Lect 4: Diagnostic Technologies Flashcards
Flouresence In Situ Hybridization
molecular probes (fragments of DNA) are hybridized to chromosomes
Goal of FISH
to determine if a gene, a mutation or a chromosome rearrangement is present or absent. So the molecular probe used must be well characterized to the specific locus being examined
How do you make a probe?
a unique gene is chosen and a fragment of DNA is isolated. One stand is labeled with a fluorescent dye. The DNA on the slides is then denatured and the fluorescently labeled single-stranded molecular probe is allowed to hybridize to the chromosomal DNA. The rest of the DNA is counterstained with another fluorochrome to allow visualization of the entire chromosome using a fluorescent microscope.
Cells in which phase can be used for FISH analysis?
metaphase or interphase (S-phase)
FISH detecting deletions
A person who does not have a deletion will have TWO signals, one for each chromosome…the probe can hybridize. There is no signal on the deleted chromosome, because
there is no DNA sequence present that is complementary to the probe, and no hybridization can
occur.
In order to be certain that the error is not due to technicality or anything
there are dual colored probes where you have internal control..if this doesn’t hybridize just like our probe doesn’t hybridize then we can count it as a true deletion. There are now two signals per chromosome in an individual
What are the paramaters of your probe?l
locus specific–if your probe is detecting errors at a certain locus, then it will only pick up the ones that are there and no where else.
chromosome specific–duh!
Fish probe 1: Repeat Sequence DNA
chromosomes usually isolated from telomeres or centromeres; not useful it will confirm the presence or absence of these telomeres–CAN BE USED FOR DISOMY, TRISOMY, TURNER SYNDROME (X)
Fish probe 2: Single copy DNA
target site and control site; two signals per chromosome; can detect deletions
Fish probe 3: Subtelomere FISH
targets DNA sequences from the distal ends of the chromosomes in regions proximal to the actual telomere regions; v
==>very important for diagnosing because it allows us to identify very small (cryptic) deletions and rearrangements that cannot be seen by standard karyotype
Whole chromosome paints
cocktail of many unique DNA fragments from along the entire length of a chromosome such that following
hybridization, the entire chromosome fluoresces.
What is WCP used for
most useful in identifying complex rearrangements and TRANSLOCATION and marker chromosomes. If a patient has an abnormal
chromosome with extra material of unknown origin, it may be possible to use chromosome
painting to identify the source of the extra DNA.
FISH helps to identify Coniguous Gene Syndromes (eg Williams and VCFS
regions with a cluster of closely assoc genes whose normal functions are generally unrelated. A deletion of this region will give multiple phenotypes. FISH can detect microdeletions (3MB) and microduplications
==>example is William’s Syndrome: contiguous gene syndrome involving deletion of elastin deletion
FISH can help pick up VCFS
VCFS is due to an interstitial 3MB deletion on chrom 22
==>results from: there are repeat sequences that flank the gene. During meiosis the homologous chromosomes should pair evenly. Because the repeat sequences have similar sequences, it is possible that the wrong repeats will pair giving rise to misalignment. If a recombination even takes place, deletions or duplications occur.
Why is it that most patient have a deletion of the same sets of genes but hv different phenotypes AND they hv parents who are mildly affected with the same deletion?
the combination of alleles inherited by the affected child will be different than that in normal parent. In the parent they have alleles that might compensate for the missing chromosome. And the child will inherit a set of chromosomes from the other parent that may not even compensate for the deletion so that is how they can hv different phenotypes.
Microarray
gene chip technology include gene arrays that look at DNA sequences of interest or look at the gene products to understand which genes are being expressed in a particular cell at a particular time. They look for copy number/ gain or loss
What is the basic principle?
a test DNA is compared to a reference DNA that has a known genetic complement. The DNAs are hybridized and the resulting fluorescent signal is identified and recorded
yellow=equal amounts of red/green
red=more signal in pt than control: duplication
green=less signal in pt than control: deletion
Gene arrays
Depending on the DNAs placed on the chip, the analysis can identify genetic polymorphisms,
specific mutations, or copy number variation (CNV). This type of assay will usually not detect
balanced rearrangements because if the rearrangement is completely balanced, the total
amount of DNA is conserved and no change in the relative amounts will be detected. If,
however, the rearrangement is unbalanced and happens to break within one of the tested
genes, the results would show a loss of sequence for that gene.
Gene arrays can tell you
which specific mutation of CF your patient has or which mitochondrial mutation they have.
Expression arrays
for looking at what DNA is being overexpressed in cells. can create a fingerprint of the tumor
Chromosome Microarray
THE BEST!!!!
instead of reading the data out per gene you get the data out PER CHROMOSOME: can see deletions and duplications…chromosome laid on the side…..does not pick up balanced translocation
What technology should be used to diagnose Williams Syndrome (a contiguous gene syndrome)
FISH
What technology should be used to diagnose Kleinfelter’s Syndrome?
Karyotype analysis
What technology should be used to diagnose Turner Syndrome?
Karyotype analysis
