Microarray Technology and Applications

1
Microarray Technology and Applications

• Katie Bishop
• Biochem 660
• Pike Lab
• 09/21/09

2
Microarrays

• Introduction to DNA microarray technology
• Gene Expression Arrays
• Chromatin immunoprecipitation microarray analysis
  (ChIP-chip)
• Protein microarray technology

3
Why are microarrays important?

• High throughput method to query millions of DNA,
  proteins, or metabolites
• Key applications
• Gene expression patterns
• Chromatin immunoprecipitation
• Protein microarrays

4
Microarrays

• Introduction to DNA microarray technology
• Gene Expression Arrays
• Chromatin immunoprecipitation microarray analysis
  (ChIP-chip)
• Protein microarray technology

5
DNA Microarray Basic Design

• Microarray setup
• Array 1 cm x 1 cm
• 103-106 of features per array, 105-106 of probes
  per feature
• all probes within a feature contain the same
  sequence
• each feature has a different sequence

6
Types of DNA Arrays

• Affymetrix Arrays
• probes 25 nucleotides long
• Nimblegen Arrays
• probes 60-mers
• Spotted Arrays
• cDNA probes up to 1 kb in length

7
Affymetrix Arrays
8
Deprotection of Nucelotides
Gao et al. Nucleic Acids Research 2001, Vol 29.
No. 22, 4744-4750.
9
Affymetrix Arrays
10
Affymetrix Arrays
11
Nimblegen Maskless Array Synthesis
http//www.nimblegen.com/technology/manufacture.ht
ml
www.nimblegen.com
12
Maskless Array Synthesis
http//www.nimblegen.com/technology/manufacture.ht
ml
13
NimbleGen Arrays
14
Types of DNA Arrays

• Affymetrix Arrays
• probes 25 nucleotides long
• Nimblegen Arrays
• probes 60-mers
• Spotted Arrays
• cDNA probes up to 1 kb in length

15
DNA Microarray Questions
Single nucleotide polymorphisms (SNP) can be
detected using 25bp nucleotide probes.
atgtcaTggta
Allele 1
atgtcaAggta
Allele 2
What type of microarray technology could be used
to identify alleles? Why?
16
Microarrays

• Introduction to DNA microarray technology
• Gene Expression Arrays
• Chromatin immunoprecipitation microarray analysis
  (ChIP-chip)
• Protein microarray technology

17
Gene Expression Arrays One Color
http//www-microarrays.u-strasbg.fr/images/spotted
/spottedExpressAmplif.jpg
18
Gene Expression Arrays Scatter Plot
Two fold line
15
13
Treated (log2 ratio)
11
9.5
9
7
5
5
7
9
11
13
15
Linear Best Fit Line
Untreated (log2 ratio)
5.5
19
Gene Expression Arrays Two Color
Treated
Untreated
http//www-microarrays.u-strasbg.fr/images/spotted
/spottedExpressAmplif.jpg
20
Gene Expression Arrays Two Color
21
Gene Expression Arrays Question
What happens to gene expression following 24hr
OSM treatment?
15
C
D
13
11
No change? Supressed? Induced?
OSM 24hr (log2 ratio)
9
7
B
A
5
5
7
9
11
13
15
Untreated (log2 ratio)
22
Microarrays

• Introduction to DNA microarray technology
• Gene Expression Arrays
• Chromatin immunoprecipitation microarray analysis
  (ChIP-chip)
• Protein microarray technology

23
Chromatin Immunoprecipitation (ChIP) overview
Massie CE and Mills IG EMBO reports 9, 4, 337343
(2008)
24
Chromatin Immunoprecipitation Microarray
(ChIP-chip) Analysis
25
Chromatin Immunoprecipitation (ChIP) - chip assay
overview
26
Ligation Mediated PCR
Linear amplification Amplify all fragments
with one primer
27
Chromatin Immunoprecipitation (ChIP) chip
assay overview
28
Tiled Microarrays
Probe Size 51bp Probe Overlap 18bp Probe
Spacing 15bp
Resolution 32bp
What type of microarrays would you use?
29
ChIP-chip Data
Ab vs IgG
30
AcH4 ChIP-chip Results
How would you assess where AcH4 levels changed
with OSM treatment?
31
Other Types of DNA Array Methodology
Affymetrix Microarrays Gene Expression ChIP-chip S
NP Genotyping CNV Analysis Custom Microarrays
Nimblegen Microarrays Gene Expression
ChIP-chip Comparative Genomics Seq Comparative
Genomic Hybrid Sequence Capture DNA
Methylation Custom Microarrays
32
Microarrays

• Introduction to DNA microarray technology
• Gene Expression Arrays
• Chromatin immunoprecipitation microarray analysis
  (ChIP-chip)
• Protein microarray technology

33
Basic Design - Protein Microarrays
Hall, Ptacek, Snyder 2007 Mech Ageing Dev. 128
161-167
34
Protein Attachment Methods
Covalently attach via primary amines or
adsorption onto slides with nitrocellulose
His-tag protein on nickel-derivatized
slide Biotinylated-protein on strepavidin-coated
slide
35
Identification of DNA-binding protein
36
Cognate Site Identifier (CSI) Arrays
Warren et al., PNAS 103 867-872
37
References
DNA Microarrays lthttp//www.nimblegen.com/gt lthttp
//www.affymetrix.com/gt lthttp//smd.stanford.edu/r
esources/resinfo.shtmlgt Gene Expression Schulze
et al. Nat Cell Biol. 20013(8)E190-5. ChIP-chi
p Massie et al. EMBO Rep. 2008 April 9(4)
337343. We et al. Cancer Res 2006 66(14)
6899-6902. Protein Microarrays/CSI Hall et al.
2007128(1)161-7 Warren et al., PNAS 103
867-872