Introduction to CLC Main Workbench20 June, 2012

Ansuman Chattopadhyay, PhDHead, Molecular Biology Information ServicesHealth Sciences Library SystemUniversity of Pittsburghansuman@pitt.edu

Sequence Analysis Software Suits Wisconsin GCG VectorNTI DNA STAR-LaserGene GeneiousCLC 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: http://www.hsls.pitt.edu/molbio/clcmain

Access via Pitt - Network Connect Instruction video: http://goo.gl/JNjMt

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_002660 FASTA file Raw sequence

CLC features:

Search, Import, Create new sequence

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

Import nucleotide and protein sequences into CLC Main workbench

Link to the video tutorial:http://media.hsls.pitt.edu/media/clres/.swf

Resources

•CLC Main workbench: http://www.hsls.pitt.edu/molbio/clcmain

CLC DNA sequence

Import a Protein Sequence

Protein Sequence

Human PLCg1 Refseq no: NP_002651 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

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

www.biologyreference.com

Restriction Digestion

Protein Primary Structure Analysis

Antigenicity Plot

Protein Analysis Report

Protease Digestion

Proteolytic Cleavage

Primer Design

Primer Analysis & Design

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

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

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1983-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

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

PCR – non specific bands

Christiane B etal., http://goo.gl/KVCxI

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Design PCR Primers to amplify the region covering exons 4-5 in human PLCg1 mRNA sequence

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

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

Design PCR primers to amplify a region present in a DNA sequence

Link to the video tutorial:http://media.hsls.pitt.edu/media/molbiovideios/PCR.swf

Resources

•CLC Main workbench: http://www.hsls.pitt.edu/molbio/clcmain

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

Design primers for TaqMan real-time PCR

Link to the video tutorial:http://media.hsls.pitt.edu/media/molbiovideios/pcr2-clc-ac0112.swf

Resources

•CLC Main workbench: http://www.hsls.pitt.edu/molbio/clcmain

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

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

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

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

In silico cloning

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

Sequence Alignment

Pair-wise Alignment Global Local

Multiple Sequence Alignment

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Sequence Alignment

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Pair-wise Sequence Alignment

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

Multiple Sequence Alignment

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

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

Thank you!Any questions?

Carrie Iwema Ansuman Chattopadhyayiwema@pitt.edu ansuman@pitt.edu 412-383-6887 412-648-1297

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