Diagnostic Technologies

Question 1

What is involved in fluorescence in situ hybridization? How can you observe the results of the experiment?

Answer 1

Molecular probes are hybridized to chromosomes;

Fluorescent microscope

Question 2

What is the goal of FISH? What must the probe be?

Answer 2

Determine if a gene, specific mutation, or particular chromosomal rearrangement is present/absent; specific to the locus being examined

Question 3

On which cells can FISH be performed? How can the probe work?

Answer 3

Metaphase and interphase cells;

DNA is denatured and fluorescently labeled single-stranded molecular probe can hybridize to the chromosomal DNA

Question 4

In FISH, what will someone with no deletions on chromosomes appear? What is a problem with this train of logic?

Answer 4

He/she should have two signals; if one signal is missing, it might be due to chromosome deletion or loss of technical error

Question 5

What can be used to confirm hybridization with FISH?

Answer 5

Control probe localizes to different region of same chromosome with a different color; 2 signals per chromosome

Question 6

Where will a FISH probe specific to chromosome 7 at band q11.23 bind to? What are the parameters of this probe?

Answer 6

Detects abnormalities of that locus but NO others;

locus and chromosome specific

Question 7

What are the 3 types of FISH?

Answer 7

1. repeat sequences;
2. single copy DNA (subtelomere FISH)
3. Chromosome painting (multi-color)

Question 8

Where are repeat sequences isolated from? What do these particular probes tell you?

Answer 8

1. Centromeres (chromosome enumeration)

2. telomeres (confirm presence or absence of the telomeric regions)

Question 9

What does a unique sequence or single copy probe help identify?

Answer 9

Identify the presence or absence of the gene, gene region, or chromosomal rearrangement of interest

Question 10

What do subtelomere FISH probes bind to? What properties must this DNA have?

Answer 10

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

Question 11

Where are some of the gene rich regions found on chromosomes?

Answer 11

subtelomere regions

Question 12

What is the basis for chromosome painting (WCP)? When is this probe most useful?

Answer 12

After hybridization, the entire chromosome fluoresces; this type of probe is most useful in identifying complex rearrangements or marker chromosomes

Question 13

For VCFS, what is a problem with respect to chromosomes?

Answer 13

3 MB deletion on chromosome 22

Question 14

How much does a FISH probe cover? What could be missed?

Answer 14

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

Question 15

What can help determine if you should use FISH?

Answer 15

1. Karyotype analysis could give you info on chromosomes;

2. Clinical information helpful?

Question 16

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?

Answer 16

We could expect to see multiple phenotype anomalies if the region is deleted; closely associated genes but functions generally unrelated

Question 17

What are three examples of contiguous gene syndromes that she goes into depth on?

Answer 17

WAGR (11p); Williams syndrome (7q); VCFS (22q)

Question 18

What does WAGR stand for?

Answer 18

Wilm’s tumor; aniridia; genitourinary; mental retardation

Question 19

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?

Answer 19

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

Question 20

What are some features of VCFS? What is the deletion causing VCFS?

Answer 20

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

Question 21

What happens with chromosome 22 in VCFS patients?

Answer 21

Interstitial microdeletion on chromosome 22, with approx 40 genes and 8 pseudogenes

Question 22

What leads to a VCFS as opposed to someone with a completely different phenotype?

Answer 22

Error due to unequal crossing over leading to deletion; duplication 22q syndrome is also due to unequal crossing over

Question 23

If a parent carries a deletion, how are they not clinically abnormal?

Answer 23

Parent might have alleles on the chromosome he/she has that can compensate for the deletion on chromosome 22

Question 24

What is the basic principle of a microarray and comparative genomic hybridization (CGH)?

Answer 24

A test DNA (e.g. labeled in red) is compared to a reference DNA (labeled in green possibly); DNAs are hybridized

Question 25

Given that test DNA is labeled red and reference DNA is labeled green, what does too much red mean? What about too much green?

Answer 25

Duplication in the test DNA; deletion in the test DNA

Question 26

What are gene arrays (DNA) not able to detect? What can they detect?

Answer 26

balanced rearrangements;

genetic polymorphisms, specific mutations, copy number variation (CNV)

Question 27

What occurs with expression arrays in terms of finding something to hybridize DNA fragments? What indicates increased expression and decreased gene expression?

Answer 27

Take RNA from tissues, generate cDNA, and hybridize the selected DNA fragments; red can indicate increased expression, green decreased expression, black median expression

Question 28

What do peaks and valleys indicate on a chromosome microarray?

Answer 28

Gain (duplication) of segments; loss (deletion) of DNA

Question 29

What is seen normally with chromosome array in terms of pattern? What indicates duplication and deletion?

Answer 29

Straight lines; up and down

Question 30

What is microarray analysis the first tier study for?

Answer 30

1. Developmental delay; 2. intellectual disability 3. autism spectrum disorders 4. multiple congenital abnormalities

Question 31

What can be next generation sequencing be used for?

Answer 31

Genetic disorders, oncology, infectious disease, repro health, population genetics, forensics, agricultural

Question 32

What are things to keep in mind when ordering sequencing?

Answer 32

Is the laboratory accredited and the test validated? Time (2-3 days for analysis, 1-2 weeks for interpretation); $3000-5000?; insurance coverage!