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BIOCHEMICAL FOLLOW UP OF GENETIC VARIATIONS AMONG DIFFERENT BREEDS OF SHEEP USING MICRO-SATELLITE ANALYSIS
By: Adham Alkhadrawi
Under The Supervision Of
Prof. Dr. Yaqut Abd El Fattah ElsenosyHead of biochemistry department, faculty of Vet Medicine, Benha University.
Prof. Dr. Khaled Abd El Aleim KahiloHead of biochemistry department, faculty of Vet Medicine, Kafrelsheikh University.
Prof. Dr. Mohamed Galal AgourChief researcher, Director of animal health research institute
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 (Ob gene) is the gene responsible for fat deposition and body weight gain in animals and humans through the production of Leptin.
Fecundity gene
Obese gene
In this study, Three microsatellites were selected for Fec gene
Primers were designed by Sun and his co-workers, 2010
OarHH35 BM143 OarAE101
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.
Two microsatellites were selected for Ob gene
OARHH35 BMS648
Primers were designed by Ye-Liang and his co-workers, 2006
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.
What are MicrosatellitesAlso 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
Microsatellite typesBased on repeat pattern
Perfect
imperfect
Compound
Complex
Based on number of base pairs
Mono
Di
Tri
Tetra
CACACACACACACACACACACACA
CACACACACA CACACACACACA
CACACACACA TGTGTGTGTGTGT
CACACACACA
CACACACACA
TATATATATATATATA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
GTGTGTGTGTGTGTGTGTGTGTGTGTGGTGTTGTGT
AATAATAATAATAATAATAATAATAATAATAATAATAATAAT
CATGCATGCATGCATGCATGCATGCATGCATGCATG
Co-dominant
Microsatellite properties
Abundant in the eukaryote genome (~103 to 105 loci dispersed at 7 to 10100 kilobase pair (kb) intervals)
Microsatellite properties
Polymorphic loci with allele number as high as 14 – 15 per locus
5 repeats
7 repeats
Microsatellite properties
Mostly reported from non-coding region, hence can be independent of selection
CACACACACACACACACACACACA
GENE1GENE1 GENE2
Flanking region is highly conserved in related species
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
GTGTGTGTGTGTGTGTGTGTGTGTGT
How Microsatellites are amplified
CACACACACACACACACACACACACA
Primer
AT
CG
dNTPsTaqpolymerase
Primer annealing Extension Denaturation
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
Animals & Samples
40 female sheep from 4 different breeds are studied for body weight and litter size
Blood samples are collected in vacutainer tubes through jugular vein puncture
10 Rahmani10 Osimi10 Finnish10 Rahmani-finnish hybrid
DNA extraction using commercial kits
Serum isolated for LH & LEP measurement
Blood samples are used for
BloodSerum
Polymerase Chain Reaction & Automated Sequencing
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'
dNTP ddNTP
PP
T
P
HOH
2O
G
P
H
T
P
HO
Chain Termination
1- dNTPs interact with OH group at position3'
2- PPi and H2O are released
3- ddNTPs interact in the same manner
However4- Subsequent nucleotides cannot bind at position 3' due to lack of OH group which leads to ….
Automated Sequencing
PMT
3- Laser beam scans the gel
1- PCR products loaded
2- gel run starts
4- PMT sends digital signal to computer
5- Data interpreted by software
Measuring LH and Leptin levels ELISA
LHLEP
TMB
HRP
TMB
Blue color (Tetramrthylbenzidine diamine)
1- Antibodies specific for LH & LEP attached to solid phase (well)2- LH & LEP bind specific antibodies3- Nonspecific proteins washed4- Antibodies specific for LH & LEP linked to HRP bind5- TMB added and oxidized by HRP to give ….6- Color intensity measured by spectrophotometer
HRP
THANK YOU