subjects and methods - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/44297/9/09...subjects...
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Subjects and Methods
3.1 Introduction to Area
3.2 Subject
3.3 Methods
3.4 Laboratory Methods
3.5 Isolation of Genomic DNA from Human Blood
3.6 Wet DNA Lab Analysis: Polymerase Chain Reaction (PCR)
3.7 DNA Markers Studied
3.8 DNA Molecular Weight Marker used as Standard ladder
3.9 Agarose Gel Electrophoresis
3.10 Statistical Analysis
Chapter III
50
J.l lntrutluctiun tn Area
].I. I Chhattisgarh State
I .ocakd in ( 'cnlrallndia. ( 'hhallisgarh stale was born, through carving out of about
.n percent area of eastern pari of Madhya Pradesh. on November I. 2000. lo become lh~:
26'" slate oflhc lndianl!nion. Chhallisgarh extends southeast of Madhya Pradesh from
I 7°46'N lo 24°06' N latitude and fromlW0 IS'E to ll4°5I'F longitude Figure].!. Th~: land
of the stale is unique in the sense \hal it has no sea and no connection with llimalaya and yet
it has hills and mountains with big rivers. With an area of 1.35.133 sq.kms. The region has a
population of2.07,95.956 according to Census 2001. As its name implies. the region is
covered by thirty-six forts and is often referred to as "howl of rice". Uttar Pradesh in north,
Jharkhand in the northeast, Orissa in the southeast, Andhra Pradesh in the south. Maharashtra
in the southwest and Madhya Pradesh in the northwest bound it. The state comprises three
Commissioner's Division namely Raipur. Bilaspur and Bastar including sixteen districts. These
districts are 13astar, Kanker (North Bastar), Dantewada (South Bastar), Bilaspur, Janjgir
Champa, Korba, Raigarh, Jashpur, Sarguja, Koriya, Durg, Raipur, Dhamtari, Mahasamund,
Rajnandgaon, and Kabirdham.
3.1.2 Bas tar Division: Geographical location
The Bastar division situated at the extreme southeast corner ofChhattisgarh in India
lies between latitudes 17°46' to 20°34' Nand longitudes 80°15' to 82° 12' E with an area
of about 39,171 sq. kms Figure 3.2.
In the year 1999, the Bastar division has been divided into three districts namely
Bastar, Kanker and Dantewada. All these three districts come under Bastar division with the
divisional head quarter at Jagdalpur, which is also the district head quarter ofBastar district.
The data for the present study was collected from all the three districts of Bas tar division.
The beauty ofBastar division lies in its natural forest area and various types of
tribals. Of the total population more than 70% are tribals like Gonds, Abhujmaria, Bison
horn Maria, Muria, Raja Muria, Jhoria Muria, Doria, Dhruva, Bhatra, Halba etc. Figure 3.3
shows ethnic map ofBastar.
Table 3.1 Area, population, number of villages etc. in three districts of Bas tar division
Bastar District Geographical location Latitude 18.4 North to 19.9North. LDngitude 80.5 East to 82.1 East
Total Geographical Area 8755.79 sq.kms.
Population (As per 200 I Total-1302253 Census) Male - 648068
Figure. 3.1 Map of India showing new Chhattisgarh State
GUJRAT
ARABIAN SEA
LAKSHADWEEP {INDIA)
,,,
GOA
JAMMU AND KASHMIR
MAHARASHTRA
ANDHRA PREDESH
NAIDU
\
SRI\ LANKA)
I N D I A N
-,, "'
IIIIIOOMimmlmiiM~ T 21789
0
"''
INDIA SHOWINGCHHATIISGARH
l'ITATE
,_ MYANMAR (BURMA)
BAY
OF
BENGAL
·~) IJ
AN DAMAN SEA
,,
c E A N
'" '12" 9(,',
"'
M
-
~
'
- CHHATTISGARH STATE
51
Figure. 3.2 Map showing Bastar Division in Chhattisgarh State
''
"'
• State Capital "" District Headquarters • Tahsil Headquarters '~ Area under study
S2
D G
• D D
Figure 3.3 Ethnic Map of Bas tar
' 'b (,' ,c 0
,'t.l\ " 0
01~11 Durg
\)' . 0 Bhonuprotappur
•' '1- Kanker
Gond - Bison Horn Muria
Muria IIliilliiJ] Raia Muria
Choria [[]]]] Dhurwa
Hill Maria Outside Abujhmarh lim Bhathra
Hill Maria of Abujhmarh [EJ Doria
53
0
I h:malc 1>541 X5
!
SC( llJlJI)- XJ4JJ (6.41%) Sl( llJ'II )- 742799 ( 57.(!4"!.>)
lchsils Jagdalpur. Kondagaon Kcshkal. Narianpur
No. of' lllocks (.ian pad l'anchyat) Darhha. llastanar. Makdi. Kondagaon. l.ohandiguda. l'arasgaon. Kcshkal. llastar. Bakawand. Tokapal. lladcrajpur. Narayanpur. Jagdalpur. Abujhmarh (Orcha).
- ------~-- ------ -- ---- ---------- -------
'il>tal Villages 1343 1--- - ...
No. of'Litcrates Total - 488887, Male- 305540, Female- 18334 7
Kankcr Geographical location Latitude 20°6' North to 20°24' North Longitude 80°48' East to 81 °48'East
Total Geographical Area 1285.01 sq. kms
Population (As per 2001 Census) Total- 651333 Male- 3,24678 Female- 3,26655 SC-24130(3.70%) ST- 296584 (45.55%)
Tehsils Kanker, Charama, Bhanupratappur Narharpur, Antagarh, Pakhanjoor
No. of Blocks (Janpad Panchyat) Kanker, Charama, Sarona Bhanu pratappur ,N arharpur, Antagarh, Durgu Kondal, Koyali beda
Total Villages 1074
No. ofLiterates Total- 400709, Male- 225598, Female-175111
Dantewada Geographical location Latitude 18-46-48 N-to- 19-28-30N Longitude 80-15-42 N-to- 81-58-43E
Total Geographical Area 10,238.99 sq. kms
Population (As per 200 I Census) Total-7, 19,065 Male- 3,56,502 Female- 3,62,563 ST -4,76,945 (66.33%)
Tehsils Dantewada, Bijapur, Bhopalpatnam Sukma
No. of Blocks (Janpad Panchyat) Dantewada, Kuwakonda, Katekalyan. Geedam. Sukma.
< 'hhattisgarh 'tate ar~ in Jla,tar. (iond is th~
principal trib~ of Dravidian fiunily. and p~rhaps
th~ most important of non-Aryan offl>r~st trib~s
in dia. Th~y ar~ represented as one of the
Schcdukd Tribes of India according to article
46 of the lnuian Constitution.
The woru 'Uonu' has led to many speculations
and there is some confusion as regarus the origin
of the word 'Gond'. It has been either derived
from 'Kond', which in Tdgu means 'a mountain'
region spread over Western Bihar and Eastern
Bengal. The name by which the Gonds called
themselves is Koi or Koi tur. According to
Grigson (1938) all the Koitur of the Bastar were
officially classed as Gonds, though the name
Gond was hardly known in Bastar. According
l•'igure 3.4 Distribution of Good population in central India
to Russell and Hiralal ( 1916) the derivation of word 'Gond' is uncetiain. 'Gond' is the term
given to the tribe by the Hindus or Mohammedan (Russell and Hiralal, 1975). General
Cunningham considered that the name Gond is probably came from "Gauda' the classical
term for part of United Provinces and Bengal. The word 'Gond" in central province is
frequently or usually pronounced Gur, which is practically as the same sound as god. The
language spoken by them is 'Gondi' a Central Dravidian language of the same family as
Tamil, Canarese, and Telgu and therefore, it is I ikely that they come from the south into the
Central province.
What had distinguish Gonds was not merely their vast numbers, by far the largest tribal
group, but also because of the political power they ruled over a kingdom with fifty-two
garhs, the dynastic through which they continued to rule before they succumbed to Mogul,
and Later Maratha military assaults. Muslim writer described this territories as Gondwana
with the Maharaja exercising control over a number of petty chiefs and administering area
covering Satpura plateau toN armada valley to the South, which now comprises the district
of Jabal pur, Mandla, Seoni, Balaghat, Chhindwara, Betul etc.
The Gond trace their origin and ancestry to Pandava Prince and his famous tribal spouse,
Hindamba (Chhattophadhya, 1978). Among the Gond proper there are two-aristrocratic
subdivisions- the Raj-Gond and Khatolas but out of Gond tribe a number of separate group
have naturally developed. Among them are several occupational castes such as Agarias or
iron workers, Pradhans or priests etc. There are several other small local sub-divisions of
Gonds viz. Muria, Maria Abuj hmaria etc. Chhatopadhyay ( 1978) say in fact it would be
more correct to describe them as various groups of Gond, for each has acquired its own
language and mode of life.
S7
'I h.: thr.:<: r.:gions happ.:n to h.: th<: habitat ofth..:s..: trib..:s r..:pr..:s..:nting tho: thr<:<: ditkr.:nt
habitab ofthc·s.: 1·ast <iond soci<:t) including- tho: Bastar groups: the Orissa group: and. tho:
Satpura group in th.: Chota Nagpur and M.l' r..:gion ..:xcluding Bastar (one.: upon a tim.:
Jlastar h.: longed to the Orissa political region (Mehta. 1984 ).
Th.: ..:ntire region which has been the habitat of the Oonds. historically known as
'(iondwana' contains the northern back of the Godavari river. the Mahanadi and its tributaries.
esrccially the Tel and the Ai. and the Narmada. the Wardha and the Waiganga. the western
<ihats and the Satrura ranges including the Meykul range. The ancestors of the present day
Oonds might have lived in some of these areas even two million years ago. The· Bas tar
group' in the north of the Godavari river appears to be the original home of the Gond tribal
complex (Mehta, 1984 ).
The present study refers to the Gond tribes ofBastar who form the largest single tribal
group. In Bastar Maria, Moria and Doria are the three major sub-tribes ofGond. Among
Maria also there are two sub-sects (I) Hill Maria or Abujhmaria and (ii) Plain Maria or
Dandami Maria or Bison-horn Maria. The present study was confined to the M uri a,
Abujhmaria and Bison-horn Maria ofBastar.
3.2.2 Abujhmaria (ABM)
(Figure 3.5) Ethnically homogenous population- the Abujhmaria, inhabits Abujhmarh
region. Grigson (1938) called the Abujhmaria as Hill Maria and considered it to be most
backward tribe between Godavari and Ganga. Elwin ( 193 8) considered them to be a lowly
section of the Gond tribe. Abujhmaria is one of the primitive tribal groups (PTG) of our
country identified in the Fifth Five-Year Plan. Abujhmaria is considered to be in an extremely
underdeveloped stage by the Scheduled Areas and Scheduled Tribe Commission (1961 ).
This tribe is at very low stages of acculturation. The Government of India identifies it as
PTG on the following criteria:
1 . Very low level of agricultural technology.
2. Level ofliteracy below 5 %.
3. Habitat in remote and isolated areas.
4. Stagnant or declining population growth.
Abujhmaria fulfils the all above requisites to fall into the category ofPTG.
In the general appearance a Hill Maria is like any other tribe of the district, but there
are certain distinctive features by which he can be always identified as a Hill Maria. Like the
ordinary Gond type. he is hard skinned. but has also copper complexion. with straight black
hair (some time curly hairs are found) the growth of the hair on face is much less than what
58
3.5 Photograph showing theAbujhmaria tribe
3.5 Photograph showingtheAbujhmaria tribe
IlL' find \\lth liS.
In< irant's < ia/~llL'~r th~ t~rntus~d l(u·this area is .. Madian" or "Ahujhmarh". According
to llislop "Mar" is th~ (iondi word lill· a tree and they thought. "the name of the Madya suh
d1\ JSJon of(ionds s~~ms to h~ derived from Mara, the (iondi term for a tree". According to
this Madya or Maria means "men ofjungle". Mad or mar meaning Jungle.
Sherring in his "Hindu Trihal and Castes" published in 1876 again used the term
i\hujmard and referring to the Maria (ionds he says:
"These singular people live in a remote and hospitable tract of country known as Mad ian
or Ahujhmard". The term Ahujhmarh is a mixture of Hindi and a Gondi word- the llindi
word bci ng "Abujh" meaning mysterious or unknown and "Marh" meaning jungle or hills. If
that were so this term has obviously not originated in the land to which it refers hut has been
to it hy the outsiders (ADA Report, 1993).
This view is substantiated by the fact that the Hill Marias donot refer to their land as
Abujhmarh and it is only the outsiders who use this term. The Hill Maria refers to their land
as Metabhum- "Meta" meaning Hills and "Bhum" meaning land and thus Metabhum means
"lands of the hills" which is exactly whatAbujmarh is. The people of Abujhmarh refers to
themselves as Meta Koitor or simply as Maria and they are not aware of the term Hill Maria
which is contained in the Scheduled Castes and Scheduled Tribes Lists Amendment Act
1976. That is why the population of Hill Maria is not available in the Census.
Maria is divided into several clans, namely Mandavi, Bagh etc. They use clan names
as surnames. Adult marriage is the prevalent rule. Divorce is allowed. Widows and widowers
are permitted to marry. Women are allowed to plough the land. Marriage takes place at the
bride's place. They burry the dead and observe death rituals and pollution for two days.
Both men and women wear only a piece of loin cloth and some men-folk wear short
coats throughout the year. Young girls also donot cover the breasts but as the people of the
borders are now coming in contact with the outsiders, while working on the roads or by
coming in weekly market they have taken a long piece of loin cloth. On an average, they
spend about Rs.l5/- per year family for purchasing clothes. Their women wear tattoo marks
and ordinary ear rings.
They are non-vegetarian using rice, pulses as well. They practice shifting cultivation
and can grow fruits like bananas or their own tobacco. In the shifting cultivation coarse
grains and rice are generally grown. They are engaged in bee-beeping and other cottage
industry on which they depend upon their livelihood. They are engaged in fishing as well.
They celebrate local festivals like Nowa. A mush Parav. Hareli. Kaksai. Div.ali etc.
6(1
J.Z .. , Uisnn-lwrnMaria (Uti I~) (ligur~ \.(>) Bisonllorn rv1aria trih~ IS aiHllh~r suh
1v p~ of< ionds trihe and they call th~mselves as Maria or l>andami Maria. Their cultur~ and
living vvays is almost similar to Ahhujhmarias.They reside in the south oflndravati river .
.lagdalpur. Dantcwada, and Bijapur Tehsil. (irigson ( 1938) has studied them in less detai I
than the II ill Maria. Their characteristic dance is marriage dance with a splendid Bison-horn
headdress.
The Bison-! lorn Maria adorns the hi son horns during their dancing rituals. They collect
minor i(Hest produce. The Bison I lorn Maria docs not plough the earth, as it would mean
ini'licting pain on her body. Pointed wood pieces arc used for piercing earth crust for
cultivation.
Stone implements arc used for harvesting produce. They perform the marriages through
Ghotul system. Divorces and remarriages are common but adultery is not allowed. Marriages
amongst the offsprings of blood relations are common
3.2.4 Muria (MUR) (figure 3.7) The home of the Muria is the Bastar district.
Like the Marias, they are lumped together with the Gonds and therefore, their exact number
is not known. The word Muria is used in Bastar to mean, generally an aboriginal, Elwin
( 194 7) has emphasized their reference to "Kala Muria". In this sense it has long been
applied by the state officials to all the primitive tribes except the Marias of Abujhmarh. But
Muria actually refers to a particular tribe ofKondagaon and Narayanpur Tahsils.
The name Muria has been derived from Mur, the palas tree or from Mur, a root. The
Muria people are very colorful and lively tribe of Bastar district who has been immortalized
by Elwin in his book "The Muria and Their Ghotul". The Murias are divided into three well
marked division- the Raja Muria, the Ghotul Muria and Jhoria Muria. The Raja Murias
are found around Jagdalpur. The Ghotul Muria are found around in Kondagaon and
Narayan pur tehsils and are so-called because they have combined dormitory or Ghotul for
unmarried boys and girls of village. Grigson believes that the most important mark of cultural
separation is a great importance that the Muria have given to the Ghotul system or the role
of the youth-hall in the social structure. This seems to have created the legendary god
Lingo. The Jhoria Murias are perhaps the prettiest and most livelier of them all. The Jhoria
Murias. are commonly known as Johrias are found to the west of Bare Donger in Narayanpur
Tehsil and are considered to have originated by the union of Hill Maria and the Muria (Sharma
and Tiwari. 2002). Elwin has sometimes suggested that the Murias come from the northern
region of the Narmada valley. They evidently occupied a more beautiful and hospitable
region compared to Abujhmarh hills, and the region is endowed with rivers and prosperous
hunting grounds. They have built their culture in the west and north of the mass of Ahujhmarh
Mountains. According to Elwin (1947) these people seem to have been Hill Marias in the
past and then came under strong alien influence from across the Chanda border
Figure 3.6 Photograph showing the Bison-Hom Maria Tribe
Figure 3.6 Photograph of Bison-Horn Maria Tribe
6.1
62
Figure 3. 7 Photograph showing the Muria Tribe
Figure 3. 7 Photograph showing the Moria Tribe
6J
l'h~ Lrih~s ha1~ sn~ral ~:lans. hut 11011 Lh~ dan sysl~lll '~~Ills to he gr~atly con!'us~d.
I h~ Murias pr~s~n~ th~ old traditiuns hut in a ..:on!'us~d and disorderly way. I h~ Muria
clans'''" roughly in !'iv~ phartri~s. 11hich arc again divided into number of clans. nam~ly
Mandavi. Bag h. etc. Adult marriag~ is th~ rule butt he cngagcm~nt (mahala) is pcrl(mn~d at
the age of live or six y.:ars. l'h.:y practic~ cross cousin. junior sororal<:, and junior !.:viral<:
marriages. Divorc~ is allow.:d and compensation is paid either to wife's parent or to th<:
husband. Widows and widow.:rs ar.: p<:rmilted to marry. Marriage tak.:s place at the brid.:'s
hous<: and feast is hosted by the brid.:groom's parents. lh<:y burry tho: dead and obs.:rv<.:
death rituals and pollution f(Jr two days.
Muria women wear a necklace (hansli). The hansli is heavy solid Neck ring of thick
twisted metal. finely engraved with triangle and diamond shaped, also wear a chain of black
beads. Muria men wear a choker around their necks. The Muria comb used by both men
and women is a symbol of romance and friendship.
The economy of the Murias includes agriculture, food gathering, and hunting. In some
places they practice axe cultivation, which resembles the Marias in many ways. But despite
the fact that shifting axe-cultivation plays a very important part in their lives, many Murias
have permanent fields, which they use for paddy cultivation. The Murias are good cultivators.
They celebrate local festivals like Nowa, Amush Parav, Hareli, Diwali etc. Watt (1970)
writes that no festival among the Muria is complete without dormitory (Ghotul) group giving
a dance performance (Singh, 1994).
3.3 Methods
3.3.1 Sample selection Three tribal groups viz. Abujhmaria, Bison-horn Maria and
Muria have been sampled from their primary regions of habitat. Blood samples (5-lOml)
were collected from healthy unrelated individuals with individual informed consent (Figures
3.8.1, 3.8.2). All sampled individuals were unrelated at least to the first cousin level. Collection
of blood samples was carried out from several villages ofBastar Division, Chhattisgarh. The
location of sampling is given in Table 3.3 and Figure 3.9. Blood samples were transported in
ice to the Human Genomics Lab of School of Studies in Anthropology, Pt Ravishankar Shukla
University, Raipur for further analysis.
3.3.2 Collection of socio-demographic data
In addition to the biological samples, a minimum set of socio-demographic data was
collected in a consistent way from all individuals and populations. To ensure that all individuals
contributing samples to the work central repositories and database remain anonymous, each
sample was given a unique identification rather than being recorded by the name of the
3.8.1 Photograph showing collection of Biological Sample of Bison-horn Maria Tribe
3.8.2 Photograph showing collection ofBiological Sample ofMuria Tribe
64
ind11 idual. Ini(Jrlllation about thc: namc:s o!'thc: indi1 idual and thc:ir parc:nb 11ill hc: routJnc·l~
colkcll'd in thc: lidd but th1, inlormatJon has h..:..:n tr..:at..:d as..:onlid..:ntial.
It 11as important that thc:rc: v.a, willing and in!'ormc:d participation in th<.: 1\0I'k and th.:
wnsc:nt o!'indiYiduals to partic:ipat.: was ohtain.:d in a culturally appropriate manner. l'.:oplc
w.:re informed about the overall objectives of the work in a manner they can understand
within th.:ir culturalcont<.:xl.
Tahlc :l.] Areas of sampling. sample sizc,linguistk aJ'Jiliation. occupation. and couc
usc:d for the populations includcu in the stuuy
Population Sample Geographical Linguistic Occupation (Code) Size Distribution Affiliation
1------ --- -------
Dandami Maria 60 Village: Dugampara, Language- llunting. Foou or (M-31) llhamata. Chingitari, Dmvidian Gathering. Bison-hom (F-29) Malcvai. Gccdam, Dialect-Gondi Agriculture Mm·ia Darbha (BHR) District: Dantewara
and Bastar
Hill Maria 54 Village: Mumar, Language- Shifting or (M-27) Kutul, Pangur, Orchcha, Dmvidian Cultivation Abujhmaria (F-27) Jharawahi, Kihkad, Dialect-Gondi (Penda), (ABM) Aalnar, Kachchapal, Hunting
Toyanar, Kohkarneta, Kundla, Chotadongar, Garpal, Irakbhatti, Gudali, Nainar, Sarngpal, Kalmanar, Hatlanar, Amkot, Gulrnari, Kuakodi, Mardel,Metana District : Dantewara and Bas tar
Muria 49 Village : Amabeda,Arra, Language- Hunting, (MUR) (M-22) Shuklapal, Om vi dian Gathering,
(F-27) Tewda, Salam, Kottura, Dialect-Gondi Agriculture Badepinjori, Bade Tewda, Semargaon, Neeljar, Pupgaon, Bharritola, Surdongar, Chikli, Surdongar, Bhaismundi, Sode District: Kanker and Bas tar
Figure 3. 9 Map showing the locations ofsampling of populations included in the study.
--------=-====----===--~====================-;--l
II
II II II
II
KOELIBEOA II II II II
II _, II 6' ()
II v v II II
II -5-II
II -;.
II _, II
(\ ?> II
(} -5- II
I)
BHAIRAMGARH
'
BIJAPUR
(}
I) 0 I)
' DANTEWARA
' '
' ' ' ' '
' ' ' ' '
' '
'
'
KESHKAL I '
PHARASGOAN
KONDAGOAN
BHANRURI BAKAWAND
CHITRAKOT
'
JAGDALPUR KESHLOOR
0 Abujhm:u Ja
'\ Hi-..on-lwrn /\.Lilla
+ 1\htria
J..l Lahoralol")· Methods
J .. t I l'rqmralion of Solution for Isolation of Genomic J)NA
J..t. I. I L}·sis I Buffer (lOX)
( 'omponcnts (/\mmonium ( 'hlorid~)
NH-'Cl ( l'olassi um hicarbonat.: )KIICO LDT/\ O.SM Snl. (pll-lUl)
JA.l.2 Lysis II Buffer
l
lOOm I
X.2'>g,m O.X4gm 200~tl
Components lOOm!
I M Tris (pi-1-S.Q) 1m1 5M Nacl 8.0m1 ED'! A 0.5M Sol. (pl-!-8.0) 400 fll
Note: For making Lysis ( 1 X solution) 1 Oml of Lysis l (1 OX)+ add 90ml. Distilledl-!
20
3.4.1.3 SX TE Buffer
Components
2M Tris HCI (pH-8.0) 0.5M Tris EDTA (pl-!-8.0)
3.4.1.4 6M Nacl (Saturated)
Components
Nacl
3.4.1.510% SDS
Components
SDS
3.4.1.6 Proteinase K Solution
Components
10%SDS 0.5M EDTA(pl-!8.0) (2mM)
10m!
3.52gm (adjust pHwithi-!Cl) 1.8612gm (adjust pH with NaoH)
lOOm!
35.1gm
lOOm\
10gm
lOOm!
1m!
J.S Isolation of Genomic DNA from Human Blood (Miller eta!., 1988)
The DNA was extracted by the salting out procedure of Miller et al (1988).
Genomic DNA from human blood was isolated from following procedure:
67
•!• 5ml-l Oml of venous blood was collected from individual in a I Om\ Tarson tube.
which contained 1 Omg of EDTA.
•!• Santpk-; \\L'n: kL'pl in ~0 (·until us~.
•:• I X 1-)'is I hurkr \\as adckd in ~ac:h tube and volume was ntade up lim!.
•:• Tubes werc kept in 00(' l<>r ~0 minutes until all RBC wcre hrokcn.
6!1
•:• Alh:r that tuhcs werc shaken vigorously f(>r c:ompktc hcmolysis and c:entriruged in
cold condition at 3000 rpm t<>r 20 minutc:s to settle down all WB( ·.
•:• Supc:rnatant part was discardcJ, and same process was repeated ror two more
ti llll.'S.
•:• 3m! of Lysis II buffer wc:rc added in each tube and shakcd vigorously.
•:• 20~tl of I O'X, SDS solution and 250f.tl of Proteinase K solution was added in each
tube at a time.
•:• All solutions in each tube mixed properly and tubes were kept in gentle shaker for
over night at room temperature.
•:• Next day lml of 6M Nacl solution (super saturated) was added in each tube and
vortex for mixing properly about 20 seconds.
•!• After mixing, tubes were centrifuged at room temperature at 4000 rpm for 30 minutes.
•:• Double volume of alcohol was added in each tube, and DNA came out as a thread
like appearance.
•:• DNA was taken in 1.5ml micro tube with some amounts of alcohol.
•:• DNA was dried in a speed vac machine for removing alcohol and then lml ofTE
buffer was added in each tube to dissolve the DNA.
3.6 Wet DNA Lab Analysis: Polymerase Chain Reaction (PCR)
Each DNA sample was analyzed for polymorphisms at 15 biallelic loci of which 8
were Insertion/deletion polymorphisms (lOPs) and remaining were RFLPs. (Figure
3.10,3.11)
3.6.1 Steps Involved during PCR Analysis
Polymerase Chain Reaction (PCR) is a molecular biological technique for amplifying
(creating multiple copies of) DNA without using a living organism, such as E. coli or yeast.
PCR is commonly used in medical and biological research labs for a variety oftasks, such as
the detection of genomic diversity, hereditary diseases, the identification of genetic fingerprints,
the diagnosis of infectious diseases. the cloning of genes, and paternity testing etc.
Mullis was awarded the Nobel Prize in Chemistry in October 1993 for this achievement,
only sewn years after he first published his ideas. The idea was to develop a process by
69
Figure 3.10. Human Genomic Lab of School of Studies in Anthropology
Figure 3.11 Human Genomic Lab of School of Studies in Anthropology: One of tbe DNA isolation step
70
11 l11d1 Ill\: A nnJid he artllicially multiplied through n:peated cycks of duplication driven by
.111 c'lll) me calkJ llNA polymerase. One oft he lirst thcrnwstablc DNA-Polymcrases was
Phtai11ed from lhamu1 <ICflltllicu.l a11d called liuf
1 )N A polymerase occurs natllrally i11 I i ving organisms. where it functions to duplicate
llNA 11hen cells divide. It works by binding to a singk DNA strand and creating the
compkmentary strand. In Mullis's original PCR process. the cn;ymc was used in vitro (in
a co11trolkd ..:nvironment outsid..: an organism). The douhlc-stranded DNA was separated
into two singk strands by heating it to '!6"C. At this temperature. however. DNA Polymerase
11as destroyed so that the enzyme had to be replenished alkr the heating stage of each
cycle. Mullis's original PCR process was very inefficient since it required a great deal of
time. vast amounts of DNA Polymerase, and continual attention throughout the PCR process.
Later. this original PCR process was improved by the use of DNA-Polymerase taken
from thermophilic (heat-loving) bacteria that grow in geysers at a temperature of over II ooc. The DNA-Polymerase taken from these organisms is thermostable (stable at high
temperatures) and, when used in PCR, did not break down when the mixture was heated to
separate the DNA strands. Since there was no longer a need to add new DNA Polymerase
lor each cycle, the process of copying a given DNA strand could be simplified and automated.
PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single
gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy
only short DNA fragments, usually up to I 0 kb (kb=kilo base pairs= I 000 base pairs) DNA
is double-stranded, and therefore, it is measured in complementary DNA building blocks
(nucleic acids) called base pairs. Certain methods can copy fragments up to 40 kb in size,
which is still much less than the chromosomal DNA of a eukaryotic cell-for example, a
human cell contains about three billion base pairs.
PCR, as currently practiced, requires several basic components. These components are:
• DNA template, which contains the region of the DNA fragment to be amplified
• Two primers, which determine the beginning and end of the region to be amplified
• DNA-Polymerase, which copies the region to be amplified
• Nucleotides, from which the DNA-Polymerase builds the new DNA
• Buffer, which provides a suitable chemical environment for the DNA-Polymerase
The PCR reaction is carried out in a Thermal cycler.
3.6.2 Principle of the PCR
The cycling reactions:
71
1 hen: are three major steps in a l't 'R, whid1 are repeated for 30 or 40 ~ycks. This is
done <lll an automated cycler. whicl1 .:an heat and cool the tubes with the reaction mixture in
;1 \cry short time.
-- wanted l:l'lll.!
<=<== ;='~<-
= \"''''"<=< =<== ====< h!~Hk --. -<=< =<- ~ -< -,'- '- --<--- 2 -- =
.. (.:Op!l'" X <.UJ'IC'> J(l (Oj)ll!\ 3:! l"OPI~
· ··· ····· .. ._ 15!1H.:ydc
" 2 ,.. fiX hrlli'm CIIJ'IC'
Figure 3.12 The steps involved during Polymerase Chain Reaction (PCR)
Denaturation at 94°C: During the denaturation, the double strand melts open to single
stranded DNA, all enzymatic reactions stop (for example: the extension from a previous
cycle).
Annealing at 54°C: The primers are jiggling around, caused by the Brownian motion.
Ionic bonds are constantly formed and broken between the single stranded primer and the
single stranded template. The more stable bonds last a little bit longer (primers that fit exactly)
and on that little piece of double stranded DNA (template and primer), the polymerase can
attach and starts copying the template. Once there are a few bases built in, the ionic bond is
so strong between the template and the primer that it does not break anymore.
Extension at 72°C: This is the ideal working temperature for the polymerase. The
primers, where there are a few bases built in, already have a stronger ionic attraction to the
template than the forces breaking these attractions. Primers that are on positions with no
exact match get loose again (because of the higher temperature) and don't give an extension
of the fragment.
The bases (complementary to the template) are coupled to the primer on the 3' side
(the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases
are added complementary to the template)
3. 7 DNA Markers Studied: Total 15-biallelic polymorphic loci have been studied.
These are the "core set" of DNA markers approved by the Department of Biotechnology.
Government oflndia to study Human Genomic Diversity (HGD) in the context of indian
populations in order to generate data base.
72
Out of 15 DNA markers. eight arc insertion/deletion polymorphic loci: Alu ACE
(chn>llH>Somc: 17q~J ); Alu I'V92 (chromosome: I 6); Alu ('1)4 (chromosome 12p 12): Alu
I'LAT (chromosome: 8); Alu I> I (<:hromosome: .1 ); Alu AI'O (o:hromosomc: I I); Alu FXIIIB
(chromosome: 6p25-24) and one mitodmndrial DNA (mt-NlJC) insertionilkktion locus on
chromosome II. Along with this seven autosomal unlinked restriction site polymorphism
(RSI's). that arc ESIVPv111/ (chromosome: 6q25.1 ); NAT/ Kpnl (chromosome: 8p22);
LI'L!Pv11ll (chromosome: 8p22); ALH/Hae/11 (chromosome: 4qll-13); I'SCR/Taql
(chromosome: 21 q 11.2); CVI'I AIM.\p I (chromosome: 15q22-q24 ): T2/Mspl (chromosome:
Llql4-q24);. Their brief description arc given below such as primer sequence. PCR
protocol. Band size etc.
Table 3.4. I' rimer sequences used for I'CR amplification, their corresponding
annealing temperatures, and restriction endonuclease digestion protocols (where
necessary) for Alu insertion/deletion and Autosomal unlinked RFLI' markers
Locus Primer sequences Annealing Restriction Reference Temp Digestion
Protocol
Alu 5'-ACAAAG TCC AGG TIT CIA ACA G-3' 63"C Not Z1schler et mtNU( 5'-AGT CrT GCT TAT TACAATGATGG-3' Applicable al. t995
Atu 5'-CTG GAG ACC ACT CCC ATC err TCIC3' 58"C Not Stoneking et ACE 5'-GAT GTG GCC ATC ACA "I TC GTC AGAT-3' Applicable all997
Atu 5'-AAGTGCTGTAGGCCA TTTAGATTAG-3' SO"C Not Stoneking et APO 5'-AGT CTTCGA TGACAG CGTATA CAGA-3, Applicable al 1997
Atu 5'-AGGCCTTGT AGGGTTGGTCTGATA-3' 58"C Not Edwards CD4 5'-TGC AGC TGC TGA GTG AAA GAA CTG-3' Applicable and Gibbs 1992
Atu 5'-TGC TGA TGCCCA GGGTTA GTAAA-3' 66"C Not Stoneking et Dl 5'-TIT CTG CTA TGC TCT TCC CTC TC-3' Applicable all997
Alu 5'-TCAACTCCA TGA GATTTT CAGAAG 1C3' 56"C Not Stoneking et FX3B 5'-CTG GAAAAAATGTAT TCAGGT GAG"!C3' Applicable al1997
Alu 5'-AACTGGGAAAATTTGAAGAGAAAG'IC3' 54"C Not Stoneking et PV92 5'-TGA GTT CTC AAC TCC TGT GTG TTA G-3' Applicable all997
Atu 5'-GTGAAAAGCAAG GTC TAC CAG-3' 60"C Not Tishkoff et PlAT 5'-GAC ACC GAG TTC ATC TTG AC-3' Applicable al. 1996
ESR 5' CTG CCA CCC TAT CTG TAT C-3' 63"C 5 units of Pvull Anderson et 5'-CTC TGC CAC CCT GGC GTC-3' in appropriate al. 1994
buffer was added to the tube. Incubated at 37°C for 2 hours
NAT 5'-GACATTGAAGCA TATTTTGAAA-3' 56"C 5 units of Kpnl Cascorbi et 5'-GATGAAAGTATTTGA TGTlTA-3' in appropriate al. 1996
buffer was added to the tube. incubated at 3 7°C for 2 hours
CYPIA 5'-CTGACT GGC TTCAGC AAG TT-3' 56"C 5 umts of Mspl Hayashi ct 5'-lAG GAG TCT TGT CTC ATGCCT-3' in appropriate al. 1991
buffer was added to the tuhc. incubated at 37°C for 2 hour~
I I
73 ~- ------ -----
' -..ruH..,..,L'II and I'~< K ~ ~' -< .<, « ; I I ( . I:\.\ \ < ;<; <, \ \ C \ \ \-3' (,() 'l .:; urut.., ol /Uij/
~'-{'('I,\,\(' ,u;,\(;(; I C\l'.\ \(; <;._\' HI •lPI'IOPil·lt~ l\rm:(l...htll\L'll,
buller \\ ,,.., I t)l) J
added to thL' llihL'.
inudmtL"d at 6)"( ·
lor 2 horn'> ~--~--- - ~ - -- -- - --- ------ -- -· -
I Z ~·-c I(; ('A(; 1'1 I Ill ('I{' lAC <;(;.J' tS'(' _c; unit.., or ,\/.\'/'' l.)ll/1 Jorde
~'-{'(;( ('I(;('IA('AACII ("I'(; <;I' I 1-3' rn appropnatc tl'l·•-.nnal hulkr \\il'> <. 'ormmmil:ation)
;\ddcd to the tuhc, im:uhall:d at 65"( · l~1r 2 hr.., -r----~-~-----~ - -~ ~-------- -- ------- -- --~-
I JJI, 5'-AGG C I I ('A(' I ('A I('(' 1; I(; ('{'I I'C-3' 62°(' 5 umt.., of' hw// Stcpanov
5'-I'IA'IGC'II;(' Ill A(;A('I(' 111; 1('-3' in appropriate and huf'll:r wa~ I ,cnu.a. added to the tube, 1993 incuhah:d at 65°C for 2 hrs
ALII 5'-G'IA GG I GGACI I GGAGAAGG-3' 63°(' 5 units of' 1/ae/1/ I ,ynn. Jorde 5'-GATATACII GGCAAGG t'CC-3' inappropnatc (Personal
bufiCr was Cornmunication) added to the tube, mcubatcd at 65°C
for 2 Hours
3. 7.1 Alu -mt NUC (Subfamily: Human Specific -1 on chromosome number 11)
PCR Protocol
WC(I') 94 oc (15'), 63 oc (30'), 72°C ( 1.30'), X 30 Cycles noc (2')
PCR Product protocol
H20 = 7.25 fll 1.5mM PCR Mix= 1.0~1
Primers
mtNUC 1 = 0.25~-tl mt NUC 2 = 0.25~-tl a-Taq = 0.25fll Genomic DNA= 0.5~-tl
Gel-1.5% Product Size+ 684 bp
-ISO bp
KCI(J M)-500 J,tl, TrisHCI(lM)-1 OOJ,tl, MgC12(1M)-15J,tl, dNTPs(25mM)-80fll, Distilled H20-3 77 fll.
3.7.2Aiu-ACE (Subfamily: Human Specific -1 on chromosome number 17)
PCR Protocol
94°C(4') 94°C (I'), 58°C ( 1 '), 72°C (1 '),X 30 Cycles 72°C(3)
PCR Product protocol
/12<) 7.l>fd I '>m\ll'l'l{ Mix I .Up!
At T I 0.3pl /lt'/'2 !Up! <L-laq 0.3pl tienomic DNA 0.5ftl
( id 1.5'Vo Product Size+ 490 bp
- 190 bp
Kt 'It I M J-500 pl. Irislll 'II I M l-1 ODpl. Mgt 'I,{ I M l-l5ftl. dN II'' i~5mM )-HOpi. Distilkd II ,0-1 77pl.
J.7.3 Alu -AI>() (Subfamily: Human Specific-] on chromosome number II) . ------
PC I{ l>rotocol
•we (3') <WC ( l '), 50°C (I'), 72°C (I'), X 30 Cycles 72°C (3 ')
PCR Product protocol
!120= 7.6fd l.SmM PCR Mix= 1 .Ofll KC1(1 M)-500 fll, TrisHCI(lM)-1 OOfll, MgCI
2(1M)-15pl,
Primers
APOI =0.3pl APO 2 = 0.3fll a-Tag= 0.3fll Genomic DNA= O.Spl
Gel-2.0% Product Size+ 400 bp
- 100 bp
dNTPs (25mM)-80pl, Distilled H,O-3 77 pl.
3. 7.4Aiu- CD 4 (Subfamily: Human Specific-1 on chromosome number 12)
PCR Protocol
94°C (5') 94°C (!'), 58°C (1.30'), 72°C (1.30'), X 30 Cycles 72°C (3')
PCR Product protocol
H20 = 7.6pl l.SmM PCR Mix= 1 .Ofll KCI(1 M)-500 fll, TrisHCI(lM)-1 OOpl, MgCI,(l M)-lSfll,
dNTPs (25mM)-80pl, Distilled H, 0-3 77 pl.
Primers
CD4 1 = 0.3pl CD4 2 = 0.3fll a-Tag= 0.3pl Genomic DNA= O.Spl
----
(,d 1.) tJ,j)
!'11>duct Si;.: t I )00 hp - I 200 hp
.'.7.5 Alu -IH (Subfamily: lluman Specific -I on chromosome number 3)
I'< 'I{ l'rutocul
<J.j"(. (5')
'i.J"(' ( 1'). 66"(' (I'). 72"(' ( 1'). X JO Cydcs
~~~ --
72"(' (J') -------~------------------1
I'( 'R l'roduct protocol
1120- 7.2~d
I.SmM I'CR Mix= l.O~d
!'rimers
------~-------
KCI( 1 M)-500 )..II, TrisiiCI( I M)-1 OOfll. MgCI,( I M)-l5f11, dNTI's (25mM)-80f1l. Distilled II,0-377j..tl.
~----------------------------~ Dl = 0.3fll D2 = 0.3fll u-Taq = 0.3~tl Genomic DNA= 1.0 ~tl
Gel-1.5% Product Size+ 650 bp
-350 bp
3. 7.6 Alu FXIII B (Subfamily: Human Specific -1 on chromosome number 1)
PCR Protocol
94"C (3') 94"C (I'), 56"C (I'), 72"C (I'), X 32 Cycles 72"C (3')
PCR Product protocol
H20= 7.6)..11 1.5mM PCR Mix= I.Ofll
Primers
FXlll B 1 = 0.3)..11 FXIII B 2 = 0.3 J.!l a -Taq = 0.3)..11 Genomic DNA= 0.5fll
Gel-1.5% Product Size+ 700 bp
- 500 bp
KCI(IM)-500 )..II, TrisHCI(IM)-IOOfll, MgCI2(1M)-I5f1l, dNTPs (25mM)-80J.!l, Distilled H,0-377).!1.
-~- 7. 7 Alu - 1'\' tJ2 (Suhfamih: II uman Spel·ific -I nn chrnmnsnme number I (J)
I'( 'I{ l'rutucnl
•>-1 "(. I -l'i
76
•I~ "( , I I '), 'i~ "(' I I ' ). 7 ~"(' I I ' ). X 30 C) c ks 72"('11')
----------- -------------- ___ ____, J'('J{ l'ruduct prntnrnl
11:~1 > u,1ti l..'imM PCR Mix LOrd
Primers
I'V I IUfd I'V 2 ~0.3rLI a-·I~KI - 0.3rtl
KCI( I M)-500 rt!JrisiiCI( I M )-IIHlrtl. MgCI,( I M)-15pl. JNTPs (25mM)-l!Opl, Distilled II ,0-377rtL
-- ---------1
-~cn_on_Jic_D_N_A~ ~-5-__,r::_Li _____________________ ______1
(lei- 2.0% Product Size + 400 hp
- 110 hp
3. 7.8 Alu- PLAT (Subfamily: Human Specific -I on chromosome number 8)
PCR Protocol
95°C (4') 95°C ( 1 '), 60°C (I'), 72°C ( 1 '),X 30 Cycles 72°C (3')
PCR Product protocol
H20 = 7.6)-ll 1.5mM PCRMix = l.Opl
Primers
PLAT 1 = 0.3 pl PLAT2=0.3pl a-Tag = 0.3 pl GenomicDNA=0.5pl
Gel-1.5% Product Size + 400 bp
- 110 bp
3. 7.9 ESR !Pvull
PCR Protocol
we (5')
KC1(1 M)-500 )ll, TrisHCl(IM)-1 OOpl, MgCl1(1M)-15pl,
dNTPs (25mM)-80)ll, Distilled H10-377pL
95°C (30'), 63 oc ( 1 '), 72°C ( 1 '). X 30 Cycles 72°C (3')
PCR Product protocol !----~-_:____ __________________ -
H20 = 7.75fll 1.5mM PCR Mix~ 1 .Ofll KCI( I M)-500 rd. Trisi!CI( 1 M)-1 OOpl. \lgCI,( I\ 1 1-l.'ipl.
c__ ___________ ~d.._N:..'T_._P_.,_s ~__-( 2::::5."m".'M"-'l':-8".'00pi_J)~~ti_llcd 112 0-3 77 p L
l'rimt·r.
I SR I 0.25111 I SR 2 0.25pl tt·laq ti.25pl ( icnomic J>N J\ 0.5 111
Di~-:esliun l'rutucul (l>i~-:cstion with J>vu II at 37°(' fur 2-4 hours)
1120 1.0fd Jlulli:• R.:action · 1.5pl !'I'll If O.Sftl
tiel 1.5 •y., Product Size ( 1/t) 900,480 hp. ( -/-) 1350 bp, (+I-) 1350, 900,450bp
3.7.10 NAT/ Kpn/ ·--
I'CR l'rotocol
94°C (4') 94°C (I'), 56°C (I'), 72°C ( 1.30'), X 30 Cycles 72°C (2')
I'CR Product protocol
ll20 = 7. 75itl I.SmM PCR Mix= I.Of.ll KCI(I M)-500 f.ll, TrisHCI(I M)-1 OOf.ll, MgCI2(1M)-15f.ll,
dNTPs (25mM)-80pl, Distilled H20-377pl.
Primers
NAT 1 = 0.25f.ll NAT 2 = 0.25f.ll a-Taq = 0 .. 25f.!l Genomic DNA= 0.5 f.l]
Digestion Protocol (Digestion with Kpnl at 37°C for 2-2112 hours)
H20 = 3.0pl Buffer Reaction =l.5f.l1 Kpnl = 0.5 p1
Gel-1.5% Product Size ( +/+) 570,480 bp, (-/-) I 050 bp, (+/-) 1 050,570,480bp
3.7.11 CYPlA!Mspl
PCRProtocol
94°C ( 4') WC ( 1 '), 56°C ( 1 '), 72°C ( 1.30'), X 30 Cycles 72°C ( 2')
PCR Product protocol
H20 = 6. 9 f.ll l.SmM PCR Mix= l.Of.ll KCI( I M)-500 f.ll, TrisHCI( I M)-1 OOf.ll. MgCI,( I M)-l5fd.
dNTPs (25mM)-80f.ll, Distilled H,O-3 771LL .
77
CYJ>I,\ I O.:ifd
l"YI'L\ 2 O.:ipl
11- I aq 0.1> pi < ic-nomic DNA 0.5 pi
lli~-:~stion l'rotncoi (lli~-:cstion with M.~pl at 37°C for 2-3 hours)
112<> .\.Ofd Jlufli:r Reaction i.5pi MIJ!I-1 0.5fd
(icJ I. 5 •y., Product Size: ( 1 I+) 878, 200bp, ( -/-) I 07Hbp, ( +/-) 878, 200, I 078bp
4.7.12 I'SCR/ Taql --- ------
I'CR l'rotocol
94°(' (5') 94°C (30'), 60°C (45'), 72°C (45'), X 30 Cycles noc (4')
PCR Product protocol
1120 = 7.6pl I .5mM PCR Mix= l.Of.tl KCI(IM)-500 pi, TrisHCI(I M)-1 OOpl, MgCI,(l M)-l5f.tl,
dNTPs (25mM)-80f.1l, Distilled H,0-377f.ll.
Primers
PSCR I = 0.4pl PSCR 2 = 0.4pl a-Taq = 0.1 f.ll Genomic DNA= 0.5 pi
Digestion Protocol (Digestion with Taql at 65°C for 3-4 hours)
H20 = 6.4f.ll Buffer Reaction= 1.5pl Taq-1 = O.lpl
Gel-1.5% Product Size (+/+) 313,76 bp, (-/-) 389bp, (+/-) 389,313,76bp
4.7.13 T2/ Mspl
PCR Protocol
94°C (!') 94°C (1'), 65°C (I'), 72°C (1.15'), X 30 Cycles 720C (4')
PCR Product protocol
H20 = 7.7f.ll 1.5mM PCR Mix= l.Opl KCI(l M)-500 f.tl, TrisHCI(l M)-1 OOf.IL MgCIJ I M)-l5f!l.
dNTPs (25mM)-80J-LL Distilled H,0-377f.tl.
7H
- - -
Prinu.·~
I.' I II ..':ipl I.'..' II ..':iftl
<t- I aq tU pi l i,·,HmJic I lNI\ 0.5 ftl
Uigcslion l'rolocol (Uigcslion with M.~p-1 at 37°C for 3-4 hours)
11..'0 .1.0ftl llulkr Reaclion 1.5pl
\/lfl-1 0.5fd
(icl ..'.O'X, Product Size (+I 1 ) 220.120 hr. ( -/- ) .142bp. ( +/-) 342.220.120br
3.7.14 LI'L! Pvu/J - ---- --
PC R Protocol -- -------
'WC (3') 'WC (I'). 62°C (I'), 72°C( I'), X 30 Cycles 72°C(3')
I'CR Product protocol
1120 = 7.25!-!1
--
I.SmM PCR Mix= l.Of!l KCI(IM)-500 fll, TrisHC1(1 M)-1 OOfll, MgC12
( I M)-15fll, dNTPs (25mM)-80fl1, Distilled H,O-3 77 fll.
Primers
LPLI = 0.25f.-t1 LPL 2 = 0.25 J.L1 a-Taq = 0.25f.-t1 Genomic DNA= 1.0 f-11
Digestion Protocol (Digestion with Pvull at 37°C for 3-4 hours)
H20 = 3.0!-!1 Buffer Reaction =l.5J.L1 Msp-1 = 0.5f.-t1
Ge1-2.0% Product Size(+/+) 171,148bp, (-/-)319bp, (+/-)319.171,148bp
3.7.15ALB/ Haelll
PCR Protocol
94°C (4') 94°C (1'), 63°C (40'), 72°C (!'),X 30 Cycles 72°C (4')
PCR Product protocol
H20 = 7.75f11 1.5mM PCR Mix= 1.0fll KC1(1M)-500 !-!I. TrisHCI( 1 M)-1 OOf!l. MgCI,( 1 M)-15!-!l.
dNTPs (25mM)-80!-!l. Distilled H,0-377!-!l.
79
------- --'
l'rim•·"
\I II I O.~:ipl
\I 1\ .' ll.2:ipl u-laq O . .::':ipl l ic·notllic I >NA O.'i fd
l)ig•·stiun l'rutucol (lligcstiun with llaclllut 37°(' for 2-4 hours)
II~() '\.()p\
llulkr Rca<:! ion 1.5pl
HO
"""III-I o.sr'=-ti~-------------------------___J tiel 2.o•y,, !'n>ducl Si.ce ( t I 1) 350.250. 75bp, ( -/-) 425.250bp, ( t /-) 425,350.250.75bp.
3.!! BNA Molecular Weight Marker used as Standard Ladder - --- - ----
q> X 174 DN/\ Cleaved with llae lll --~--. ---
Size Rw1ge 72-1 353bp. ----
Fragment: II (72,118.194.234,271,281,310,603,872, 1078, 1353bp)
Reference + Ostrovsky de spicer, P and Maloy, S. 199!. Focus 13:268.
3.9 Agarose Gel Electrophoresis
The amplified DNA products were mixed I X loading buffer and run on 1.5% or 2%
agarose gel containing ethdium bromide (0.1 pg/ml). Electrophoresis was carried out in I X
TBE buffer for approximately 90min at 60V. Observed under UV transilluminator and
documented by photography.
3.9.1 Preparation of SX TBE (Stock Solution)
S.No. Components Amount
I T ris (T rizma Base) 54gm
2 Boric Acid 27.5gm
3 EDT A. Na Salt 3.72gm
4 H20 940ml
Tris base and EDTA and Boric acid were dissolved in 940 ml of de-ionized water
and made up the final volume ( 1 DOOm!) by adding autoclaved distilled water.
3.9.2 Preparation of SX Gel Loading Dye
S.No. Components Amount
I 50% Glycerol SmloflOO%
2 2mMEDTA 40ul ofO.SM (Ph-8) --
3 l%SDS 0.5 ml0f20% -----
4 0.1% Bromophenol blue lmlofl% ·-----
0.1 "., .\) knd ·)anol
Iota!
J.lJ .. ' l'reparulinn nf Aguruse gel
HI
lml ot'J'''"
10m!
i\garn'~ powd~r (Sigma). 2gm or 1.5 gm was weighed ami dissolved in diluted I X
1 J\1· huller. "''lume made to ltHlml and the mixture was heated in a microwave oven until
agaro'c was compktely dissolv~d. i\garose solution was allowed to cool until it reached a
tempera tun: oJ' 50-55°C'. !-'!ask was swirled \o keep the contents a\ a uni f(>nlllempera\urc
l<> prcvcnlthc agarosc !'rom gelling a\ the boltom ol'lhe flask. Swirling was also used lo
prevent the bubble formation in the gel mixture. To this, Sftl of cthidium bromide solution
1 I Omg/ml) was added.
3. 9.3.1 Custing of Gel
The plastic \ray was kept on to the horizontal tray. Comb was placed 1.0 mm above
the plate and warm agarose solution was poured into the mould. The thickness of the gel
was 4-5 mm and formation of air bubbles were avoided while pouring. After gel was
completely set (30-40 min at room temperature), the comb was removed gently.
3. 9.3.2 Loading of the gel
Mounted gel in electrophoresis tank was flooded with fresh running buffer (IX TBE)
to cover the gel to a depth of about lmm. The DNA samples were mixed with the gel loading
buffer (Bromophenol Blue, Xylene cyanol FF) and then carefully loaded these under the
buffer in their respective marked wells.
3.9.3.3 Starting of Gel
After loading samples, the lid of the gel tank was closed, electric lids were attached
and the power supply (50V) was turned on. The formation of bubbles was indicative of
beginning of electrophoresis. Within a minute, bromophenol blue started migrate towards
the anode. Gel was run until the bromophenol blue and Xylene Cyanol FF migrated to
appropriate distance through the gel.
3.9.3.4 Detection of Gel
The running buffer was poured off from the top of the gel and the intensities of DNA
band (s) were examined and the migration distances were observed directly by the gel on
the UV Trans-illuminator plates.
Hl
.~.l O.l Allcl~ Fr~qucncy ("alcuhttions
t\lkk i"r.:qu.:ncics and th.:ir standard errors w.:rc calculated by simpk and hig.hly cflicient
IIJL"Iilod oi" g.:m: counting .
.1.1 0.2 The Chi-sq uarc test
'1(, t.:st f(Jr the statistical signilicance of the diiTcrcnces between the observed phenotype
numbers and that expected under panmixia, Chi-square testi(Jr the departure from the I lardy
Wcinherg equilibrium was performed.
3. I 0.3 Measure of (;enctic Variation
The extent of genetic variation in a population can be calculated efficiently by the use
of speci fie measures that combine several aspects of genotype frequency distribution in a
single value.
Heterozygosity (h) gives an estimate of the genic variation at a single locus and is
defined as
Where x, is the frequency of the i'" allele. Average heterozygosity (H) is the average
heterozygosity (h) over all loci (Nei, 1973).
3.10.4 Measures of genetic differentiation
Measure of genetic differentiation are measures of genetic variability among populations
with respect of known gene frequencies. There are many such measures available in the
literature (Wahlund, 1928; Wright, 1943,1951; Nei, 1973). However, for the present study,
Nei 's gene diversity estimate was calculated.
Nei (1973) defined gene diversity for a single locus as the heterozygosity expected
under Hardy-Weinberg equilibrium, disregarding the actual genotype frequencies in population.
That is, gene diversity is a measure of the degree of genic variation in a population and has
nothing to do with the frequency of heterozygote except in a Hardy-Weinberg population.
Using the above definition, Nei showed that the gene frequency variation in a substructure
population can be analyzed in terms of gene diversity of the total population. It can be
partitioned into its components, for example, intra- subpopulation and inter-subpopulation
gene diversities. Thus the gene diversity of the total population (H 1) can be expressed as:
H -H +D T- ~ ST
HJ
1),1
til<: av.:ra~<: ~<:ll<: di\ <:rsity h<:tW<:<:Il sobpopulatinns.
1 h.: absolute g.:ne d i ll<:ren t iati on among su hpopulat ions may he rneasun:d hy I\ 1• while
the· gc·nc· differentiation relative to the total population is giv.:n hy u,,. The coefficientnf
g.:\1<: dilkrentiation, deli ned as:
3.1 0.5 Measure of {;cnctic Distance (I> A)
The genetic distance measure (D J between populations (Nei, 1987) is defined as:
M' ~~cc. L(l-I,'>/XikYik) k'= I I =(
DA=~~~~~-
Where MK and rare the number of alleles at the the k'" locus and the number of loci
examined respectively. This measure takes a value between 0 and 1, the latter value being
obtained when the two populations share no common alleles. Since the maximum ofD A is I,
DAis non linearly related to the number of gene substitutions. When D A small, however, it is
roughly linear with evolutionary time (Nei eta!., 1983).
3.1 0.6 Test of Significance
Pairwise comparison between population has been made for all the biallelic loci by
Chi-square test following the method of Workman and Niswander (1970).
Population I
Population 2
N, p,
N, P,
q, = 1-p,
q, = 1-p,
Where N 1
and N2
are number of chromosomes studied for the population! and
population 2. p 1 and p2 are allele frequencies in population 1 and population 2.
p = ~X p, N
+ ~_2_ X p2 N
Where N = Nl+N2
q = 1-p
x'(Idf)= (2N
1p1
2 + 2N2p,J)- P (2Nl
1 + 2N,P,)
pq df= degree of freedom
H4
J.l II. 7 l'h~·logenclic tree ( Uentlogram)
l'h1 Jogc:nc:tic: tree: (Dendrogram) was construc:tcd by using the neighbor_joining (N.I)
lllc'thod.
J>ISI'i\N software (Ota, 1993) has been used to compute average heterozygosity.
gL·nc di 1 crsity and its associate parameters (Nei, I 973 ). genetic distance between populations
1 Nci. I 'IX7) and to construct phylogenetic trees.
3.1 0.8 Gene flow among populatioons (Harpending & Ward Model, 1982)
To assess the relative amount of gene flow experienced by each population, we have
used regression model originally proposed by Harpending and Ward( 1982). For this, the
hctcrozyosity of the ith population was plotted against the distance of the population from
the centroid (r ), calculated as '
r=(p P)'/P(l-P), ' ,_
where P, and Pare, respectively, the frequency of the insertion/deletion allele in
population i and the total population. Under the island model of population structure,
Ilarpending and Ward have shown that there should exist a linear relationship between
heterozygosity and distance from the centroid.
h, = H(l-r,)
where hi and H denote respectively the heterozygosities of population i and the total
population.
To assess the relative amount of gene flow graph was constructed by SAS software.