carcinomenormal

inflammation

DKFZ Genomics Activities

Basic Level of Functional Analysis

DNA RNA ProteinDNA RNA Protein

Enzymatic activity

DNA RNA ProteinDNA RNA Protein

Structure

Organisation

Splicing

RNAi

Localisation

Combination

ModificationInteraction

Copy numberStructure

DegradationComposition

Transcription ExpressionMethylation

SNPs

Basic Level of Functional Analysis

DNA RNA ProteinDNA RNA Protein

Structure

Organisation

Splicing

RNAi

Localisation

Combination

ModificationInteraction

Copy numberStructure

DegradationComposition

Transcription ExpressionMethylation

SNPs

Basic Level of Functional Analysis

Enzymatic activity

Hoheisel, J.D. (2006) Nature Rev. Genet., in press.

Microarrays

Robotic Production

Drosophila Microarray

Hild et al. (2003)Genome Biol. 5, R3.Boutros et al. (2004)Science 303, 382-385.Hollich et al. (2004)Biotechniques 37, 282-284.

Drosophila Microarray

In-House In Situ Synthesis of Oligomer Chips

Oligomer Synthesis Within the Channels

Features (Microscopic View)

Mask-Free Control of Oligonucleotide Synthesis

1. Molecules bound to glass surface

2. Cleavage of protection groups at illuminated spots

3. Condensation of amidites results inoligomerelongation

Light

Amidites

Digital micromirror system

Photolabileprotection group

1. 2. 3.

Oligomer Synthesis Within the Channels

Epigenetic Measurements

methC methC CC U T

bisulfite PCR

Typinganalysis

healthyhealthy

tumourtumour

Functional Genome AnalysisEpigenetics

DFG

5'...-GA

GC

AG

TGG

AC

G...-3'

5'...-GA

GC

AG

CG

GA

CG

...-3'

sequence polymorphism

type 1

type 2

type 1type 2

JHGCCAAGCTGGAGCCGAGCTG

GAGCCCAGCTG

GAGCCTAGCTG

AAGCTATACGT

AAGCTGTACGT

AAGCTCTACGT

AAGCTTTACGT

CGTCGAATGCT

CGTCGGATGCT

CGTCGCATGCT

CGTCGTATGCT

CCAGGAGTCTC

CCAGGGGTCTC

CCAGGCGTCTC

CCAGGTGTCTC

ab

c d

ab

c d

TypingS

ingle Nucleotide

Polym

orphisms

Oligomers for methylated DNAOligomers for unmethylated DNA

PCR-product made from unmethylated TIMP3 promoter DNA

Mund et al. (2005) Nucleic Acids Res. 33, e73.

Detection by Mismatch Discrimination

Transcriptional Profiling

cDNA microarrayscanned image

sample 1 sample 2

gene 1 gene 2 gene 3

RNA

ManMouseDrosophila melanogasterArabidopsis thalianaSaccharomyces cerevisiaeHydractinia echinata

Bacillus subtilisTrypanosoma bruceiNeurospora crassaPseudomonas putidaRhizobium meliloti

EU

EU

EU

Transcriptional Profiling Projects

pancreas

EU

Profiling Pancreatic Cancer

Pancreatic Cancer vs.

Chronic Pancreatitis

carcinomenormal

inflammation

Correspondence Analysis; Fellenberg et al. (2001) PNAS 98, 10781

patient

differentiallytranscribed gene

M-CHiPSData Processing Software

Target Selection

Buchholz et al. (2005) Clin. Cancer Res. 11, 8048-8054.

Target Selection

carcinomenormal

M-CHiPSData Processing Software

Correspondence Analysis; Fellenberg et al. (2001) PNAS 98, 10781

differences between individualsdifferences between individuals

inflammation-specific genesinflammation-specific genestumour-specific genestumour-specific genes

inflammation

Buchholz et al. (2005) Clin. Cancer Res. 11, 8048-8054.

M-CHiPSData Processing Software

Transcript Profiling Social Behaviour

healthycontrol

� MCA� MCAC

� SCA� IPMT

carcinomenormal

inflammation

normal tissue

ductal adenocarcinoma

cystic tumours

case tumour

cystictumours

Complex Antigen-Antibody InteractionsAntibody Microarrays

illustration

Kusnezow & Hoheisel (2002) BioTechniques 33 (suppl.), 14-23.Kusnezow et al. (2003) Proteomics 3, 254-264.Kusnezow & Hoheisel (2003) J. Mol. Recognit. 16, 165-176.Kusnezow et al. (2004) Protein Microarrays (Schena, M., ed.), 247-284.Kusnezow et al. (2005) Handbook of Immunohistochemistry

(Hayat, M.A., ed.), 23-37.

real experiment

EU

Kusnezow et al. (2006) Expert Rev. Mol. Diagn. 6, 111-124Kusnezow et al. (2006) Proteomics, in press.

Selection of Antibodies

TranscriptionalProfiling

Expression Profiling

• DNA sequences of 900 genes/ESTsexhibiting differential transcription

• Peptide selection from sequences according to 12 selection criteria

• Peptide synthesis

• Immunisation of rabbits

• Affinity purification of polyclonal antibodies

• Characterisation by in situ analyses

Antibody Microarrays

ProteinBinder Consortium

EU

Serum Sample AnalysisAntibody Microarrays

EU

Analysing Pancreatic Cancer

Transcript Profiling

pancreas

EU

Epigenetic Analysis

Protein Expression

www.dkfz.de/funct_genome

Functional Genome Analysis, DKFZ, Heidelberg