Exam #2 Mean = 73% Median = 74% Mode = 90% A range: | | | | | | | | | B range: | | | | | | | | | C...
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Transcript of Exam #2 Mean = 73% Median = 74% Mode = 90% A range: | | | | | | | | | B range: | | | | | | | | | C...
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Exam #2
Mean = 73%Median = 74% Mode = 90%
A range: | | | | | | | | |B range: | | | | | | | | |C range: | | | | | | |D range: | | | | | | | | | |Failing: | | | | | | | | |
For Final:
Final - Exam #22
- or -
Final - Final Average2
…whichever is higher.
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How are we different?…at the RNA level.
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Northern Analysis
DNA hybridizing to RNA,
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DNA Arrays and Expression
…grid gene-specific ASOs onto the DNA chip, or “known” cDNAs onto microarrays,
– extract mRNA from a specific tissue,
– make fresh cDNA from the new mRNA,• and label it,
– bind to the array for display.
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http://www.bio.davidson.edu/courses/genomics/chip/chip.html
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Genes and Targets• With many genome projects finished done, most, if not all of
the genes identified can be gridded,
– presently, several completely sequenced genomes have been gridded,
• H. sapiens (>20,000 genes)• Arabidopsis (>26,000 genes),• C. elegans (>22,500 genes),• Drosophila (>13,500 genes),• yeast (>6000 genes),• more,
• drug identification, fundamental research, etc.
www.affymetrix.com
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Gene/Drug Discovery
…genes involved in cancer and other diseases have been identified through a variety of techniques,
– genome expression analysis provides a means of discovering other genes that are concomitantly expressed,
– genome expression analysis provides a means of monitoring drug/treatment regimes.
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Applications
• Can study the role of more than 1700 cancer related genes in association with the (rest) of the genome,
• Define interactions and describe pathways,
• Measure drug response,
• Build databases for use in molecular tumor classifications,
– benign vs. cancerous, slow vs. aggressive.
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Extended Applications
• Water quality testing (4 hours vs. 4 days),
• Environmental watchdogs,
• Fundamental research on non-human subjects,
• Direct sequencing of related species for evolutionary studies,
• Comparisons of gene regulation between closely related species,
• etc.
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Monday
•Human and chimp DNA is ~98.7 similar,
•But, we differ in many and profound ways,
•Can this difference be attributed, at least in part, to differences in gene expression, rather than differences in the actual gene and gene products?
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How are we different?…at the RNA level.
…and at the protein level.
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Gene Expression Technologies
• General Scheme: Extract mRNA, synthesize labeled cDNA, Hybridize with DNA on the array,
– DNA Chips (Affymetrix) and MicroArrays can measure mRNA concentration of thousands of genes simultaneously,
– look for genes that are expressed similarly (clustering).
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Linear Round
What is clustering?
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T (RESOLUTION)
Cluster Analysis Yields Dendrogram
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What’s the Question
• Human and chimp DNA is ~98.7 similar,
• But, we differ in many and profound ways,
– can this difference be attributed, at least in part, to differences in gene expression, rather than differences in the actual genes and gene products? Especially in the brain.
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Huh?
• Prevailing notion (though fading fast):
– a structural gene is mutated, – alleles that code for favorable adaptive proteins survive,– and, in fact, out-compete old alleles…evolution marches
on.
• Paper’s hypothesis: it’s not just the genes that are changing, but the REGULATION of the genes,
– cis and trans acting factors are considered here.
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Why Brain?
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Why Regulation?
• The human genome contains about 20k - 25k genes,
– Drosophila, 13,061,
– Arabidopsis, 26,027,
– C. elegans, 19,099 genes.
• One hypothesis: “many” of the additional genes found in complex organisms, are transcription factors,
• more complex control of transcription leads to more complex organisms.
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First
...What does it mean that our genomes are 98.7% similar at the DNA level, and how do we know this?
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Human/Chimp BES SimilarityBAC End Sequences
This represents coding and non-coding regions of the genome.
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Are our Phenotypes 98.7% Similar?
• Some apparent differences,
– HIV susceptibility, epithelial neoplasms (cancers), malaria, and Alzheimers,
• In fact, there is only one well understood biochemical difference,
– A 92 bp deletion in a gene that codes for a hydroxylase, results in an un-hydroxylated secretion protein in our immune system.
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• Check patterns of gene expression level,
– using DNA Chip arrays,
– for >10,000 genes in humans, chimps, orangutans, and macaques,
• Compare brain, liver, and blood specifically,
• Perform cluster analysis and compare the amount of change in expression levels across the genome.
The Experiment Part I: Transcriptome Analysis
T (RESOLUTION)
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Affymetrixprobe
• U95A array...
63,000 probe set10,000 Human genes
5 arrays
Affymetrix DNA Chip
Part I: Plan A
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Targets
• Labeled Human cDNA, Chimp cDNA, Macaque cDNA,
– collect tissue (from cadavers),
– brain, blood, liver,
– extract RNA, make cDNA
– label cDNA.
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Cluster Analysis
Distances represent the relative differences in gene expression changes.
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So What?
• Changes in gene expression are greatest in the Human brain gene cluster.
Primates
Mice
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Probably Rejected by the Journal
• Why?
– Probe was human, target chimp and ape mRNA sequence may slightly differ,
• at the “allele specific oligonucleotide” level, single base changes may skew the data.
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Microarraybetter probe
• Spotted 17,997 PCR products onto nylon, probed with labeled cDNAs,
– PCR primers are available, in kits, that will amplify just about any part of the human genome,
– 1,000 bp fragments were generated,
• Base pair differences won’t affect probe sensitivity over this large a target.
Part I: Plan B
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Microarrays
...denatured, double stranded DNA (500 - 5000 bp) is dotted, or sprayed on a glass or nylon substrate,
...up to tens of thousands of spots per array,
quill technology...
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Microarray Data17,997 genes
5:1 difference in expression profiles.
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• Check for differences in protein expression using 2-D gel analysis,
– differences in location on the gel indicate qualitative differences,
– differences in amount indicate quantitative differences,
• Controls,
– Rodent tests.
The ExperimentPart II: Proteome Analysis
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Proteomics(2d-gels)
• Proteins separated by mass, then by charge.• Qualitative (positions), Quantitative (amount), Present/absent (+/-)
Human Chimp
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8500 Protein Spots
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What do You Think?
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Microarray Data1b
What does this figure demonstrate?
How is the data different from Fig.
1a?
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Proteomics(2d-gels)
• What does this figure show?• What conclussions were drawn from the data?
• Does it support the microarray data?
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Final Review• Friday: Pheromone paper.
• Friday December 7th. Lecture time will be used for review,
• Final Tuesday 10:30 - 12:30,
• Office Hours: as usual.