FISH abbreviation stands for… + alternative designation (Bezeichnung)
F - fluorescence
I - in
S - situ
H - hybridisation
alternative: molecular cytogenetics
FISH workflow
A: DNA to be analyzed
B: DNA probe (= DNA fragments of sequence of interest)
1: fluorescent labeling
2: denaturation
3: hybridization
—> usually use glass slides and add DAPI
Types of FISH probes
synthetic oligonucleotides
single-copy sequences from genomic sequence w/o repetitive elements -> prevent cross-hybridization
adding fluorescence to probes
(bacterial) vector-based DNA fragment amplification
insert probe into bacterial vactor (e.g. F-plasmid/BAC)
extraction followed by fluorescent labelling
Kind of FISH probes - centromeric probes (CEP)
Kind of FISH probes - locus-specific probes
FISH - microdeletion syndromes - characteristics
small particular deletions of gene clusters or single genes
deletions = frequent below the resolution limit of G-banding -> detected reliably via FISH
mostly lead to complex but distinct clinical phenotypes
usually sporadic
FISH - microdeletion syndrome - most frequent cause
= non homologous recombination between repetitive sequences (LCRs = low copy repeats) during meiotic cross over
LCRs = predisposed for non-homologous recombination => mis-alignment
=> deletion (more severe) or duplication (not always phenotypic) syndromes
FISH - microdeletion syndromes - deletion 22q11.2
FISH - microdeletion syndromes - deletion 7q11.23 (Elastin-gene; ELN)
DiGeorge Syndrome
= deletion 22q11.2
phenotype (facial)
narrow upslanted palpebral fissures
long nose with broad nasal bridge and hypoplastic nares
small mouth
low set small ears with thick overfold helix
additional:
heart defects
intellectual disability
muscular hypotony
frequent short stature
Williams-Beuren-Syndrome
= deletion 7q11.23 (Elastin gene; ELN)
prominent forehead
periorbital fullness
frequently strabism
thick, broad everted lower lip
full cheeks and round nasal tip in young children
small hypoplastic milk teeth
clinical:
cardiovascular alterations
hyperacusis
intellectual disability (50-60 %)
abilities:
musical sense
face recognition
eloquence = “cocktailparty behavior”
FISH advantages
FISH disadvantages
array-CGH abbreviation and what to detect
Array-based Comparative Genomic Hybridization
detection of deletions and duplications (any imbalanced copy numberm but no unbalanced translocation & polyploidies)
Array-CGH workflow
red label = genomic reference DNA (usually pool from 10 people)
green label = genomic test DNA (DNA of interest)
then hybridization to DNA-microarray with oligonucleotides (25 or 60-85 bp)
—> green < red
—> green > red
types of arrays
targeted
for certain genes
targeted with backbone
lower resolution than whole genome
whole genome
randomly investigation of sequence
in diagnostics: 180,000 oligonucleotides
array-CGH - evaluation with Log2 ratio (control:probe) signal
—> negative value
—> positive value
Log2 ratio
-> every single dot = one probe on array
-> left: 44,000 oligo probes; right: 1,800,000 oligoprobes
homozygous deletion
precise breakpoint mapping
heterozygous deletions
array-CGH - impact factors on results
DNA quality
labelling
hybridization
washing
array-CGH advantages
array-CGH disadvantages
Last changeda year ago
