Molecular Cytogenetics

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Molecular Cytogenetics
Fluorescence In Situ Hybridization to Human
Chromosomes
Metaphase FISH
Interphase FISH
Fiber FISH
Chromosome sorting and microdissection
Spectral Karyotyping
FISH as a genomic analysis and positional
cloning tool.
Comparative Genomic Hybridization
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Why should you care?
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Much progress on single nucleotide variation of
the estimated 15 million SNPs we have described
3.4 million genotyped SNPs But we are just
starting to describe structural variation in the
human genome This includes insertions,
deletions, inversions, duplications, and
translocations of DNA sequences Over the last
two years, several genome-wide surveys
have described large-scale (gt100 kb),
intermediate-scale (500 bp-100 kb) and
fine-scale (1bp -500 bp) structural variation in
the human genome.
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Copy Number Variation Project to
comprehensively identify CNVs in the 269 samples
analyzed by the International HapMap
Project Over 1,447 copy-number variant regions
spanning12 of the reference DNA sequence
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Array-based technologies dependent on the
detection of copy -number differences are unable
to detect structural variation events that have
arisen due to balanced chromosomal
rearrangements (such as inversions or reciprocal
translocations of chromosomal segments).
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Fluorescence In Situ Hybridization to Human
Chromosomes
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Fluorescence In Situ Hybridization (FISH) One
can, in theory, build a de novo physical map from
just STS content maps or finger print data
supplemented with radiation hybrid
frameworks, but you need FISH to rivet it back to
reality. FISH is also a valuable tool in
investigating genome evolution, disease gene
hunting and in many clinical applications.
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Metaphase FISH
A direct and fairly rapid method for deriving
framework physical maps
Resolution at the gtMb level
Requires fairly long regions of genomic
continuity..several kb. Overall probe length is
short (a few hundred bp)
Cosmids, BACs, YACs work well..usually amplified
by either inter ALU PCR or by DOP PCR
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Alu PCR
The human genome contains 500,000 Alu elements
which are quasi-randomly dispersed. The average
frequency of an Alu element is 1 per 5kb..
Use PCR primers that face outwards from both ends
of the reference Alu sequence and PCR the
intervening DNA.
Results in representations of a large genomic
clone.also contains repetitive elements and
must be repeat suppressed before hybridization.
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Padlock Probes
A 90 base long single stranded nucleotide probe
that has 30 bases of homology with the
target. The 30 nucleotides are positioned at each
end of the 90mer.
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Padlock Probes
A 90 single stranded nucleotide probe that has
30 bases of homology with the target. The 30
nucleotides are positioned at each end of the
90mer.
Ligation of the probe
The padlock probe is actually wrapped around the
target
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Hybridizing individual exons
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Myb amplifications
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Chromosome 20q amplifications
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Interphase FISH
Useful for higher resolution ordering of markers
Resolves in the 100kb range
Requires many more observations and detailed
measurements than metaphase FISH
cDNAs are in general very poor probes (small
regions of contiguos genomic homology)
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Detecting deletions or clone coverage gaps by
simultaneous hybridization with two probes
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Fiber FISH
Mapping at the 10kb level.
Very useful for detecting deletions in clones
(esp. YACs)
Used for estimating gap closure distances in
large scale sequencing projects.
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Optical Mapping
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Spectral Karyotyping
Chromosomal and subchromosomal painting probes
that make use of sorted or microdissected
chromosomes
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Chromsome purification by using flow sorting.
Chromosomes stained with a fluorescent dye are
passed through a laser beam. Each time, the
amount of fluorescence is measured and the
chromosome is deflected accordingly. The
chromosomes are then collected as droplets.
(Fig 6-25)
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Spectral Kartyotyping
Mixtures of fluorophores used to separately label
chromosome-specific probes.
These are mixed and hybridized en masse
Interpreted via spectral interferometer.
Tremendously useful in detecting insertions and
translocations, especially in cancers.
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Metaphase FISH to investigate segmental genomic
duplications
Courseaux et al (2003) Genome Research 13 369
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Can balanced chromosomal rearrangements be used
to gain insights into complex disease?
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The fate of a million implanted human zygotes.
(Robertsonian translocations are due to fusion or
dissociation of centromeres.)
(Fig 18-23)
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How to identify genetic components for complex
disease?

• Linkage/association studies - Large collection of
  individuals and families

Problem - heterogeneity
- very frequent
disorder

• Early onset diseases or highly penetrant alleles
• Extreme phenotype - insight

• Gene expression studies to identify possible
  major players
• Difficult because we dont really know what
  tissue expresses this particular gene

• Chromosomal rearrangements to identify regions
  in the genome

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Balanced chromosomal rearrangements have
frequently been critical to gene
identification
Mendelian Traits - NF1 - Polycystic kidney
disease - Duchenne muscular dystrophy -
Lowes Syndrome Translocations,
inversions, breaks involved in all of the above
Balanced chromosomal rearrangements to identify
positional candidates
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Identifying YACs and BACs crossing Breaks
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Idiopathic scoliosis

• Affects 4 of the population
• Approximately six times more frequent in females
• A clear genetic component, but complex and
  heterogeneous

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Summary
Molecular Cytogenetic methods have been a
valuable genomics tool in map assembly and
validation
FISH and SKY have become standard clinical
diagnostic tools.
Chromosome imaging methods continue to be used to
explore basic questions about chromosome
mechanics, evolution, genome rearrangments and as
positional cloning tools.
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• Comparative genomic hybridization (CGH) as
  originally configured is a molecular cytogenetic
  technique that allows detection of DNA sequence
  copy number changes throughout the genome in a
  single hybridization. CGH is based on
  co-hybridization of two differentially labeled
  DNAs (eg tumor and normal) to human metaphase
  chromosome spreads.

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• In a typical experiment
• The tumor DNA is labeled with biotin-dUTP and
  normal DNA with DIG-dUTP prior to hybridization.
• Three images of the hybridization, representing
  both fluorescent labels as well as DNA
  counterstain (DAPI), are acquired using
  epifluorescence microscopy and a CCD camera
  interfaced to a computer.
• Subsequently, an image analysis software
  program is used to calculate green-red
  fluorescence intensity ratio profiles along all
  chromosomes.
• Regions of the genome that are either gained or
  lost in the tumor are indicated by the
  differences between hybridization of the two
  labeled DNAs as reflected by intensity ratio
  profiles.

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Sensitivity

• The sensitivity is approximately 2-20 Mb. For
  example, a 10-fold amplification of a 200 kb
  region should be detectable under optimal
  hybridization conditions.

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Limitations

• Best for overt amplifications.

CGH does not detect balanced chromosomal
translocations or inversion. It is difficult to
detect small interstitial deletions.
If Loss of Heterozygosity and subsequent
chromosome endo- reduplication has occurred, then
LOH will produce a false negative.
Sensitivity..all that repetitive DNA.
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• Nevertheless, a highly successful and widely
  adopted method, especially in cancer genomics.
  Several thousand publications on CGH over the
  past few years.

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• Comparative genomic hybridization by arrays
• (Array CGH)

1. BAC arrays.
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BAC arrays for CGH
BAC tiling paths on a microarray.
Low DNA yields from BACs, so use DOP PCR to
produce enough to spot.
Resolution limited by the size of a BAC
Need redundant coverage to minimize false
negatives and positives.
Independent assays to validate the BAC map.
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DOP-PCR primer 5- CCG ACT CGA GNN NNN NAT
GTGG-3
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Hyperbranched strand displacement amplification.
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F29 and Bst large fragment 5 to 3 exo
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• Comparative genomic hybridization by arrays
• (Array CGH)

2. cDNA arrays.
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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).
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Tumors frequently require LCM and samples may be
small.
Need methods to reproducibly amplify these
samples Whole genome amplifications..
By other methods, once you use it you lose it.
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• Comparative genomic hybridization by arrays
• (Array CGH)

3. Long oligonucleotide arrays
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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?
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Oligo Arrays (AffyChips)
High density oligonucleotides 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
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Affymetrix photolithography and combinatorial
chemistry
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DMD Digital Micromirror Device
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Tm matched oligos and highly reproducible, but
does everyone need this level of
resolution? Nevertheless, affordable, quick and
custom made (if necessary)..the current gold
standard for CGH