The use of microsatellite analysis for animal genotyping

1. BIOCHEMICAL FOLLOW UP OF GENETIC
VARIATIONS AMONG DIFFERENT BREEDS OF
SHEEP USING MICRO-SATELLITE ANALYSIS
By: Adham Alkhadrawi

2. Under The Supervision Of
Prof. Dr. Yaqut Abd El Fattah Elsenosy
Head of biochemistry department, faculty of Vet Medicine,
Benha University.
Prof. Dr. Khaled Abd El Aleim Kahilo
Head of biochemistry department, faculty of Vet Medicine,
Kafrelsheikh University.
Prof. Dr. Mohamed Galal Agour
Chief researcher,
Director of animal health research institute

4. Fecundity gene
Fecundity gene (Fec gene) is responsible for increasing litter size
(production of twins and triplets) in some animal species through
increasing the expression of LH
Obese gene
Obese gene (Ob gene) is the gene responsible for fat deposition
and body weight gain in animals and humans through the
production of Leptin.

5. In this study, Three microsatellites were selected
for Fec gene
OarHH35 BM143 OarAE101
Primers were designed by Sun and his co-workers, 2010
Two microsatellites were selected for Ob gene
OARHH35 BMS648
Primers were designed by Ye-Liang and his co-workers, 2006
SUN W., H. CHANG, HASSAN HUSSEIN MUSA AND MINGXING CHU (2010) Study on relationship
between microsatellite polymorphism and producing ability on litter size trait of Hu sheep in
China. African Journal of Biotechnology Vol. 9(50), pp. 8704-8711.
YE-LIANG SUN, LIU GUO-QING, WANG GANG, REN HANG-XING, DAI RONG, LIU SHOU-REN,
XIE ZHUANG (2006) Correlation Analysis Between Microsatellite Marks and Body Weight
in Meat Sheep. Scientia Agricultura Sinica 39(10):2095-2100.

6. What are Microsatellites
Also known as STR, SSR, VNTR
STR: Short Tandem Repeats
SSR: Simple Sequence Repeats
VNTR: Variable Number Tandem Repeats
Tandemly repeated DNA sequences
with the repeat/size of 1 – 6 bases
repeated several times.
Highly polymorphic; can be analyzed
with the help of PCR.
Individual alleles at a locus differ in
number of tandem repeats of unit
sequence owing to gain or loss of one
or more repeats and they can be
differentiated by electrophoresis
according to their size.
5 repeats
7 repeats

7. Microsatellite types
Based on repeat pattern
Perfect
imperfect
Compound
Complex
Based on number of base pairs
Mono
Di
Tri
Tetra
CACACACACACACACACACACACA
CACACACACA CACACACACACA
CACACACACA
TGTGTGTGTGTGT
CACACACACA
TATATATATATATATA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
GTGTGTGTGTGTGTGTGTGTGTGTGTGGTGTTGTGT
AATAATAATAATAATAATAATAATAATAATAATAATAATAAT
CATGCATGCATGCATGCATGCATGCATGCATGCATG

8. Microsatellite properties
Co-dominant
Abundant in the eukaryote genome (~103 to 105 loci
dispersed at 7 to 10100 kilobase pair (kb) intervals)

9. Microsatellite properties
Inherit in Mendelian Fashion

10. Microsatellite properties
Polymorphic loci with allele number as high as 14 – 15 per locus
5 repeats
7 repeats

11. Microsatellite properties
Mostly reported from non-coding region, hence can be
independent of selection
GENE1 GENE1 GENE2
Flanking region is highly conserved in related species
CACACACACACACACACACACACA

12. Microsatellite properties
Can be obtained from small amounts of tissues [STR analysis
can be done on less than one billionth of a gram (a nanogram)
of DNA (as in a single flake of dandruff)]
PAGE separation; silver staining/automated genotyping

13. How Microsatellites are amplified
Primer annealing Extension Denaturation
GTGTGTGTGTGTGTGTGTGTGTGTGT
CACACACACACACACACACACACACA
Taq dNTPs
Primer
G C
T A
polymerase

15. This study aims to investigate the effects of genetic differences
among four local sheep breeds using microsatellite markers on
TWO traits
Fecundity (litter size) Weight Gain

17. Animals & Samples
40 female sheep from 4 different breeds
are studied for body weight and litter size
10 Rahmani
10 Osimi
10 Finnish
10 Rahmani-finnish hybrid
Blood samples are collected in vacutainer
tubes through jugular vein puncture

18. Serum Blood
DNA extraction using
commercial kits
Blood samples are used for
Serum isolated for LH &
LEP measurement

19. Polymerase Chain Reaction & Automated
Sequencing
Template DNA
Primer
ddNTPs dNTPs

20. Polymerase Chain Reaction & Automated
Sequencing
dNTP ddNTP
T
P
HO
T
P
H
A
P
H
G
P
H
C
P
H
ddNTPs are
labeled with dyes
of different colors
OH at
position3'
Lack OH at
position3'
P P
T
P
HO
H2O
G
P
H
T
P
HO
1- dNTPs interact with
OH group at position3'
2- PPi and H2O are released
3- ddNTPs interact in
the same manner
However
4- Subsequent nucleotides
cannot bind at position 3' due
to lack of OH group which
leads to ….
Chain Termination

21. Automated Sequencing
1- PCR products loaded
2- gel run starts
3- Laser beam scans the gel
4- PMT sends digital signal to
computer
PMT
5- Data interpreted
by software

22. Measuring LH and Leptin levels ELISA
1- Antibodies specific for LH & LEP attached to solid phase (well)
2- LH & LEP bind specific antibodies
3- Nonspecific proteins washed
4- Antibodies specific for LH & LEP linked to HRP bind
5- TMB added and oxidized by HRP to give ….
6- Color intensity measured by spectrophotometer
Blue color
THANK YOU
(Tetramrthylbenzidine diamine)
TMB
HRP
TMB
HRP
LEP LH