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![Page 1: Analysis of protein-DNA interactions with tiling microarrays Srinivasan (Vasan) Yegnasubramanian Sidney Kimmel Comprehensive Cancer Center Oncology Dept.,](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649f4f5503460f94c71ae6/html5/thumbnails/1.jpg)
Analysis of protein-DNA interactions with tiling microarrays
Srinivasan (Vasan) Yegnasubramanian
Sidney Kimmel Comprehensive Cancer Center
Oncology Dept., Genitourinary Division
March 7, 2007
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Identical genetic sequence, but very different gene expression and phenotypes…
Normal Human BrainNormal Human Brain
Normal Human LiverNormal Human Liver
Normal Human KidneyNormal Human Kidney Benign Human Prostate
…These differences are due to Epigenetic changes.
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Epigenetics is the study of heritable processes that alter gene expression without an accompanying change in gene sequence
These processes are usually mediated by factors, such as proteins/ribonucleo-proteins, that bind genomic DNA
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(3.4x10-10 meters/bp) x (6x109 bp/genome) = ~2 meters/genome
Radius of the nucleus is ~ 10 µM !!!
Klug and Cummings, 1997
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[(6 x 109 bp/genome) / (195 bp/nucleosome)] = ~ 30.8 x 106 nucleosomes/genome
~ 5 % of nuclear volume
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http://www.albany.edu/~achm110/solenoidchriomatin.html
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DNA methylation occurs at CpG dinucleotides in mammalian genomes
5’…ACGT…3’
5’…ACGT…3’5-me
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DNA methylation patterns in normal and cancer cell genomes
Herman and Baylin, NEJM, 2003
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DNA methylation can lead to silencing of gene expression
Robertson and Wolffe, Nat Rev Genet, 2000
HDAC2
MBD2
HDAC1
MBD3
RbAp46RbAp48
Mi-2
MTA268kD
66kD
HDACMeCP2
Sin3A
HDAC2
MBD2
HDAC1
MBD3
RbAp46RbAp48
Mi-2
MTA268kD
66kD
HDACMeCP2
Sin3A
>2 MDalton Complex
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Struhl, Cell, 2004
http://www.berkeley.edu/news/features/1999/12/09_3dimage.html
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Diameter of DNA Double helix: 20 Angstroms
Diameter of Transcriptional machinery: >1,000 Angstroms
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Developing an understanding of epigenetic processes…
DNA Modifications(e.g. Methylation)
Gene TranscriptionalChanges
DNA-ProteinInteractions
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Characteristics of Tiling Microarrays
Microarray contains n probes of length L distributed across x base pairs on a genomic region of interest. That is, n probes are tiled across a genomic region of interest
The average resolution or sampling/window size, then, is R = x / n, or
d1 d2 d6d5d4d3 d7
1
1
1
n
dR
n
N
N
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Affymetrix Tiling microarrays Human Chromosome 21/22 microarrays
> 35 million bp of non-repetitive sequence on Chrom 21/22 represented with >1 million probe sets on three microarrays (currently on a single array). R ~ 35 bp.
ENCODE arrays representation of 1% of genome corresponding with ENCODE regions at 35 bp
resolution with single microarray. Tiled arrays of 10 human chromosomes
74,180,611 probe pairs interrogating 30% of human genome (i.e. 10 complete chromosomes) at on >90 microarrays. R ~ 5 bp.
Tiled arrays of whole genome interrogation of whole genome (1.7 Gb) on 7 microarrays (~50,000,000 PM
probes only) or 14 microarrays (~50,000,000 PM + MM probe sets). R ~ 35 bp. Promoter Tiling arrays
interrogation of all 5’ upstream regions of known genes on a single microarray
All probes are 25-mers
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Strategy
Label and Hybridize SamplesTo Tiling Microarrays
Chromatin Structure(In vivo DNA/Protein
Interactions)
Biostatistical Analysis toIdentify Genomic Regions
of Interest
DNA Methylation(In Vitro DNA/Protein
Interactions)
TranscriptomeAnalysis
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Crosslink
YLyse & Sonicate
IP Reverse crosslinks
Total Reverse crosslinks Amplify
Amplify
Label/hybridize
Label/hybridize
ChIP-Chip for “in vivo” DNA protein interactions
Other controls for IP(e.g., no antibody, non-
specific antibody)
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Current limitations for ChIP-Chip Process is very inefficient and requires large
amounts of input material Sonication step can be quite variable and
cannot be easily quality controlled with small amounts of starting material
Currently difficult to perform on clinical specimens
Labor-intensive
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Genome-wide, high-resolution DNA methylation detection by taking advantage of tiling arrays and DNA-protein interactions in vitro
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HDAC2
MBD2
HDAC1
MBD3
RbAp46RbAp48
Mi-2
MTA268kD
66kD
HDACMeCP2
Sin3A
HDAC2
MBD2
HDAC1
MBD3
RbAp46RbAp48
Mi-2
MTA268kD
66kD
HDACMeCP2
Sin3A
Endogenous methyl-CpG binding domain proteins
MECP2
MBD1
MBD2
(Anti-5mC Ab)
MBD3
MBD4
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6His-MBD2-MBD binds symmetrically methylated oligonucleotides
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
b
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
b
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
c
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
c
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
b
EC50 = 222 +/- 67 nM EC50 = 156 +/- 70 nM EC50 = 519 +/- 250 nM
EC50 >> 10 M EC50 >> 10 M
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
b
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
b
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
c
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
c
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
b
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.222 ± 0.067 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
b
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
b
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
b
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 = 0.156 ± 0.070 µM
b
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
c
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
c
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
c
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
EC50 = 0.519 ± 0.250 µM
Log [MBD2-MBD] (µM)
c
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
a
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
b
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
b
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
100
120
140
160
An
iso
tro
py
Log [MBD2-MBD] (µM)
EC50 >> 10 µM
b
EC50 = 222 +/- 67 nM EC50 = 156 +/- 70 nM EC50 = 519 +/- 250 nM
EC50 >> 10 M EC50 >> 10 M
Yegnasubramanian et al., Nucleic Acids Res, 2006
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Fe
Fragment
Enrich for densely
methylated fragments
Real-time PCR
0.0
0.1
0.2
1.0
WBC M.SssIWBC
Re
lati
ve E
nri
ch
me
nt
MBD2-MBD + – – –Anti-His-Ab + + – –
Protein G Magnetic Beads + + + –
0.0
0.1
0.2
1.0
WBC M.SssIWBC
Re
lati
ve E
nri
ch
me
nt
MBD2-MBD + – – –Anti-His-Ab + + – –
Protein G Magnetic Beads + + + –
MBD2-MBD + – – –Anti-His-Ab + + – –
Protein G Magnetic Beads + + + –
Use of 6His-MBD2-MBD for enrichment of methylated genomic DNA
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII Digestion Only
R2 = 0.962
R2 = 0.976
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssI WBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
MBD2-MBD Capture Only
R2 = 0.981
R2 = 0.538
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
0.01 0.1 1 10 1000.0001
0.001
0.01
0.1
1
10
100WBC M.SssIWBC
Input Genomic DNA (ng)
Ou
tpu
t D
NA
(n
g)
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
R2 = 0.982
R2 = 0.113
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
R2 = 0.985
COMPARE-MS Assay Performance
Ou
tpu
t D
NA
(n
g)
Input M.SssI treated WBC DNA (ng) in 20 ng of untreated WBC DNA
0.01 0.1 1 10 1000.001
0.01
0.1
1
10
100
Yegnasubramanian et al., Nucleic Acids Res, 2006
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Fe
FeFe
Whole-genome DNA methylation assay
Fragment
Enrich methylated fragments
Amplify
Fragment/label/hybridize
Amplify
Fragment/label/hybridize
Total input
![Page 23: Analysis of protein-DNA interactions with tiling microarrays Srinivasan (Vasan) Yegnasubramanian Sidney Kimmel Comprehensive Cancer Center Oncology Dept.,](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649f4f5503460f94c71ae6/html5/thumbnails/23.jpg)
SonicationRestrictionEnzyme
Fragmentation techniques
![Page 24: Analysis of protein-DNA interactions with tiling microarrays Srinivasan (Vasan) Yegnasubramanian Sidney Kimmel Comprehensive Cancer Center Oncology Dept.,](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649f4f5503460f94c71ae6/html5/thumbnails/24.jpg)
Middle ground
Pool different restrictionenzyme digests
![Page 25: Analysis of protein-DNA interactions with tiling microarrays Srinivasan (Vasan) Yegnasubramanian Sidney Kimmel Comprehensive Cancer Center Oncology Dept.,](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649f4f5503460f94c71ae6/html5/thumbnails/25.jpg)
Dynamics of amplification and fold enrichment…
Fold enrichment dependent on: Amount of each
species after enrichment
Total amount of all enriched species
Enrich
Enrich
Total
Amplify
to 20
Amplify
to 20
Amplify
to 20
![Page 26: Analysis of protein-DNA interactions with tiling microarrays Srinivasan (Vasan) Yegnasubramanian Sidney Kimmel Comprehensive Cancer Center Oncology Dept.,](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649f4f5503460f94c71ae6/html5/thumbnails/26.jpg)
Ongoing and future work
DNA Modifications(e.g. Methylation)
Gene TranscriptionalChanges
DNA-ProteinInteractions
Preprocessing
Preprocessing
Preprocessing
Analysis
Analysis
Analysis
Meta-Analysis
Cancer
Normal
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End of slides