Human geneome diversity of Chenchu tribe of Andhra Pradesh - India

1. 1

2. We are fascinated with the question of human origin.
Palaeontology and archaeology are the branches
that traditionally deal with the reconstruction of
human origin and history.
Molecular biology is contributing to this area through
DNA analysis.

3. HUMAN GENOME DIVERSITY PROJECT
HUMAN GENOME DIVERSITY PROJECT SEEKS TO UNDERSTAND
THE GENETIC RELATIONSHIP BETWEEN DIFFERENT POPULATIONS
BY USING STANDARD DNA MARKERS LIKE
mtDNA
“Y” CHROMOSOME
HGDP IN INDIA- CCMB IS ONE OF THE PIONEER INSTITUTE
WHICH IS ANALYSING PRIMITIVE TRIBES AND CASTE AT
MOLECULAR LEVEL. AROUND 6000 BLOOD SAMPLES OF DIFFERENT
POPULATIONS HAVE BEEN ANALYSED FOR THEIR GENETIC DIVERSITY

4. TWO HYPOTHESES REGARDING EVOLUTION OF MAN
OUT OF AFRICA THEORY
MULTI REGIONAL THEORY
MITOCHONDRIAL DNA IS COMMONLY USED IN THE
CONSTRUCTION OF EVOLUTIONARY TREES.
EVOLUTION OF MAN

5. MITOCHONDRIAL DNA WAS FIRST RECORDED BY
ELECTRON MICROSCOPY IN 1963 BY NASS & NASS
IT WAS ISOLATED FROM YEAST MITOCHONDRIA
BY SCHATZ
IN 1981 THE MITOCHONDRIAL GENOME WAS COMPLETELY
SEQUENCED BY ANDERSON et al.
mtDNA IS MATERNALLY INHERITED [GILES ET AL 1980]
mtDNA IS A SEMI-AUTONOMOUS MOLECULE, WHICH DOES NOT
UNDERGO RECOMBINATION.

6. CIRCULAR MOLECULE OF 16569 BP LENGTH
37 GENES
13 CODING PROTEINS
2 rRNAS, 22 tRNAS
THE ONLY NON-CODING REGION IN THE MTDNA IS
SITUATED BETWEEN THE GENES CODING FOR
tRNA-PRO AND t-RNA-PHE.
IT IS ABOUT 1120bp IN HUMANS

7. MITOCHONDRIAL DNA MAP

8. THE CONTROL REGION IS HIGHLY STRUCTURED,
WITH A CONSERVED CENTRAL REGION (CCR)
FLANKED BY TWO HIGHLY DIVERGENT
PERIPHERAL DOMAINS.
 IN HUMANS THE TWO PERIPHERAL REGIONS ARE
CALLED HYPER VARIABLE REGIONS ONE AND TWO (HVR-I
AND HVR-II).
THE CONTROL REGION IS RELATIVELY TOLERANT OF
HIGH MUTATION RATE,
THE MT GENOME IS INHERITED INTACT OVER
THOUSANDS OF GENERATIONS, WITHOUT THE
CONFOUNDING EFFECT OF CROSSOVER WITH A
PATERNAL CHROMOSOME.

9. THESE FEATURES OF mtDNA HAVE BEEN EXPLOITED FOR
EVOLUTIONARY AND POPULATION GENETIC STUDIES
MAKING IT A SUITABLE CANDIDATE FOR THE STUDY.
THIS PROJECT AIMS TO FIND GENOMIC DIVERSITY
WITHIN AND BETWEEN ENDOGAMOUS POPULATIONS BY
EVOLUTION USING A STANDARD DNA MARKER –mtDNA

10. ISOLATION OF DNA
QUANTIFICATION OF DNA
DILUTING THE DNA SAMPLES
AGAROSE GEL ELECTROPHORESIS
POLYMERASE CHAIN REACTION

11. PCR FOR 9bp
PCR Reaction Mixture
Requirement
PCR Buffer
MgCl2
dNTPs
Primer A
Primer B
Taq
Polymerase
DDW
DNA (10ng/μl)
Volume(μl)
1
0.6
0.8
1.0
1.0
0.5
1.1
4
PCR Conditions
Steps
1
2
3
4
5
6
7
8
Conditions
950
C for 5min
950
C for 1min
550
C for 1min
720
C for 1min
Go to 2, 35 times
720
C for 5min
40
C for ever
End
THE PRODUCTS WERE THEN RESOLVED IN 6% POLYACRYLAMIDEGEL .
NO DELETIONS AND INSERTIONS WERE OBSERVED.

12. POLYMERASE CHAIN REACTION
HVR I PRIMER
PCR Reaction Mixture
Requirement Volume(μl)
PCR Buffer
MgCl2
dNTPs
Primer F
Primer R
Taq Polymerase
DDW
DNA (10ng/μl)
1
0.6
0.35
0.2
0.2
0.5
3.15
4
PCR Conditions
Steps Conditions
1
2
3
4
5
6
7
8
940
C for 1min
940
C for 30sec
600
C for 45sec
720
C for 2 min30sec
Go to 2, 34 times
720
C for 7min
40
C for ever
End

13. SEQUENCING PCR
THESE PCR PRODUCTS ARE SEQUENCED IN ABI PRISM 3700
AUTOMATED DNA ANALYSER
Requirement
BigDye™
Primer F
DDW
DNA (10ng/μl)
Volume (μl)
1.8
0.1
2.1
1
PCR Reaction Mixture PCR Conditions
Steps
1
2
3
4
Conditions
950
C for 10sec
500
C for 5sec
600
C for4min,
30cycles
40
C for ever & end

14. THE SEQUENCES WERE ASSEMBLED IN AUTOMATED DNA
SEQUENCER
ANALYSIS OF FLOURESCENT LABELLED DNA MOLECULES.
THIS INSTRUMENT COMPRISES OF A ELECTROPHORETIC
APPARATUS WITH A 5000 V POWER SUPPLY.
ARGON ION LASER.
CCD CAMERA.
MACINTOSH COMPUTER WITH SOFTWARE SUCH AS
AUTO ASSEMBLER.
 THE SEQUENCES WERE ASSEMBLED AND COMPARED
WITH ANDERSON mt DNA SEQUENCE AND MUTATION SITES
WERE NOTED.

15. NOTE ALL THE MUTATION SITES AFTER ASSEMBLING THE
DNA SEQUENCES WITH THE ANDERSON SEQUENCE .
MJ NETWORK ,THE NETWORK WAS DRAWN USING
HVR 1 SEQUENCES THROUGH NETWORKING 3.0 VERSION
SOFTWARE OF FLUXUS ENGINEERING.
A TOTAL OF 15 HAPLOTYPES WERE OBTAINED .
OUT OF 15 ,10 ARE UNIQUE AND REST FORM
COMMON SETS OF HAPLOTYPES.
MJ NET WORK

16. MEDIAN JOINING NETWORK OF CHENCHU TRIBE

17. NJ NET WORK
NEIGHBOUR JOINING TREE WAS CONSTRUCTED WITH
THE DATA FROM 44 CHENCHU INDIVIDUALS WITH THE
DATA FROM THE INDIVIDUALS OF OTHER INDIAN POPULATIONS
USING CLUSTAL X, ARLEQUIN ,PHYLIP SOFTWARES .
THE CHENCHU POPULATION HAS CLOSE RELATOION
WITH DUNGRI BHILS ,DUNGRI GARASIAS, IRULA NILGIRIS

18. NJ NETWORK OF CHENCHU TRIBE