Diagnostic Technologies Flashcards
What is involved in fluorescence in situ hybridization? How can you observe the results of the experiment?
Molecular probes are hybridized to chromosomes;
Fluorescent microscope
What is the goal of FISH? What must the probe be?
Determine if a gene, specific mutation, or particular chromosomal rearrangement is present/absent; specific to the locus being examined
On which cells can FISH be performed? How can the probe work?
Metaphase and interphase cells;
DNA is denatured and fluorescently labeled single-stranded molecular probe can hybridize to the chromosomal DNA
In FISH, what will someone with no deletions on chromosomes appear? What is a problem with this train of logic?
He/she should have two signals; if one signal is missing, it might be due to chromosome deletion or loss of technical error
What can be used to confirm hybridization with FISH?
Control probe localizes to different region of same chromosome with a different color; 2 signals per chromosome
Where will a FISH probe specific to chromosome 7 at band q11.23 bind to? What are the parameters of this probe?
Detects abnormalities of that locus but NO others;
locus and chromosome specific
What are the 3 types of FISH?
- repeat sequences;
- single copy DNA (subtelomere FISH)
- Chromosome painting (multi-color)
Where are repeat sequences isolated from? What do these particular probes tell you?
- Centromeres (chromosome enumeration)
2. telomeres (confirm presence or absence of the telomeric regions)
What does a unique sequence or single copy probe help identify?
Identify the presence or absence of the gene, gene region, or chromosomal rearrangement of interest
What do subtelomere FISH probes bind to? What properties must this DNA have?
DNA sequences at distal ends of the chromosomes in regions close to telomeres; unique to the chromosome and the specific arm of the chromosome, with short arm probes in green, long arm probes in red
Where are some of the gene rich regions found on chromosomes?
subtelomere regions
What is the basis for chromosome painting (WCP)? When is this probe most useful?
After hybridization, the entire chromosome fluoresces; this type of probe is most useful in identifying complex rearrangements or marker chromosomes
For VCFS, what is a problem with respect to chromosomes?
3 MB deletion on chromosome 22
How much does a FISH probe cover? What could be missed?
Just the critical region (portion of genetic anomaly always or almost always altered during a given mutational process); could miss abnormalities occurring within same gene but outside the critical region
What can help determine if you should use FISH?
- Karyotype analysis could give you info on chromosomes;
2. Clinical information helpful?
What is characteristic of contiguous gene syndromes in terms of phenotype? How are the genes in these regions of the genome related to each other?
We could expect to see multiple phenotype anomalies if the region is deleted; closely associated genes but functions generally unrelated
What are three examples of contiguous gene syndromes that she goes into depth on?
WAGR (11p); Williams syndrome (7q); VCFS (22q)
What does WAGR stand for?
Wilm’s tumor; aniridia; genitourinary; mental retardation
What is Williams syndrome associated with? Is it a microdeletion or contiguous gene syndrome? What are features consistent with elastin absence? What other features indicated that other genes were involved with Williams?
Deletion of the elastin gene on chromosome 7; CGS;
thickening of skin, renal anomalies, skeletal and joint limitations, supravalvular aortic stenosis;
low IQ, excellent musical skills but bad math, outgoing and friendly;
blue sclera and stellate iris
What are some features of VCFS? What is the deletion causing VCFS?
Learning disabilities, hypotonia, short stature, cleft lip and/or palate, facial and cardiac anomalies, feeding difficulty at birth, weak immune system;
3 MB on chromosome 22
What happens with chromosome 22 in VCFS patients?
Interstitial microdeletion on chromosome 22, with approx 40 genes and 8 pseudogenes
What leads to a VCFS as opposed to someone with a completely different phenotype?
Error due to unequal crossing over leading to deletion; duplication 22q syndrome is also due to unequal crossing over
If a parent carries a deletion, how are they not clinically abnormal?
Parent might have alleles on the chromosome he/she has that can compensate for the deletion on chromosome 22
What is the basic principle of a microarray and comparative genomic hybridization (CGH)?
A test DNA (e.g. labeled in red) is compared to a reference DNA (labeled in green possibly); DNAs are hybridized
Given that test DNA is labeled red and reference DNA is labeled green, what does too much red mean? What about too much green?
Duplication in the test DNA; deletion in the test DNA
What are gene arrays (DNA) not able to detect? What can they detect?
balanced rearrangements;
genetic polymorphisms, specific mutations, copy number variation (CNV)
What occurs with expression arrays in terms of finding something to hybridize DNA fragments? What indicates increased expression and decreased gene expression?
Take RNA from tissues, generate cDNA, and hybridize the selected DNA fragments; red can indicate increased expression, green decreased expression, black median expression
What do peaks and valleys indicate on a chromosome microarray?
Gain (duplication) of segments; loss (deletion) of DNA
What is seen normally with chromosome array in terms of pattern? What indicates duplication and deletion?
Straight lines; up and down
What is microarray analysis the first tier study for?
- Developmental delay; 2. intellectual disability 3. autism spectrum disorders 4. multiple congenital abnormalities
What can be next generation sequencing be used for?
Genetic disorders, oncology, infectious disease, repro health, population genetics, forensics, agricultural
What are things to keep in mind when ordering sequencing?
Is the laboratory accredited and the test validated? Time (2-3 days for analysis, 1-2 weeks for interpretation); $3000-5000?; insurance coverage!
