Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System University of Pittsburgh
Sequence Analysis Software Suits Wisconsin GCG VectorNTI DNA STAR-LaserGene Geneious CLC Main
Why CLC Main ? Windows Mac Linux DNA, RNA, Protein, Microarray Data Analysis Regular Update HSLS Licensed
CLC Main Access HSLS CLC Main Registration Link: Access via Pitt - Network Connect Instruction video:
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
CLC Main Graphical User Interface (GUI)
CLC Main
Basic Navigation -DNA -Protein
Import a DNA Sequence
DNA Sequence Human PLCg1 Refseq no: NM_ FASTA file Raw sequence CLC features: Search, Import, Create new sequence
CLC DNA sequence
Import a Protein Sequence
Protein Sequence Human PLCg1 Refseq no: NP_ Uniprot Accession Number: P19174 FASTA file Raw sequence CLC features: Search, Import, Create new sequence
CLC protein sequence
Protein sequence manipulation Create a new protein with PLCg1 SH2-SH2- SH3 domains
Back Translation Reverse Translate PLCg1 SH2-SH2-SH3
Perform Restriction Digestion
Restriction Mapping
Restriction Digestion
Protein Primary Structure Analysis
Antigenicity Plot
Protein Analysis Report
Protease Digestion
Proteolytic Cleavage
Primer Design
Primer Analysis & Design A little something to get you in the mood…
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 Kary Mullis
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
PCR – non specific bands Christiane B etal.,
Design PCR Primers to amplify the region covering exons 4-5 in human PLCg1 mRNA sequence
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
In silico cloning
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
In silico cloning
Sequence Alignment Pair-wise Alignment Global Local Multiple Sequence Alignment
Sequence Alignment
Pair-wise Sequence Alignment
Multiple Sequence Alignment
PLCg1 Orthologous sequences PLCg1: Mouse: NP_ Rat: NP_ Cow: NP_ Dog: XP_ Zebra fish: NP_ Human: NP_ NP_067255,NP_037319,NP_776850,XP_542998,NP_919388,NP_
Thank you! Any questions? Carrie IwemaAnsuman Chattopadhyay