Open Journal Systems

Cover Image

Rapid PCR-SSCP Screening of Awassi Sheep Using a Short Fragment (260bp) of Leptin Gene

Adil Hussein Radhi, Allawi Luaibi Dagher Al-Khauzai, Mohammed Baqur S Al-Shuhaib


The genetic variations of Iraqi native sheep breed were described using a non-radioactive single stranded conformation polymorphism (SSCP). Only 260 bp fragment of exon II and a portion of intron II leptin (LEP) gene from forty one individuals of Awassi breed were amplified by standard PCR, using the locus specific primers. PCR products were subjected to SSCP and visualized by ethidium bromide. According to SSCP bands, six different genotypes were identified. The resulting genotypes of Iraqi Awassi sheep were AA, AB, AH, AM, AR, and AS, and their alleles were A, B, H, M, R, and S respectively.


Awassi, Leptin gene, Sheep, PCR, SSCP Polymorphism

Full Text:



Kale DS, Yadav BR, Prasad I: DNA Polymorphisms at Candidate Gene Loci and Their Relation with Milk Production Traits in Murrah Buffalo (Bubalus bubalis). Iranian Journal of Applied Animal Sci 2014, 4: 39-43.

Nassiry MR, Heravi Moussavi A, Alashawkany AR, Ghovati S: Leptin Gene Polymorphism in Iranian Native Golpayegani and Taleshi Cows. Pakist. J. Biol. Sci 2007, 10: 3738-3741.

Liefers S: Physiology and genetics of leptin in perparturient dairy cows. 2004, Wageningen University. Ph. D. Thesis.

Geary TW, McFadin EL, MacNeil DM, Grings EE, Short RE, Funston RN, Keisler DH: Leptin as a predictor of carcass composition in beef cattle. J. Anim. Sci 2003, 81: 1-8.

Chilliard Y, Deavaud C, Bonnet M: Leptin expression in ruminants: nutritional and physiological regulations in relation with energy metabolism. Domest. Animal Endocrinol 2005, 29: 3 -22.

Zhou H, Hickford JGH, Gong H: Identification of allelic polymorphism in the ovine leptin gene. Mol, Biotech 2009, 41: 22-25.

Agarwal R, Rout PK, Singh SK: Leptin: a biomolecule for enhancing livestock productivity. Indian J. Biotechnol 2009, 8: 169 – 176.

Rock FL, Altmann SW, Heek MV, Kastelein RA, Bazan JF: The leptin haemopoietic cytokine fold is stabilized by an intrachain disulfide bond. Horm Metab Res 1996, 28: 649-652.

Hiroike T, Higo J, Jingami H, Toh H: Homology modeling of human leptin/leptin receptor complex. Biochem Biophys Res Commun 2000, 275: 154-158.

Dubey PP, Sharma A, Gour DS, Prashant, Jain A, Mukhopadhyay CS, Singh A, Joshi BK, Kumar D: Leptin gene polymorphism in Indian Sahiwal cattle by single strand conformation polymorphism (SSCP). South African Journal of Animal Science 2008, 38 (2): 131-135.

Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T: Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. USA 1989, 86: 2766-2770.

Sunnucks P, Wilson AC, Beheregaray LB, Zenger K, French J, Taylor AC: SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology. Mol. Ecol 2000, 9: 1699-1710.

Hayashi K, Yandell DW: How sensitive is PCR-SSCP? Human Mutation 1993, 2: 338-346.

Jaeckel S, Epplen JT, Kauth M, Miterski B, Tschentscher F, Epplen C: Polymerase chain reaction — single strand conformation polymorphism or how to detect reliably and efficiently each sequence variation in many samples. Electrophoresis 1998, 19: 3055-3061.

Girman D: The use of PCR-based single-stranded conformation polymorphism analysis (SSCP-PCR) in conservation genetics. In: Molecular Genetic Approaches in Conservation (eds Smith TB, Wayne RK) 1996, 167–182. Oxford University Press, Oxford.

Hayashi K: PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods and Applications 1991, 1: 34-38.

Bonifacio C, Santos C, Belo C, Cravador A: Single stranded conformation polymorphism (SSCP) analysis of aS1- casein, b- casein and k- casein genes in Charnequeira Portuguese indigenous breed. Arch. Zootec 2001, 50: 105-111.

Aslaminejad AA, Nassiry MR, Farajollahi H, Mahdavi M, Abbasi H, Javadmanesh A: Polymorphism in Exon 3 of Leptin Gene in Iranian Native Cattle Breeds. J. Appl. Anim. Res 2010, 37: 159-162.

Oikonomou G, Michailidis G, Kougioumtzis A, Avdi M, Banos G: Effect of polymorphisms at the STAT5A and FGF2 gene loci on reproduction, milk yield and lameness of Holstein cows. Res. Vet. Sci 2011, 91: 235-239.

Shojaei M, Abadi MM, Foze MA, Dayani O, Khezri A, Akhondi M: Association of growth trait and Leptin gene polymorphism in Kermani sheep. J. of Cell and Mol. Res 2010, 2, (1): 67-73.

Boucher D, Palin MF, Castonguay F, Gariepy C, Pothier F: Detection of polymorphism in the ovine leptin (LEP) Gene: Association of a single nucleotide polymorphism with muscle growth and meat quality traits. Can. J. Anim. Sci 2006, 86: 31-35.

Barzehkar R, Salehi A, Mahjoubi F: Polymorphisms of the ovine leptin gene and its association with growth and carcass traits in three Iranian sheep breeds. J. Biotech 2009, 7 (4): 241- 246.

Yeh FC, Yang R, Boyle T: POPGENE: Version 1.31. Microsoft Window–based Freeware for Population Genetic Analysis, University of Alberta. Edmonton, AB, Canada. 1999.

Arranz JJ, Bayon Y, San Plimitivo F: Differentiation among Spanish sheep breeds using microsatellites. Genet. Sel. E., 2001, 33 (5): 529-542.

Hashemi A, Mardani K, Farhadian M, Ashrafi I, Ranjbari M: Allelic polymorphism of Makoei sheep leptin gene identified by polymerase chain reaction and single strand conformation polymorphism. Afr. J. Biotechnol 2011, 10: 17903-17906.

Almeida SE, Almeida EA, Moraes JCF, Weimer T: Molecular marker in the LEP gene and reproductive reproductive performance of beef cattle. J. Anim. Breed. Genet 2003, 120 (2): 106.

Fisher S, Lerman L: Separation of random fragments of DNA according to properties of their sequences. PNAS USA 1980, 77: 4420-4424.

Patino-Garcia A, Sotillo-Pineiro E, Modesto C, Sierrasesumaga L: Screening of the human tumor necrosis factor-alpha (TNF-α) gene promoter polymorphisms by PCR–DGGE analysis. Mutation Research/Mutation Research Genomics 1999, 406: 121-125.

Riesner D, Steger G, Zimmat R, Owens R, Wagenhofer M, Hillen W, Vollach S, Henco K: Temperature-gradient gel electrophoresis of nucleic acids: Analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions. Electrophoresis 1989, 10: 377-389.

Yap EPH, McGee JO'D: Nonisotopic SSCP detection in PCR products by ethidium bromide staining. Trends Genet 1992, 8: 49.

Hongyo T, Buzard GS, Calvert RJ, Weghorst CM: 'Cold SSCP': a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses. Nucleic Acids Research 1993, 21: 3637-3642.

Xie T, Ho SL, Ma OCK: High resolution single strand conformation polymorphism analysis using formamide and ethidium bromide staining. Mol Pathol 1997, 50(5): 276-278.



  • There are currently no refbacks.