Introduction to CLC Main Workbench 20 June, 2012

1. Introduction to CLC Main Workbench20 June, 2012 AnsumanChattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System University of Pittsburgh ansuman@pitt.edu

2. Sequence Analysis Software Suits • Wisconsin GCG • VectorNTI • DNA STAR-LaserGene • Geneious • CLC Main

3. Why CLC Main ? • Windows • Mac • Linux • DNA, RNA, Protein, • Microarray Data Analysis • Regular Update • HSLS Licensed

4. CLC Main Access • HSLS CLC Main Registration • Link: http://www.hsls.pitt.edu/molbio/clcmain • Access via Pitt - Network Connect • Instruction video: http://goo.gl/JNjMt

5. Topics • CLC Main GUI • Import DNA sequence into CLC • Import Protein sequence into CLC • Design PCR primers • Perform restriction enzymes digestions • Run in silicoagarose gels • Protein primary structure analysis • Protease digestions

6. CLC Main Graphical User Interface (GUI)

7. CLC Main

8. Basic Navigation -DNA -Protein

9. Import a DNA Sequence

10. DNA Sequence • Human PLCg1 • Refseq no: NM_002660 • FASTA file • Raw sequence CLC features: Search, Import, Create new sequence

12. CLC DNA sequence

13. Import a Protein Sequence

14. Protein Sequence • Human PLCg1 • Refseq no: NP_002651 • Uniprot Accession Number: P19174 • FASTA file • Raw sequence CLC features: Search, Import, Create new sequence

15. CLC protein sequence

16. Protein sequence manipulation • Create a new protein with PLCg1 SH2-SH2-SH3 domains

17. Back Translation • Reverse Translate PLCg1 SH2-SH2-SH3

18. Perform Restriction Digestion

19. Restriction Mapping www.biologyreference.com http://www.hsls.pitt.edu/molbio

20. Restriction Digestion

21. Protein Primary Structure Analysis

22. Antigenicity Plot

23. Protein Analysis Report

24. Protease Digestion

25. Proteolytic Cleavage

26. Primer Design

27. Primer Analysis & Design A little something to get you in the mood… http://www.hsls.pitt.edu/molbio

28. Polymerase Chain Reaction (PCR) 1983-Kary Mullis • very simple • exponential amplification • similar to natural DNA replication The primary reagents, used in PCR are: • TemplateDNA–DNA sequence to amplify • DNA nucleotides–building blocks for new DNA • Taqpolymerase–heat stable enzyme catalyzes new DNA • Primers–single-stranded DNA, ~20-50 nucleotides, complimentary to a short region on either side of template DNA http://www.hsls.pitt.edu/molbio

29. Things to consider for primer design… • Primer-Dimer formation • Secondary Structures in Primers • Illegitimate Priming in Template DNA due to repeated sequences • Incompatibility with PCR conditions SOURCE: NCBI http://www.hsls.pitt.edu/molbio

30. PCR – non specific bands Christiane B etal., http://goo.gl/KVCxI http://www.hsls.pitt.edu/molbio

31. Design PCR Primers to amplify the region covering exons 4-5 in human PLCg1 mRNA sequence http://www.hsls.pitt.edu/molbio

34. Design PCR primers to amplify a DNA region covering a protein domain • PCR amplification of human PLCg1 SH3 domain • CLC Main Features: • Reverse Translate • PCR Primer Design • Video Tutorials http://www.hsls.pitt.edu/molbio

35. In silicocloning

36. Molecule Construction Clone a fragment from pBR322 into pUC19 ☼Donor fragment: pBR322, 5’EcoRI—3’AvaI ☼Recipient fragment: pUC19, 5’SmaI—3’EcoRI video tutorials http://www.hsls.pitt.edu/molbio

37. http://www.hsls.pitt.edu/molbio

38. In silicocloning http://www.hsls.pitt.edu/molbio

39. Sequence Alignment • Pair-wise Alignment • Global • Local • Multiple Sequence Alignment http://www.hsls.pitt.edu/molbio

40. Sequence Alignment http://www.hsls.pitt.edu/molbio

41. Pair-wise Sequence Alignment http://www.hsls.pitt.edu/molbio

42. Multiple Sequence Alignment http://www.hsls.pitt.edu/molbio

43. PLCg1 Orthologous sequences • PLCg1: • Mouse: NP_067255 • Rat: NP_037319 • Cow: NP_776850 • Dog: XP_542998 • Zebra fish: NP_919388 • Human: NP_002651 • NP_067255,NP_037319,NP_776850,XP_542998,NP_919388,NP_002651 http://www.hsls.pitt.edu/molbio

44. Thank you!Any questions? Carrie IwemaAnsumanChattopadhyay iwema@pitt.eduansuman@pitt.edu 412-383-6887 412-648-1297 http://www.hsls.pitt.edu/molbio