Download Lctures Clinical genetics – 4

Lectures Clinical Genetics
Dr. Aneela Javed
Chapter 5:
How to study patients DNA
Two essentials
1- hybridization
2-Amplification of sequence of interest
1- hybridization for DNA testing in dif techniques:
labeling with fluorescent probes
Dot blot: point mutation or small seq change
sickle cell (SCA): point mutation in the β-globin chain of haemoglobin,
hydrophilic amino acid glutamic acid
hydrophobic amino acid
valine The β-globin gene is found on chromosome 11
Southern blotting:
Large scale changes, inversions,
deletions, that change pattern of
restriction fragments,
trinucleotide repeat expansions that
are too large to amplified by PCR.
Fragile X syndrome
expansion of the CGG trinucleotide repeat at 5’utr, affecting the Fragile X
mental retardation 1 (FMR1) gene on the X chromosome, required for
normal neural development.
length of the CGG repeat, 55 (unaffected by the syndrome), above 55
unstable a premutation (at risk of fragile X associated disorders), or full
mutation 200 or > (usually affected by the syndrome).
As gc repeats are difficult to amplify or detect by pcr so Southern blottB,
x –inactivation of repeat expansions cause methylation of FMR1
promotor thus restriction enzyme ECLx 1 is unable to cut
individuals with FXS due to missense mutations or deletions involving
FMR1 will not be diagnosed using this test and should therefore undergo
sequencing of the FMR1 gene if there is clinical suspicion of FXS.
FISH
FISH uses fluorescent probes that bind to only those parts of the
chromosome with which they show a high degree of sequence
complementarity.
Fluorescence microscopy can be used to find out where the fluorescent
probe is bound to the chromosomes.
Chk the presence or absence of seq, copy number and chromosomal
location of seq at least several kb long. or presence or absence of
chromosomes.
Deletions: probe will not bind
Change in chr no: down syndrome chromosome specific probe
1. First, a probe is constructed. The probe 1okb or so is tagged directly
with fluorophores, with targets for antibodies or with biotin. Tagging
can be done in various ways, such as nick translation, or PCR using
tagged nucleotides.
2. an interphase or metaphase chromosomes are denatured and
firmly attached to a substrate, usually glass.
3. The probe is then applied to the chromosome DNA and incubated for
approximately 12 hours while hybridizing.
4. Several wash steps remove all unhybridized or partially hybridized
probes.
5. The results are then visualized and quantified using a microscope
that is capable of exciting the dye and recording images.
6. If the fluorescent signal is weak, amplification of the signal may be
necessary in order to exceed the detection threshold of the microscope.
Fluorescent signal strength depends on many factors
• probe labeling efficiency,
• the type of probe,
• and the type of dye.
DIFFERENT TYPES OF FISH PROBES
Centromeric Probes; consist of repetitive DNA sequences found
in and arround the centromere of a specific chromosomes.
Used for rapid diagnosis of trisomies 13, 18, 21.
Chomosomes specific unique sequence probes; specific for a
particular single locus. Locus specific probes for chromosomes
13q14 and the critical region for down syndrome on
chr.21(21q22.13-21q22.2), X and Y chromosomal abnormalities.
Telomeric probes; Complete set of telomeric probes for all 24
chromosomes, used for subtelomeric abnormalities (deletions,
translocations).
Whole chromosome paint probes; consist of a cocktail of probes
obtained from different parts of a particular chromosome, used
for ring chromosomes and translocations.
M-FISH.
Multiplex in situ hybridization (M-FISH) is a 24-color karyotyping technique
for studying complex interchromosomal rearrangements.
The process involves three major steps.
1. Firstly, the multiplex labeling of all chromosomes in the genome with
finite numbers of spectrally distinct fluorophores in a combinatorial
fashion, such that each homologous pair of chromosomes is uniquely
labeled.
2. Secondly, the microscopic visualization and digital acquisition of each
fluorophore using specific single band-pass filter sets and dedicated MFISH software. These acquired images are then superimposed enabling
individual chromosomes to be classified based on the fluor composition in
accordance with the combinatorial labeling scheme of the M-FISH probe
cocktail used.
3. The third step involves the detailed analysis of these digitally acquired and
processed images to resolve structural and numerical abnormalities.
Figure 3: Spectral karyotyping and multifluor FISH paint each human chromosome
in one of 24 colors.
uses mixtures of differently colored probes.
A range of colors in mixtures of fluorescent dyes can be detected, so each
human chromosome can be identified by a characteristic color using wholechromosome probe mixtures and a variety of ratios of colors.
Although there are more chromosomes than easily distinguishable
fluorescent dye colors, ratios of probe mixtures can be used to create
secondary colors. the probe mixture for the secondary colors is created by
mixing the correct ratio of two sets of differently colored probes for the same
chromosome.
FIBER FISH
interphase chromosomes are attached to a slide in such a way that they
are stretched out in a straight line, rather than being tightly coiled, as in
conventional FISH,
This is accomplished by applying mechanical shear along the length of
the slide, either to cells that have been fixed to the slide and then lysed,
or to a solution of purified DNA. A technique known as chromosome
combing is increasingly used for this purpose.
The extended conformation of the chromosomes allows dramatically
higher resolution - even down to a few kilobases. The preparation of
fiber FISH samples, although conceptually simple, is a rather skilled art,
and only specialized laboratories use the technique routinely.
allows the visualization of individual genes or other small DNA elements on
chromosomes with a resolution of approx 1000 bp.
Applications of fiber-FISH range from the determination of numbers of
repetitive genes to establishing the physical order of cloned DNA
fragments along continuous sections of individual chromosomes. Particularly
in organisms with relatively small and gene dense genomes, such as
protozoan parasites, fiber-FISH can easily be used as a complementary
technique to classical in vitro mapping approaches.
Q-FISH
Q-FISH combines FISH with PNAs and computer software to quantify
fluorescence intensity. This technique is used routinely in telomere length
research.
labelled (Cy3 or FITC)
syntheticDNA mimics peptide nucleic
acid (PNA) oligonucleotides .
FLOW-FISH
Flow-FISH uses flow cytometry to perform FISH automatically using percell fluorescence measurements.