Cytogenetics Techniques Overview
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Questions and Answers

What is the primary purpose of the post-hybridization washing step?

  • To enhance the visibility of the counter stain
  • To increase the temperature for better hybridization
  • To remove unbound or non-specifically bound probes (correct)
  • To incubate the cells overnight

At what temperature are the cells typically incubated during the conversion of double-stranded DNA to single-stranded DNA?

  • 25 degrees Celsius
  • 50 degrees Celsius
  • 37 degrees Celsius (correct)
  • 42 degrees Celsius

What is the role of the fluorescent-labeled probe?

  • To increase the temperature during incubation
  • To stain the cells for easier observation
  • To enhance the hybridization conditions
  • To emit light and indicate binding to the target DNA (correct)

What is the main goal after completing the washing technique?

<p>To analyze hybridized sample abnormalities (D)</p> Signup and view all the answers

When analyzing the hybridized sample, what determines the patterns observed?

<p>The fluorescent signals emitted by the probes (D)</p> Signup and view all the answers

In which step do scientists apply the counter stain?

<p>After washing unbound probe DNA (C)</p> Signup and view all the answers

What biological materials can serve as target samples in this process?

<p>Cells, tissue sections, and body fluids (A)</p> Signup and view all the answers

What must occur to the cells before the fluorescent probe can hybridize effectively?

<p>They need to be incubated under specific conditions (D)</p> Signup and view all the answers

What is the primary function of probes when they hybridize with target DNA?

<p>To emit light for visualization (C)</p> Signup and view all the answers

Which hapten is commonly used in indirect labeling for signal detection?

<p>Biotin (C)</p> Signup and view all the answers

What type of probe is designed to determine the presence or absence of specific sequences?

<p>Locus-specific probes (A)</p> Signup and view all the answers

How are digoxigenin haptens detected?

<p>With anti-digoxigenin antibodies (B)</p> Signup and view all the answers

What does the category of 'probes' refer to?

<p>Broad classifications based on function (B)</p> Signup and view all the answers

Which example represents the application of probes in medical diagnostics?

<p>Identification of BCR-ABL fusion in leukemia (A)</p> Signup and view all the answers

What causes a stronger green signal in DNA labeling?

<p>Less target sequence for red probe binding (A)</p> Signup and view all the answers

What characteristic is NOT typically used to classify probes?

<p>Color (D)</p> Signup and view all the answers

What is the role of Cot-1 DNA in the labeling process?

<p>To block repetitive sequences (C)</p> Signup and view all the answers

In terms of their function, what do locus-specific probes provide information about?

<p>Specific genes or chromosomal locations (A)</p> Signup and view all the answers

What is the purpose of denaturation in the labeling process?

<p>To separate double-stranded DNA into single strands (B)</p> Signup and view all the answers

What indicates a duplication in a DNA sample during the labeling process?

<p>More copies of a DNA segment compared to the reference (C)</p> Signup and view all the answers

Why is repetitive DNA commonly found in telomere and centromere regions?

<p>They provide structural stability to chromosomes (D)</p> Signup and view all the answers

What effect does the presence of unlabeled Cot-1 DNA have on red probes?

<p>It reduces the efficiency of red probes (A)</p> Signup and view all the answers

What happens to the hydrogen bonds between complementary bases during denaturation?

<p>They are broken, separating the strands (A)</p> Signup and view all the answers

How does a missing segment of DNA affect the strength of the red signal?

<p>It diminishes the red signal strength (B)</p> Signup and view all the answers

What does a stronger red signal indicate in the context of DNA copy number?

<p>Increased copy number of a DNA segment (D)</p> Signup and view all the answers

What will indicate a deletion in the test sample?

<p>Increased green signal in the corresponding region (A)</p> Signup and view all the answers

When regions of DNA show equal copy numbers, what color signal is observed?

<p>Yellow signal (B)</p> Signup and view all the answers

What contributes to a stronger red signal in the probe binding?

<p>Multiple copies of the target sequence in the test sample (D)</p> Signup and view all the answers

How does a duplication affect the fluorescence signal interpretation?

<p>It causes an increase in red signal intensity in multiple regions (A)</p> Signup and view all the answers

In fluorescence signal analysis, what does the presence of a green signal suggest?

<p>A loss of one or more DNA segments (A)</p> Signup and view all the answers

Why is it important to analyze the fluorescence signals along chromosomes?

<p>To identify variations in DNA copy numbers (A)</p> Signup and view all the answers

What does a stronger green signal correspond to in the context of DNA analysis?

<p>Decreased presence of the target sequence (C)</p> Signup and view all the answers

What is the primary application of Array CGH?

<p>To evaluate DNA copy number alterations in cancer (D)</p> Signup and view all the answers

How does Array CGH differ from traditional CGH?

<p>Array CGH uses higher resolution and DNA microarrays (C)</p> Signup and view all the answers

What is considered a limit of using CGH in clinical settings?

<p>It should not be used when knowledge of genetic status will not affect treatment decisions (C)</p> Signup and view all the answers

What advantage does Array CGH have over traditional CGH?

<p>Greater resolution for smaller CNVs (A)</p> Signup and view all the answers

Why is Array CGH considered the gold standard for detecting CNVs?

<p>It allows for high-resolution evaluations (D)</p> Signup and view all the answers

In what context is the use of CGH considered experimental?

<p>Without symptoms or knowledge impacting treatment decisions (C)</p> Signup and view all the answers

What type of technology does Array CGH employ?

<p>DNA microarrays targeting specific genome regions (C)</p> Signup and view all the answers

What is a common application of Array CGH outside of cancer research?

<p>Prenatal genetic mutation screening (A)</p> Signup and view all the answers

What is one limitation of traditional Comparative Genomic Hybridization (CGH)?

<p>It cannot detect small copy number variations (CNVs). (B)</p> Signup and view all the answers

What advantage does automated Comparative Genomic Hybridization (aCGH) have over traditional CGH?

<p>It reduces human error and speeds up sample processing. (D)</p> Signup and view all the answers

Which type of chromosomal rearrangement cannot be detected by CGI?

<p>Balanced rearrangements like translocations (A)</p> Signup and view all the answers

What does the spectral karyotyping (SKY) technique primarily allow for?

<p>Visualization of chromosome pairs in different colors. (C)</p> Signup and view all the answers

What is a significant technical requirement for performing Comparative Genomic Hybridization (CGH)?

<p>Specialized equipment and expertise. (C)</p> Signup and view all the answers

What aspect of the aCGH technique simplifies the process as compared to traditional hybridization methods?

<p>No denaturation is required. (B)</p> Signup and view all the answers

Which of the following is a feature of aCGH in relation to traditional CGH?

<p>Offers higher resolution for smaller CNVs. (B)</p> Signup and view all the answers

Why are balanced chromosomal rearrangements undetectable by CGH?

<p>They do not change the copy number of DNA segments. (B)</p> Signup and view all the answers

Flashcards

Probes in DNA hybridization

Specific DNA segments used to detect complementary sequences in a sample.

Fluorescence Signal Detection

The signal emitted when probes bind to DNA, allowing visualization under a microscope.

Indirect Labeling (Probes)

Using haptens (e.g., biotin) as intermediate labels to detect probes indirectly.

Hapten-Streptavidin-Biotin

Method to detect probes where biotin binds tightly to streptavidin and is then linked to a detection molecule.

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Digoxigenin detection

Method using anti-digoxigenin antibodies linked to fluorescent dyes for probe detection.

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Categories of Probes (broad)

Classifications of probes based on their function/target DNA sequence in the genome.

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Locus-specific probes

Type of probe used to find a specific gene or chromosomal region to detect presence/absence.

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BCR-ABL fusion detection

Example application of probes to detect a specific chromosomal change linked to chronic myeloid leukemia.

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DNA conversion to single-stranded

The process of converting double-stranded DNA into single-stranded DNA.

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Hybridization

The process where a probe binds to its complementary target sequence.

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Post-hybridization washing

Removing unbound probe DNA from the sample.

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Target sample

Biological material (cells, tissues or fluids) used in the study.

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Counter stain

A stain used to improve visualization, enhancing contrast/clarity

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Fluorescence Microscopy

Using a microscope to view samples by looking for fluorescence emitted from labelled probes

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Fluorescence probe

Molecule that emits light when exposed to light of a specific wavelength after binding to the target.

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Washing stringency

Washing process's intensity or strength in removing unbound material.

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Deletion in DNA

A segment of DNA is missing from the test sample compared to the reference sample.

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Duplication in DNA

Extra copy of a chromosome, resulting in many regions where red probe can bind.

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Balanced hybridization

Equal copy numbers of DNA segments in both test and reference samples.

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Increased green signal

Indicates a deletion of a particular DNA segment in the test sample compared to the reference.

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Increased red signal

Indicates duplication of a particular DNA segment in the test sample compared to the reference.

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Fluorescence Signal Interpretation

Analyzing fluorescent signals along each chromosome to determine copy number variations.

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Copy Number Variations (CNVs)

Alterations in the DNA of a genome that cause cells to have an abnormal copy number of DNA segments.

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Lowered signal (deleted region)

Less target DNA for probe binding, causing the signal to decrease.

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Duplication (Cytogenetics)

A segment of DNA is present in multiple copies in the test sample compared to the reference.

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Red Signal (Cytogenetics)

Indicates a duplication in a chromosome segment, meaning more copies of DNA sequence in sample, when compared to a reference.

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Green Signal (Cytogenetics)

Indicates a reduction in a chromosome segment, meaning less copies of DNA sequence in sample, when compared to a reference.

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Chromosome Segment Duplication

A portion of a chromosome that has been copied and inserted, resulting in more copies of the target DNA sequence compared to the reference.

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Denaturation (DNA)

Separation of double-stranded DNA into single strands by breaking hydrogen bonds.

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Cot-1 DNA

Enriched for repetitive DNA sequences, used to block or suppress detection of telomere and centromere regions.

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Target DNA

The specific DNA sequence being analyzed or tested, the sample under investigation.

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CGH Technology Purpose

Initially developed to study genomic changes in cancer, CGH helps detect DNA copy number alterations related to chromosomal abnormalities.

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CGH Benefits

CGH provides high-resolution evaluation of DNA copy number changes, offering a powerful tool for diagnosis and research.

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CGH Clinical Use

CGH is used in complex diagnostics, but its use is considered experimental in the absence of strong indications or when the results won't directly impact treatment decisions.

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aCGH Advancement

Array CGH (aCGH) uses DNA microarrays to analyze genomic changes, offering higher resolution and sensitivity than traditional CGH.

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aCGH Advantages

aCGH offers increased sensitivity and resolution, allowing for the detection of smaller DNA copy number changes.

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aCGH Application

aCGH has become the standard for finding DNA copy number variations (CNVs) in clinical and research settings.

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aCGH in Prenatal Screening

aCGH is used to screen for gene mutations in fetuses without structural abnormalities or to test products of conception after an artificial insemination.

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CNV Detection Purpose

Detecting CNVs is important in various research and medical fields, providing valuable insights into genetic diseases and personalized medicine.

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What is aCGH?

aCGH stands for array comparative genomic hybridization. It's a technique used to detect copy number variations (CNVs) in DNA, like extra or missing pieces of chromosomes, which can be associated with genetic disorders.

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How does aCGH work?

In aCGH, DNA from a patient is labeled with one color and compared to DNA of a healthy person labeled with another color. Both DNAs are then applied to a microarray, a chip containing many DNA probes representing different parts of the genome. The relative amounts of each color at each probe location indicate whether the patient's DNA has extra or missing copies of those regions.

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What are the benefits of aCGH?

aCGH is a powerful tool for detecting CNVs due to its automation, which reduces human error and allows for faster processing. It's also non-invasive, making it a safer alternative to traditional methods like amniocentesis.

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What are some limitations of aCGH?

aCGH has some limitations. It cannot detect small CNVs or those located close together, and it cannot detect balanced chromosomal rearrangements, such as translocations or inversions.

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What is SKY?

Spectral karyotyping (SKY) is a technique which allows scientists to see all the pairs of chromosomes in an organism at once, each with a unique color.

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What is SKY used for?

SKY is used to identify chromosomal abnormalities, such as translocations, deletions, duplications, and other abnormalities.

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What is the main difference between aCGH and SKY?

aCGH detects copy number variations (extra or missing pieces of chromosomes), while SKY identifies the location, size, and number of each chromosome.

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What happens in CGH?

In CGH, the DNA of a patient and a healthy reference are labeled with fluorescent dyes and hybridized to a microarray, a chip containing DNA probes representing different parts of the genome. Based on the fluorescent signals, a computer analyzes the data and generates a plot, revealing copy number variations and other chromosomal abnormalities.

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Study Notes

Cytogenetics Techniques

  • Karyotyping: Examines chromosomes in cells to identify genetic problems causing disorders or diseases.
  • Process: Culture cells, arrest at metaphase, prepare slides, stain, and visualize chromosome arrangements.
  • Chromosomal Banding Techniques: Several methods used to visualize banding patterns on chromosomes:
    • G-banding
    • R-banding
    • Q-banding
    • T-banding
    • Silver staining

Molecular Cytogenetic Techniques

  • Fluorescence In Situ Hybridization (FISH): Molecular cytogenetic technique tagging genetic material with fluorescent molecules to map gene locations on chromosomes to detect small deletions and duplications.
  • Process: Denature DNA, label DNA probe, combine probe with target DNA, and visualize target sequence under a fluorescence microscope.
  • FISH Probe Types:
    • Whole chromosome
    • Unique sequence
    • Repetitive sequence
  • FISH Sample Types: Frozen sections, paraffin-embedded sections, cells in suspension (blood, bone marrow, amniotic fluid, etc.)

Comparative Genomic Hybridization (CGH)

  • Method: Analyzes DNA copy number variations by comparing test DNA with a control DNA by hybridization on chromosome spreads followed by image analysis.
  • Applications: Identify gains or losses in DNA content, e.g., in cancer.

Spectral Karyotyping (SKY)

  • Technique: Visualizes all chromosome pairs in different colors enabling the identification of chromosomal abnormalities.
  • Process: Uses fluorescent probes that label each chromosome, enabling visualization of the entire set under fluorescence microscopy.
  • Applications: Detect chromosomal abnormalities including translocations, deletions, and inversions.

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Description

Explore various cytogenetic techniques including karyotyping and molecular cytogenetics. This quiz covers cellular processes, chromosomal banding techniques, and fluorescence in situ hybridization (FISH). Test your understanding of these methods used to identify genetic disorders and their underlying mechanisms.

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