in the~case of sago

palm the environmental condition might only be the water content in the soil which has

yet to be studied and fully understood Other affecting factor might be their own

physiological changes which can include the starch content Hence the genetic

component of flowering is evident from the numerous mutations that identify genes

affecting flower morphology or function (Westhoff et al 1998) Although the majority

of the mutations are inherited as simple recessive traits and many of the mutations have

been thoroughly described morphologically and genetically (Howell 1998) the function

of the gene and the mechunism through which altered development occurs are not known

The determination of the molecular basis of such flotal mutation has been impeded by the

lack of a simple method for the isolation of the affected gene on the basis of phenotype

and mapping alone The morphogen~sis of flowers is associated with differential

expression of genes (Jordan 1993) The differentially expressed genes between

reproductive and vegetative organs are the basis of a strategy for the molecular analysis

of the genetic component of flowering The major difficulty in isolating genes involved

in the flowering process is that little is known of the identity of the proteins they encode

the tissues in which they are expressed or the time at which they are active during plant

development Methods of gene isolation which are closely based on knowledge of

genetics are therefore the most likely to be successful Thus the study of flowering

process should start with isolation of the regulatory gene of this physiological process

7

14 The isolation of flower specific gene

Prior to finding the regulatory gene and the biochemical pathway flower-specific

gene(s) must first be identified Tissue-specific gene(s) can be studied using many

approaches As they are differentially expressed the methods for identifying them can

be based on two different approaches - firstly differential screening of cDNA libraries

(St John and Davis 1979) and secondly the construction of subtracted cDNA libraries

(Sargent and David 1983) These approaches have been successfully applied in other

plants but they are rather laborious and time-conswning and require large amounts of

RNA Differential screening detects only abundant mRNAs while subtractive

hybridization is more sensitive but even more difficult to set up Finatly a major

limitation of both procedures is that only one pair of RNA between them can be

perfonned at any given time

A new method known as RNA tmgerprinting through random peR amplification

is a good alternative for studying tissue specific gene expression or any regulatory gene

expression The method is rapid and fingerprints of any tissue-specific RNA can be

easily produced This method offers numerous advantages over other methods mentioned

above including its simplicity and its ability to compare the fluctuations in gene

expression between multiple samples simultaneously using only nanograms amounts of

RNA In addition it can also yield information on the overall patterns of gene expression

between different cell types or between different physiological conditions of the same

cell type (McClelland et ai 1995)

8

141 RAP-peR and differential display of mRNA

RNA finger printing or differential display was first introduced by Liang and

Pardee (1992) It is a technique used for analyzing broad-scale gene expression patterns

and subsequently for the isolation and cloning of gene sequences with desired expression

characteristics The technique relies upon the use of RNA arbitrary primers or any

random primer and the polymerase chain reaction (peR) and it is similar to the more

established techniques such as randomly amplified polymorphic DNA (RAPD) analysis

of genomic DNA conceptually Informative patterns or fingerprints of the reamplified

products can be produced even when no previous information is available concerning

primer binding sites or expected products The fmgerprints provide the basis for

selecting and ultimately isolating differentially expressed genes and have even been

suggested as a means for identifying and classifying different RNA sources (Liang et al

1993)

142 RAP-peR technique

Figure 1 depicts the overall concept of the RAP-peR technique During first

strand synthesis a single 18-base arbitrary primer anneals and extends from sites

contained within the messenger RNA (mRNA) 1bis is where RAP-peR differs from

conventional differential display of mRNA where an oligodeoxythymidine primer

oligo(dT) is anchored at the 3tenninus by one or two specified bases Second-strand

synthesis proceeds in a similar manner during a single round of low-stringency peR

peR amplification at high stringency proceeds by virtue of having incorporated the

arbitrary primer into both ends of the peR to amplify the cDNA A template-dependent

9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r

First-strand s~ntnesis

~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC

Second-strand synthesis ~-

ACCT ACC ------------- GCT GCA

peR amplification my

Figure 1 The RAP-peR technique (Buchner 1994)

10

Pusat Khidmat MakJumat Akademik UNlVERSITI MALAYSIA SARAWAK


A DIFFERENTIAL DISPLAY TECHNIQUE PKHIOMAT MAKLUMAT AKAOEMIK

111111111 rlI~ii 111111111 1000246514

Bong Shiaw Kong

BSc (Hons) in Biotechnology

A thesis submitted in full fulfillment of the requirement for the degree of Masters of Science

Faculty ofResource Science and Technology UNIVERSITY MALAYSIA SARA W AK

2004

DECLARATION


higher learning


ii

ACKNOWLEDGEMENT




and support

III

Abstract



















tI





IV

Abstrak

I-




sagu


















vi













vii


TABLE OF CONTENT

CONTENTS PAGE









~ methods----- 19




viii









210 DNA sequencing ---------------------------------- 44




----------------------~------------------------------------51


ix






BmLIOGRAPHY------------------------~------------89

APPENDIX 1

APPENDIX II

x

LIST OF TABLES

Table No Title Page








xi

LIST OF FIGURES

Figure No Title

















Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10

DECLARATION


higher learning


ii

ACKNOWLEDGEMENT




and support

III

Abstract



















tI





IV

Abstrak

I-




sagu


















vi













vii


TABLE OF CONTENT

CONTENTS PAGE









~ methods----- 19




viii









210 DNA sequencing ---------------------------------- 44




----------------------~------------------------------------51


ix






BmLIOGRAPHY------------------------~------------89

APPENDIX 1

APPENDIX II

x

LIST OF TABLES

Table No Title Page








xi

LIST OF FIGURES

Figure No Title

















Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10

ACKNOWLEDGEMENT




and support

III

Abstract



















tI





IV

Abstrak

I-




sagu


















vi













vii


TABLE OF CONTENT

CONTENTS PAGE









~ methods----- 19




viii









210 DNA sequencing ---------------------------------- 44




----------------------~------------------------------------51


ix






BmLIOGRAPHY------------------------~------------89

APPENDIX 1

APPENDIX II

x

LIST OF TABLES

Table No Title Page








xi

LIST OF FIGURES

Figure No Title

















Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10

Abstract



















tI





IV

Abstrak

I-




sagu


















vi













vii


TABLE OF CONTENT

CONTENTS PAGE









~ methods----- 19




viii









210 DNA sequencing ---------------------------------- 44




----------------------~------------------------------------51


ix






BmLIOGRAPHY------------------------~------------89

APPENDIX 1

APPENDIX II

x

LIST OF TABLES

Table No Title Page








xi

LIST OF FIGURES

Figure No Title

















Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10

Abstrak

I-




sagu


















vi













vii


TABLE OF CONTENT

CONTENTS PAGE









~ methods----- 19




viii









210 DNA sequencing ---------------------------------- 44




----------------------~------------------------------------51


ix






BmLIOGRAPHY------------------------~------------89

APPENDIX 1

APPENDIX II

x

LIST OF TABLES

Table No Title Page








xi

LIST OF FIGURES

Figure No Title

















Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10













vii


TABLE OF CONTENT

CONTENTS PAGE









~ methods----- 19




viii









210 DNA sequencing ---------------------------------- 44




----------------------~------------------------------------51


ix






BmLIOGRAPHY------------------------~------------89

APPENDIX 1

APPENDIX II

x

LIST OF TABLES

Table No Title Page








xi

LIST OF FIGURES

Figure No Title

















Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10


TABLE OF CONTENT

CONTENTS PAGE









~ methods----- 19




viii









210 DNA sequencing ---------------------------------- 44




----------------------~------------------------------------51


ix






BmLIOGRAPHY------------------------~------------89

APPENDIX 1

APPENDIX II

x

LIST OF TABLES

Table No Title Page








xi

LIST OF FIGURES

Figure No Title

















Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10









210 DNA sequencing ---------------------------------- 44




----------------------~------------------------------------51


ix






BmLIOGRAPHY------------------------~------------89

APPENDIX 1

APPENDIX II

x

LIST OF TABLES

Table No Title Page








xi

LIST OF FIGURES

Figure No Title

















Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10






BmLIOGRAPHY------------------------~------------89

APPENDIX 1

APPENDIX II

x

LIST OF TABLES

Table No Title Page








xi

LIST OF FIGURES

Figure No Title

















Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10

LIST OF TABLES

Table No Title Page








xi

LIST OF FIGURES

Figure No Title

















Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10

LIST OF FIGURES

Figure No Title

















Xll

c -

Page

10

15

23

49

54

55

58

59

60

62

65

67

68

73

75

76

I

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10

Abbreviations

~g

JLl

2-BE

AMP

APS

bp

BPB

ddH20

DDRT

DEPC

DIG-dUTP

DNA

dNTP

EDTA

EtBr

IPTG

LB

LiCI

M

MgCh

microgram

microliter

Butoxyethanol

Ampicillin


Base pair

Bromo phenol blue




Digoxig~nin-ll-dUTP


Deoxynucleotide


Ethidiwn bromide



Lithiwn chloride

molar

Magnesium chloride

xiii

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10

MMLV-RT

OMT

PAGE

peR

pSI

PVPP

RAP

RNA

RNase

rpm

SA-PMP

SDS

TAE

TBE

TE

TENfED

V

vv

W

X-gal


O-methyltransferase






Ribonucleic acid

Ribonuclease






Trist-EDTA


Volt

volume over volume

Watt


xiv


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10


11 Introduction




















tonnes












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10












































3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10























3



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10



district



















4












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10












Johnson and











5























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10























6























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10























7






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10






















8













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10













1993)










9

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10

RAP-peR

------------- - -AAA ~CC TCCA

_ pt olf04r


~ RNA -------------- AAA

eDNA ---- CCATCCA

W7

ACGTACC~ eDA CCT GC





10

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