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Biochemistry 412
2005
5 April Lecture
DNA Microarrays
Microarrays
• DNA Arrays
• Protein Arrays
• Other
Microarrays
• DNA Arrays
• Protein Arrays
• Other
Microarrays Have Led to an Explosion in mRNA Profiling Studies
Stolovitky (2003) Curr. Opin. Struct. Biol. 13, 370.
Two Main Types of DNA Microarrays
Grünenfelder & Winzeler (2002) Nature Rev. Genet. 3, 653.
Lockhart & Winzeler (2000) Nature 405, 827.
Affymetrix Gene Chips - In Situ Synthesis
Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5022.
Note: 4N masks required to
make an array of oligonucleotides,
each of length N.
Note: this is the photolabile blocking
group, “X”, indicated schematically
in Figure 1.
Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5022.
Key feature: known oligo
sequence at each “address”
on the chip.
Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20.
In situ synthesized (Affymetrix) microarrays:
Advantage - massive coverage of sequences possible
Disadvantage - expensive and not easily customized
Spotted arrays (oligonucleotides or dsDNA):
Advantage - flexibility and cost; can be “home-made”
Disadvantage - big initial investment; less standardization
Note:
Not all arrays have to be on chips…!
- Illumina, Inc.
Caveat….Caveat….Caveat…!!
• Results from the different
DNA microarray methods
don’t always agree!
• And results for mRNA abundance
differences don’t always agree
with protein abundance data!
Stages in the Life Cycle of a
Technological Innovation
1. Unveiling (first publication)
2. Hype (lots of follow-on publications,
meetings, venture capital, etc.)
3. Disillusionment (more paper opportunities!)
4. Promise eventually fulfilled (years later)
5. Enters routine use
Applications of DNA Microarrays
• Genotyping
• mRNA profiling and
“transcriptome” analysis
• Genome analysis (cancer and
evolutionary studies)
• Genome-wide splicing analyses
• Etc.
Applications of DNA Microarrays
• Genotyping
• mRNA profiling and
“transcriptome” analysis
• Genome analysis (cancer and
evolutionary studies)
• Genome-wide splicing analyses
• Etc.
Comparative mRNA Analyses Using Microarrays
Bryant et al (2004)
Lancet Infect. Disease
4, 100.
Technical Proficiency & Experimental Design are Key to Reproducibility
Lockhart & Barlow (2001)
Nature Rev. Neurosci. 2, 63.
However, if your technique isn’t good enough, the data must be “normalized”...
Forster et al (2003)
J. Endocrinol. 178, 195.
Microarray data:
Analysis of a hypothetical time course of mRNA abundances
for several genes
Leung & Cavalieri (2003) Trends in Genetics 19, 649.
Note: caloric restriction gene chip experiment w/ rats.
Ref: Lee et al (1999) Science 285, 1390.
Lee et al (1999) Science 285, 1390.
Common Genes Induced in Immature Dendritic Cells in Response to Infection with Diverse Pathogens
Huang et al (2001) Science 294, 870.
Bassett et al (1999) Nature Genet. (suppl.) 21, 51.
Applications of DNA Microarrays
• Genotyping
• mRNA profiling and
“transcriptome” analysis
• Genome analysis (cancer and
evolutionary studies)
• Genome-wide splicing analyses
• Etc.
Microarrys Can Also be Used to Analyze Chromosomal Rearrangments
CGH - “Comparative genomce hybridization
LOH - “Loss of heterozygosity”
Albertson & Pinkel (2003) Human Molec. Genet. 12, R145.
Applications of DNA Microarrays
• Genotyping
• mRNA profiling and
“transcriptome” analysis
• Genome analysis (cancer and
evolutionary studies)
• Genome-wide splicing analyses
• Etc.
Microarrays can be used to detect tissue-specific alternative splicing
Blue - tissue 1
Purple - tissue 2
Yellow - pooled sample (average)
Le et al (2004) Nucleic Acids Research 32, e180.
Microarray probe elements specifically designed to detect alternative splicing
Barrass & Beggs (2003)
Trends in Genetics 19, 295.
Applications of DNA Microarrays
• Genotyping
• mRNA profiling and
“transcriptome” analysis
• Genome analysis (cancer and
evolutionary studies)
• Genome-wide splicing analyses
• Etc.
RNA as a repository of past generations’ genetic information??
Weigel & Jürgens (2005) Nature 434, 443.