Introduction to CLC Main Workbench 20 June, 2012 Ansuman Chattopadhyay, PhD Head, Molecular Biology Information Services Health Sciences Library System.

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Presentation transcript:

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