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Overview research topics and techniques 1. clinical, biochemical and molecular studies of hereditary connective tissue disorders 2. genetic studies of.

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Presentation on theme: "Overview research topics and techniques 1. clinical, biochemical and molecular studies of hereditary connective tissue disorders 2. genetic studies of."— Presentation transcript:

1 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

2 Nadine Van Roy Mireille Van Gele Jo Vandesompele Katleen De Preter Jasmien Hoebeeck Filip Pattyn Bruce Poppe Stefan Vermeulen

3 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 Overview research topics and techniques

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5 MYCN copy number

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

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9 f

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

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

12 Comparative genomic hybridisation - CGH

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

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

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

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

18 MPE600 Chromosome 1 Copy Number Profiles Measured by Chromosome and Array CGH kb log2ratio

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

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

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

22 MPE600 Chromosome 1 Copy Number Profiles Measured by Chromosome and Array CGH kb log2ratio

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

24 '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 Overview research topics and techniques

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

26 laser 'capture' microdissectie (LCM):

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29 laser 'capture' microdissection (LCM):

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

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35 microarrays

36 microarras

37 microarrays

38 Sestri Levante 2000 Alizadeh et al. Nature

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