I.
Fluorescence in-situ hybridization
1. What types of chromosomal abnormalities can be detected using unique sequence FISH probes, and
why are these probes particularly useful for these abnormalities?
The unique sequence of FISH probes enables the detection of various chromosomal abnormalities,
including deletions, amplification, translocation, duplication, aneuploidy, and loss of a chromosomal
region. These FISH probes are useful for detecting these abnormalities because they bind to specific DNA
sequences, they are highly sensitive and specific, and the assays can be performed quickly, which might
be too small to be detected by traditional karyotyping or other cytogenetic techniques.
2. Explain the difference between using whole chromosome probes and repetitive sequence probes in
FISH. When would each be most appropriate?
The whole chromosome probes are probes that cover the entire length of a chromosome, and they are
used to visualize the entire chromosome, making it easier to detect large structural changes like
translocation and complex rearrangements. On the other hand, the repetitive sequence probes are
probes that target repetitive DNA sequences that are found throughout the genome. Like centromere
and telomere that contain highly repetitive sequence. Also, it is used in chromosome enumeration and
to identify specific chromosomal regions. Whole chromosome probes are most appropriate when
analyzing complex rearrangements or entire chromosomal complements, while repetitive sequence
probes are ideal for quick assessments of chromosome count or structural stability in defined regions.
3. Based on the illustration of the FISH procedure, briefly describe the purpose of the denaturation
step.
Denaturation is crucial, allowing probes to bind their complementary DNA sequences during
hybridization. Samples in denaturation are often heated in a controlled environment, which disrupts the
hydrogen bond between DNA strands that causes two strands to separate and become single-stranded
DNA.
4. Explain the significance of the yellow signal in a normal break apart signal pattern.
When two fluorescent probes, each labeled with a different color, are closely positioned and overlap, the
yellow signal in a normal break apart signal pattern shows that the targeted chromosomal region is
intact and has not experienced any structural rearrangement or breakage.
II. Comparative Genomic Hybridization (CGH)
1. After reviewing the CGH procedure illustration, describe the role of the test and reference DNA in
detecting copy number variations (CNVs).
By comparing the fluorescent signal strength of the labeled test DNA to that of the reference DNA, the
test and reference DNA can identify copy number variation. In order to identify genomic imbalances in
the sample, regions with a greater test DNA signal indicate gains in copy number, whereas regions with a
lower signal indicate losses.
2. What conclusions can be drawn when the test DNA shows a higher fluorescence signal compared to
the reference DNA?
An amplification of that particular genomic region in comparison to the normal reference genome is
suggested when the test DNA exhibits higher fluorescence signal than the reference DNA, indicating that
a copy number duplicates in the test DNA.
3. How does CGH complement the findings from FISH when analyzing complex chromosomal
abnormalities?
CGH analyzes complex chromosomal abnormalities by providing a genomic wide overview of copy
number variations and identifying gains or losses across the entire genome. While FISH enables the
precise localization and visualization of particular chromosomal regions or genes that aid in validating
the structural details of abnormalities detected by CGH.
