Overview research topics and techniques 1. clinical, biochemical and molecular studies of hereditary connective tissue disorders 2. genetic studies of neurofibromatosis type I, BPES and familial breast cancer 3. genetic studies of pediatric tumors (incl neuroblastoom) and Merkel cell carcinoma, genetic study of leukaemia

http://www.come.to/neuroblastoma Nadine Van Roy Mireille Van Gele Jo Vandesompele Katleen De Preter Jasmien Hoebeeck Filip Pattyn Bruce Poppe Stefan Vermeulen

Overview research topics and techniques 1. basic technology for DNA and RNA work - biochemistry of collagen 2. karyotyping 3. FISH : detection of DNA targets in cytological preparations) one or two DNA targets CGH (comparative genomic hybridisation) : detection of genomic imbalances of chromosomal regions fiber FISH : FISH on stretched chromatin fibers FICTION : FISH combined with immunophenotyping M-FISH (multicolor FISH) : 24 or more DNA targets

MYCN copy number

Fluorescence in situ hybridisation DNA Commercial Cot1 DNA Labeling: nick translation Mix 10ml culture Denaturation and incubation at 37°C Wash, detection and counterstain Denaturation Hybridisatie o/n

f

Comparative genomic hybridisation - CGH Tumor DNA Normaal DNA Fluorescentie intensiteit Ratio 1.0 Normal Amplified Loss Groen Rood Normale metaphase

Comparative Genomic Hybridization (CGH) to Chromosomes and Arrays Test Genomic DNA Reference Genomic DNA Cot-1 DNA Ratio Ratio Position on Chromosome Position on Sequence

Comparative genomic hybridisation - CGH

Comparative genomic hybridisation - CGH

CGH profiles for chromosomes 1, 6, 8, 15 en 17 in a neuroblastoma case

Comparative Genomic Hybridization (CGH) to Chromosomes and Arrays Test Genomic DNA Reference Genomic DNA Cot-1 DNA Ratio Ratio Position on Chromosome Position on Sequence

Low and High Resolution Arrays for the Analysis of Copy Number Changes on 20q in Breast Tumors

Human Array CGH with ~ 1 Mb Resolution ~2500 BACs Triplicate spots 130 mm centers 864 well plates 0.5 mg DNA Random Prime Cy3 and Cy5 16 hr Hyb. 12 mm

MPE600 Chromosome 1 Copy Number Profiles Measured by Chromosome and Array CGH -2 -1.5 -1 -0.5 0.5 1 1.5 2 50000 100000 150000 200000 250000 300000 kb log2ratio

Assembly of microarrays for genome-wide measurement of DNA copy number Snijders et al. Nature Genetics november , 263-264

Low and High Resolution Arrays for the Analysis of Copy Number Changes on 20q in Breast Tumors

Human Array CGH with ~ 1 Mb Resolution ~2500 BACs Triplicate spots 130 mm centers 864 well plates 0.5 mg DNA Random Prime Cy3 and Cy5 16 hr Hyb. 12 mm

MPE600 Chromosome 1 Copy Number Profiles Measured by Chromosome and Array CGH -2 -1.5 -1 -0.5 0.5 1 1.5 2 50000 100000 150000 200000 250000 300000 kb log2ratio

Assembly of microarrays for genome-wide measurement of DNA copy number Snijders et al. Nature Genetics november , 263-264

Overview research topics and techniques 'real time' quantitative PCR using SYBR I Green: quantification of gene expression laser 'capture' microdissectie (LCM): dissectie van cellen uit weefselcoupes 'suppression subtractive hybridisation (SSH): comparative gene expression analysis for two cell populations microarray-analysis: 'high throughput' gene expression analysis

laser 'capture' microdissectie (LCM): isolation of single cells or small islets of cells from cytological preparation

laser 'capture' microdissectie (LCM):

laser 'capture' microdissectie (LCM):

laser 'capture' microdissection (LCM):

Lab-on-Chip - Bioanalyser Agilent technologies for analysis of minute amounts of DNA, RNA or protein

microarrays

microarras

microarrays

Alizadeh et al. Nature Sestri Levante 2000