1. PCR amplification of ovine somatotropin gene and its partial characterization Moazzam Ali & Dr. Waheed Akhtar

2. Somatotropin Somatotropin or growth hormone is a peptide hormone, it causes growth of almost all tissues of body, increases protein synthesis, mobilization of fatty acids and decreases glucose utilization GH has remarkable biotechnological applications.

4. Primer designing Collect sequences of growth hormone gene of sheep, goat and bovine animals (complete gene, partial gene, mRNA) All the sequences were aligned using Clustal X software The alignments were studied for conserved regions Primers were designed in those regions using primer 0.5 and primer 3 softwares. Finally the primers were tested using BLAST for there performance.

6. PCR amplification There is no universal protocol for PCR amplification and one has to optimize reagent and temperature cycling steps for each set of primers

7. PCR Results of First Primer Pair Lanes 2 to 7 contain PCR products amplified with increasing magnesium conc (2 – 4.5 mM) and lane 10 is size marker PCR product size is 483bp 1 2 3 4 5 6 7 8 9 10 11 12

8. PCR Results Using Second Primer Pair Lanes 3 to 9 contain PCR products amplified by increasing Mg++ conc (2 - 5.0 mM) and lane 11 is size marker mispriming bands below the actual bands in lanes 7, 8 and 9 PCR product size is 414bp PCR product size is 414bp 1 2 3 4 5 6 7 8 9 10 11 12

9. Use of PCR enhancers for amplification by second primer pair BSA concentrations 10, 25 and 50µl/ml Triton X100 concentrations 0.05% and 0.1% BSA Triton X100

10. Use of PCR enhancers for amplification by second primer pair Betaine Conc. 1.2M DMSO Conc. 5% Glycerol Conc. 5% Betaine DMSO Glycerol

11. Nested PCR

12. Amplification of 1.32kb fragment (external round of nested PCR) Gel image showing 1321 bp PCR products amplified from two different sheep samples

13. Amplification of 414 bp fragment (internal round of nested PCR) Gel image showing 414 bp PCR products amplified from using second primer pair and 1.32kb PCR product as template from three different sheep samples

14. PCR Results Using Third Primer Pair Lanes 2-7 contained PCR products amplified under increasing magnesium conc (2 - 4.5 mM with 0.5 mM increments) and lanes 8-9 under increasing betaine (0.5 and 1 M) Nested PCR improved amplification, results not shown here PCR product size is 567 bp 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

15. Restriction digestion of amplified products Theoretical patterns of restriction digests were determined using appropriate softwares and then the patterns were confirmed by restriction digestion Some complications in restriction Gene duplication at growth hormone locus and it is an allelic phenomenon Partial digestion and star activity

16. Restriction Digestion of PCR Product, Amplified by First Primer Pair Uncut, lane 2 Mva I, lanes 3, 4, 5 Pvu II, lanes 6, 7, 8 Bsu RI, lanes 9, 10, 11 Size marker, lanes 1, 12 Three different samples were studied 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

17. Restriction Digestion of PCR Product, Amplified by Second Primer Pair Uncut, lane 2 Apa I, lanes 4,5,6 Bsu RI, lanes 7,8,9 Size marker, lane 11 Three different samples were studied 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

18. Restriction Digestion of PCR Product, Amplified by Second Primer Pair Uncut, lane 2 Mva I, lanes 4,5,6 Pst I, lanes 7,8,9 Size marker, lane 11 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

19. Restriction Digestion of PCR Product, Amplified by Third Primer Pair Uncut, lane 2 Sma I, lanes 4,5,6 Mva I, lanes 7,8,9 Size marker, lane 11 Three different samples were studied 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

20. Restriction Digestion of PCR Product, Amplified by Third Primer Pair Uncut, lane 2 Apa I, lanes 4,5,6 Bsp 1431, lanes 7,8,9 Size marker, lane 11 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

21. Restriction Digestion of PCR Product, Amplified by Third Primer Pair Uncut, lane 2 Bsu RI, lanes 4,5,6 Pst I, lanes 7,8,9 Size marker, lane 11 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

22. Restriction Map of Ovine Growth Hormone Gene

23. Thank you