Biochemistry 4122005

5 April Lecture

DNA Microarrays

Microarrays

• DNA Arrays

• Protein Arrays

• Other

Microarrays

• DNA Arrays

• Protein Arrays

• Other

Stolovitky (2003) Curr. Opin. Struct. Biol. 13, 370.

Microarrays Have Led to an Explosion in mRNA Profiling Studies

Grünenfelder & Winzeler (2002) Nature Rev. Genet. 3, 653.

Two Main Types of DNA Microarrays

Lockhart & Winzeler (2000) Nature 405, 827.

Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5022.

Note: 4N masks required tomake an array of oligonucleotides,each of length N.

Affymetrix Gene Chips - In Situ Synthesis

Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5022.

Note: this is the photolabile blockinggroup, “X”, indicated schematicallyin Figure 1.

Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20.

Key feature: known oligosequence at each “address”on the chip.

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 differentDNA microarray methodsdon’t always agree!

• And results for mRNA abundancedifferences don’t always agreewith 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. Disease4, 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.

Ref: Lee et al (1999) Science 285, 1390.

Note: caloric restriction gene chip experiment w/ rats.

Lee et al (1999) Science 285, 1390.

Huang et al (2001) Science 294, 870.

Common Genes Induced in Immature Dendritic Cells in Response to Infection with Diverse Pathogens

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

Albertson & Pinkel (2003) Human Molec. Genet. 12, R145.

CGH - “Comparative genomce hybridizationLOH - “Loss of heterozygosity”

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 1Purple - tissue 2Yellow - 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.