Microarrays: Theory and Application

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Microarrays Theory and Application

• By Rich Jenkins
• MS Student of Zoo4670/5670
• Year 2004

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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

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Enter Microarrays
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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

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Types of Arrays

• cDNA arrays
• - Commonly called DNA microarrays
• - 500-5000nt per probe
• - 1 probe per gene
• - lt10,000 probes possible
• - Attached usually by solution treatment,
  followed by an incubation period

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Attachment Strategies
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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

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What goes on chips?

• cDNA libraries
• ESTs
• Sequence variants
• homologs

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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 wells probes is indicated by black.
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Reading the Chips

• Affymetrix GeneChip array

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Oligo Vs. cDNA
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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

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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

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Microarray uses

• Transcriptome analysis
• New gene discovery through function and the
  guilt-by-association principle
• Polymorphism analysis
• Proteomic analysis
• Toxin effect identification
• Pharmacological development

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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.

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Databases

• So you have all this data
• searching with NCBI

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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.

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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.

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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.

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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 chemists periodic table.
• New technologies will continue to rise from the
  need to process and store the masses of
  information gathered from microarray analysis.

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Thank you