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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)


Inherit in Mendelian Fashion


Polymorphic loci with allele number as high as 14 – 15 per locus

5 repeats

7 repeats


Mostly reported from non-coding region, hence can be independent of selection

CACACACACACACACACACACACA

GENE1GENE1 GENE2

Flanking region is highly conserved in related species


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

Template DNA

Polymerase Chain Reaction & Automated Sequencing

Primer

dNTPsddNTPs

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

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