overview research topics and techniques 1. clinical, biochemical and molecular studies of
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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 - PowerPoint PPT PresentationTRANSCRIPT
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