Sequence polymorphism of the COBRA gene of Kelampayan

(Neolamarckia cadamba)

Aaron Bong Sing Woei

(20575)

This project is submitted in partial fulfilment of the requirements for the degree of

Bachelor of Science with Honours

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2011

i

ACKNOWLEDGEMENT

First of all, I would like to thank God for His blessing upon the completion of this project.

My deepest gratitude to my supervisor, Dr. Ho Wei Seng, for his encouragement, guidance

and support in making this project possible. Besides that, my grateful thanks to my co-

supervisor, Dr. Pang Shek Ling for guiding me in experimental processes and providing

me information on this project. I would like to extend my gratitude and appreciation to my

colleagues, Mr. Phui Seng Loi (Msc. Student), Mr. Liew Kit Siong (Msc. Student), Ms.

Tchin Boon Ling (Msc. Student) and all my laboratory members for guiding me patiently

and providing a supportive environment throughout the whole project. Lastly, I would like

to thank my friends and family for giving me encouragements, supports and tolerances

through this project.

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DECLARATION

I hereby declare that this thesis is based on my original work except for quotations and

citation, which have been duty acknowledged. I also declare that it has not been previously

or concurrently submitted for any other degree at UNIMAS or other institutions.

___________________________________

Aaron Bong Sing Woei

Resource Biotechnology Programme

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

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TABLE OF CONTENT

ACKNOWLEDGEMENT

DECLARATION

TABLE OF CONTENTS

LIST OF ABBREVIATIONS

I

II

III

V

LIST OF TABLES AND FIGURES

ABSTRACT/ABSTRAK

VI

1

INTRODUCTION 2

LITERATURE REVIEW

2.1 Neolamarckia cadamba (Kelampayan)

2.2 Wood Formation

2.3 COBRA Gene

2.4 Single Nucleotide Polymorphism (SNP)

2.5 Application of Single Nucleotide Polymorphisms (SNPs)

4

4

5

7

9

10

MATERIALS AND METHOD

3.1 Plant Materials

3.2 DNA Extraction and Purification

3.2.1 Chemicals and Reagents

3.2.2 Total Genomic DNA Isolation

3.2.3 DNA Purification

3.3 DNA Quantification

3.3.1 Agarose Gel Electrophoresis

3.3.2 Spectrophotometric Quantification of DNA

3.4 Data Mining and Primer Design

3.5 Polymerase Chain Reaction (PCR)

3.6 PCR Optimization with MgCl2

3.7 PCR Product Purification

3.8 Cloning of PCR Products

3.8.1 Ligation

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12

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14

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15

16

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18

18

19

19

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3.8.2 Transformation and Blue/White Screening

3.9 Confirmation of the Desired Insert

3.10 DNA sequencing and analysis

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20

20

RESULTS AND DISCUSSION

4.1 Collection of Plant Samples

4.2 DNA Extraction and Purification

4.3 DNA Quantification

4.4 Primer Design

4.5 Polymerase Chain Reaction (PCR)

4.6 PCR Optimization

4.7 PCR Product Purification

4.8 Cloning of PCR Products

4.9 Confirmation for Desired Insert

4.9.1 Colony PCR

4.10 Plasmid Purification

4.11 DNA Sequencing and Data Analysis

4.11.1 Evaluation of sequence variation in COBRA gene

among N. cadamba

4.11.2 Single nucleotide polymorphism discovery

4.11.3 Determination of non-synonymous mutation and

synonymous mutation

4.11.4 Identification of possible restriction enzymes for

SNP site

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27

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31

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33

33

37

39

41

CONCLUSIONS

REFERENCES

43

45

v

LIST OF ABBREVIATIONS

[K+] Concentration of salt

AFLPs Amplified fragment length polymorphisms

Bp Base pair(s)

CTAB Cetyltrimethylammonium bromide

ddH2O Double distilled water

DNA Deoxyribonucleic acid

EDTA

EST

Ethylenediamine tetraacetic acid

Expressed Sequence Tag

GAP Glycosylphosphatidylinositol (GPI)-anchored protein

GPI Glycosylphosphatidylinositol

IPTG Isopropyl β-D-1-thiogalactopyranoside

LB Luria broth

LD Linkage disequilibrium

MAS Molecular marker-assisted selection

NaCl Sodium chloride

ORF Open reading frames

PCR Polymerase chain reaction

PVP Polyvinylpyrrolidone

QTL Quantitative trait locus

RFLP Restriction fragment length polymorphism

SFC SARAWAK FORESTRY Cooperation

SNP Single nucleotide polymorphism

TE Tris-EDTA

Tm

X-gal

Melting temperature

5-bromo-4-chrolo-3-indolyl-β-galactopyranoside

ΔH Enthalpy for helix formation

ΔS Entropy for helix formation

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LIST OF TABLES AND FIGURES

Figures Page

2.1 N. cadamba (a) The tree of N. cadamba, and (b) Cylindrical

trunk with coarse surface

5

2.2 Model of wood formation 6

2.3 Structure of glycosylphosphatidylinositol (GPI) anchor 7

3.1 Description of plots at Landeh Reserve Nature 12

4.1 Gel electrophoresis of purified genomic DNA samples on a 0.8%

agarose gel

24

4.2 Gel electrophoresis of PCR products on a 1.5% agarose gel 27

4.3 Gel electrophoresis of PCR products on a 1.5% agarose gel. 29

4.4 Gel electrophoresis of purified PCR product on a 1.5% agarose

gel

30

4.5 Gel electrophoresis of colony PCR products on a 1.5% agarose

gel

31

4.6 Gel electrophoresis of purified plasmid on a 1.0% agarose gel 32

4.7

4.8

4.9

4.10

4.11

4.12

4.13

Alignment result of Populus Tomentosa DNA (GU053565) and

sample COBRA A

Alignment result of Populus Tomentosa mRNA (GQ410777)

and sample COBRA A

Comparison of sequence structure of Populus Tomentosa

genotype 38 COBRA-like 4 protein gene (GU053565), Populus

Tomentosa COBRA-like 4 protein mRNA (GQ410777) and

sequence of COBRA gene from sample A

Alignment result of six partial sequences of COBRA gene using

ClustalW software

Alignment of the protein sequences using ClustalW software

Possible restriction enzymes for SNP site from COBRA W using

NEBcutter V2.0

A close up of possible restriction enzymes for SNP site from

COBRA W using NEBcutter V2.0

34

35

36

39

40

42

42

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Tables

Page

3.1 Ligation reaction mixture and volume 19

4.1 Plot of the collected trees 22

4.2 Characteristics of collected Kelampayan trees 23

4.3 Estimated purified DNA concentration of a N.cadamba based on

band intensity of samples and marker on a 0.8% agarose gel

25

4.4 Estimated DNA concentration and DNA purity of the six

purified N.cadamba samples

25

4.5 Primer pairs designed to amplify the COBRA gene 26

4.6 PCR reaction mixture used to amplify the COBRA gene 27

4.7 PCR reaction mixture in PCR optimization used to amplify the

COBRA gene

28

4.8 BLASTn output for amplified partial 517 bp COBRA gene 33

4.9

4.10

BLASTp output for COBRA gene sequence with intron removed

Position of sequence variations within the sequenced samples

37

39

4.11 The non-synonymous and synonymous mutation occurred in the

samples

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1

SEQUENCE POLYMORPHISM OF THE COBRA GENE OF KELAMPAYAN

(Neolamarckia cadamba)

AARON BONG SING WOEI

Resource Biotechnology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Neolamarckia cadamba or locally known as Kelampayan is a fast growing deciduous tree with great

commercial values. COBRA gene is involved in cellulose deposition and orientation of cell expansion. Any

single nucleotide differences may influence the functions of the gene. In this study, the DNA sequence

polymorphisms in COBRA gene from N. cadamba were detected. The targeted DNA sequence of COBRA

gene was amplified with the designed primer by using Polymerase Chain Reaction (PCR) technique. The 517

bp of COBRA gene amplicons were subjected to BLASTn analysis to search for homology sequence and

validate the identity of the obtained sequences through NCBI. Multiple alignment was carried out by

ClustalW for manual detection of Single Nucleotide Polymorphisms (SNPs). Five SNPs were detected in the

exon region and two SNPs in the intron region of COBRA gene amplicons. The sequence of six partial

COBRA gene was then subjected to in silico restriction analysis. One possible restriction enzyme, HpyCH4III

was detected for SNP at nucleotide 384 bp of a sample which could be useful for genetic marker

development.

Key words: Neolamarckia cadamba, polymerase chain reaction (PCR), COBRA gene, single nucleotide

polymorphisms (SNPs), molecular markers

ABSTRAK

Neolamarckia cadamba atau lebih dikenali sebagai Kelampayan merupakan species pokok kayu yang cepat

membesar dan mempunyai nilai komersial yang tinggi. Gen COBRA memainkan peranan dalam deposisi

selulosa and orientasi dalam pengembangan sel. Sebarangan pembezaan nukleotida akan mempengaruhi

fungsi gen. Dalam kajian ini, polimorfisme nukleotida tunggal (SNP) dalam gen COBRA DNA dari N.

cadamba telah ditemui. Jujukan DNA bagi gen COBRA diamplifikasi oleh penanda molekul dengan

menggunakan teknik tindakbalas berantai polimerase (PCR). Produk PCR bersaiz 517 bp gen COBRA

dianalisa dengan BLASTn untuk mencari urutan homologi dan mengesan identiti untuk jujukan yang

diperolehi dalam pangkalan data NCBI. Penjajaran jujukan oleh ClustalW dilakukan untuk mengesan SNP

secara manual. Lima SNP telah dikesan dalam bahagian ekson dan dua dalam bahagian intron bagi gen

COBRA. Enam jujukan gen COBRA dianalisa oleh enzim penyekatan secara in silico. Penemuan satu

pengkhususan enzim, HpyCH4III bagi SNP pada nukleotida 384 bp adalah amat berguna dalam penghasilan

penanda molekul.

Kata kunci: Neolamarckia cadamba, tindakbalas berantai polimerase (PCR), COBRA gene, polimorfisme

nukleotida tunggal (SNP), penanda molekul

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1.0 INTRODUCTION

Neolamarckia cadamba, also locally known as Kelampayan is one of the fast growing

deciduous trees. It is an indigenous species for areas from India through Southeast Asia to

Papua New Guinea. Due to the fast growth rate and great economic value of its timber,

plantations of N. cadamba have been raised in India, Sri Lanka, Myanmar, Indonesia,

Malaysia and the Philippines. It grows well in freshwater swamps and it is commonly

found in logging area of lowland dipterocarp forests (Nair, 2007). The timber is generally

used for light-weight purposes like moulding, skirting, frames and so on (Lim et al., 2005).

Besides that, N. cadamba is one of the selected plant species for planted forestry in

Sarawak.

The increase of global demand for wood requires an increase in forest production.

It is essential for research and development (R&D) of faster growth, high-yield and short

rotation plantation forests as compared to the conventional breeding which is time-

consuming and costly. The role of forest biotechnology to provide adequate tools for tree

breeders and production of high quality plantation are required to augment traditional tree

improvement activities. In Sarawak, SARAWAK FORESTRY Corporation (SFC) in

collaboration with UNIMAS has ventured in the development of forest genomics,

biotechnology and tree improvement research for development of planted forest and

conservation of forest resources. The forest biotechnology researches are to develop

adequate tools to produce trees that adapt to the country’s conditions and significantly

achieve economic benefits. Other than that, application of forest genomics will enhance

tree productivity and wood quality. Development of highly informative and polymorphic

genetic markers such as single nucleotide polymorphisms (SNPs) can be used in the

selection of better trees for plantation and genetic improvement activities. According to

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Gupta et al. (2001), the application of SNPs at molecular level can be used in selective

breeding with desired traits by plant breeders.

Plant cell wall plays a vital role in human life as it provides large scale of dietary

fibers and raw materials for textile, lumber, pulping, and potentially for biofuels. In

economic point of view, understanding the regulatory mechanisms involved for

biosynthesis of cell wall and identifying regulatory genes such as cellulose, hemicelluloses

and lignin are of obvious important (Zhang et al., 2009). Cellulose that composes 40% to

50% of wood is an essential component of the load-bearing network by providing strength

required in cell walls through cellulose deposition. The physical properties of cellulose also

determine the orientation of cell expansion (Brown, 2003). The presence of COBRA gene

in the woody plant species is essential as it plays the role in the regulation of cellulose

deposition and orientation of cell enlargement.

Identification of COBRA gene and sequence polymorphism controlling wood

quality traits are vital especially in forest tree breeding programs. An improvement of

percentages in traits such as pulp yield can produce large gains for a pulp and paper mill

industry. Therefore, the objective of this study was to design primers for the amplification

of the partial COBRA genomic fragment in the six samples of different N. cadamba trees

through PCR. Moreover, single nucleotide polymorphisms (SNPs) were detected in this

study by comparing the genotype of the samples. Non-synonymous or synonymous

mutations due to single base differences in partial COBRA genomic sequence which may

result in changes of phenotypic trait or encoding gene were identified as well. Restriction

enzyme that recognizes and restricts at the identified SNP site in the COBRA gene was

identified which is necessary in developing marker for selection of planting material at the

seedling stage.

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2.0 LITERATURE REVIEW

2.1 Neolamarckia cadamba (Kelampayan)

Neolamarckia cadamba or locally known as Kelampayan is under the family of Rubiaceae.

N. cadamba is a medium to tall growing deciduous tree that grows up to 40 m to 45 m tall.

Its bole is straight and cylindrical. This species usually starts branching after 25 m and a

diameter of up to 100 cm with buttresses to 2 m tall. It is widely dispersed in lowland to

mountain forests of up to 1000 m altitude where it is often found by streams, rivers and in

open sites of the forests (Lim et al., 2005).

According to a study done by Lim et al. (2005), N. cadamba is classified as one of

the light-coloured timbers with low density. Due to the presence of large vessels, the

timber of N. cadamba is white with coarse surfaces. Light and soft properties of the timber

promote the usage for light-weight purposes such as moulding, picture frames, skirting,

disposable chopstick, wooden sandals, veneer, plywood and general utility furniture.

Besides that, the seeds have great medicinal value as it cures astringent, ulcer, digestive,

diarrhoea, expectorant, fever and vomiting (Peter, 2007).

N. cadamba leaves is largely ovate, whole margin of elliptic-oblong with smooth

surface and pinnate venation. The length of the leaves varies from 7.5 to 18 cm with 4.5 to

16 cm breadth. Thick prominent midrib and uniform thin lamina is observed in the leaf

through microscope. Short and thick wall of unicellular trichomes are observed on the

epidermis layer of the leaves. The presence of unicellular, lignified trichomes, paracytic

stomata, simple starch grains and sandy balls of calcium oxalate crystals are observed

through the microscopic study of N. cadamba leaf powder (Patel and Kumar, 2008).

5

(a) (b)

Figure 2.1 N. cadamba (a) The tree of N. cadamba, and (b) Cylindrical trunk with coarse surface. (Adapted

from (a) http://www.flickriver.com/photos/tags/anthocephaluscadamba/interesting/ (b)

http://www.flickr.com/photos/dinesh_valke/2065669915/)

2.2 Wood Formation

Wood is also known as secondary xylem which is constructed by four major ecological

sequences, including cell division, cell expansion, cell wall thickening, and programmed

cell death (Demura and Fukuda, 2006). Primary wall formation and modification are

involved in the expansion of procambial cells and the cambial daughter cells. These two

cells produced by cambial initials will differentiate into phloem cells and xylem cells

(tracheary elements and fibre). Components of tracheary elements are tracheids and

vessels. Vessels such as protoxylem vessels have annular and spiral secondary wall

thickenings while metaxylem vessels have reticulate and pitted thickening (Demura and

Fukuda, 2006). The expansion of cell is followed by the thickening of cell wall that forms

the secondary wall. Formation of secondary wall is driven by the coordination of numerous

genes that involved in the biosynthesis of cell wall proteins and polysaccharides (cellulose,

6

hemicelluloses), and formation of lignin. Lastly, an empty tube is formed with secondary

walls when programmed cell death happens (Demura and Fukuda, 2006).

Figure 2.2 Model of wood formation. Adapted from (a) Demura and Fukuda, 2006) (b)

http://www.bcb.uwc.ac.za/ecotree/trunk/Formationwood1.htm

According to Plomion et al. (2001), wood is made up of between 40% and 50% of

cellulose. Cellulose is composed of 1,4-β-glucan chains that are synthesized by a cellulose

synthase complex. This multi-protein complex simultaneously synthesizes 18 to 36 glucan

chains that bond together in a single microfibril (Liepman et al., 2010). This basic

structural crystallized microfibril formed as the result of a strong association of intrachain

and interchain hydrogen bonds between the different chains of β-linked glucose residues

(Plomion et al., 2001). In order to provide strength to cell walls, cellulose microfibrils have

to be deposited in a specific orientation. Cellulose microfibrils are usually deposited in

analog arrays transverse of growing cells to the direction of growth.

(a)

(b)

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2.3 COBRA Gene

The COBRA gene in the woody species is responsible for the regulation of cellulose

deposition and orientation of cell enlargement. This gene encodes for putative

glycosylphosphatidylinositol (GPI)-anchored protein (GAP) targets the cell surface of the

plant (Zhang et al., 2009).

COBRA protein is fixed by GPI to the external plasma membrane leaflet and

inserted into the endoplasmic reticulum. The addition of GPI anchor involved the cleavage

of a hydrophobic C-terminal peptide and followed by linkage of a preassembled GPI

anchor through an amide bond onto the last amino acid residue which is the remains of the

cleavage. This attachment site is called ω-site. Specific phospholipases cleave GPI anchor

and causes the release of the protein from the membrane (Roudier et al., 2002). The

modified protein is transported to the cell surface and attached to the plasma membrane

aided by the fatty acid chains of the GPI anchor (Cooper, 2000).

Figure 2.3 Structure of glycosylphosphatidylinositol (GPI) anchor (Cooper, 2000).

+NH3-CH2-CH2

C terminus

Ethanolamine

Protein

N-acetylgalactosamine

Mannose

Glucosamine

Inositol

8

In a study done by Zhang et al. (2009), a COBRA-like protein was successfully

isolated and identified as PtCOBL4 when comparison of nucleotide sequence was

compared with the known full-length of Arabidopsis COB gene sequences. It is confirmed

as one of the members of COBL family, PtCOBL4 shared 72.7%, 64.8%, and 65.3%

similarity with Arabidopsis COBL members, rice BC1 members and maize BK2 proteins

respectively. Besides that, PtCOBL4 was examined for these characteristics: CCVS motif,

an N-terminal signal peptide sequence for secretion, a highly hydrophobic C terminus, and

specific features around the ω-site. With all these criteria, PtCOLB4 is classified as COBL

protein.

In Arabidopsis, AtCOBL4 is identified to be implicated in the cellulose biosynthesis

in the secondary wall through mutant analysis (Brown et al., 2005). The expression of

AtCOBL4 is found to be similar with Arabidopsis cellulose synthase (AtCesA) genes that

are involved in secondary cell wall synthesis (Persson et al., 2005). According to Zhang et

al. (2009), PtCOBL4 is suggested to be an ortholog of AtCOBL4 as they shared high

nucleotide homology (70.9%) and amino acid identity (72.7%). Other than that, PtCOBL4

gene is found to be expressed dominantly in the mature xylem fiber cells during the late

stage of cell wall thickening. This showed that PtCOBL4 is involved in the secondary cell

wall deposition in the stem of trees (Zhang et al., 2009).

Studies by Roudier et al. (2005) proved that COBRA gene is found to be distributed

in a banded pattern that is perpendicular to the longitudinal axis via a microtubule-

dependent mechanism. Mutants of COBRA gene are defective in elongation of cell growth

that occurs after the initial cell expansion and this indicates the involvement of COBRA

gene in morphogenesis (Roudier et al., 2005). According to Roudier et al. (2005),

Arabidopsis mutants of COBRA gene has shown to reduce cellulose content and cause the

cells to swell due to disorganize cellulose microfibril deposition. However, the

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disorganization of cellulose occurs before the depletion of cellulose content in the cell

wall. This shows that COBRA is involved in the orientation of deposition of cellulose

microfibrils while the reduction in cellulose content is likely caused by a feedback

mechanism that reflects the already disordered deposition of cellulose microfibrils

(Roudier et al., 2005).

2.4 Single Nucleotide Polymorphism (SNP)

Before the emergence of molecular marker as an indicator of sequence polymorphism,

plant breeders select the plant based on the phenotypic agronomic traits. With the

development of technologies and genetic markers, approaches such as DNA

fingerprinting, mitochondrial DNA sequencing or restriction fragment length

polymorphism (RFLP) analysis, amplified fragment length polymorphisms (AFLPs),

nuclear gene sequencing and genotyping of various types of nuclear loci are adopted

(Aitken et al., 2004). However, some of these approaches are time-consuming and labour

intensive in large-scale genome analysis (Edwards and Mogg, 2001). Approaches like

RFLPs are laboured intensive and costly. Larger quantities of DNA are needed yet it

produces only low quantity of polymorphism (Williams et al., 1991). Besides that, these

approaches are confronted with technical and analytical issues that limit their application.

For example, the highly variable mutation rate of gene limits the analysis of genome

(Aitken et al., 2004).

According to Aitken et al. (2004), an ideal genetic marker for population and

evolutionary studies should possess at least three properties. Firstly, biases associated with

single locus analysis can be avoided by using markers that are abundant and distributed

widely across the genome. To facilitate analysis and interpretation, well-understood and

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well-characterized models of evolution should be used. Lastly, technical applications must

allow data acquisition from numerous loci marked in large population samples, while the

data have to be comparable in laboratories using different genotype scoring procedures or

technologies (Sunnucks, 2000). All these ideal marker characteristics are fulfilled by single

nucleotide polymorphisms (SNPs). Single Nucleotide Polymorphisms (SNPs) are referred

to single DNA base differences between homologous DNA fragments including small

insertions and deletions of multiple bases. Polymorphisms of single base are usually

biallelic (Dietrich et al., 1999). These variations in DNA bases can be used as genetic

markers that can be identified in the neighboring of virtually every gene (Rafalski, 2002).

These molecular markers are essential in genetic localization, map-based positional

cloning, detection of marker-trait gene relationship through linkage and linkage

disequilibrium (LD) mapping and the assessment of genetic association between

organisms.

2.5 Application of Single Nucleotide Polymorphisms (SNPs)

In the study of functional genomics where expressed sequence tags predominate, the use of

SNPs as molecular markers for individual genotyping are needed for molecular marker-

assisted selection (MAS) (Gupta et al., 2001). In the genome, SNPs may occur in the

coding, non-coding and intergenic region. SNP in plant increase our knowledge to

understand the genetic basis of important agronomic traits. The presence of SNPs in the

coding region will demonstrate the association with the trait even though the mutant

phenotype may or may not able to be determined. However, recognition of SNPs that

associate with trait is useful for MAS and for gene isolation. In DNA, SNPs are recognized

as the most frequent type of variation found in DNA. Recent estimation has shown that one

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SNP occur every 100-300 bp in any genome. This shows that SNPs are recognized as the

most abundant molecular markers in the present time (Gupta et al., 2001).

Detection of associations between allelic forms of a gene and phenotypes especially

in common diseases with multifactorial genetics can be done with SNPs too. The study of

linkage disequilibrium (LD) able to provide information regarding positional cloning,

linkage and evolution by constructing LD map. Positional cloning of genes for disease

susceptibility is dependent on linkage and allelic association (Maniatis et al., 2002). This

showed that LD is a promising tool for fine mapping of gene diseases in a population.

According to Moon et al. (2007), this molecular marker, SNPs are the most

essential forms of DNA variation for quantitative trait loci (QTL) mapping. QTL is defined

as the genes influencing heritable quantitative trait, a phenotypic characteristic that is

attributed by two or more genes (Grisel, 2000). By implementing QTL mapping, the

underlying genes in a quantitative trait can be discovered with the utilization of molecular

markers like SNPs. The greater number of polymorphic DNA markers used, the greater

chances of discovering closely linked genes or the relevant genes (Grisel, 2000). The

association analysis between the markers and the quantitative trait contributes to the

identification of phenotypic variation as it helps researcher to derive a better insights into

the genetic basis of a complex trait (Wu et al., 2007). QTL mapping can be carried out by

comparing same genotype strains of various inbred and the interactions of two or more

genes with their environment (Grisel, 2000). Hence, the utilization of SNPs markers

enables the identification of heritable beneficial quantitative trait which can be used in

selective breeding programme.

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3.0 MATERIALS AND METHODS

3.1 Plant Materials

Fresh young leaves were collected from six randomly selected Neolamarckia cadamba

trees for total genomic DNA extraction from the Kelampayan Trial Plot at Landeh Nature

Reserve, Semengok, Sarawak (Figure 3.1).

Figure 3.1: Description of plot at Landeh Nature Reserve

3.2 DNA Extraction and Purification

3.2.1 Chemicals and Reagents

The reagents used in this study are liquid nitrogen, CTAB extraction buffer (100 mM Tris-

CL pH 8.0; 1.4 M NaCl; 20 mM EDTA; 2% CTAB; 1% polyvinylpyrrolidone (PVP); 2%

(v/v) β-mercaptoethanol), isopropanol, TE buffer, chloroform:isoamy alcohol (24:1) and

sterile distilled deionized water (ddH2O).

Tree 1 – Tree 16

Tre

e 1

– T

ree

9

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3.2.2 Total Genomic DNA Isolation

Total genomic DNA of N. cadamba was extracted by using the modified CTAB method

from Doyle and Doyle (1990).

Six thousand micro liters of CTAB extraction buffer was added with 2% of β-

mercaptoethanol in a 15 ml snap cap Falcon tube. The tube was then preheated for 30

minutes at 65°C. The sterilized mortar, pestle and spatula were pre-chilled in liquid

nitrogen. Plant material of approximately 1.0 g was ground to powder form in liquid

nitrogen. During the grinding process, the tissue should be kept frozen. It is essential to

grind the tissue to a very fine powder in order to obtain high yield of molecular weight

DNA. The ground tissue powder was then scraped and placed into the preheated buffer and

swirled gently to mix. The sample was then incubated at 65°C for 1 hour with shaking.

After incubation, the sample was cooled down for 5 minutes. Then, 750 µl of the

mixture in the Falcon tube was transferred into 1.5 ml microcentrifuge tubes with excised

wide-bored tips. Equal volume of chloroform/isoamyl alcohol (CIA) (24:1; v/v) was added

and mixed gently. The sample was centrifuged at 13,000 rpm for 10 minutes at room

temperature. Later, the aqueous phase was transferred to new 1.5 ml microcentrifuge tubes.

Equal volume of CIA was added to the tubes and the tubes were centrifuged again. This

process is to ensure any carbohydrates impurities were removed. Following centrifugation,

the aqueous phase was transferred to clean microcentrifuge tubes. About 2/3 volume of

cold isopropanol was added to the tubes and mixed gently. Precipitation of nucleic acids

was observed. The sample was then stored overnight at -20°C.

The next day, the sample was thawed and centrifuged for 15 minutes at 13,000 rpm.

Pellet was formed and the supernatant was carefully poured off. Five hundred micro liters

of 70% cold ethanol was added and centrifuged for 10 minutes at 13,000 rpm. The

14

supernatant was decanted and air dry for 10 – 15 minutes. The pellet was resuspended by

adding 80 µl of ultra pure water. The tubes were flicked gently to dissolve the pellet and

later on stored for an hour at -20°C.

3.2.3 DNA Purification

The isolated DNA was purified by using Wizard® Genomic DNA Purification Kit

(Promega, USA) based on the manufacture’s protocol.

The sample was top up with ultra pure water to 600 µl. RNase of 3 µl was extracted

from the kit and added into the sample. The sample was incubated for an hour at 36.5°C.

An hour later, 200 µl of Protein Precipitation Solution was then added. The tube was

flicked gently and centrifuged for 3 minutes at 13,000 rpm. Six hundred micro litres of

supernatant was transferred to a new tube containing 600 µl isopropanol. The tube was

mixed gently and centrifuged for 10 minutes at 13,000 rpm. The pellet was formed and the

supernatant was carefully poured off. Later, 70% of ethanol was added and centrifuged for

5 minutes at 13,000 rpm. The ethanol was then discarded and the tube was left to air-dry

for 15 minutes. The pellet of DNA was resuspended in 30 µl of ultra pure water. It was

then incubated for an hour at 36.5°C and stored at -20°C for further analysis.

3.3 DNA Quantification

3.3.1 Agarose Gel Electrophoresis

The presence and concentration of isolated genomic DNA from the leaf was determined by

running a 0.8% agarose gel. Agarose powder of 0.32g was added into a conical flask

15

containing 40ml 1X TAE Buffer and the mixture was heated at 300°Cfor 2 minutes. Later,

the mixture was cooled while swirled gently and poured into gel casing. Bubble was poked

and comb of 16 wells was inserted. The gel was left to cool and solidify for 45 minutes at

room temperature.

Five micro liters of genomic DNA was mixed with 2 µl of 1X loading dye. Three

micro liters of Lambda-HindIII (Promega, USA) was used as DNA marker and mixed with

1 µl of 1X loading dye. The gel was run under 50V, 40A for 90 minutes. Later, the gel was

stained with ethidium bromide for 10 seconds. It was then followed by de-staining with

distilled water for 30 minutes. Presence of any bands were detected by visualized the gel

under a UV transilluminator.

The concentrations of the genomic DNA samples were checked by comparing the

intensity of samples’ band with standard DNA of known concentration. An estimation of

the DNA concentration was made by comparing the brightness of the known band with the

sample bands. The concentration of DNA was calculated based on the formula below:

DNA Conc. (ng µl)

= Marker frag. size

Marker total sizeX Marker Conc. (µg µl) X

Marker vol. (µl)

DNA vol. (µl)X 10³

3.3.2 Spectrophotometric Quantification of DNA

The concentration of purified DNA was quantified using Lambda 25 UV/VIS

Spectrophotometer (Perkin Elmer, USA). Three micro liters of DNA sample was mixed

with 2,997 µl of ultra pure water in a quartz cuvette. The absorbance reading was taken at

wavelength of 230nm, 260nm, and 280nm. The absorbance ratio of A260/A280 and A260/A230

16

were calculated to account the purity of the DNA samples. The concentration of each DNA

sample was calculated based on the formula below:

[DNA] (ng/µl) = A260 x OD260

The OD260 unit for dsDNA correspond to a concentration of 50 µg/ml. The dilution factor

has automatically counted by the programme while giving the value of A260.

3.4 Data Mining and Primer Design

Primer was designed using Primer Premier 6.0 (PREMIER Biosoft International, USA).

The forward and reverse primer pairs were designed based on the EST of Neolamarckia

cadamba with the nucleotide sequence length of 536 bp that is obtained by Forest

Genomics and Informatics (fGiL).

In PCR, primers should be designed so that there are no inverted repeats or

homopolymeric regions. There should be approximately 50% of GC contents in the base

composition. At the 3’ end of primers, there should be more than 1 G or C residues. Any

shortage of above criteria will affect the stability of primer-template interaction

(Middendorf et al., 2005).

In designing primers, it is important to have optimal length of the primer as it

controls the specificity. Longer primers are prone to form secondary structures like

hairpins, dimmers, and self-complementation. It is vital to avoid the forming of secondary

structure as it would limit the number of primers available for bonding. It is best to use

shorter primer that would provide the best balance between specificity and efficiency.

Optimally, primers length should be between 18 and 24 bases. Besides that, melting

temperature (Tm) can be calculated based on the following formula: