1. Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, 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/clcmainhttp://www.hsls.pitt.edu/molbio/clcmain Access via Pitt - Network Connect  Instruction video: http://goo.gl/JNjMthttp://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 silico agarose 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 http://www.hsls.pitt.edu/molbio www.biologyreference.com

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 http://www.hsls.pitt.edu/molbio A little something to get you in the mood…

28. Polymerase Chain Reaction (PCR) very simple exponential amplification similar to natural DNA replication The primary reagents, used in PCR are: Template DNA–DNA sequence to amplify DNA nucleotides–building blocks for new DNA Taq polymerase–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 1983-Kary Mullis

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/KVCxIhttp://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 silico cloning

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

38. In silico cloning 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 IwemaAnsuman Chattopadhyay iwema@pitt.eduansuman@pitt.edu 412-383-6887412-648-1297 http://www.hsls.pitt.edu/molbio