This Lecture

1
This Lecture More on array-based CGH cDNA
arrays oligonucleotide arrays Representational
difference analysis Representational
Oligonucleotide Microarray Analysis
(ROMA) Exploring large scale copy number
polymorphisms by ROMA Genomic tools for
exploring epigenetic modifications Methylation
-specific PCR Restriction landmark genomic
scanning mcr BC
2

• Comparative genomic hybridization by arrays
• (Array CGH)

2. cDNA arrays.
3
cDNA arrays for CGH

• Originally attempted by Botstein and Brown labs
  in early 1990s

Generally regarded as lacking the sensitivity of
genomic arrays too few probes across a given
interval and too short an average contiguous
probe length (one exon).
4

• Microarray analysis reveals a major direct role
  of DNA copy number alteration in the
  transcriptional program of human breast tumors
• Jonathan R. Pollack, Therese Sørlie, Charles
  M. Perou, Christian A. Rees_, Stefanie S.
  Jeffrey, Per E. Lonning,Robert Tibshirani,
  David Botstein_, Anne-Lise Børresen-Dale, and
  Patrick O. Brown

5
Array of 6,700 cDNAs arranged in the Golden
Path.
44 primary breast tumors (at least 50 tumor) and
10 cell lines.
Correlations of previously derived expression
data with CGH data.
6
(No Transcript)
7
Still need compilations of probes to reliably
call an alteration
Of 117 high level DNA amplifications (over 91
different genes) 62 (54 genes) are upregulated
gt2-fold.
42 (36 genes) are upregulated gt4-fold.
So, high correlation between amplifications and
increased gene expression.
8
Tumors frequently require LCM and samples may be
small.
Need methods to reproducibly amplify these
samples.
By conventional methods, once you use it you lose
it.
9

• Comparative genomic hybridization by arrays
• (Array CGH)

3. oligonucleotide arrays
10
Oligo Arrays (AffyChips)
High density oligonucleotides (25mers)
synthesized on a chip.
These oligos are used like a large set of
allele-specific oligos to interrogate patient DNA
Can test 500,000 SNPs
11
Oligo arrays for SNP typing have multiple levels
of redundancy.
12
Illumina etched fiber optic cables with oligos
linked to beads, massively parallel Expression
profiling or SNP typing.
13
(No Transcript)
14
Whole-Genome Expression BeadChips, Six or eight
whole genomes on a single microarray. Over 10
million features.
Sentrix Universal-16 BeadChip. Each array can
genotype 1536 SNP's or gene targets
Sentrix Array Matrix. Microplate-compatible. 96
arrays in parallel, with1536-multiplex array
protocol.
15
Long oligonucleotide arrays
70 mer tiling paths across the genome (or sampled
every few kb)
High sensitivity
Can remove that repeat suppression problem.
However, is this quick and inexpensive?
16
DMD Digital Micromirror Device
17
Key Technology Features
Nimblegen

• High-density oligo arrays
• New array design every 3 hours
• Iterative redesign for array improvement
• Open-systems approach

18
Array Options

• 1 - 70mers
• Up to 390,000 features
• per array

19
Tm matched oligos and highly reproducible, but
does everyone need this level of resolution?
20
Representational Difference Analysis
A comparative genomic hybridization method of a
different sort.
Based upon subtractive cloning methods plus
differential PCR of one population of DNAs.
Technically demanding, but successful in the
right hands.
21
(No Transcript)
22
Genomic representations are just randomly chosen
subsets of a genomic DNA digest, amplified via
ligated linkers. See Lucito et al., (1998) PNAS
954487
Can start with 1,000 cells (1ng genomic DNA).
Claimed to yield linear representations.
23
The idea is quite straightforward
BglII
Cut and purify size range
A representation of the genome
24
(No Transcript)
25
(No Transcript)
26
Lucito et al., (2000) Genome Research 10 1726
Arraying representations, then hybridizing
representations to these.
Reduce the probe and target complexity to boost
signalnoise.
Arrays of 2,000 BglII (LCR) fragments, hybridized
with BglII LCRs.
27
(No Transcript)
28
When we compare a differentially labeled
sample with itself, the ratio of channel
intensity for each feature ideally should be a
constant. In fact, there is a deviation from this
ideal, which is virtually independent of
intensity (Fig. 1B). Possible sources for
this variation include the differential behavior
of the DNA samples after they are labeled with
different fluorochromes, variation in
hybridization and washing conditions over the
surface of the array, gross regional changes in
the physiochemical properties of the
array surface, machine fluctuations, and software
artifacts, among others.
29
(No Transcript)
30
(No Transcript)
31
However, we dont know the sequence identity of
each of these BglII fragments.
But now we have the genomic DNA sequence, so we
can design and build a long oligonucleotide
array that will specifically interrogate our sets
of BglII fragments. Sampled at 35kb intervals.
Representational Oligonucleotide Microarray
Analysis (ROMA)
32
Exploring large scale copy number polymorphisms
by ROMA
33
(No Transcript)
34
ROMA with one probe per 35kb and 85,000 long
oligonucleotides.non repetitive and better
signal to noise.
BglII representations of the human genome for 20
individuals.
35
(No Transcript)
36
(No Transcript)
37
Future applications of CGH

• The continued phasing-out of BAC arrays.

RDA fragments as probes for genomic arrays
Representational oligonucleotide arrays across
the genome
Screening for large additions/deletions across
the genome and their relationship to complex
disease.
38
Genomic tools for exploring epigenetic
modifications

• Restriction landmark genomic scanning
• Bisulfite sequencing
• Methylation-specific PCR
• mcrBC

39
(No Transcript)
40
Methylation as an epigenetic modification
The attachment of a CH3 group to the 5 carbon of
the cytosine residue in a CpG dinucleotide is a
universal and tightly regulated epigenetic
modification found in mammals.
CpG suppression methylation of CpG found in
vertebrates represents a mutational hot spot
through deamination of methylcytidine to thymidine
41
Deamination of cytosine can be repaired
Deamination of 5-methylcytosine cannot be repaired
42
90 of this CpG is in repetitive elements. The
remainder is largely confined to dense clusters
called CpG islands. Most CpG island are
unmethylated.
In cancer the genome in general is
hypomethylated.but some specific areas are
hypermethylated e.g.inactivation of p16
by aberrant methylation of CpG islands is a
frequent event in stomach carcinomas.
43
Restriction Landmark Genome Scanning (RLGS)
A two dimensional gel electrophoresis method for
resolving changes in methylation.
Step 1. Digest with a labeling restriction
enzyme e.g. NotI (GCGGCCGC) which marks CpG
islands
Step 2. Fill in the site overhang with 32P
label.
Step 3. If necessary cut with an additional
enzyme to make the average fragment size
shorter.
Step 4. Resolve the fragments in the first
dimension.
Step 5. Digest in situ with another enzyme.
Step 6. Run second dimension and capture pattern
by autoradiography.
44
NotI
HinfI
EcoRV
45
RLGS issues
Very difficult to develop standardized
electrophoretic methods
About 2,000 spots resolved per gel..good but
not comprehensive.
Even when a spot is seen to move in a tumor
sample.what is the identity of that spot?
Libraries of fragments from particualr digestions
have been produced and sequenced to derive
reference fragments with sequence IDs.
With a complete genomic sequence one can derive a
virtual RLGS profile.
46
Virtual RLGS superimposed with a real
NotI/EcoRV/Hinf1 RLGS
47
Bisulfite Sequencing
The chemical modification of cytosine to uracil
by bisulfite treatment provides another method
for the study of DNA methylation.In this
reaction, all cytosines are converted to uracil,
but those that are methylated (5-methylcytosine)
are resistant to this modification and remain as
cytosine. This altered DNA can then be amplified
and sequenced, providing detailed information
within the amplified region of the methylation
status of all CpG sites. However, this is a
technically demanding method.
48
Could bisulfite conversion be used as the basis
for methylation-specific CGH?
There are usually lots of unmethylated CpGs
clustered together.
e.g. ------TAGCGGGCCCGTTGTCGGTCG-----------
Becomes -------TAGUGGGUUUGTTGTUGGTUG---------- u
pon bisulfite treatment
If this site was methylated in a tumor and then
bisulfite treated it becomes-----TAGCGGGUUCGTTGTC
GGTCG-------
Could these be compared on a whole genome array?
49
Methylation-specific PCR (MSP)
A combination of bisulfite treatment with PCR
primers that hit specific CpG sites. This can be
designed nicely around specific sites of
methylation, but it is not a de novo discovery
tool.
50
mcr BC
mcrBC cuts at (G/A)mC (N40-3000) (G/A)mC
This can be exploited to cut up fragments or
clones that have methylated regions. For
examplegene threshing in plants
51
BglII
BglII
mcrBC
Apply these by CGH to ROMA.
MOMA
52
Summary
Array-based genomic scanning tools are
increasingly being used to monitor large scale
sequence variation between genomes, and
disease-related copy number alterations within
genomes.
These tools are applicable to genome-scale
scanning of epigenetic modifications.
Validation of these observations often includes
resorting back to fluoresence in situ
hybridization methods.