Diagnostic Technologies - RM Flashcards
What technique hybridizes molecular probes to chromosomes?
FISH
What are fragments of single stranded DNA homologous to region of DNA sequence of interest labeled with fluorochrome?
molecular probes
What phase in the cell cycle is FISH performed at?
metaphase
Why do you need an internal control in FISH? What does the control probe do?
to confirm hybridization is occuring (otherwise can’t tell if missing signal is due to disease related issue or technical error of probe not binding)
-control probe binds to different region of same chromosome that should be unaffected
How many FISH signals indicates a normal individual?
2 signals per chromosome, 1 for the molecular test probe, 1 for the control probe indicating hybridization worked
What are centromere probes used for?
chromosome enumeration (to see if there’s gain/loss)
What are telomere probes used for?
confirms presence or absence of telomeric regions
What does a single copy probe do?
identifies presence/absence of a gene or chromosomal rearrangment of interest specific to gene locus
Why are subtelomere FISH probes important?
identifies very small (cryptic) deletions and rearrangments in DNA sequences in distal ends of chromsomes proximal to the telomeres that can’t be seen by standard karyotype
-3-5% of unexplained mental retardation due to cryptic terminal deletions
What does chromosome painting do?
probes for many DNA fragments along chromosome make entire chromosome fluoresce so you can identify complex rearrangements and translocations due to different colors in a chromosome
What is multicolor FISH good for detecting?
chromosomal translocations, duplications, deletions in multiple chromosomes using 1 hybridization
What can’t multicolor FISH identify?
inversions, small deletions or duplications in chromosome
What is the “critical region” in FISH?
portion of genetic anomaly that is always or almost always altered in mutational process
-what the probe is based off of since it can’t cover the entire deletion
What are the disadvantages of FISH?
can’t detect diseases that FISH probe isn’t designated for, can’t screen all chromosomes or all loci
What may be associated with subtelomeric microdeletion?
developmental delay
What are contigous gene syndromes? What technique is commonly used to detect it?
deletions in regions in genome with clusters of closely associated genes whose normal functions are generally unrelated, causing multiple phenotypic anomalies
FISH used to detect these microdeletions
What does WAGR stand for? What chromosome and arm of the chromosome is it on?
Wilms tumor, aniridia, genitourinary defects, mental retardation
11p
What syndrome results from deletion of elastin gene on proximal long arm of 7? What are the features of it?
Williams syndrome
-coarse hair/skin, lack of flexibility in aorta, supravalvular aortic stenosis, skeletal and joint limitations, renal anomalies, excellent music skills, bad at math, blue sclera, stellate iris
What is caused by 3 MB deletion on 22q?
VCFS
What symptoms are associated with VCFS?
learning disability, hypotonia, short stature, cleft lip/palate, facial anomalies, cardiac anomalies (conotruncal heart defects), feeding difficulties at birth, weak immune system
How is presentation of VCFS so variable?
- depends on complement of alleles in homologous chromsome (may be able to compensate for deletion)
- new combo of alleles for normal chromosome may cause different phenotype
- deletions can range in size and genes deleted
What compares relative amounts of DNA from test source and reference source with known genetic complement?
microarray
What does it mean if there’s an excess of test DNA signal in microarray compared to reference DNA signal?
duplication in the test DNA
What does it mean if there’s an excess of reference DNA signal in microarray compared to test DNA signal?
deletion in test DNA
What do gene arrays identify? What can’t they identify?
genetic polymorphisms, specific mutations, copy number variation (duplication/deletion)
-can’t identify balanced rearrangements
What do expression arrays identify? How is it visualized?
- what DNA is actually being expressed in particular cells
- visualized as heat map of high level of expression/low level expression/median level expression based on colors
Why are expression arrays important for cancer studies?
- expression of genes in tumors were assayed and upregulated genes for each tumor type were logged as the “fingerprint” for that type
- established clinical test to diagnose tumor type by running this panel of standards against the test specimen
What do peaks in chromosome microarray results reveal? valleys?
peaks–>gain of segments–>duplications
valleys–>loss of segments–>deletions
What is the best technique for cases of unexplained developmental delay, intellectual disability, autism spectrum disorders, and multiple congenital anomalies?
microarray
What is the best for genome wide, relatively large numerical and structural abnormality testing?
karyotype analysis
What is molecular diagnostics best for?
well defined, specific, very small (1-300 base pair) mutations
-targeted testing
What size mutation is FISH best for detecting?
10 kb - 10 MB
What can’t microarrays detect?
balanced rearrangements
What two techniques are best for detecting mosaicism?
FISH, microarray
What is best at detecting cosanguinety/identity by descent?
microarray
If clinical findings suggest KNOWN numerical or structural disorder, what techniques are best?
FISH or karyotype (specific and targeted testing)
