Diagnostic Technologies Flashcards
1
Q
Fluorescence In Situ Hybridization (FISH)
A
- combines the best of cytogenetics and molecular diagnostics to obtain information that cannot be obtained by either technology alone
- molecular probes
- used to be tritium then fluorescent dyes
- the most common goal is to determine if a gene, a specific mutation, or a particular chromosomal rearrangement is present or absent, so the molecular probe(s) used must be well characterized and specific to the locus being examined
2
Q
FISH technology
A
- to create a probe a target like DNA gene or location is chosen and a fragment of DNA is isolated
- this should be DNA that is unique to the region. One strand of the fragment is labeled with a fluorescent dye
- to confirm that the probe works, it will be hybridized to a metaphase cell preparation and it should bind to the same location from which it was derived. Once an appropriate probe is chosen, testing can take place
- FISH can be performed on either metaphase or interphase cells, and slides are prepared just as they would be for a karyotype analysis
- the DNA on the slies is then denatured and the fluorescently labeled singe stranded molecular probe is allowed to hybridize to the chromosomal DNA
- the rest of the DNA is counterstained with another flurochrome to allow visualization of the entire chromosome complement using a fluorescent microscope
3
Q
FISH probes and multiple loci
A
- the original FISH probes hybridized to a single locus, but this created difficulty in interpretation of the results
- the was never clear if the missing signal was due to a disease related issue, such as chromosome deletion or if the loss was a technical error such as failure of probe to bind to target
- a control probe was localized to a different region of the same chromosome in all the assays as a hybridization control
- usually test and control labeled with different colored fluorochromes
- a person with a deletion should have two signals on one chromosome but only a single signal on the deleted chromosome
4
Q
Parameters of probe
A
- it only detects the site from which it was derived
- therefore if the probe is specific to chromosome 7 at band 11.23, it will detect abnormalities of that locus but NO others
- thus, a single FISH assay does not provide the same type of genomic information available by karyotype analysis, but it may provide more detailed information about one specific locus
5
Q
3 Types of FISH
A
- repeat sequences
- single copy DNA- subtelomere FISH
- chromosome painting- multi-color
6
Q
Repeat Sequence Probes
A
- usually isolated from telomere or centromere regions
- centromere probes are usually used in chromosome enumeration (to detect the gain or loss of specific chromosomes)
- a true telomere probe recognizes the six base repeat present at the ends of all chromosomes and will confirm the presence or absence of the of the telomeric regions
7
Q
Single copy probe
A
- isolated from cloned DNA of a disease-causing gene or a fragment of DNA of known location associated with a particular gene
- this type of probe is used to identify the presence or absence of the gene, gene region, or chromosomal rearrangement of interest
8
Q
Subtelomere TISH
A
- DNA sequences from the distal ends of the chromosomes in regions proximal to the actual telomere regions
- telomeric sequences themselves cannot be used since these are repeat sequences that are the same of all chromosomes
- the DNA used must be unique to the chromosome and to the specific arm of the chromosome
- the short arm probes are green, red is long arm
- important because it allows us to identify very small (cryptic) deletions and rearrangements that cannot be seen by standard karyotype analysis
- it is known that the subtelomere regions of the chromosomes are gene rich, and that 3-5% of unexplained mental retardation is due to cryptic terminal deletions
9
Q
Chromosome painting
A
- 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
- this type of probe is more useful in identifying complex rearrangements or marker chromosomes
- if the patient has an abnormal chromosome with extra material origin, it may be possible to use chromosome painting to identify the source of the extra DNA
10
Q
Multicolor RISH
A
- type of chromosome painting that is used to detect multiple chromosomes with one hybridization
- this is done with special probes using a fluorescence microscope and computer with specialized software
- for a typical fluorescent microscope, the maximum number of useable color is three (one target sequence, one control sequence, and one counter stain)
- combinations of fluorochromes have been developed that allow detection of each of the 24 different chromosome followed by a unique color assignment by the computer. Although this technology is very good at detecting chromosome rearrangements (translocations, large duplications or deletions), it cannot identify inversions, small deletions, or small duplications
11
Q
FISH facts
A
- probes do not cover the entire deletion- just the critical region
- for example for a 3 MB deletion, the probe may be only 10 KB (velocardiofacial syndrome)
- therefore, a deletion may be present that cannot be detected by the FISH probe designed for that disease
12
Q
What FISH when?
A
- you can’t screen all chromosomes or loci
- maximize your results
- if you think you know the disease- start there (unique sequence)
- if karyotype analysis has given you chromosomes- use that information (whole chromosome paint or unique sequence will identify a particular region of a chromosome)
- does clinical info helps?- developmental delay may be associated with a subtelomeric microdeletion
13
Q
Contiguous Gene Syndromes
A
- regions in the genome with clusters of closely associated genes whose normal functions are generally unrelated
- deletion of that region results in multiple phenotypic anomalies that can be described as a particular syndrome
- WAGR-11p
- Miller-Dieker/Lissencephaly-17p
- Williams syndrome-7q
- Velocardiofacial syndrome-22q
- 1p-syndrome
- Prader Willi/Angelman syndromes
14
Q
WAGR
A
- one of the best known contiquous gene syndromes
- located on the short arm of chromosome 11
- can affect one or more of the genes aligned in tantem
- Wilms tumor
- aniridia
- genitourinary
- retardation
15
Q
Williams syndrome
A
- associated with the deletion of the elastin gene on the proximal long arm of chromosome 7
- deletion involving several adjacent genes
- absence of elastin: coarse skin and hair, lack of flexibility in the aorta, supravalvular aortic stenosis
- they have developmental problems and cannot live on their own
- skeletal and joint limitations
- renal anomalies
- usually low IQ
- excellent musical skills but terrible with math
- outgoing and friendly
- blue sclera
