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PCR amplification of ovine somatotropin gene and its partial characterization

 

 

 

      Abstract

 

Ovine growth hormone gene was characterized using PCR amplification. Three primer pairs were designed using freeware; Clustal X for gene alignments and primer 0.5 and primer 3 for primer designing. The primers were capable of amplifying overlapping fragments from somatotropin genes of ovine, caprine and bovine animals. Using these primers three fragments 483 bp, 414 bp and 567 bp were amplified, respectively from ovine somatotropin gene. A 1321 bp fragment was also amplified using left primer of first primer pair and right of third, which was 81% of the total gene. The amplification conditions were optimized for each primer pair and it was found that first primer pair worked well in standard PCR conditions whereas second pair required a nested PCR approach using the 1321 bp fragment as template, to get a single band in reasonable quantity. The third primer pair worked satisfactory with slight mispriming in excess reagent conditions while nested PCR was used to improve amplification. The reagent conditions were almost same for all primers including 10x amplification buffer with (NH4)2SO4, 400µM dNTPs, 1µM each primer, 2 units of Taq polymerase, 10µg/ml BSA, 3.5 mM magnesium chloride and 1 µg genomic DNA template or 1µl of 500x dilution of nested PCR template, for a 50µl reaction. By restriction digestion of the amplified fragments 26 restriction sites of six different restriction enzymes were identified in the studied portion of gene, including two star activity sites of Pvu II and one polymorphic Bsu RI site. The study was done on three different samples of local breed of sheep.