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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. |