Trudeau Institute Molecular Biology Core Facility

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Trudeau InstituteMolecular Biology Core Facility

• Scottie Adams, Manager

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Bioinformatics Applications in Immunological
Molecular Biology
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Trudeau Aerial
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Trudeau Addition
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Immune System
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MBCF provides

• Services
• Reagents
• Special Projects
• Expertise/Consulting

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MBCF Services

• Automated DNA Sequencing
• Fragment Analysis (Spectratyping)
• Primer/probe design
• Mycoplasma testing
• Real time Quantitative Fluorescent PCR
• RNase Protection Assay (RPA)
• Databank Searches/Bioinformatics
• Recombinant Protein Production

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MBCF Reagents

• Class I Tetramers (FACS staining reagent)
• DNA/RNA probes for
• In Situ Hybridization (ISH)
• Methylation Assays (Southern blots)
• Mycoplasma testing
• Real time Quantitative PCR
• Stock Primers
• Class II Multimers (FACS staining reagent)

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DNA Sequencing

• De novo
• Mutation Verification
• Recombinant Protein Construct Verification

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DNA Sequence Contig
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Sequence Contig
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Mutation Detection
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Codon Usage
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Fragment Analysis

• Genotyping
• Spectratyping
• DNA fingerprinting

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Recombinant Protein Expression

• MHC Class I Staining Reagents - CD8 cells
• MHC Class II Staining Reagents - CD4 cells
• Vaccines
• Protein Analysis

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Class I Tetramer
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General Scheme
Preparation of construct Clone by phone Protein
production Heavy chain B2microglobulin Folding
Heavy and light chain with peptide Purification
of monomer S300 column Biotinylation of
monomer S300 column Tetramerization with PE/APC
strepavidin Add in small aliquots to insure
tetramers
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Heavy Chain Construct
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Protein Expression
pET Inducible Expression Vectors E. Coli system
BL21(DE3) Grow bacteria (6 L) Induce protein
production (IPTG) Purify exclusion bodies
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Protein Production
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Purification
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Biotinylation Test
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Tumor Immunity

Donor
(Thy 1.2)
8.78
0.09
6.24
0.06
6.15
0.06
0.01
2.25
Untreated
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RPA Screening
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Quantitative Real Time PCR

• Gene Expression
• Bacterial/Viral Quantitation
• Allelic discrimination (SNPs)

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QF-PCR Assay Design

• Design Primers/Probe for Gene of Interest
• Extract RNA from tissues
• Generate cDNA by reverse transcription
• Run QF-PCR reaction with gene of interestusing
  AbiPrism 7700
• Analyze Results

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Double Stranded DNA
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Denaturation
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Annealing
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Extension/Signal Release
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QF PCR Schematic
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Northern Blot Analysis
Disadvantages Subjective Quantitation Requires
10 µg RNA Time-Consuming Uses Radioactivity
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In vitro stimulated Th2 cells
Day 1 Day 8
GAPDH
0.355 cycles
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In vitro stimulated Th2 cells
Day 1 Day 8
IL-4
5.5 cycles
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In vitro stimulated Th2 cells
Day 1 Day 8
IFNg
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QF-PCR vs Northern
Advantages Objective relative quantitation Conser
ves sample Time-Efficient Simple quantitation of
multiple targets Fluorescent technology
59-fold By TaqMan
60-fold By TaqMan
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QF-PCR Assay Design

• Design Primers/Probe for Gene of Interest
• Extract RNA from tissues
• Generate cDNA by reverse transcription
• Run QF-PCR reaction with gene of interestusing
  AbiPrism 7700
• Analyze Results

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Conclusions

• Bioinformatics is your best friend
• Learn to make it work for you

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Thanks

• Dolan DNA Learning Center
• Dave Micklos
• Uwe Hilgert
• Trudeau Institute
• Susan Cooper
• Peter Sayles
• Tim Miller