Genotyping of various buffalo breeds for k-casein using PCR-RFLP technique
Pipalia, Devendrakumar L. (1999) Genotyping of various buffalo breeds for k-casein using PCR-RFLP technique. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
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The B variant of k-casein is reported to be favorable for milk quality and considered to be included in breeding strategies of dairy cattle. The present study was undertaken with primary objective to genotype four different Indian buffalo breeds (Jaffarabadi, Mehsani, Surti and Pandharpuri) for k-casein type by Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP). A 379 bp k-casein fragment was amplified using primers K1 (5’- CAC GTC ACC CAC ACC CAC ATT TAT C-3’) and K2 (5’-TAA TTA GCC CAT TTC GCC TTC TCT GT – 3’). DNA was extracted from blood and semen samples collected from 20 Jaffarabadi, 44 Mehsani, 29 Surti and 5 Pandharpuri using phenol – chloroform method. The quality of DNA was assessed by UV spectrophotometry and agarose gel electrophoresis. PCR was carried out in a final reaction volume of 15 μl and the reaction mixture was subjected to initial denaturation at 940C – 4 minutes, annealing at 560C – 1 minute, and extension at 720C for 1 minute and final extension step of 720C for 10 minutes. Increase in the stringency during initial cycles of PCR reaction increased the sharpness and brightness of the bands. PCR products (379 bp) was restriction enzyme (RE) digested by Hinf I and Hind III. Kcasein /Hinf I analysis yielded two fragments of 288 and 91 bp only for all the DNA samples and hence, all the samples were typed BB. While k-casein/Hind III yielded two fragments of 225 and 154 bp for all the samples. Thus, PCR-RFLP using Hinf I and Hind III revealed all the samples to be monomorphic for this locus. Absence of polymorphism at this k-casein locus in buffaloes may be a species characteristic. However, this require conformation by analyzing large many samples from different buffalo breeds.
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