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 GeleJo Vandesompele Katleen De Preter Jasmien Hoebeeck Filip PattynBruce Poppe Stefan Vermeulen

1. basic technology for DNA and RNA work - biochemistry of collagen2. karyotyping3. FISH : detection of DNA targets in cytological preparations)

one or two DNA targetsCGH (comparative genomic hybridisation) : detection ofgenomic imbalances of chromosomal regionsfiber FISH : FISH on stretched chromatin fibersFICTION : FISH combined with immunophenotyping

M-FISH (multicolor FISH) : 24 or more DNA targets

Overview research topics and techniques

MYCN copy number

Fluorescence in situ hybridisation

Labeling: nick translation

DNA

Denaturation and incubation at 37°C

Mix

Denaturation

Hybridisatie o/n

Commercial Cot1 DNA

Wash, detectionand counterstain

10ml culture

f

Comparative genomic hybridisation - CGH

Tumor DNA Normaal DNA

Fluorescentieintensiteit

Ratio 1.0

Normal Amplified Loss

GroenRood

Normale metaphase

Comparative Genomic Hybridization (CGH) to Chromosomes and Arrays

Test Genomic DNA Reference Genomic DNA

Rat

io

Rat

io

Position on Chromosome Position on Sequence

Cot-1 DNA

Comparative genomic hybridisation - CGH

1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 x y

Comparative genomic hybridisation - CGH

n = 21

n = 22

n = 23

n = 24

n = 25

n = 26

n = 27

n = 28

n = 29

n = 210

n = 211

n = 212

n = 213

n = 214

n = 215

n = 216

n = 217

n = 218

n = 219

n = 220

n = 221

n = 222

n = 2x

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

Comparative Genomic Hybridization (CGH) to Chromosomes and Arrays

Test Genomic DNA Reference Genomic DNA

Rat

io

Rat

io

Position on Chromosome Position on Sequence

Cot-1 DNA

GT 849 NABC1*

Low resolution scanning array of chromosome 20

~1 MbHigh resolution array of 20q13.2

p arm q arm

D20

S48

0

847g7

D20

S10

20

P4067

D20

S90

2

RM

C20

C00

1

P4039B4166

B4104, B4123

845f3

SG

C31

010

WI-1

6697

P4030

P4009

D20

S21

1

A4185

B4103

B4097

D20

S87

6

B4109

D20

S10

44

P4028

P4010

B4099

WI-9

227

afm

224z

d12

D20

S60

9

B4195

B4130

D20

S85

4

P4187

D20

S72

0

D20

S91

3

B4018

NABC1

PIC 1- like

ZNF218

100 Kb

820f5

D20

S18

3

L39 20q::22qW

I196

42

B4198

D20

S12

0

B4132

WI-1

1116

EW

I-157

02E

STS

G25

428

D20

S70

8E

931h6

D20

S77

6

B4201

dJ1191N16

P4007

B4121

B4087 B4090

B4208

CRP

ZNF217

B4188

P4069

P4016

P4041

Proc. Natl. Acad. Sci. USA, 95, 8703-8708 (1998)

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 m centers

864 well plates

0.5 g 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

0.5

1

1.5

2

0 50000 100000 150000 200000 250000 300000

kb

log

2rat

io

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

GT 849 NABC1*

Low resolution scanning array of chromosome 20

~1 MbHigh resolution array of 20q13.2

p arm q arm

D20

S48

0

847g7

D20

S10

20

P4067

D20

S90

2

RM

C20

C00

1

P4039B4166

B4104, B4123

845f3

SG

C31

010

WI-1

6697

P4030

P4009

D20

S21

1

A4185

B4103

B4097

D20

S87

6

B4109

D20

S10

44

P4028

P4010

B4099

WI-9

227

afm

224z

d12

D20

S60

9

B4195

B4130

D20

S85

4

P4187

D20

S72

0

D20

S91

3

B4018

NABC1

PIC 1- like

ZNF218

100 Kb

820f5

D20

S18

3

L39 20q::22qW

I196

42

B4198

D20

S12

0

B4132

WI-1

1116

EW

I-157

02E

STS

G25

428

D20

S70

8E

931h6

D20

S77

6

B4201

dJ1191N16

P4007

B4121

B4087 B4090

B4208

CRP

ZNF217

B4188

P4069

P4016

P4041

Proc. Natl. Acad. Sci. USA, 95, 8703-8708 (1998)

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 m centers

864 well plates

0.5 g 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

0.5

1

1.5

2

0 50000 100000 150000 200000 250000 300000

kb

log

2rat

io

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

'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

laser 'capture' microdissectie (LCM):

isolation of single cells or small islets of cellsfrom cytological preparation

laser 'capture' microdissectie (LCM):

laser 'capture' microdissectie (LCM):

laser 'capture' microdissection (LCM):

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

BCAT1ASCL1MYCNNCLLAP18GAP43NEUROD1VHLCHD3SAM68KPNA2HMG2CDC25BTBP_run1bTBP_run1aTBP_run2NPYTMODCALM2RGS4HAND2NTRK1MAB21L1HPRT1_run1bHPRT1_run1aHPRT1_run2S100A6TGFBIMMEUBC_run1aUBC_run1b18s_rRNA_run1b18s_rRNA_run1aRLP11DDOSTCDC42Hs.12842Hs.120931NAGDDX1PHBHMBS_run1aHMBS_run1aHMBS_run2RPL13ARPS25TNFRSF10BGAPD_run2Hs.28462CCND1MEIS2ATBF1-AATBF1-BMEIS1TCF12G10D12DVL2DKK3MMP2Hs.182594MSTPO32RGS5IGF2G4D5UBC_run2ACTB_run2ACTB_run1aACTB_run1bCD44PMX1MYCLTBP1HXBSRPRINHBATGM2SOS1YWHAZ_run2ID2SDHA_run2NTRK2CTNNB1PIM1B2MHOXA9

SK-N-B

ESJNB-1

0N206

NMB

NGP

LA-N-6

SK-N-A

SNBL-S

SK-N-S

HSJNB-1

SK-N-F

ICLB-GA

CHP 90

1TR-14

UHG-NP

LA-N-1

SMS-KA

NSK-N-M

CSJNB-6

IMR-32

SJNB-1

2SJNB-8

CHP 13

4STA-NB

-3NLF

LA-N-5

STA-NB

-10

STA-NB

-12

SMS-KC

NRH brai

nH feta

l bra

inH uter

usH mamm

ary g

land

H lung

H trac

hea

H kidn

eyH live

rH hear

tSTA-NB

-9NN-1

NB-3

STA-NB

-8GI-ME-

N

microarrays

microarras

microarrays

Sestri Levante 2000

Alizadeh et al. Nature