Microarrays: Theory and Application By Rich Jenkins MS Student of Zoo4670/5670 Year 2004.
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Microarrays: Theory and Application By Rich Jenkins MS Student of Zoo4670/5670 Year 2004
Genetics in Motion -Prior techniques were sufficient for limited studies -But completion of genomic studies opened more avenues -A need for the ability to examine how many genes relate to many other genes
Types of Arrays Oligonucleotide arrays - Commonly called DNA chips - Affymetrix and others - 5-50 mer oligos per probe - multiple probes per gene - 260,000+ probes per chip - Attachment usually by photolithography, followed by an incubation period
Types of Arrays cDNA arrays - Commonly called DNA microarrays - 500-5000nt per probe - 1 probe per gene - <10,000 probes possible - Attached usually by solution treatment, followed by an incubation period
cDNA Synthesis Issues Normal PCR does not decently reflect the initial concentrations of mRNA in the cell. Several rounds of linear amplification based on cDNA synthesis and a template-directed in vitro transcription reaction (cDNA/IVT) are now being used, in a system that does not produce the same inconsistencies as PCR
What goes on chips? cDNA libraries ESTs Sequence variants homologs
Reading the Chips One sample will fluoresce red when hybridized with on chip probes, the other will fluoresce green. If both hybridize, then the well will fluoresce yellow. Neither sample hybridizing with a given well’s probes is indicated by black.
Oligo Vs. cDNA Part II DNA chip drawbacks: Possibly provide too much data Require fairly expensive equipment or must be sent to a commercial firm DNA microarray drawbacks: Do not measure as great a variety of hybridization Often use only one probe to test a given gene
Oligo Vs. cDNA Part III DNA chips Common uses: -Expression profiling -New gene identification -Polymorphism analysis -Large scale sequencing DNA microarrays Common uses: -Expression profiling -New gene identification -Polymorphism analysis
Microarray uses Transcriptome analysis New gene discovery through function and the guilt-by-association principle Polymorphism analysis Proteomic analysis –Toxin effect identification –Pharmacological development
Guilt by Transcription cDNA created from library of cells currently active mRNA (partial cellular transcriptome). Genes without known function that express consistently with known genes are often assumed to be similar in function to the known gene.
Databases So you have all this data… searching with NCBI
Proteomic Analysis Chips are under development to supercede current Western blot procedure for protein analysis. Current protein information from chips is gleaned mostly from transcriptome analysis.
Proteomic Analysis Toxin effects may be measured by their effects on the current levels of mRNA in a cell, compared to the mRNA profile of either a normal, healthy cell and/or a cell that has a non-sense mutation in the same region as that affected by the toxin.
Proteomic Analysis Using a process similar to that of toxin analysis, the pharmacological industry has begun using microarrays to determine the area of protein synthesis affected by a given drug. This type of research may cheapen and speed drug development greatly, since hit or miss approaches with poorly understood biochemical pathways might be avoided.
Conclusions Microarrays will play a very important role in the near future of genetics, and biology as a whole, and may provide the genetic equivalent of the chemist’s periodic table. New technologies will continue to rise from the need to process and store the masses of information gathered from microarray analysis.