Advances in Animal and Veterinary Sciences
PCR Amplification Protocol for GC Rich Protamine Gene from Chicken Testis cDNA
Sanjeev Kumar Sharma1, Chathathayil Muhammedali Shafeeque2, Jag Mohan1, Parappurath Abdul Azeez2, Ram Pratap Singh2
1Physiology and Reproduction Division, Central Avian Research Institute, Izatnagar 243122, Bareilly, India; 2Sálim Ali Centre for Ornithology and Natural History Anaikatty 641108, Coimbatore, India.
*Correspondence | Ram Pratap Singh, Sálim Ali Centre for Ornithology and Natural History Anaikatty, India; Email:email@example.com
Amplification of GC rich templates using PCR is usually difficult compared to non-GCrich targets. GC rich regions produce complex inter and intra strand folding (hairpins and loops) due to higher hydrogen bonding with neighbouring cytosine and guanine. These secondary structures in DNA are resistant to melting and cause Taq DNA polymerases to stall; they also hamper primer annealing, resulting in incomplete or non-specific amplifications. The aim of this study was to develop an easy PCR protocol to amplify very high GC rich (88% in the coding region) protamine gene of chicken. The cDNA used for amplification was synthesised from testis RNA and PCR products were detected by agarose gel electrophoresis. Protamine amplicon successfully amplified only with Go Taq DNA polymerase in the presence of DMSO. Optimization of MgCl2 concentration, buffer strength, annealing temperature, DMSO and the DNA template concentration, were important in the PCR reaction resulting in successful amplification. In addition, PCR started directly at hot plate is also important to get rid of primer-dimer formation.
Keywords | PCR, GC rich gene, DMSO