Biochemistry 412. DNA Microarrays. March 30, 2007

Transcription

1 Biochemistry 412 DNA Microarrays March 30, 2007

2 Put a hex on you! Saturn s north pole, as seen from the Cassini spacecraft

3 Microarrays Have Led to an Explosion in mrna Profiling Studies Stolovitky (2003) Curr. Opin. Struct. Biol. 13, 370.

4 Two Main Types of DNA Microarrays Grünenfelder & Winzeler (2002) Nature Rev. Genet. 3, 653.

5 Oligonucleotide microarray Spotted DNA microarray Lockhart & Winzeler (2000) Nature 405, 827.

6 How labeling is done to make probes for use with oligonucleotide microarrays Fluorescence detection is made possible by staining the hybridized array with an avidin-fluorophore conjugate, which binds to the biotin moieties on the hybridized nucleic acids. Dalma-Weiszhausz et al (2006) Meth. Enzymol. 410, 3.

7 Affymetrix Gene Chips - In Situ Synthesis Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, Note: 4N masks required to make an array of oligonucleotides, each of length N.

8 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.

9 Key feature: known oligo sequence at each address on the chip. Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20. See also:

10 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

11 Note: Not all arrays have to be on chips! - Illumina, Inc.

12 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!

13 Applications of DNA Microarrays Genotyping (cf. first lecture) mrna profiling and transcriptome analysis Genome analysis (cancer and evolutionary studies) Genome-wide splicing analyses Etc.

14 Applications of DNA Microarrays Genotyping (cf. first lecture) mrna profiling and transcriptome analysis Genome analysis (cancer and evolutionary studies) Genome-wide splicing analyses Etc.

15 Dalma-Weiszhausz et al (2006) Meth. Enzymol. 410, 3.

16 Dalma-Weiszhausz et al (2006) Meth. Enzymol. 410, 3.

17 Comparative mrna Analyses Using Microarrays Note: this experiment shows competititve hybridization for a spotted array. Bryant et al (2004) Lancet Infect. Disease 4, 100.

18 Technical Proficiency & Experimental Design are Key to Reproducibility Lockhart & Barlow (2001) Nature Rev. Neurosci. 2, 63.

19 However, if your technique isn t good enough, the data must be normalized... Forster et al (2003) J. Endocrinol. 178, 195.

20 Note: caloric restriction gene chip experiment w/ rats. Ref: Lee et al (1999) Science 285, 1390.

21 Lee et al (1999) Science 285, 1390.

22 Applications of DNA Microarrays Genotyping (cf. first lecture) mrna profiling and transcriptome analysis Genome analysis (cancer and evolutionary studies) Genome-wide splicing analyses Etc.

23 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.

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25 Nature 444, 444 (2006)

26 Redon et al (2006) Nature 444, 444.

27 Redon et al (2006) Nature 444, 444.

28 Applications of DNA Microarrays Genotyping (cf. first lecture) mrna profiling and transcriptome analysis Genome analysis (cancer and evolutionary studies) Genome-wide splicing analyses Etc.

29 Dalma-Weiszhausz et al (2006) Meth. Enzymol. 410, 3.

30 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.

31 Microarray probe elements specifically designed to detect alternative splicing Barrass & Beggs (2003) Trends in Genetics 19, 295.

32 Applications of DNA Microarrays Genotyping (cf. first lecture) mrna profiling and transcriptome analysis Genome analysis (cancer and evolutionary studies) Genome-wide splicing analyses Etc.