Introduction to CLC Main Workbench20 June, 2012
Ansuman Chattopadhyay, PhDHead, Molecular Biology Information ServicesHealth Sciences Library SystemUniversity of [email protected]
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: 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
http://www.hsls.pitt.edu/molbio
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
http://www.hsls.pitt.edu/molbio
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
Sequence Alignment
Pair-wise Alignment Global Local
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 [email protected] [email protected] 412-383-6887 412-648-1297
http://www.hsls.pitt.edu/molbio