UNIVERSITI PUTRA MALAYSIA

DEVELOPMENT OF A PCR - CYCLE SEQUENCING SYSTEM FOR ANIMAL MITOCHONDRIAL DNA ANALYSIS

LAU CHIN HOON

FSAS 1994 10

DEVELOPMENf OF A PCR - CYCLE SEQUENCING SYS'l'F)f

FOR ANIMAL MI'l'OClIOODRIAL DNA ANALYSIS

By

LAU anN HN

--0'" - - _ ... ... _'-" ... _ ... _ ... .....,_ ... __ .. _- .L .... .... ,,- ... \.6 ..... ...,."' .. J

Science and Environmental Studies, Universiti Pertanian Malaysia

March 1994

Dedi cated to the readers :

Be t rue and s imp l e . If not , p l ease l eave sci ence a l one .

My heartiest gratitude to:

Dr. Khat i jah Yusoff, Professor Dr. Yukio Yamada and Associate

Professor Dr. Tan Soon Guan for their support in this research and supervision of this thesis;

Associate Professor Dr. Norani A. Samad for access to her facility at the Virology Laboratory, and also her capacity as the Head of Department of Biochemistry and Microbiology during the period of my

M.S. candidacy;

Ms Lau Pin Sian, Mr. Kok Kah Mun and Mr. Lau Lam Kwee for their company at Serdang and for their assistance which rid the worry of daily life from my mind, without whom I could not possibly be able to concentrate on my research;

Associate Professor Dr. Gan Yik Yuen for introducing population

genetics of mitochondrial DNA to me;

Dr. Hiroshi Yasue and his officers a t the Laboratory of Cell Genetics, National Institute of Animal Industry, Tsukuba, for

providing a 2-month training in 1992; the stint was sponsored by the

Society of Techno-innovation in Agriculture, Forestry and Fisheries (STAFF), Japan;

Mr. Ng Ban Kim, Mr. Tan Wen Siang and all other researchers at Lab

142 / 143 whose enchanting interest in molecular genetics has liven my life in the lab;

Mr. Julian Lim of Bio-diagnostic for regular updates on advances in

molecular techniques.

This study was supported by Intensification of Research in Priority Areas (IRPA) grant no. 1-07005-041 and 1-07005-027

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

LIST OF F IGURES

L I ST OF PLATES

LIST OF ABBREV IAT IONS

ABSTRACT

ABSTRAK

CHAPTER

I

I I

INTRODUCT ION

L ITERATURE REV I EW

Introduction

Structure and Characteristics of Mitochondrial DNA . . . .

Approaches in the Analysis of Mitochondrial DNA Variation

Restriction Analysis .

Sequencing of Mitochondrial DNA

Polymerase Chain Reaction . . . .

Methods of DNA Sequence Analysis

iv

PAGE

iii

vi i

viii

ix

xi

xiii

1

5

5

5

8

8

12

14

1 6

I I I MATERIALS AND METIIODS 22

IV

Blood and DNA Samples 22

Chicken Livers Mitochondrial DNA . 22

Chicken Blood 23

Water Buffalo 23

Other Animals 24

Analysis of DNA Samples by Electrophoresis . . . . 24

Extraction of Blood Mitochondrial DNA . 25

Construction of Oligonucleotide Primers 25

#1. D-loop Primers UPM257 and UPM258 25

#2. Cytochrome b Primers KY1 and KY2 26

#3. D-ioop Primers LCH2-UP and LCH4-DO 26

Polymerase Chain Reaction . 28

Purification of PCR Products 29

By Column Chromatography 29

By Gel Electrophoresis . 29

Selective Alcohol Precipitation 30

DNA Sequencing 30

RESULTS and DISCUSSIONS 32

Polymerase Chain Reaction 32

D-loop Amplification 32

Cytochrome b Amplification 32

v

Pur i f icat ion and Sequencing of PCR Product s . . . . . . . . . . . . • 48

Pur i f icat i on of PCR Fragmen t s by Low Me l t ing Point Ge l E l ectrophores i s and by Co lumn Chromatoghaphy 48

Pur i f i cat i on by Se l ective Ethanol Precipi tat i on . 51

Genera l Di scuss ion 54

Rat iona l e in Method Deve l opment 54

Po lymerase Cha in Reaction 55

DNA Sequence Fide l i ty . 5 5

Sequence Qua l i ty 56

Sequence Out put . 57

Temp l ate Concent ration 58

Limi tat ion of CUrrent Met hod and Recommendat i ons for Future Pract i ce 59

CONCLUSION 65

B IBLI OORAPHY 68

APPENDI CES 75

VITA

A . Protoco l for Choma Spin Co l umn Chromatography . . . . 76

B. Protoco l for Therma l Cyc l e Sequencing w i t h Labe led Incorporat ion . . . . . . . 77

C . Par t i a l Cytochrome b DNA Sequence of Bovine ( cytb Bovine) and Chi cken ( Cytb Leghorn ) 78

D. Chi cken D- l oop DNA Sequence 79

81

vi

F i gure

1

2

3

4

LIST OF FIGURES

The Genomi c Organ i sat ion of t he Chi cken MtDNA Compared wi t h t hat of other Vertebrates . . . . .

Compar i son of DNA Sequencing Protoco l s between ( l )Di rect Sequenc i ng of Denatured Doubl e-St randed DNA; ( 2) Sequenc ing of Sing l e-St randed Asymmet r i c PCR Product and ( 3 ) Cyc l e Sequenc ing . . . . . . . .

Locat ion of D- l oop and Cyt ochrome b PCR Pr i mers . . . . . . . . . . . .

A l ignment of Pr imer KYl Leghorn Sequence w i t h t hose of Human ( Hum ) , Bovi ne ( Bov ) , Frog ( Xen ) and Kocher ( Koc ) Un iversa l Pr imer Showing Conservat i on at 3' Region .

v i i

Page

7

19

26

40

LIST OF PLATES

Plate Page

1 The Results of PeR Amplification of Chicken D-loop from Liver MtDNA . . . . . . . . . . 33

2 Analysis of Chicken D-Ioop Amplification from Liver MtDNA 34

3 (a)Amplification of Chicken D-Ioop 3' Region from Blood by UPM 258/ LCH2-UP and (b)PeR Products from Extended 15 Cycles of Amplification . . . . . . . . . . . . . 38

4 Amplification of D-loop 5'-Region from Blood by Primer UPM257/LCH4-DO . . . . . 39

5

6

7

8

Amplification of Cytochrome b from Chicken Liver MtDNA Samples .

Analysis of Amplified Cytochrome b from Buffalo . . . . . . . . . . .

Analysis of Amplified Cytochrome b from Other Farm Animals . . . . .

PeR of Cytochrome b from Chicken Blood . . . . . . . . . . . .

9 Sequence Autoradiogram of Column Purified (Panel 1 and 2) and Agarose Gel Purified (panel 3 ) Cytochrome b PeR

10

Product

Comparison of Sequence Quality Obtained from DNA Purified with Chroma Spin-lOO and Selective Precipitation . . . . . . . . .

viii

41

43

44

45

50

53

bp

cyt. b

dNTP

ddNTP

Kb

min

roM

ug

ul

ng

O D

PCR

pmol

s

TEE

TE

v/v

w/v

LIST OF ABBREVIATIONS

base pair

cytochrome b

deoxyribonucleotides

dideoxyribonucleotides

ki lobase

minutes

millimolar

microgram

microlitre

nanogram

optical density

Polymerase Chain Reaction

picomole

seconds

Tris-borate-EDTA buffer

10 roM Tris.Cl, 1 roM EDTA

volume/volume

weight/volume

ix

Abstract of thes i s subm i tted to the Senate of Un i vers i t i Pertanian Malaysia in fulfilment of the requirements for the degree of Master of Science .

DEVELOPMENT OF A PCR - CYCLE SEQUENCIOO SY� FOR ANIMAL MITOCHONDRIAL DNA ANALYSIS

By

LAU CHIN HN

March, 1994

Chairperson: Khatijah Yusoff, Ph. D.

Facul ty Science and Environmental Studies

A system for the study of sequence polymorphisms in animal

mitochondrial DNA was developed . This system consists of two

candidate markers which are mitochondrial cytochrome b and D-loop .

Six novel primers were constructed for their amplifications by

polymerase chain reaction (PeR) . The primers KYl and KY2 amplified

a 359-bp port ion of the cytochrome b gene from chicken, water

buffalo, horse, goat and sheep . Primers UPM257 and UPM258 were used

to synthesize a 1 . 4 Kb-fragment containing the complete chicken

mi tochondrial D-loop . Primers LCH4-DO and LCH2-UP paired wi th

primers UPM257 and UPM258 respectively to amplify a 355-bp 5' region

and 395-bp 3' region of the D-loop . These primers were specific in

their amplifications . The mitochondrial DNA could be amplified from

x

heterogeneous or degraded DNA preparat i ons , as we l l as d i rect l y from

b l ood i ncubated in proteinase K t hus bypass i ng l engthy DNA iso l at i on

steps . On ly 5 u l of b l ood was requi red for a lOO-u l PCR .

For t he pur if i cat i on of PCR produ c t s , e t hano l sele c t i ve

preci p i t at ion was found to be bot h e f f i c i ent and econom i cal . Despi te

i ts l ower recovery compared to the Chroma Spin co l umn , suff i c ient

quant i ty of DNA cou ld be obtained for subsequent sequencing.

The sequence of the amp l i f i ed doubl e-st randed cytochrome b was

effect ive l y determined by h i gh temperature cyc l e sequencing whi ch

does not requ i re t he product i on of s i ng l e-s t randed DNA or pre

react i on denaturat ion of temp late DNA and pr imer anneal ing . Clean

sequence l adder was obtained us ing 35S dATP incorporat i on .

The system deve loped i n t his research i s s i mp l e , w i th the gene

amp l i f i cat ion and sequencing be i ng hand l ed by a t hermal cyc ler . The

semi -automat i c nature of t h is system wou l d make the product i on of

dat a on mtDNA polymorphisms faster wi t h m i n i mum operat ion errors .

These features are part i cu l ar l y usefu l for anal yz ing l arge numbers

of sampl es in populat ion stud i es . Moreover , a cyc l e-sequencing based

met hod w i l l undoubtedly dominate sequence anal ys i s work in mo l ecu l ar

b i o logy .

xi

Abs t rak tes i s yang d ikemukakan kepada Senat Univers i t i Per t an i an Ma l ays i a bagi memenuhi keper l uan unt uk I j az ah Master Sains.

PERKf)f8ANGAN SATU SIS'fF)f PCR - PENJUJUKAN KITARAN UNTUK ANALISIS OOA MITOKONDRIA BINATAOO-

Oleh

LAU CHIN HOON

March, 1994

Pengerus i : Khat i j ah Yusoff , Ph . D .

Faku l t i Sains dan Pengaj i an A l am Seki tar

Sat u s i s tem un t uk mengka j i po l i mo r f i s ma j u j ukan da l am DNA

m i t okondr i a ( mt DNA ) t e l ah d i ma jukan . S i s tem i n i mengandung i dua

cal on penanda iaitu s i tokrom b dan ge lung-D mi tokondr i a . Enam pr imer

baru t e l ah direkabina dal am kaj i an i n i bagi t i ndak ba l as rantaian

po l i merase ( t po lymerase cha i n react ion ' , PCR) gen i t u. Pr imer KYl

dan KY2 d igunakan dal am amp l i fi kas i sebahagi an ( 359 pasangan bes)

gen s i tokrom b dar ipada ayam , kerbau , kuda , kambing dan b i r i -bi r i .

Pr imer UPM257 dan UPM258 digunakan untuk ampl i f i kas i serpi han 1 . 4-

ki l obes yang mengandung i ge l ung-D lengkap m i t okondr i a ayam . Pr imer

LCH4-DO dan LCH2-UP berpasangan dengan pr i mer UPM257 dan UPM258

mas ing-mas ing untuk me l ipat gandakan bahagian 5 ' ( 355 pasangan bes )

dan 3' ( 395 pasangan bes ) dar i ge lung-D. Kesemua pr imer i tu adal ah

spes i f i k da l am ampl i f ikas i . DNA mi tokondr ia bo leh

xi i

diampl ifikasi daripada penyediaan DNA yang t idak tulen ataupun

sempurna, di samping daripada darah yang dieramkan dalam proteinase

k secara terus dan ini dapat mengelakkan Iangkah-langkah dalam

pengasingan DNA . Hanya 5 ul darah diperlukan bagi setiap lOO-ul PCR.

Untuk penulenan produk peR, pemendakan pilihan etanol telah

dikenal pasti sebagai cara yang berkesan dan ekonomik . Walaupun

pemulihannya adalah lebih rendah daripada turus Chroma Spin,

ku a n t i t i DNA y a n g cukup b o l e h d i p e r o l e h i untuk penjujukan

selanjutnya.

Penjujukan bebenang-dua sitokrom b telah dijalankan dengan

penjujukan kitaran (tcycle-sequencing') suhu tinggi yang tidak

memer lukan penghasi Ian DNA bebenang-tunggal dan pra-t indak balas

penguraian templat DNA dan pelekapan primer . Tangga jujukan yang

bersih dapat diperolehi daripada penjujukan sitokrom b yang

menggunakan penyatuan 35s dATP .

Kaedah yang dimajukan ini adalah mudah, dengan amplifikasi dan

penjujukan gen disediakan oleh pemutar haba (tthermal cycler') .

Ciri separa automat ik ini menjadikan penghasi Ian data pol imorfisme

mtDNA lebih cepat dengan kesilapan yang minimum. Semua ini amatlah

berguna untuk anal isis bilangan sampel yang banyak . Selain daripada

xiii

ini , kaedah yang berasaskan penjujukan k i t aran tanpa keraguannya

akan mengaruh anal i s i s jujukan dal am kebanyakan bidang penye l idikan

b i o l og i mo leku l .

xiv

aIAPTER. I

INTRODUCTION

A research programme ent i t l ed tGenet i c Studies of Domest ic An i

mal s ' has been s tarted at t he Univers i t i Per tani an Ma lays i a. The

f i rs t phase of wor k i n t he pro ject was t o i dent i fy a su i tab l e

approach t o t he s tudy o f DNA po l ymorph i sms . As t h i s l ine of research

was new in Ma lays ia , the log i ca l s tart ing po int of t he research was

t he deve lopment of research me t hodo l ogy wh i ch cons i s ted o f t wo

component s . The f i rst component was t he se l ect ion of candidate gene

loci t hat could funct ion as markers for probing genet i c d i fferences .

The second , which was the deve lopment of a technique appropr iate

for det ect ing sequence var i at ions at t hese loc i , const i tutes t he

subject mat ter of this t hes i s . This Chapter gives a review on t he

evo l ut i on of t he research d i rect i on whi ch l ater l ed to t he adopt i on

of t he current approach in genet i c ana lys i s .

The i n i t i a l obj ect i ve was t o per form Res t r i ct i on Fragment

Length Po lymorphi sms (RFLP) ana lys i s of pou l t ry growth hormone and

pro l ac t i n genes . The sources o f bot h t he bov i ne growth hormone

( Gordon et ai . , 1 983 ; Woychi k et a l . , 1 982 ) and pro lact in ( Cooke and

Baxter , 1 98 1 ) genom ic and cDNA probe were located . However , two

1

2

possible pitfalls of applying these probes i n t he hybri d i zat ion

expe r i ment s were ant i c i pated . F i rs t l y , use of t he he t e r o l ogous

probes ( from bovine ) to detect pou l t ry DNA necess i t at ed the l owering

o f h yb r i d i za t i on s t r i ng e n c y and t h i s wou l d r e d u c e t a r g e t

spec i f i c i ty . Second l y , cross hybr idizat ion o f growth hormone to

chor ion i c somatomammot ropin in human had been report ed . A s imi l ar

event ( i . e. cross hybr i d i za t i on w i t hi n t he pro l ac t in fam i l y of

genes and w i t h other growth hormone- l ike genes ) cou l d t ake p l ace in

pou l t ry RFLP anal ys i s . In bot h cases, t here is t he poss ibi 1 i ty of

p i c k i n g u p f a l s e po s i t i ve s wh i c h c o u l d l e ad t o e r r o n e o u s

interpretat ion o f exper imental dat a . Know l edge on the gene s t ructure

of t hese hormones and the l eve l of l oca l techn i cal know-how at that

t ime was cons idered to be i nsuff i c i ent for qua l i t y assurance in th i s

work .

Informat ive res t r i ct ion enzymes and e thni c speci f i c mi tochond

ria l DNA ( mtDNA ) types have been ident i f ied in human . I t is thought

t hat s imi l ar f ind ings cou l d be obtained in domestic an ima l s; this

be ing the a l ternat ive approach for det ect ing genet ic po lymorph isms.

For this purpose, th e research then concen t ra t ed on deve l oping

t e c h n i q u e s for i s o l a t i on of m t DNA f r om ch i c k e n l ive r s and

pur i f i cat ion of the mt DNA by cae s i um ch l or ide dens i ty grad i en t

u l t racen t r i fugat ion.

3

As the understanding and ins ight for the popu l at i on geneti cs of

m t DNA g r ew deepe r , t he n e ed f o r an a l t e rnat i ve s t r a t egy was

gradua l l y fe l t . Some informati on and exposure had been ins t rument a l

in the shi ft o f research st rategy away from the RFLP-based method .

First l y, using the mtDNA-RFLP method, Wakana et al ( 1986 ) found only

one restriction variant from 1 6 chi cken var iet ies examined. Such a

low l eve l of polymorphism was indeed unexpected for mtDNA . It is

on l y by DNA sequencing that a more thorough screening of the genome

could be made to resolve whether this l ow l evel of variation was due

to a technical drawback of restriction analysis or to a resu l t of an

evolut ionary bott l eneck. Second l y, A PCR-based method wou l d be a

more superior way of studying genetic variation. Furthermore, the

comp l ete chicken mi tochondrial sequence became avai lable and this

made possible the construct ion of PCR 01 igonucleot ide primers. The

then arrival of a therma l cycler at the l aboratory cata l yzed the

adopt ion of peR-sequencing strategy in the research, which formed

the core of this thesis.

In ana l yzing mitochondria l DNA po lymorphisms. DNA sequencing

undoubtedly yields the f inest l eve l of reso l ution and suits most

of the problems. However, sequenc ing of large numbers of samp l es

needed for popu l ation stud ies can be tiresome and time-consuming.

The obj ective of t his thesis research was to a l l eviate these prob

l ems by improving DNA templ ate generation and the reaction process.

4

To achieve this end, two cur rent technological advances - the

p o l ymer ase chain r eaction (peR) coup l ed to cycle o r l inear

amp l ificat ion sequencing were ut i l ised.

comprised the fo l l owing:

This research, therefore,

1. Design of suitabl e PeR and sequencing primers;

2 . se l ection of a DNA extraction method ;

3 . devel opment of a PeR condition for mtDNA ampl ification

4. identification of a suitabl e method for purifying amp l ified

product, and

5 . deve l opment of high temperature sequencing. The mito

chondria l cytochrome b and D- l oop were se l ected as markers for

futu r e studies of genet i c va riation in l o cal chicken. water

buffa l oes and other domest ic animal s. Natura l l y, the DNA fragments

from these loci were used as temp l ates in the devel opment of PeR and

cyc l e sequencing .

It is anticipated that the technica l foundation bui l t up in

this present work wi l l be capab l e of carrying the overa l l research

into the next phase which is the co l lection of data on mitochondrial

DNA po l ymorphisms. Furthermore, the simpler. semi-automated PCR-se

quencing method deve l oped in this pro j ect wou l d a l l ow more person

time to be re-a l l ocated from mechanica l data generation into conso l

idating research planning, data analysis and app l ication and explo

rat ion of new research area which wou ld improve the management of

l oca l anima l genetic resour ces.

CHAPfER II

LITERATURE REVIEW

Introduction

Animal mitochondrial DNA (mtDNA ) had been studied extensively to

address problems in populat ion genet ics and systemat ics of closely

related species and individuals within a species. It had been ap

plied to the study of matrilineal relationships in a population. the

h i story of speciat ion. and geographic populat ion structure. These

studies had been thoroughly reviewed by Avise et 81. ( 1986; 1987 )

and Moritz et 81. ( 1 98 7) . In animal breeding, mtDNA has been used as

a molecular marker in the study of productive traits and the degree

of genet ic variabi 1 i ty (Hecht , 1990) . This chapter would give an

overview of the major techniques in populat ion genet ic analysis of

mtDNA which was considered in the course of methodology selection.

The emphasis would be on the PCR and DNA sequencing technology.

Structure and Characteri sti cs of Mitochondrial DNA

Animal mitochondrial DNA (mtDNA) is a covalently closed circu

la r mole cule (Walla c e. 19 8 6) . Th e c o m p l e t e genome in many

vertebrates had been sequenced. It contains genes for 13 protein

5

6

subuni ts funct ioning in ATP pro duct ion, 2 ribosomal RNAs (12s and

16s) , 22 t ransf er RN A s an d a non-co ding region known as

d isplacement-loop (D-Ioop) (it is common that the tD-loop ' and the

tnon-coding region' a re used interchangeably). The gene order is

conserved in human ( Anderson et ai., 1981 ) , mouse ( B ibb et al.,

1981) and bovine ( Anderson et ai., 1982). Some rearrangement s were

present in chicken, quail, Guinea fowl, pheasant and t urkey ( Desjar

d ins and Mora is, 1990) , in wh ich t he cont iguous t RNAGlu and ND6

genes are locat ed adjacent t o the D-Ioop (F igure 1) .

There are unique features wh ich make mtDNA an ideal molecular

syst em f or populat ion analysis . Fi rs tly, i t s ub iqu i t y enables

comparison of data between species. Secondly, the complicat ion of

genet ic recombinat ion or rearrangement is bypassed due to it s ma

t ernal inheritance. In m ice, only 10-4 of the mtDNA is paternally

inheri t ed relat ive to the maternal contribut ion (Gyllensten et ai.,

1991) . Th irdly, the mtDNA has a base subst itut ion rat e of 1% per 106

years ( Brown et a1., 1979) which is 1 t o 10 t imes faster than in

s ingle-copy nuclear DNA ( Av ise et a1., 1987) . Th is result s in

cons iderab Ie sequence variat ion between individuals. Fourthly, the

number of cop ies of mtDNA molecules present was est imated to be 1 000

t o 8000 per f ibroblast (Reis and Goldste in, 1983), and 2.6 x 105

per oocyt e (M ichaels et aI., 1982 ) in bovine . This high copy number,

along w i th the ir compart mental i zed locat ion separat e from nuc lear

DNA, have made mt DNA relat ively s imple t o isolate and assay.

12 S ,RNA. 014

Mammal, �d Xtnopus lonis mtDNAs

AlP ....

NOl

12S,RNA r

N02

Q I Chicken mt DNA -j

AlP ....

Figure 1. The Genomic Organisat ion of the Chicken MtDNA Compared with that of other Vertebrates (Desjardins and Morais, 1990) .

NOS

l S

H

-J

8

Approaches in the Analysi s of Mi tochondrial DNA Variat ion

Mitochondria l DNA had been mainl y studied as a who l e by restric

t ion ana l ysis and in part by DNA sequencing. The choice of either

method depends on the level of resolution requ ired in solving the

issue ( s) at hand, the amount of DNA that can be extracted from the

animals or b io l ogical spec imens and budget expediency. Between the

two approaches, the restr iction ana l ys is method is more commonly

used mainly because it is technical l y less demanding and less costly

compared to DNA sequenc ing .

Restriction Analysis

In the method involv ing restr ict ion analys is ( also known as

trestr ict ion fragment length po l ymorph ism ' , RFLP ) , the sequence

var iat ion is revea l ed by the number and s ize of DNA fragments

produced by restr iction endonuclease digestion. The var iations arise

f r om base subst itut ion, add it ion or delet ion. In order to be

detected and become informat ive, the sequence var iat ion must be

located in the recogn ition sequence or within two cleavage sites

( in the case of l ength mutat ion ) of the restrict ion enzyme being

used in the study. The restricted fragments are separated by ge 1

e l ectrophoresis and can be detected by b l ot hybridization,

radioactive end- l abe l l ing or fl uorescence using ethidium bromide.

9

In the blot hybridi z ation method (Southern, 1975). the

restricted DNA is transferred onto a membrane after electrophoresis.

The immobilized DNA is then detected by a labelled probe prepared

from purified mtDNA. In this method, 2-7 ug of total cellular DNA

extracted from blood (Ferris et a1., 1981) or about 0 . 2 ug of

platelet DNA (Denaro et a1 . • 1981) is required. Since the fragment

1 abe II i n g is mt DNA probe-d i rect ed. the samp 1 es need not be

homogeneous. The paper of Johnson et a1. (1983) studying divergence

of human ethnic groups is an elegant classic example of applying

this technique: restrict ion endonuc lease morphs (banding patterns)

were sorted into mtDNA types. and subsequent ly genet ic distances.

time of divergence and evolutionary rates were calculated. In

Malaysia, the blot hybridization approach had been applied to a

preliminary

a1., 1991).

study of buffalo restriction cleavage patterns (Gan et

In contrast to the tolerance to sample impurities in the hy

bridization method, the other two methods of detecting restriction

fragments (Brown. 1980� Cann and wi Ison, 1983) requires that the

mtDNA be intact and of high purity. These criteria are meant to

prevent false positive and high background which complicate band

scoring. Choice of either one of the identification methods depends

on the amount of mtDNA available. They both normally require 1 or 2

rounds of caesium chloride density gradient centrifugation. The

10

radioactive based methods are sensitive in that picogram quantity of

DNA can be detected and restriction analysis can be carried out in a

situation where the amount of DNA is limiting. The ethidium bromide

staining method, on the other hand, requires at least 3-5 ng of DNA

per band for detection. and as much as 8 ug of mtDNA is required for

HaeIlI digestion (Horai and Matsunaga. 1986). However. due to the

requirement for special containment and the heal th hazard, the

radioisotope based methods are not always favourable. In addition.

the blot hybridization has the added disadvantage of requiring too

many steps and too many days for DNA detection.

Abundant DNA had been isolated from liver or brain in cow

(Hauswirth et a1., 1984), heart or pectoral muscle from birds (Ball

et a1., 1988) and whole bodies of fish (Bermingham et a1., 1991). In

addition. 200 to 300 ug closed circular mtDNA had been recovered

from individual placenta (Horai et a1., 1984). The availability of

mtDNA isolated from some of these sources makes feasible the use of

the direct fluorescent detection method which is fast, inexpensive,

and justifies the labou r and expense in DNA extr action and

purification. As the experimental procedure is simple, confirmation

or duplication of results or trial of new restriction enzymes can be

easi Iy done long after the original experiment. Use of the other

methods require more effort in setting-up and material preparation.