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

Enter Microarrays

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

Attachment Strategies

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 ChipsOne 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.

Reading the Chips

Affymetrix’ GeneChip array

Oligo Vs. cDNA

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 chipsCommon 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.

Thank you