164 J Pub Health Bio Sci. 2013;2(1):164-168 Journal of Public Health and Biological Sciences Vol. 2, No. 1 Jan – Mar 2013, p.164-168 ISSN 2305-8668 (Print) 2307-0625 (Online) URL: http://www.jphbs.com PCR OPTIMIZATION AND RESTRICTION ANALYSIS OF ST cDNA FROM VARIOUS COW AND GOAT BREEDS Sumbul Khalid *1 , Sardar Faisal Mahmood 2 , Muhammad Jadoon Khan 3 , Mirza Imran Shahzad 4 , Azra Khanum 5 1 Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan; 2 CNRS, UMR 144, Institut Curie, 26 rue d’Ulm, 75248 Cedex 05, Paris, France; 3 Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan; 4 Department of Biochemistry, University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Bahawalpur, Pakistan; 5 Department of Biochemistry, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan. *Corresponding Author: Sumbul Khalid (sumbul.khalid@iiu.edu.pk), Department of Bioinformatics and Biotechnology, Women Campus, International Islamic University, Islamabad, Pakistan. Abstract Introduction: Keeping in mind the importance and uniqueness of Pakistan’s indigenous livestock breeds, PCR conditions for optimum amplification of somatotropin (ST) cDNA were optimized for cow and goat breeds. Material and Methods: ST cDNA was synthesized from total RNA extracted from pituitaries of 6 breeds of cow and 2 goat breeds, all animals native to Pakistan. Optimization was done for annealing temperature, concentrations of MgCl 2 , Taq DNA polymerase and dNTPs. Results: Results showed that optimum annealing temperature for amplification of ST cDNA from both animal species was 54°C. Optimum MgCl 2 concentration for goat samples was found out to be 2.0mM while for cow samples it was 1.5mM. Taq polymerase was checked for 0.5U to 1.5U and all samples showed similar intensities, therefore 0.5U of polymerase were used. In case of dNTP concentration 0.15mM was optimum for both animal species. The amplified fragments were cloned in T/A vector and further characterized by restriction analysis before sequenced. Conclusion: Six sequences were submitted to NCBI GenBank with following accession numbers: DQ307368, EU086738, DQ307369, DQ307370 and DQ307371. Key words: PCR, restriction analysis, cow somatotropin, goat somatotropin Introduction The polymerase chain reaction (PCR) is a biotechnology technique to amplify a single copy of DNA generating thousands to millions of copies of the particular DNA sequence. 1 PCR was invented by Kary Mullis in 1983 and it is such a powerful method for the rapid amplification of target nucleic acid sequence that it won, for the inventor, a Noble Prize in the field of chemistry. 2 A single PCR reaction includes multiple cycles of nucleic acid heat denaturation, annealing and extension. PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. 3,4 Exogenous administration of somatotropin (ST) has been shown to increase lactation performance in mammals. 5 In case of farm animals, an increase in milk yield response with ST has been demonstrated in pigs 6 , sheep 7 , goats 8 and cows. 9 The study reported in this paper was a part of a project where ST cDNA was amplified, cloned, expressed and the protein was purified. In this paper the results for optimization of PCR conditions for amplification of ST cDNA from two animal sources are reported. The successfully amplified right sized fragments were then cloned in T/A vector and characterized by limited restriction analysis. Original Article