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Chromosomes:
Molecular cytogenetics
Radioactive ISH
y
(
Taylor, 1957
)
Nick translation
DNA probe
pApGpCpTpApCpGpTpApT
pTpCpGpApTpGpCpApTpA
DNasi I
pApGpCpT…ApCpGpTpApT
pTpCpGpApTpGpCpA…TpA
DNApolymerase I
+ dATP,dCTP,dGTP,dTTP
+ Biotine-16-dUTP
pApGpCpT ApCpGpTpApT
pTpCpGpApTpGpCpA TpA
)
)
A
A
FISH
fluorescent in situ hybridization: (FISH) A technique used to identify
the presence of specific chromosomes or chromosomal regions
through hybridization (attachment) of fluorescently-labeled DNA
probes to denatured chromosomal DNA.
Step 1. Preparation of probe. A probe is a fluorescently-labeled segment of
DNA complementary to a chromosomal region of interest.
Step 2. Hybridization. Denatured chromosomes fixed on a microscope slide are
exposed to the fluorescently-labeled probe. Hybridization (attachment) occurs between
the probe and complementary (i.e., matching)
chromosomal DNA.
Fluorescent in situ Hybridization (FISH)
ƒ Hybridization of complementary gene‐ or region‐
specific fluorescent probes to chromosomes.
Interphase or metaphase
cells on slide (in situ)
Probe
Microscopic
signal (interphase)
Probes
wcp
whole chromosome painting
pcp
partial chromosome paonting
band specific
band-specific pcp
alphoid sequences
centromere-specific
YAC (yeast art. chrom)
600Kb-2Mb
BAC (bacterial art. chrom) 150-180 Kb
telomeres
repeats
subtelomeric
specific sequences
plasmid
100Kb
cosmid
40Kb
FISH Probes
• Chromosome‐specific centromere probes (CEP®)
– Hybridize to centromere region
– Detect aneuploidy in interphase and metaphase
• Chromosome painting probes (WCP)
– Hybridize to whole chromosomes or regions
– Characterize chromosomal structural changes in metaphase cells
• Unique DNA sequence probes (LSI®)
– Hybridize to unique DNA sequences
– Detect gene rearrangements, deletions, and amplifications
Uses of Fluorescent in situ Hybridization (FISH)
• Identification and characterization of numerical and structural chromosome abnormalities.
• Detection of microscopically invisible deletions.
• Detection of sub‐telomeric aberrations.
• Prenatal diagnosis of the common aneuploidies (interphase FISH).
FISH
Step 3. Visualization. Following hybridization, the slide is examined
under a microscope using fluorescent lighting. Fluorescent signals
indicate the presence of complementary chromosomal DNA; absence
of fluorescent signals indicate absence of complementary
chromosomal DNA.
Green signal =
Normal control
Pink signal =
Chromosome region of interest
Normal control:
Two green signals
Two pink signals
Patient with deletion:
Two green signals
One pink signal
Painting 8
Painting 17
iso (17q)
Translocation by Metaphase FISH
WCP Probe (Whole‐Chromosome Painting)
t(7;10;11)
Genetic Abnormalities by Interphase FISH LSI® Probe
• Greater or less than two signals per nucleus is considered abnormal.
Cell
nucleus
Normal diploid signal
Trisomy or insertion
Monosomy or deletion
Structural Abnormality by Interphase FISH LSI®
Probe (Fusion Probe)
Structural Abnormality by Interphase FISH LSI®
Probe (Break Apart Probe)
t(5;17) LSI CSF1R(5q)/D5S721(5p)
Alfoide 8
r(20)
BAC CTD-3248C18
t(12;15)
FISH Probes
• Telomere‐specific probes (TEL)
– Hybridize to subtelomeric regions
– Detect subtelomeric deletions and Probe binding site
Unique sequences
rearrangements
Telomere
100–200 kb 3–20 kb
Telomere associated repeats
(TTAGGG)n
Multipainting
FISH on DNA fibers
Comparative Genome Hybridization
CGH
Melanoma
Spotter
Scanner
• 244,000 probes
• source NCBI genome (Build 34; July,
2003)
• genome-wide survey
• coding and noncoding sequences with
an average spatial resolution of ~8kB
Next future: 488,000 probes
Male,
Microcephaly ,
Malformaions.
Female, 20 monts, Developmental delay, Dysmorphic features
1 st allele: deletion
2 nd allele: mutation
>> recessive disorder
Next,
Test parents to verify if the anomalies
are inherited or de novo