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Plan A
Topics?
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Bypassing Calvin cycle
Making vectors for Dr. Harms
Making vectors for Dr. Lucent
Cloning & sequencing antisense RNA
Studying ncRNA
Engineering biodiesel, hydrogen or electricity production.
Something else?
Plan A
http://biophotovoltaics.wordpress.com/
Photosynthetic biofilms in pure culture harness solar energy in
mediatorless bio-photovoltaic cell (BPV) system.
Here we report on light-driven electrical power generated with
biofilms grown from photosynthetic fresh water or marine species
without the addition of an artificial electron-shuttling mediator.
Green alga (Chlorella vulgaris, Dunaliella tertiolecta) or
cyanobacteria (Synechocystis sp. PCC 6803, Synechococcus sp. WH
5701) strains were grown directly on a transparent, conductive
anode (indium tin oxide-coated polyethylene terephthalate) and
power generation under light and dark conditions was evaluated
using a single-chamber bio-photovoltaic cell (BPV) system.
Increased power outputs were observed for all strains upon
illumination, with the largest light effect observed for
Synechococcus (maximum 10.3 mW m−2 total power output
recorded under 10 W m−2 white light).
Analyzing your clone
1) FISH
2) “Restriction mapping”
3) Southern analysis : DNA
4) Northern analysis: RNA
5) Sequencing
Genome projects
1) Prepare map of
genome
Genome projects
1) Prepare map
of genome
• To find genes
must know
their location
Sequencing Genomes
1) Map the genome
2) Prepare an AC library
3) Order the library
FISH to find their
chromosome
Sequencing Genomes
1) Map the genome
2) Prepare an AC library
3) Order the library
• FISH to find their chromosome
• identify overlapping AC using ends as probes
• assemble contigs until chromosome is covered
Sequencing Genomes
1) Map the genome
2) Prepare an AC library
3) Order the library
4) Subdivide each AC into
lambda contigs
Sequencing Genomes
1) Map the genome
2) Prepare an AC library
3) Order the library
4) Subdivide each AC into
lambda contigs
5) Subdivide each lambda
into plasmids
6) sequence the plasmids
Using the genome
Studying expression of all genes simultaneously
Microarrays (reverse Northerns)
•Attach probes that detect genes to solid support
Using the genome
Studying expression of all genes simultaneously
Microarrays (reverse Northerns)
•Attach probes that detect genes to solid support
•cDNA or oligonucleotides
•Tiling path = probes for entire genome
Microarrays (reverse Northerns)
•Attach probes that detect genes to solid support
•cDNA or oligonucleotides
•Tiling path = probes for entire genome
•Hybridize with labeled targets
Microarrays
•Attach cloned genes to solid support
•Hybridize with labeled targets
•Measure amount of target bound to each probe
Microarrays
Measure amount of probe bound to each clone
Use fluorescent dye : can quantitate light emitted
Microarrays
Compare amounts of mRNA in different tissues or
treatments by labeling each “target” with a different dye
Using the genome
Studying expression of all genes simultaneously
1.Microarrays: “reverse Northerns”
• Fix probes to slide at known locations, hyb with
labeled targets, then analyze data
Using the genome
Studying expression of all genes simultaneously
1.Microarrays: “reverse Northerns”
2.High-throughput sequencing
Using the genome
Studying expression of all genes simultaneously
1. Microarrays: “reverse Northerns”
2. High-throughput sequencing
• “Re-sequencing” to detect variation
Using the genome
Studying expression of all genes simultaneously
1.Microarrays: “reverse Northerns”
2.High-throughput sequencing
•“Re-sequencing” to detect variation
•Sequencing all mRNA to quantitate gene expression
Using the genome
Studying expression of all genes simultaneously
1.Microarrays: “reverse Northerns”
2.High-throughput sequencing
•“Re-sequencing” to detect variation
•Sequencing all mRNA to quantitate gene expression
•Sequencing all mRNA to identify and quantitate splicing
variants
Using the genome
Studying expression of all genes simultaneously
1.Microarrays: “reverse Northerns”
2.High-throughput sequencing
•“Re-sequencing” to detect variation
•Sequencing all mRNA to quantitate gene expression
•Sequencing all mRNA to identify and quantitate splicing
variants
•Sequencing all RNA to identify and quantitate ncRNA
Using the genome
Studying expression of all genes simultaneously
1.Microarrays: “reverse Northerns”
2.High-throughput sequencing
3. Bisulfite sequencing to detect C methylation
Using the genome
Bisulfite sequencing to detect C methylation
Using the genome
Bisulfite sequencing to detect C methylation
ChIP-chip or ChIP-seq to detect chromatin modifications:
17 mods are associated with active genes in CD-4 T cells
Using the genome
• various chromatin modifications are associated with
activated & repressed genes
•Acetylation, egH3K9Ac, is associated with active genes
Using the Genome
•various chromatin modifications are associated with
activated & repressed genes
•Acetylation, egH3K9Ac, is associated with active genes
• Phosphorylation of H2aS1, H2aT119, H3T3, H3S10 &
H3S28 shows condensation
Using the Genome
•Acetylation, egH3K9Ac, is associated with active genes
• Phosphorylation shows condensation
• Ubiquitination of H2A and H2B shows repression &
marks DNA damage
Using the Genome
•Acetylation, egH3K9Ac, is associated with active genes
• Phosphorylation shows condensation
• Ubiquitination of H2A and H2B shows repression
• Methylation is more complex: H3K36me3 = on
•H3K27me3 = off
Using the Genome
Methylation is more complex:
•H3K36me3 = on
•H3K27me3 = off
•H3K4me1 = off
•H3K4me2 = primed
•H3K4me3 = on
Using the genome
Many sites provide gene expression data online
• NIH Gene expression omnibus
http://www.ncbi.nlm.nih.gov/geo/ provides access to
many different types of gene expression data
Using the genome
Many sites provide gene expression data online
• NIH Gene expression omnibus
http://www.ncbi.nlm.nih.gov/geo/ provides access to
many different types of gene expression data
•Many different sites provide “digital Northerns” or other
comparative analyses of gene expression
• http://cgap.nci.nih.gov/SAGE
• http://www.weigelworld.org/research/projects/geneexpr
essionatlas
Using the genome
Many sites provide gene expression data online
• NIH Gene expression omnibus
http://www.ncbi.nlm.nih.gov/geo/ provides access to
many different types of gene expression data
•Many different sites provide “digital Northerns” or other
comparative analyses of gene expression
• http://cgap.nci.nih.gov/SAGE
• http://www.weigelworld.org/research/projects/geneexpr
essionatlas
• MPSS (massively-parallel signature sequencing)
http://mpss.udel.edu/
Using the genome
Many sites provide gene expression data online
Many sites provide other kinds of genomic data online
• http://encodeproject.org/ENCODE/
Primer/probe design
Crucial for successful DNA & RNA analysis!
• Main source of specificity for PCR
Using the genome
Studying specific genes identified by WEB search
• Using PCR to focus on specific genes/conditions
Primer/probe design
Crucial for successful DNA & RNA analysis!
• Main source of specificity for PCR
•good primers only bind your sequence
Primer/probe design
Crucial for successful DNA & RNA analysis!
• Main source of specificity for PCR
•good primers only bind your sequence
• Also important for microarrays, sequencing, Southerns
Primer/probe design
• Also important for microarrays, sequencing, Southerns
• Concerns
•Specificity: only want them to bind at one place
Primer/probe design
• Also important for microarrays, sequencing, Southerns
• Concerns
•Specificity: only want them to bind at one place
•Main concern: 3’ end should not bind
Primer/probe design
• Also important for microarrays, sequencing, Southerns
• Concerns
•Specificity
• Complementarity
Primer/probe design
• Also important for microarrays, sequencing, Southerns
• Concerns
•Specificity
• Complementarity:
•Hairpins: may not melt (problem for RT) or may
reform
Primer/probe design
• Also important for microarrays, sequencing, Southerns
• Concerns
•Specificity
• Complementarity:
•Hairpins
• homoduplexes
may not melt
May be extended by DNA polymerase
Primer/probe design
• Also important for microarrays, sequencing, Southerns
• Concerns
•Specificity
• Complementarity:
•Hairpins
• homoduplexes
• heteroduplexes
may not melt
May be extended by DNA polymerase
Primer/probe design
• Also important for microarrays, sequencing, Southerns
• Concerns
•Specificity
• Complementarity:
• Melting T
• Should match!
Primer/probe design
• Also important for microarrays, sequencing, Southerns
• Concerns
•Specificity
• Complementarity:
• Melting T
• Should match!
• Every site calculates them differently!
Primer/probe design
• Also important for microarrays, sequencing, Southerns
• Concerns
•Specificity
• Complementarity:
• Melting T
• Targeting specific locations
• amplifying specific sequences
Primer/probe design
• Also important for microarrays, sequencing, Southerns
• Concerns
•Specificity
• Complementarity:
• Melting T
• Targeting specific locations
• amplifying specific sequences
• creating mutations: need mismatch towards 5’ end so
3’ end binds well
Primer/probe design
• Also important for microarrays, sequencing, Southerns
• Concerns
•Specificity
• Complementarity:
• Melting T
• Targeting specific locations
• amplifying specific sequences
• creating mutations: need mismatch towards 5’ end so
3’ end binds well
•Add restriction sites at 5’ end: may need to
reamplify an amplicon