Transcript Biochips: Genomic Assessment Under Glass

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 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.
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 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.
Thank you