Study of prolactin gene polymorphism using PCR-RFLP and SSCP in different buffalo breeds of Gujarat
Kumar, Rakesh (2004) Study of prolactin gene polymorphism using PCR-RFLP and SSCP in different buffalo breeds of Gujarat. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
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Prolactin (PRL) is one of the most versatile hormones of the pituitary gland in terms of its biological activities. More than 100 different and district effects of the hormone have been documented. Prolactin plays an important role in initiation and maintenance of lactation. It is also responsible for the synthesis of milk proteins, lactose and lipid components of milk in bovines and other mammalian species. The present study was undertaken with the objective to genotype three different buffalo breeds of Gujarat viz., Surti, Mehsani and Jaffrabadi for PRL locus (exon 3) using Polymerase Chiain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP) and Single Strand Conformation Polymorphism (SSCP) techniques. Bovine PRL gene specific primers, forward:5’-CGA GTC CTT ATG AGC TTG ATT CTT – 3’, reverse: 5’ GCC TTC CAG AAG TCG TTT GTT TTC – 3’, were used to amplify 156bp of the buffalo PRL gene homologues to the exon 3 of the bovine PRL covering 50 Surti, 30 Mehsani and 30 Jaffrabadi buffaloes by phenol:chloroform method. PCR volume of 25μl was subjected to 32 cycles each, comprising of 950C for 1 minute, 580C for 1 minute and 720C for 1 minute followed by 10 minutes of final extension at 720C. The amplified products (20μl) was digested with 10 units of RSA I restriction enzyme for 3 hrs at 370C and electrophoresed on 2% agarosegel for 1 hour. All the samples studied from the three breeds comprised of single fragment of 156 bp indicating the monomorphic nature, showing AA genotype of the locus. This result was in contrast to the earlier reports indicating the polymorphic nature of the same in the same buffalo breeds under study. SSCP of the amplified PRL fragment was performed to detect any mutation that might be present out side the restriction site in the amplicon. The PCR samples were denatured by heating and then snap chilled on ice and was run on 0.5% MDE gel at a constant voltage of 5W. All the bands that appeared on the gel were showing monomorphic pattern, indicating the probable absence any mutation and suggesting high degree of sequence conservation across different buffalo breeds in buffaloes.
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