PCR-Rflp of growth hormone gene in Deoni cattle

The present work was undertaken to study the Growth hormone gene polymorphism in Deoni cattle by using PCR – RFLP. Genomic DNA was isolated from blood samples collected randomly from 25 Deoni cows from the Central Breeding Farm of Deoni, Udgir, Dist.-Latur (Maharashtra) by using the phenol chloroform method. The quality and the quantity of the isolated DNA was checked by electrophoresis and spectrophotometer respectively. The _260/280 ratio was found in between 1.8 –

1.9. polymerase chain reaction (PCR) were performed to amplify GH1 loci of growth hormone. The best results were obtained to a master mix comprising of Taq. DNA polymerase (recombinant) 0.05 units/μl., MgCl₂ 4mM, DNTPs (dATP, dGTP, dCTP, dTTP) 4mM. Amplified PCR products were electrophoresed on 2% Agarose gel at 80 V. these DNA products were visualized by using gel documentation system.

Amplified DNA products of GH1 loci were digested with Alu I restriction enzyme by incubating at 37°C for 2 hours. Digested PCR products were run on 2.5% of the Agarose gel at 80 volts (V) for 60 to 120 minutes according to the expected fragment size of the DNA. Genotyping was performed according to the band pattern of digested PCR products.

Three restriction sites on both the strands resulted into four bands of 264,96,51 and 16 bp. Expected fourth band of 16 bp could not be visualized probably due to its small size. This genotype was referred as AA type. No different band pattern was observed other than that of AA genotype. In this study only one genotype i.e. AA was observed in Deoni population. Hence the frequency of the AA genotype was found to be 1.00. It indicates that Deoni cattle are found monomorphic for this locus.