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Biochemistry 412 2004 6 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. Affymetrix Gene Chips - In Situ Synthesis Note: 4N masks required to make an array of oligonucleotides each of length N. Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5022. 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. Lockhart & Winzeler (2000) Nature 405, 827. 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 two different DNA microarray methods don’t always agree! • Results for mRNA abundance differences from either method 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. 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 Albertson & Pinkel (2003) Human Molec. Genet. 12, R145.