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Introduction to CLC Main Workbench 20 June, 2012

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. Sequence Analysis Software Suits. Wisconsin GCG VectorNTI DNA STAR- LaserGene Geneious

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Introduction to CLC Main Workbench 20 June, 2012

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  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

  11. CLC DNA sequence

  12. Import a Protein Sequence

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

  14. CLC protein sequence

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

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

  17. Perform Restriction Digestion

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

  19. Restriction Digestion

  20. Protein Primary Structure Analysis

  21. Antigenicity Plot

  22. Protein Analysis Report

  23. Protease Digestion

  24. Proteolytic Cleavage

  25. Primer Design

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

  27. 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

  28. 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

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

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

  31. 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

  32. In silicocloning

  33. 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

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

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

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

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

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

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

  40. 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

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

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