wan nor haslinda binti wan azman - universiti malaysia … · wan nor haslinda binti wan azman ......

GENETIC CHARACTERIZATION OF DOMESTIC GOAT (Capra hircus) USING MITOCHONDRIAL DNA

WAN NOR HASLINDA BINTI WAN AZMAN

This project is submitted in partial fulfillment of the requirements for the degree of Bachelor of Science with Honours

(Biotechnology)

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARA W AK

2011

ACKNOWLEDGEMENTS

Alhamdulillah and grateful to Allah SWT because I had been completed this thesis Firstly I

would like to express my sincere gratitude and thanks to my research supervisor Dr Yuzine

Bin Esa for his guidelines attention kindness encouragement and moral support gave to me

to finish my Final Year Project

I express my gratitude to my co-supervIsor Dr Hairul Azman for his comment

guidance and recommendations during completing my thesis

The special thank go to all lab assistants postgraduate students my laboratory mate

and all my dear friends for their invaluable moral support motivation and companionship

during preparation of this thesis

Finally I declare my best appreciation to my parents and all my family for their

endless help support and confidence

TABLE OF CONTENTS

Acknowledgement I

Table of Contents II

List of Abbreviations IV

List of Tables and Figures V

Abstract

Introduction 2

Literature Reviews 5

Domestic Goat 5

Polymerase Chain Reaction (PCR) 7

Mitochondrial DNA 8

Cytochrome Oxidase I (COl) 9

Genetic Studies on Domestic Goat and Other Ovine 10

Materials and Methods 12

Sample Collection of Goat 12

DNA Extraction 13


Agarose Gel Electrophoresis 15

Purification 15

DNA Analysis 16

II

Results 18

DNA Extraction and Isolation 18

PCR Amplification 190

Purification 21

DNA Sequencing Analysis 22

Phylogenetic Inference 23

Neighbour-joining (NJ) 24

Maximum Parsimony (MP) 26

Discussions 29



Purification 31



Conclusion and Future Work 34

References 35

Appendix A 38

III

LIST OF ABBREVIATIONS

Chircus

CTAB

COl

d-loop

cyt b

DNA

dNTP

PCR

MEGA

MgCl2

mtDNA

NaCI

NaOAc

Etbr

NJ

MP

PCR

Rpm

bp

~I

degC

Capra hircus

Cetyl trimethyolammonium Bromide

Cytochrome Oxidase I

Displacement Loop

Cytochrome b

Deoxyribonucleic Acid

Deoxynucleoside tryphosphate

Polymerase Chain Reaction

Molecular Evolutionary Genetic Analysis

Magnesium Chloride

Mitochondrial DNA

Sodium Chloride

Sodium Acetate

Ethidium Bromide

Neighbour-joining

Maximum Parsimony


Rotation Per Minute

Base pair

Microliter

Degree Celcius

IV

LIST OF TABLES

Table 1 Scientific classification of goat 5

Table 2 Description of the goat used in this study 12

Table 3 Primers that were used in PCR amplification 14

Table 4 PCR parameters Number of cycle 35 cycles 19

Table 5 The average total nucleotide composition in goat 23

Table 6 Description of the Capra hircus in group based on Neighbor-joining 25

Table 7 Description of the Capra hircus in group based on Maximum Parsimony 27

Table 8 Pairwise distance or genetic distance among the Capra hircus and Bos Taurus (out-group) 28

Table 9 Aligned nucleotide sequences of the Cytochrome Oxidase I (COl) among Capra hircus and Bos taurus (out-group) Dote indicates nucleotide that are the same as those found in RATEN Dash indicates an insertion event 38

v

LIST OF FIGURES

Figure 1 Goat (Capra hircus) (Source Handalas farm Matang Sarawak) 5

Figure 2 Mitochondrial DNA (Source Wikipedia) 8

Figure 3 DNA extraction of20 samples goat M = 100-bp ladder (Fermentas) Lane 1 = K02l lane 2 = K032 lane 3 = L395 lane 4 = P466 lane 5 = 9369172 lane 6 = B303 lane 7 = P206D2 lane 8 = P454A lane 9 = H054 lane 10= L387 lane 11 = T452 lane 12 = G313 lane 14 = K014 lane 15 = K008 lane 16 = TA82A lane 17 = K003 lane 18 = R223 lane 19 = MOKLI lane 20 = RATEN 18

Figure 4 PCR products of 6 samples of extracted DNA M = 100-bp ladder (Fermentas) lane 1 = K003 lane 2 = K032 lane 3 = L395 lane 4 = MOKLI lane 5 = P466 lane 6 = 9369172 Lane 2 to 6 represents samples of extracted DNA that success in PCR Lane 1 represents sample of extracted DNA that failed in PCR reaction 20

Figure 5 PCR products of7 samples of extracted DNA Lane 1 = RATEN lane 2 = K02l lane 3 = B303 lane 4 = P206D2 lane 5 = P454A lane 6 = H054 lane 7 = L387 M = l-Kb ladder (Fermentas) Lane 1 to 7 represents samples of extracted DNA that success in PCR

reaction 20

Figure 6 PCR products of 7 samples of extracted DNA Lane 1 = T452 lane 2 =

T478 lane 3 = G313 lane 4 = K014 lane 5 = R223 lane 6 = K008 lane 7 = TA82A M = 1-Kb ladder (F ermentas) Lane 1 to 4 and lane 6 to 7 represents samples of extracted DNA that success in PCR reaction Lane 5 represent sample of extracted DNA that failed in PCR reaction

21

Figure 7 Purified PCR products Lane 1 = K032 lane 2= L395 lane 3= MOKLI lane 4 = P466 lane 5 = 9369172 lane 6 = RATEN lane 7 = K02l lane 8 = B303 lane 9 = P206D2 lane 10 = P454A lane 11 = H054 lane 12

= L387 lane 13= T452 lane 14 = T478 lane 15 = G313 lane 16 = K014 lane 17= K008 lane 18 = TA82A M= l-Kb bp ladder (Fermentas)

22

Figure 8 Neighbour- joining tree ofpartial COl mtDNA gene sequences relationship

VI

among Capra hircus and Bos taurus (out-group) 24

Figure 9 Maximum Parsimony tree of partial COl mtDNA gene sequences relationship among Capra hircus and Bos taurus (out-group) Numbers at branches are the percentage of bootstrap values at 1000 replicates 26

VII

11

Genetic Characterization of Domestic Goat (Capra hircus) Using Mitochondrial DNA

Wan Nor Haslinda Binti Wan Azman

Resource Biotechnology Programme Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

This study examines the genetic characteristic of domestic goat Capra hircus using sequence analysis of mitochondrial DNA Cytochrome Oxidase I (COl) gene A total of 20 domestic goat samples from the Handalas Farm Matang Sarawak were examined The sequence data were analyzed using both character (Maximum Parsimony) and distance (Neighbour-joining) methods Phylogenetic tree produced showed all the goats from the Handalas Farm Matang Sarawak is clustered into a single clade The result showed that all the sequences are closely related thus all samples used in this study are possibly originated from a single gene pool of domestic goat population Overall analysis from this study showed that the COl gene is limited use in examining the genetic structure of goat population in the Handalas Farm Matang Sarawak

Key words Capra hircus mitochondrial DNA Cytochrome Oxidase I (COl) gene genetic structure

ABSTRAK

Kajian ini dijalankan untuk mengkaji ciri-ciri genetik kambing domestik iaitu Capra hircus dengan menggunakan analisis jujukan gen mitokondria DNA Sitokrom Oksides I (COl) Sejumlah 20 sampel kambing domestik dan Ladang Handalas Matang Sarawak dikaji Jujukan data dianalisis menggunakan kaedah pencirian (Maximum Parsimony) dan jarak (Neighbour-joining) Filogenetik pokok yang terhasil menunjukkan ke semua kambing dari Ladang Handa las Matang Sarawak telah dikumpulkan kedalam satu clade Keputusan menunjukkan ke semua jujukan berkait rapat dan ke semua sampel yang digunakan adalah berkemungkinan berasal dari satu kolam gen populasi kambing domestik Keseluruhannya analisis dari kajian ini menunjukkan penggunaan gen COl adalah terhad dalam mengkaji struktur genetik populasi kambing di Ladang Handalas Matang Sarawak

Kata kunci Capra hircus motokondria DNA gen Sitokrom Oksides I (COl) struktur genetik

1

INTRODUCTION

The domestic goat (Capra hircus) is one of the domesticated ruminant livestock species The

goat is a member of the Bovidae family and is closely related to the sheep (Table 1) Both are

in the goat-antelope subfamily Caprinae (Table 1) There are over three hundred distinct

breeds of goats (Anom 2010 a) Goats most likely descend from the wild bezoars (Capra

aegagrus) and the origins have been confirmed by genetic studies based on mitochondrial

and nuclear DNA (Naderi et at 2007 Groeneveld et at 2009)

The classification of goat breeds is determined based on their production of goat

status such as milk and meat production or both type The domestic goat is breeds in captivity

tend to have different anatomies and behaviour from their wild ancestor Breeding of

domestic goat is controled by human either by using natural breeding or assisted reproductive

techniques (ARTs) Current stocks of goats in Malaysia are made up from Katjang Boer

JarnnapariEttawah British Alphine Saenen Toggenburg breeds as well as their crosses

(Anom 2010 b) Goat contributes income for economic importance either from natural or

renewable product such as meat milk fine leather fertilizer gelatin surgical supplies

medicine soaps luggage and others

The levels of genetic variability of domestic goat are low within a population due to

variation in performance trait An assessment of genetic variability of the breed is a step

forward conservation and improvement of genetic resources for maintaining breeding

options The assessment in animal breeding and genetics was applied for identification

animal andparentage testing gene mapping and identifying marker for performance traits As

the structure of the mitochondrial DNA in livestock species varies evaluations through

2

genetic study are needed in order to gain a better understanding about genetic variation

among domestic goat

Application of molecular marker are wide spread and has been found to be a powerful

indicator to measure genetic differences within and between individuals populations and

species Mammalian mitochondrial DNA have been used to identify variation because of

their advantage features such as it has represented the most informative genomic element

whereby it has highly informative polymorphism fast mutation rate highest stability

absence of recombination evolves less rapidly and display maternal mode of genetic

transmissions Genetic marker can also determine the relationship among breed using

calculating genetic distance and construct trees (Rout ei ai 2008) Thus this study was

carried out in a purpose to characterise the genetic variation of domestic goat by using

sequence analysis of Cytochrome Oxidase I (COl)

3

The Objectives of This Study are

1 To extract DNA from samples goat

2 To amplify Cytochrome Oxidase I (COl) gene using PCR

3 To characterize the genetic structure of goat population from the Handa1as

farm Matang based on mitochondrial DNA Cytochrome Oxidase I (COl)

sequences

4

2 Ii i at

LITERATURE REVIEW

Domestic Goat (Capra hircus)

Figure 1 Capra hircus (Source Handalas farm Matang Sarawak)

Table 1 Scientific classification of goat

Common name Domestic goat

Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra

Species Capra hircus

5

Domestic goat is a social animal and it also known as grazing specIes grass Mileski amp

Myers (201 0) stated that domestic goat is classified as sedentary because they live under

human control They live in group called herds and the size of captive herd could be

controlled by human Herds sizes in the wild tend to be 5 to 20 members but they can be as

high as 100 The herds can contain only males only females and young or a mix of both

Goats can survive on bushes trees scrub and aromatic herbs (Haenlein 1992) and eat any

kind of plant material such as grass leaves twigs as well as stems of woody plants

Goat come in varies colour like solid black white red brown spotted two and three

coloured blended shades distinct facial stripes black and white saddles depending on

breeds (Haenlein 1992) Figure 1 show a goat from the Handalas Farm Matang Sarawak

with a combination of two colours of white and black Goat has digestive tract with four

stomach compartments of ruminants consisting of the rumen the reticulum the omasum and

the obamasum The intestinal canal of the goat is about 100 feet long (11 liters) or 25 times

the length of a goat (Haenlein 1992)

The breeding behaviour of goat is usually control by human which follows

polygynous reproductive system that involves mating of one male with more than one female

(Mileski amp Myers 2010 Anom 2010 c) The females are then inseminated either directly by

those males or by novel reproductive biotechnologies which also known as assisted

reproductive techniques (ARTs) (Mileski amp Myers 2010 Rahman et at 2008) These ARTs

include artificial insemination(AI) embryo transfer (ET) multiple ovulation embryo transfer

(MOET) estrus synchronization and superovulation laparoscopic ovum pick-up (LOPU) in

vitro maturation (lVM) in vitro fertilization (lVF) and in vitro culture(lVC) collectively

6

i

known as in vitro production (IVP) intracytoplasmic sperm injection (ICSI)

cryopreservation of oocytes and embryos sperm and embryos sexing embryo splitting

embryo cloning nuclear transfer (NT) gene transfer and marker-assisted selection (MAS)

By using ARTs more offspring will be produced compared to natural breeding (Rahman et

al 2008)

Polymerase Chain Reaction (PCR)

PCR is a technique for amplifying DNA in vitro by incubating with a specific primer DNA

polymerase molecule and nucleotide (Campbell amp Reece 2005) PCR technique produces

many copies of a specific DNA sequences without having to clone the sequence in a host

genome (Russell 2006) The starting material for PCR is double stranded DNA containing

the target nucleotide sequence to be copied a heat resistant DNA polymerase all four

nucleotide and two short single-stranded DNA molecules that serve as primer One primer is

complementary to one strand at one end of the target sequence and the second primer is

complementary to the other strand at the other end of the sequence Applications of PCR

allow any specific segment of the target sequences within a DNA sample to be copied many

times completely in vitro and in a much shorter time (Campbell amp Reece 2005 Jain et al

2007)

(

7

125 -RNA

22 tRNA n~od ng gen

13 prot~n-encOdlnQ reil ons

Ojtochrnm

Mitochondrial DNA

Control r eIoC rgt or d4oo bull

NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase

Cytochrome Oxidase ~ubunlt~

Figure 2 Mitochondrial DNA (Source Wikipedia)

Mammalian mitochondrial genome is a closed circular double-stranded mitochondrial

genome about 15000 base pairs long The mitochondrial genome of animal encodes product

whereas the mitochondrial genome for fungi and plants have extra DNA that does not code

for any product (Russell 2006 Sutarno 2010)

mtDNA is maternally inherited and the offspring are clones for mitochondrial DNA

genes (Jiang amp Ott 2010 Russel 2006) This pattern of inheritance allows matriarchal

lineages to be traced and provides a mean for examining family structure in some population

This in conjunction with the rapid and regular rate of accumulation of nucleotide sequence

differences has allowed mitochondrial DNA to become a valuable tool for comparing closely

related lineage (Russell 2006)

8

~ubunll~

ATP Synthase subunits

Mitochondrial DNA is represented the most infonnative genomIc element

polymorphic sites and simple maternal inheritance without recombination in purpose to study

the population of many organism or livestock domestication (Joshi et ai 2004 Machugh amp

Bradley 2001 N aderi et ai 2007) In addition polymorphism accumulates approximately

10 to 17 times faster in mtDNA compared with nuclear DNA (Jiang amp Ott 2010) Thus

DNA is able to provide more infonnation compare to the protein because it has many non

coding regions and declining of genetic code

Cytochrome Oxidase I (COl)

Cytochrome Oxidase I (COl) is encoded by mitochondrial genome mtDNA and was exhibit

one of the most heterogeneous rates of amino acid replacement among placental mammals

COl is important enzyme which is found in all organisms that perfonn aerobic respiration

The COl gene is short genetic sequence of 648 base pairs which can be easily extracted from

cells the most slowly evolving gene and conservative protein-coding genes in the

mitochondrial genome of mammals which was preferable for the evolutionary (Maderankova

amp Provaznik 2010 Simon et ai 1994)

Besides that mitochondrial gene COl also was applied in analysis of DNA bercoding

in screening the mtDNA gene of all species and thus creating databases of COl for assigning

of unknown individuals to species and discovery of new species COl gene is suitable for

identification of animals especially birds fish and insect but it is not suitable for plants As

COl is mitochondrial gene it evolves quickly and therefore it is possible to identify close

related species and new species (Maderankova amp Provaznik 2010)

9

Genetic Studies on Domestic Goat and Other Ovine

Genetic diversity is the best measure for genetic variation within a population There are

many previous studies on genetic diversity of goat by using different kind of DNA markers

for example a study by Rout et al (2008) which analysed Indian goat using microsatellites

marker showed higher level of gene diversity compared with the European and Asian goats

breeds Studies on goat genetics by using random amplified polymorphic DNA (RAPD)

molecular marker have also proven to be an efficient tool for quantification of genetic

diversity at the population levels (Gaali amp Satti 2009 Oliveira et al 2005)

Report by Mannen et al (2001) using the displacement loop (d-loop) and cyt b gene

revealed that the domestic goats are genetically affected by two subspecies of bezoars The

HVI of the mtDNA control region have also been used in study of phylogenetic history of

human and domestic animal because it showed higher level of polymorphism (Luikart et al

2001 Naderi et al 2007) Analysis of genetic distance mismatch distribution and

comparison with wild sheep has been studied by Meadows et al (2007) where the analysis

was carried out by using the DNA sequencing consisted of mitchondrion control region (525

bp) tRNA phe and l2s rRNA (535 bp) and cyt b gene (967 bp) According to Mackay et al

(1985) and Jiang amp Ott (2010) mammalian mitochondrial DNA repeatedly contains a short

DNA d-loop at the heavy strand origin of replication and has been completely sequence in

related species to farming and agriculture

Joshi et al (2004) studied of phylogeography and origin in Indian domestic goat

using mtDNA sequence data from HVRI regions They found the evidence for population

structure and novel lineage in Indian goat that cannot merge the genetic diversity were found

within the major lineage with domestication starting 10000 years ago from a single mtDNA

10

ancestor Naderi et aI (2001) used also HVI segment of the mtDNA d-Ioop in their studied

to characterize the domestic goat mtDNA diversity based on a worldwide sampling Genetic

variations were found among goat haplogroups with the weak phylogeograpfic structure The

weak phylogeographic structure has been explained by a high mobility of goat species in a

relation to human migration and commercial trade Ruo-Yu et aI (2006) have been studied

about genetic diversity of mtDNA d-Ioop and the origin of Chinese goats for protection of

goat breed resources and goat breeding

11

1iii

MATERIALS AND METHODS

Sample Collection of Goats

A total of 20 samples goat (Capra hircus) used in this study (Table 2) were collected from

the Handalas farm Matang Sarawak Samples goat was sampled using buccal cell collection

The samples of buccal cells were categorised based on their gender lineage and also

morphological identities The buccal cells samples were collected by using cotton swab and

the cotton swab were preserved in each tube containing Phosphate Buffered Saline (PBS)

buffer The buccal cells were stored in -20De freezer upon returning to the laboratory for

DNA extraction

Table 2 Description of the goat used in this study

Tag name Description

RATEN Male (mating)

K021 K032 L395 MOKLI Female (mating)

P4669369172B303P206D2P454A

H054

Female offspring (mating)

L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction

DNA was extracted from the goat samples using the CTAB method (Grewe et al 1993) The

buccal cells sample was transfer into new 15 eppendorf tube 100 III CTAB buffer and 20 III

proteinase K was added into the eppendorf tube Another 600 III CTAB buffer was added into

the eppendorf tube and the solution was mixed well Then the eppendorf tube was incubated

in water bath for 2 to 3 hours until the DNA samples dissolve After the DNA samples was

dissolved 600 III chlorofonn-isoamyl alcohol (24 I) was added and the solution was shaken

for 2 to 3 minute followed by centrifugation of the eppendorf tube for 20 minutes at 13 000

rpm The upper layer of supernatant was transfer into a new 15 eppendorf tube

Cold absolute ethanol was added into eppendorf tube and the solution was mixed

well The eppendorf tube was sit on the bench for a few minutes followed centrifugation for

20 minutes at 13 000 rpm The ethanol was discarded carefully and the pellet was washed and

precipitated with an equal volume of ethanol and 25 III of 3M NaOAclNaCI The eppendorf

tube then was undergone centrifugation process for 20 minutes at 13 000 rpm The ethanol

was discarded carefully to ensuing the pellet is not poured out

Small droplet of liquid that was present on the eppendorftube was left for evaporation

or sucked by using tissue The pellet was suspended in 30 III distilled water (ddH20) After

DNA had been extracted quality and approximate yield was detennined by electrophoresis in

a 1 agarose gel containing ethidium bromide at 90 Volts for 30 minutes The gel was

photographed under UV light Isolated genomic DNA was used for mtDNA analysis was

place at -20degC

13

T


Cytochrome Oxidase I (COl) of the mtDNA gene was amplified by using Polymerase Chain

Reaction (PCR) The amplification was done on a Gradient Thermo Cycler (Biorard)

machine for 35 cycles with 25 III of reaction volume For PCR reaction the following

components were mixed in the order that containing of lOX PCR Buffer (Promega) 50 mM

Magnesium Chloride (MgCI2) 2mM Deoxyribonucleotide triphosphates mixture (dTTP

dA TP dCTP and dGTP) 1 III of template DNA molecule of each sample sterile distilled

water (ddH20) 10 pmolllli forward primer 10 pmolIll reverse primer and 5ulll Taq DNA

Polymerase

Two universal primers of COl sequence that were used in the PCR amplification below

Table 3 Primers that were used in peR amplification

Primers sequence Sequences 5 to 3

COl Forward (VRld tt) 5-TGT AAA ACG ACG GCC AGC TCT CAA

CCA ACC ACA ARG A Y A TYG G-3

COl Reverse (VRld tt) 5-CAG GAA ACA GCT ATG ACT AGA CTT

CTG GGT GGC CRA ARA A YC A-3

In every amplification one negative control was used to monitor the validity of the

PCR reaction Temperature profile for 35 amplification cycles was pre-denaturation (94degC

for 2 minutes) denaturation (94 degC for 1 minute) annealing (51degC for 1 minute) extension

(72 degC for I minute) and final extension (72 degC for 5 minutes) After amplification the PCR

14

1

products were run on 1 agarose gel stained with ethidium bromide at 90 volts for 40

minutes and photographed under UV light in order to determine the best annealing

temperature after optimization peR products were purified prior to sequencing using

purification kit (Promega)

Agarose Gel Electrophoresis

The 05g of 1 agarose gel was prepared and mixed with Tris-Acetic acid-EDTA (IX TAE)

buffer The solution was heated for 1 minute until the agarose powder totally dissolved The

solution was poured into the clean conical flask and 1 ~l Etbr was added into it The conical

flask was shakes slowly Then the solution immediately poured into the electrophoresis tank

The solution was cooled down for 10 minutes until the gel solidify 1 ~l a Fermentas ladder

was loaded at front of the well followed 2 ~l each peR products The peR products were run

on 1 agarose gel stained with ethidium bromide at 90 volts for 40 minutes and

photographed under UV light

Purification

peR amplified products were extracted and purified before send for DNA sequencing The

purification process was done by using the purification kit (Promega) according to the

manufacturers instruction A brief summary of the protocol is as follow An equal volume

(23 ~l) of membrane binding solution was added into the peR reaction A mini column was

inserted into the collection tube and prepared peR product was transfer into the mini column

assembly The mini column was undergone centrifugation at 14000 rpm for 3 minutes and

the supernatant was discarded Then the mini column was reinserted into the collection tube

15

T

700 fll of membrane washing buffer (within ethanol added) was added into tube and

undergone centrifugation at 14000 rpm for 2 minutes The supernatant inside tube was

discarded and the mini column was reinserted into collection tube

The DNA pellet was washed by using 500 fll washing buffer and was undergone

centrifugation at 14000 rpm for 5 minutes in order to dry the mini column and eliminate the

excess ethanol inside the mini column The mini column was transferred into new 15 ml

eppendorf tube and 28 fll of Nuclease-free water was added into mini column followed by

incubation for 1 minute Thus the mini column has undergone centrifugation at 14000 rpm

for 5 minute 2 fll of the purified PCR product was checked by running 1 agarose gel to

ensure the sufficiency recovery of the purified PCR obtained The purified DNA was then

stored at -20 0 C and sent to a commercial company First Base (First BASE Laboratories Sdn

Bhd Malaysia) for DNA sequencing Only forward primer was used to sequencing the DNA

Data Analysis

The data from DNA sequencing process was analysed using CHROMAS LITE software The

sequences then were exported to fasta format The sequence was opened in Notepad and all

the N s and ambiguities were deleting before combining all the sequences After that the

DNA sequences were aligned using CLUSTAL X (Thompson et al 1994) and the data were

save as aln and nxs file

The sequences then were further analysed in MEGA 4 using phylogenetic test The

MEGA software has been to provide tools for exploring discovering and analysing DNA

16

ACKNOWLEDGEMENTS

Alhamdulillah and grateful to Allah SWT because I had been completed this thesis Firstly I

would like to express my sincere gratitude and thanks to my research supervisor Dr Yuzine

Bin Esa for his guidelines attention kindness encouragement and moral support gave to me

to finish my Final Year Project

I express my gratitude to my co-supervIsor Dr Hairul Azman for his comment

guidance and recommendations during completing my thesis

The special thank go to all lab assistants postgraduate students my laboratory mate

and all my dear friends for their invaluable moral support motivation and companionship

during preparation of this thesis

Finally I declare my best appreciation to my parents and all my family for their

endless help support and confidence

TABLE OF CONTENTS

Acknowledgement I




Abstract

Introduction 2


Domestic Goat 5


Mitochondrial DNA 8





DNA Extraction 13



Purification 15

DNA Analysis 16

II

Results 18



Purification 21





Discussions 29



Purification 31




References 35

Appendix A 38

III


Chircus

CTAB

COl

d-loop

cyt b

DNA

dNTP

PCR

MEGA

MgCl2

mtDNA

NaCI

NaOAc

Etbr

NJ

MP

PCR

Rpm

bp

~I

degC

Capra hircus



Displacement Loop

Cytochrome b





Magnesium Chloride

Mitochondrial DNA

Sodium Chloride

Sodium Acetate

Ethidium Bromide

Neighbour-joining

Maximum Parsimony


Rotation Per Minute

Base pair

Microliter

Degree Celcius

IV

LIST OF TABLES










v

LIST OF FIGURES






reaction 20



21



22


VI



VII

11





ABSTRACT



ABSTRAK



1

INTRODUCTION






















2


among domestic goat











3






sequences

4

2 Ii i at

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16

TABLE OF CONTENTS

Acknowledgement I




Abstract

Introduction 2


Domestic Goat 5


Mitochondrial DNA 8





DNA Extraction 13



Purification 15

DNA Analysis 16

II

Results 18



Purification 21





Discussions 29



Purification 31




References 35

Appendix A 38

III


Chircus

CTAB

COl

d-loop

cyt b

DNA

dNTP

PCR

MEGA

MgCl2

mtDNA

NaCI

NaOAc

Etbr

NJ

MP

PCR

Rpm

bp

~I

degC

Capra hircus



Displacement Loop

Cytochrome b





Magnesium Chloride

Mitochondrial DNA

Sodium Chloride

Sodium Acetate

Ethidium Bromide

Neighbour-joining

Maximum Parsimony


Rotation Per Minute

Base pair

Microliter

Degree Celcius

IV

LIST OF TABLES










v

LIST OF FIGURES






reaction 20



21



22


VI



VII

11





ABSTRACT



ABSTRAK



1

INTRODUCTION






















2


among domestic goat











3






sequences

4

2 Ii i at

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16

Results 18



Purification 21





Discussions 29



Purification 31




References 35

Appendix A 38

III


Chircus

CTAB

COl

d-loop

cyt b

DNA

dNTP

PCR

MEGA

MgCl2

mtDNA

NaCI

NaOAc

Etbr

NJ

MP

PCR

Rpm

bp

~I

degC

Capra hircus



Displacement Loop

Cytochrome b





Magnesium Chloride

Mitochondrial DNA

Sodium Chloride

Sodium Acetate

Ethidium Bromide

Neighbour-joining

Maximum Parsimony


Rotation Per Minute

Base pair

Microliter

Degree Celcius

IV

LIST OF TABLES










v

LIST OF FIGURES






reaction 20



21



22


VI



VII

11





ABSTRACT



ABSTRAK



1

INTRODUCTION






















2


among domestic goat











3






sequences

4

2 Ii i at

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16


Chircus

CTAB

COl

d-loop

cyt b

DNA

dNTP

PCR

MEGA

MgCl2

mtDNA

NaCI

NaOAc

Etbr

NJ

MP

PCR

Rpm

bp

~I

degC

Capra hircus



Displacement Loop

Cytochrome b





Magnesium Chloride

Mitochondrial DNA

Sodium Chloride

Sodium Acetate

Ethidium Bromide

Neighbour-joining

Maximum Parsimony


Rotation Per Minute

Base pair

Microliter

Degree Celcius

IV

LIST OF TABLES










v

LIST OF FIGURES






reaction 20



21



22


VI



VII

11





ABSTRACT



ABSTRAK



1

INTRODUCTION






















2


among domestic goat











3






sequences

4

2 Ii i at

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16

LIST OF TABLES










v

LIST OF FIGURES






reaction 20



21



22


VI



VII

11





ABSTRACT



ABSTRAK



1

INTRODUCTION






















2


among domestic goat











3






sequences

4

2 Ii i at

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16

LIST OF FIGURES






reaction 20



21



22


VI



VII

11





ABSTRACT



ABSTRAK



1

INTRODUCTION






















2


among domestic goat











3






sequences

4

2 Ii i at

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16



VII

11





ABSTRACT



ABSTRAK



1

INTRODUCTION






















2


among domestic goat











3






sequences

4

2 Ii i at

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16





ABSTRACT



ABSTRAK



1

INTRODUCTION






















2


among domestic goat











3






sequences

4

2 Ii i at

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16

INTRODUCTION






















2


among domestic goat











3






sequences

4

2 Ii i at

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16


among domestic goat











3






sequences

4

2 Ii i at

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16






sequences

4

2 Ii i at

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16

LITERATURE REVIEW





Kingdom Animalia

Class Mammalia

Order Artiodactyla

Suborder Ruminantia

Family Bovidae

Subfamily Caprinae

Genus Capra


5























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16























6

i





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16





al 2008)












2007)

(

7

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16

125 -RNA

22 tRNA n~od ng gen


Ojtochrnm

Mitochondrial DNA


NAOH D hydroj n s

iubunlt

NADH Dehydrogenase

s u_bunts

NADH Dehydrogenasa

5U nits

Oxidase













8

~ubunll~























9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16






















9
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16
























10








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16








11

1iii









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16









DNA extraction



RATEN Male (mating)


P4669369172B303P206D2P454A

H054


L387 T452 T478 0313 K014 K008

TA82A K003 R223

Female (hybrid)

12

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16

DNA Extraction




















place at -20degC

13

T









Polymerase












14

1














Purification








15

T













Data Analysis








16

T









Polymerase












14

1














Purification








15

T













Data Analysis








16

1














Purification








15

T













Data Analysis








16

T













Data Analysis








16

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