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.
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
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.
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.
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.
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?
How are we different? …at the RNA level. …and at the protein level.
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).
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.
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.
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.
First...What does it mean that our genomes are 98.7% similar at the DNA level, and how do we know this?
Human/Chimp BES Similarity BAC End Sequences This represents coding and non-coding regions of the genome.
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.
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)
Affymetrix probe U95A array... 63,000 probe set 10,000 Human genes 5 arrays Affymetrix DNA Chip Part I: Plan A
Cluster Analysis Distances represent the relative differences in gene expression changes.
So What? Changes in gene expression are greatest in the Human brain gene cluster. Primates Mice
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.
Microarray better 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
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...
Microarray Data 17,997 genes 5:1 difference in expression profiles.
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 Experiment Part II: Proteome Analysis
Proteomics (2d-gels) Proteins separated by mass, then by charge. Qualitative (positions), Quantitative (amount), Present/absent (+/-) Human Chimp