Transcript CLC Main Workbench - University of Pittsburgh

Introduction to CLC Main
Workbench
20 June, 2012
Ansuman Chattopadhyay, PhD
Head, Molecular Biology Information Services
Health Sciences Library System
University of Pittsburgh
[email protected]
Sequence Analysis Software Suits
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Wisconsin GCG
VectorNTI
DNA STAR-LaserGene
Geneious
 CLC
Main
Why CLC Main ?
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Windows
Mac
Linux
DNA, RNA, Protein,
Microarray Data Analysis
Regular Update
HSLS Licensed
CLC Main Access
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HSLS CLC Main Registration
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Link: http://www.hsls.pitt.edu/molbio/clcmain
Access via Pitt - Network Connect
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Instruction video: http://goo.gl/JNjMt
Topics
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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
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CLC Main Graphical User Interface
(GUI)
CLC Main
Basic Navigation
-DNA
-Protein
Import a DNA
Sequence
DNA Sequence
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Human PLCg1
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Refseq no: NM_002660
FASTA file
Raw sequence
CLC features:
Search, Import, Create new sequence
Import nucleotide and protein sequences into
CLC Main workbench
Resources
•CLC Main workbench:
http://www.hsls.pitt.edu/molbio/clcmain
Link to the video tutorial:
http://media.hsls.pitt.edu/media/clres/.swf
http://www.hsls.pitt.edu/molbio
CLC DNA sequence
Import a Protein
Sequence
Protein Sequence
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Human PLCg1
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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
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Create a new protein with PLCg1 SH2-SH2SH3 domains
Back Translation
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Reverse Translate PLCg1 SH2-SH2-SH3
Perform Restriction
Digestion
Restriction Mapping
www.biologyreference.com
http://www.hsls.pitt.edu/molbio
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…
http://www.hsls.pitt.edu/molbio
Polymerase Chain Reaction (PCR)
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very simple
exponential amplification
similar to natural DNA replication
1983-Kary Mullis
The primary reagents, used in PCR are:
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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
Things to consider for primer design…
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Primer-Dimer formation
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Secondary Structures in Primers
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Illegitimate Priming in Template DNA
due to repeated sequences
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Incompatibility with PCR conditions
SOURCE: NCBI
http://www.hsls.pitt.edu/molbio
PCR – non specific bands
Christiane B etal., http://goo.gl/KVCxI
http://www.hsls.pitt.edu/molbio
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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
Design PCR primers to amplify a region present in a
DNA sequence
Resources
•CLC Main workbench:
http://www.hsls.pitt.edu/molbio/clcmain
Link to the video tutorial:
http://media.hsls.pitt.edu/media/molbiovideios/PCR.swf
http://www.hsls.pitt.edu/molbio
Design primers for TaqMan real-time PCR
Resources
•CLC Main workbench:
http://www.hsls.pitt.edu/molbio/clcmain
Link to the video tutorial:
http://media.hsls.pitt.edu/media/molbiovideios/pcr2-clc-ac0112.swf
http://www.hsls.pitt.edu/molbio
Design PCR primers to amplify a DNA
region covering a protein domain
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PCR amplification of human
PLCg1 SH3 domain
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CLC Main Features:
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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
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Pair-wise Alignment
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Global
Local
Multiple Sequence Alignment
http://www.hsls.pitt.edu/molbio
Sequence Alignment
http://www.hsls.pitt.edu/molbio
Pair-wise Sequence Alignment
http://www.hsls.pitt.edu/molbio
Multiple Sequence Alignment
http://www.hsls.pitt.edu/molbio
PLCg1 Orthologous sequences
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PLCg1:
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Mouse:
Rat:
Cow:
Dog:
Zebra fish:
NP_067255
NP_037319
NP_776850
XP_542998
NP_919388
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Human:
NP_002651
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NP_067255,NP_037319,NP_776850,XP_542998,NP_919388,NP_002651
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http://www.hsls.pitt.edu/molbio
Thank you!
Any questions?
Carrie Iwema
[email protected]
412-383-6887
Ansuman Chattopadhyay
[email protected]
412-648-1297
http://www.hsls.pitt.edu/molbio