introduction - cbs · 16 °c 2 h 70 °c 10 min ... pm mm cgatcaattgcactatgtcatttct...

INTRODUCTIONThe Technology of Microarrays

January 2009 - Hanne Jarmer

The Concept

gene specific DNA probes labeled target

gene

mRNA

Spotted arrays

High-density arrays

5 - 11 micron features

13 -16 micron features ~60 micron features

The spotting

Coating glass slides

Deposition of probes

Post-processing

Hybridization

The spotting

mRNAmRNA

cDNAcDNA

Cy3-cDNACy5-cDNA

SAMPLE CONTROL

Spotted microarrays

mRNAmRNA

cDNAcDNA

Cy3-cDNACy5-cDNA

SAMPLE CONTROL

Spotted microarrays

The Output

Cy5 Cy3

Affymetrix GeneChip® oligonucleotide array

• 11 to 20 oligonucleotide probes for each gene• Expensive to make• On-chip synthesis of 25 mers• ~20,000 genes per chip• High-quality data• Up to 6.4 M features

Affymetrix GeneChip® oligonucleotide array

The Chip

5 - 11 micron features

Photolithographyin situ synthesis

Spacers bound to surface with photolabile protection groups

Photolithographyin situ synthesis

Spacers bound to surface with photolabile protection groups

TTT

Photolithographyin situ synthesis

TTT AAA

Photolithographyin situ synthesis

The Eberwine Protocol

The Eberwine ProtocolSAMPLE

The Eberwine Protocol

RNA

SAMPLE

The Eberwine Protocol

RNA

T7

SAMPLE

70 °C10 min

The Eberwine Protocol

RNA

T7

SAMPLE

ssDNA

+ Reverse Transcriptase

42 °C2 h

70 °C10 min

The Eberwine Protocol

RNA

T7

dsDNA

SAMPLE

ssDNA

+ Reverse Transcriptase

+ RNase H+ Polymerase

42 °C2 h

16 °C2 h

70 °C10 min

The Eberwine Protocol

RNA

T7

dsDNA

SAMPLE

ssDNA

+ Reverse Transcriptase

+ RNase H+ Polymerase clean up dsDNA

42 °C2 h

16 °C2 h

70 °C10 min

The Eberwine Protocol

RNA

T7

dsDNA

T7 pol

SAMPLE

ssDNA

+ Reverse Transcriptase

+ RNase H+ Polymerase

42 °C2 h

16 °C2 h

70 °C10 min

The Eberwine Protocol

RNA

T7

dsDNA

T7 pol

SAMPLE

ssDNA

+ Reverse Transcriptase

+ RNase H+ Polymerase

+ Biotin-labeled nucleotides

42 °C2 h

16 °C2 h

37 °C6 h

70 °C10 min

The Eberwine Protocol

RNA

T7

dsDNA

T7 pol

SAMPLE

ssDNA

+ Reverse Transcriptase

+ RNase H+ Polymerase

+ Biotin-labeled nucleotides

aRNA

42 °C2 h

16 °C2 h

37 °C6 h

70 °C10 min

Detection of Biotin (Affymetrix)

Streptavidin Phycoerythrim = SAPE ( )

Detection of Biotin (Affymetrix)

Streptavidin Phycoerythrim = SAPE ( )

Detection of Biotin (Affymetrix)

Streptavidin Phycoerythrim = SAPE ( )

anti-SAPE IgG

Detection of Biotin (Affymetrix)

Streptavidin Phycoerythrim = SAPE ( )

anti-SAPE IgG

biotinylated anti-anti IgG

Detection of Biotin (Affymetrix)

Streptavidin Phycoerythrim = SAPE ( )

anti-SAPE IgG

biotinylated anti-anti IgG

Each gene is represented like this:

- Perfect Match (PM)- MisMatch (MM)

PMMM

CGATCAATTGCACTATGTCATTTCT CGATCAATTGCAGTATGTCATTTCT

PM:MM:

Roche-NimbleGen

Roche-NimbleGen

• High density: 70,000 to 2.1 M features

• Low design costs

• Probe length: 20-70 mers

• Multi-well system

• Full service!

Photolithography - micromirrors

13 - 16 micron features

Agilent

Agilent

• Density: 15-244 K features

• No design fee

• Probe length: 20-70 mers

The output

Cyanine5

Overlay

Cyanine3

Ink-Jet Printing

< 1 msec

Resistor on/off - vapourizing liquid

Multi-well formats

• Low Density, Low Cost• Whole/Partial Genome• Single Probe Per Gene• 4 x 44K • 8 x 15K

• Medium Density• Premium Partial Genome • Replicate Features• 2 x 105K

• High Density• Premium Whole Genome • Replicate Features• High Performance 244K

• Gene expression• SNP analysis• ChIP-chip (detection of TF-binding sites)• Comparative Genomic Hybridization (CGH)• Whole genome tiling arrays

Applications

Comparative Genomic Hybridization

Comparing two genomes

ChIP-chip

ChIP = Chromatin Immuno Precipitation

SNP analysisPatients

Controls

Association study

Sample PreparationHybridization

Array designProbe design

QuestionExperimental Design

Buy Chip/Array

Statistical AnalysisFit to Model (time series)

Expression IndexCalculation

Advanced Data AnalysisClustering PCA Classification Promoter Analysis

Meta analysis Survival analysis Regulatory Network

ComparableGene Expression Data

Normalization

Image analysis

The DNA Array Analysis Pipeline

Coffee BreakBe back at 10:30