50 Questions
What is the main purpose of capturing cells in division during tissue culture?
To ensure cells are in their most active phase for maximum yield
Why is Colchicine/Colcemid used in the process of capturing cells in division?
To interfere with spindle formation and prevent cell division
When is the best time to harvest bloods (for constitutional studies) in tissue culture?
After 3 days (72-hour cultures)
Why are suitable nutrients and growth enhancers essential in tissue culture?
To maximize the number of dividing cells
What is the aim of harvesting cells in tissue culture according to the provided text?
To conduct chromosome analysis
Which technique uses a DNA fragment as a probe for nucleic acid hybridization?
DNA Microarray
Which genetic conditions are microarrays routinely used for in pediatric cases?
Autism
What is the principal feature of G-banding pattern on chromosomes?
Reflects tissue-specific genes
Which technique is used to investigate chromosome rearrangement near centromeres and polymorphism?
FISH
What is the main purpose of QFPCR (quantitative fluorescent PCR)?
Rapid prenatal aneuploidy diagnosis
What is the key characteristic of C-bands in cytogenetic analysis?
Marked polymorphism in size
How are telomeres most effectively visualized?
Using FISH with terminal repeat sequence probes
What are the two main categories of cells discussed in the text with respect to their origin?
Constitutional and acquired cells
What are the key components required for suitable tissue culture conditions?
Tissue culture media, fetal calf serum, amino acids, growth enhancers, antibiotics, and antifungals
What is the purpose of capturing cells in division during tissue culture?
To maximize cell yield from actively dividing tissue and achieve a high mitotic index
How is the cell cycle important in the process of harvesting cells?
Understanding the cell cycle is fundamental to good harvesting
What is the ideal time frame for harvesting bloods in tissue culture for constitutional studies?
Three days (72-hour cultures)
How long does MLPA processing of 96 samples take in terms of hands-on time?
Less than three hours
Explain the difference between G-Dark bands and G-Light bands in G-banding.
G-Dark bands replicate mid to late in S phase, condense early in mitosis, and are gene poor. G-Light bands replicate early in S phase, condense later in mitosis, and are gene rich.
What is the significance of C-banding in cytogenetic analysis?
C-banding is used to investigate chromosome rearrangement near centromeres and to study polymorphism. It is located at specific chromosome regions and replicates late in S phase.
Describe the process and purpose of Fluorescence In Situ Hybridisation (FISH).
FISH uses fluorescent probes to bind to specific chromosome regions based on sequence similarity. It is used to detect microdeletions, duplications, and specific parts of the genome.
Explain the utility of Multiplex Ligation-dependent Probe Amplification (MLPA) in genetic analysis.
MLPA is a technique used for detecting DNA copy number changes with relative quantitation of up to 48 different nucleic acid sequences. It is cost-effective, rapid, and allows for the detection of subtelomeric deletions/duplications.
Match the genetic testing method with its primary application:
MLPA = Detection of subtelomeric deletions/duplications Microarray = Genome-wide screening of DNA copy number changes FISH = Investigating chromosome rearrangements near centromeres QF-PCR = Quantitative analysis of genetic material
Match the genetic testing technology with its characteristic feature:
MLPA = Reproducible with low coefficient of variability Microarray = Cannot detect balanced rearrangements FISH = Visualizing specific DNA sequences in chromosomes QF-PCR = Rapid and quantitative analysis
Match the following chromosome banding techniques with their characteristics:
G-banding = Reflects both structural and functional composition of the chromosome C-banding = Located at the centromeres of chromosomes, replicates late in S phase NOR Staining = Contains genes for 18S and 28S rRNA on specific chromosomes Telomere Banding = Selective staining of terminal bands or telomeric regions
Match the following molecular methodologies with their descriptions:
FISH (Fluorescence In Situ Hybridisation) = Uses fluorescent probes to bind to specific parts of the genome QFPCR (Quantitative Fluorescence PCR) = Rapid prenatal diagnosis of chromosome copy number changes MLPA (Multiplex Ligation-dependent Probe Amplification) = Allows relative quantitation of up to 48 different nucleic acid sequences CGH (Comparative Genomic Hybridisation) = Technique for detection of DNA copy number changes
Match the following cytogenetic techniques with their applications:
C-Banding = Investigation of chromosome rearrangement near centromeres and polymorphism NOR Staining = Selective staining of regions containing actively transcribed genes Telomere Banding = Visualization of terminal bands or telomeric regions T-Banding = Specialised case of R-banding used for selective staining
What are the key components required for suitable tissue culture conditions?
Tissue culture media, fetal calf serum, amino acids, growth enhancers, antibiotics, antifungals
How is the cell cycle important in the process of harvesting cells?
Understanding the cell cycle is fundamental to good harvesting; cells should be actively dividing
What are the two main categories of cells discussed in the text with respect to their origin?
Constitutional cells and acquired cells
What are the principal groups of banding techniques used in cytogenetic analysis?
Banding techniques resulting in bands along the whole chromosome and those that stain specific bands or structures
How does G-banding pattern reflect the composition of the chromosome?
G-banding pattern reflects both structural and functional composition of the chromosome
How is Quantitative Fluorescence PCR (QF-PCR) used in genetic testing?
QF-PCR is used for rapid prenatal aneuploidy diagnosis
Explain the process of non-allelic homologous recombination (NAHR) and its potential consequences on chromosome structure.
NAHR is a DNA recombination process between non-identical but similar DNA sequences on non-homologous chromosomes. It can lead to genetic imbalances or syndromes if one chromosome undergoes deletion and the other receives an extra copy of the sequence.
What are the key characteristics of G-banding patterns on chromosomes and how do they reflect the composition of the chromosome?
G-banding patterns show light and dark bands on chromosomes, reflecting regions of different base pair densities. The banding pattern helps identify different chromosome regions based on their unique banding patterns.
Describe the utility of Multiplex Ligation-dependent Probe Amplification (MLPA) in genetic analysis.
MLPA is used to detect copy number variations in DNA sequences, making it valuable for identifying deletions or duplications in the genome.
Why are suitable nutrients and growth enhancers essential in tissue culture?
Nutrients and growth enhancers are crucial for cell survival, proliferation, and maintaining their normal functions in tissue culture. They provide the necessary components for cellular growth and division.
How is Quantitative Fluorescence PCR (QF-PCR) used in genetic testing?
QF-PCR is utilized to detect aneuploidies and copy number variations quickly and accurately. It is commonly used in prenatal and postnatal genetic testing for rapid results.
What type of DNA sequences are typically found in pericentromeric regions?
Highly repetitive DNA sequences, such as satellite DNA, transposons, and other repetitive elements
Where are pericentromeric regions located in relation to the chromosome structure?
Close to the centromere of a chromosome
What is the structural feature that pericentromeric regions are close to on a chromosome?
The constricted region that joins the two sister chromatids (centromere)
What is the main characteristic of pericentromeric regions in terms of DNA composition?
Highly repetitive DNA sequences
Explain the difference between reciprocal and non-reciprocal translocation in terms of chromosome segments exchange.
Reciprocal translocation involves exchange of segments between two chromosomes, while non-reciprocal translocation involves a segment moving to another chromosome without any material being received in return.
How does Fluorescence In Situ Hybridisation (FISH) contribute to genetic analysis?
FISH is used to visualize and map the genetic material in an individual's cells, aiding in the detection of chromosomal abnormalities.
How does Multiplex Ligation-dependent Probe Amplification (MLPA) contribute to genetic analysis?
MLPA is used to detect copy number variations in genes, helping in identifying genetic disorders and diseases.
Explain the importance of capturing cells in division during tissue culture.
Capturing cells in division allows for the analysis of chromosomes at a specific stage, aiding in genetic testing and research.
Explain how U-type exchange can lead to genetic disorders.
U-type exchange can lead to genetic disorders by causing a specific segment of DNA to invert in the opposite direction, disrupting the normal genetic sequence.
Describe the mechanism of U-type exchange on chromosomes.
U-type exchange occurs when two highly similar regions on the same chromosome incorrectly align and form a loop, leading to the inversion of a specific DNA segment.
What happens when two highly similar regions on the same chromosome undergo U-type exchange?
During U-type exchange, a loop is formed which causes the inversion of a specific segment of DNA on the chromosome.
How do chromosomal rearrangements occur in U-type exchange?
Chromosomal rearrangements occur in U-type exchange when a specific segment of DNA on the chromosome undergoes inversion due to the formation of a loop.
Study Notes
Cell Types and Tissue Culture
- Constitutional cells: obtained from normal tissues or passed down from parents to offspring (e.g., blood, skin biopsies, amniotic fluid)
- Acquired cells: undergone changes in genetic makeup due to environmental factors or disease (e.g., tumor biopsies, bone marrow samples, peripheral blood)
Tissue Culture Requirements
- Suitable culture conditions
- Suitable nutrients
- Maximise numbers of cells in division
- Determine optimum time to harvest
- Method of capturing cells in division
- Culture media (basal media and supplements)
- Fetal calf (or bovine) serum
- Amino acids (L-glutamine)
- Growth enhancers
- Antibiotics
- Antifungals
Harvesting Cells
- Collection of cultured cells to analyse chromosomes
- Cells washed to remove culture medium
- Cells subjected to hypotonic solution to swell cells
- Cells washed to remove excess cytoplasm
- Cell suspensions fixed and spread onto glass slides for analysis
Chromosome Banding
- Two principal groups of banding techniques: those resulting in bands along the length of the whole chromosome and those that stain specific bands or structures
- G-banding: generally preferred for routine staining of chromosomes
- G-banding pattern reflects structural and functional composition of the chromosome
- C-banding: used to investigate chromosome rearrangement near centromeres and polymorphism
Fluorescence In Situ Hybridisation (FISH)
- Uses fluorescent probes that bind to specific parts of the chromosome
- Types of FISH probes: repetitive sequence probes and unique sequence probes
- FISH with probe TUPLE1 used for detection of VCFS
Molecular Methodology
- QFPCR (quantitative fluorescent PCR): rapid prenatal diagnosis of chromosome copy number changes
- MLPA (multiplex ligation-dependent probe amplification): detection of DNA copy number
- CGH (comparative genomic hybridisation)
- Cytogenetic analysis (karyotype)
- Next generation sequencing (NGS)
Cytogenetic Techniques
- Nucleolar Organiser Region (NOR) Staining: selectively stains regions with actively transcribed genes using silver nitrate
- Telomere Banding: selectively stains terminal bands or telomeric regions using T-banding or FISH with terminal repeat or sub-telomeric repeats sequence probes### Cytogenetic Techniques
- Removal of red blood cells is required for blood and bone marrow cultures
- Cell suspensions are "fixed" to be spread onto glass slides and stained for analysis
- Two principal groups of banding techniques:
- Those resulting in bands along the length of the whole chromosome
- Those that stain specific bands or structures
Chromosome Banding
- G-banding:
- Generally preferred for routine staining of chromosomes
- Pattern reflects both structural and functional composition of the chromosome
- G-Dark bands:
- Replicate mid to late in S phase
- Condense early in mitosis
- Gene poor with tissue-specific genes, A-T rich DNA, and enriched in LINES
- G-Light bands:
- Replicate early in S phase
- Condense later in mitosis
- Gene rich with housekeeping genes, C-G rich DNA, and enriched in SINES
- C-banding:
- Used to investigate chromosome rearrangement near centromeres and polymorphism
- Darkly staining C-bands located at centromeres of chromosomes, Yq, 1q, 9q, and 16q
- Marked polymorphism present in the size of the C-bands, relatively stable, and inherited
- Replicates late in S phase, lacks active genes
- FISH (Fluorescence In Situ Hybridisation):
- Uses fluorescent probes that bind to specific parts of the chromosome
- Probes:
- Repetitive sequence probes (centromeric or alpha satellite DNA probes)
- Unique sequence probes (target regions not repeated in the genome)
- Microdeletions/duplications can be detected
- Sub-telomeres specific for each chromosome end
- Break-apart and fusion probes used in leukaemic investigations
Molecular Methodology
- FISH:
- Probes for specific parts of the genome from whole chromosomes to microdeletion sites
- QF-PCR (Quantitative Fluorescence PCR):
- Rapid prenatal diagnosis of chromosome copy number changes
- Detects chromosome 13, 18, 21, X, and Y copy number
- Used for pregnant women with increased risk of chromosome abnormality
- Preliminary QF-PCR result confirmed by karyotyping
- Advantages: rapid, robust, accurate, detects maternal cell contamination
- MLPA (Multiplex Ligation-dependent Probe Amplification):
- Technique for detection of DNA copy number
- Allows relative quantitation of up to 48 different nucleic acid sequences
- Commercial MLPA kits available for detection of over 135 different genetic diseases
- Used for detection of subtelomeric deletions/duplications
- Advantages: rapid, simple, reproducible, low cost per data point
- DNA Microarray:
- Whole genome view by chromosome microarray
- DNA fragment used as a probe to find complementary sequences in a target
- BAC and oligonucleotide probes used
- mRNA, DNA, cDNA used as targets
- High resolution: CNV detection of 20kb (depending on platform used and backbone spacing)
- Used for genome-wide screening of DNA copy number changes/variants
- Does not detect balanced rearrangements
- Routinely used for paediatric cases and prenatal analysis
Nucleolar Organiser Region (NOR) Staining
- NORs contain genes for 18S and 28S rRNA on chromosomes 13, 14, 15, 21, and 22
- Regions with actively transcribed genes can be selectively stained using silver nitrate
- Stain identifies a protein adjacent to the NOR rather than the NOR themselves
- Chromosomes from different individuals will stain more or less intensely in a consistent manner
- Specific staining pattern is a heritable characteristic
Telomere Banding
- Terminal bands or telomeric regions can be selectively stained by T-banding
- Specialised case of R-banding, in which a more destructive treatment results in diminished staining except at terminal bands
- Telomeres are more effectively visualised using FISH with terminal repeat or sub-telomeric repeats sequence probes
Constitutional and Acquired Cells
- Constitutional cells:
- Obtained from normal tissues or cells passed down from parents to offspring
- Examples: blood, skin biopsies, and amniotic fluid
- Acquired cells:
- Undergone changes in their genetic makeup due to factors such as environmental exposure or disease
- Examples: tumour biopsies, bone marrow samples, and peripheral blood
Tissue Culture Requirements
- Suitable culture conditions:
- Suitable culture media (basal media and supplements)
- Fetal calf (or bovine) serum
- Amino acids (L-glutamine)
- Growth enhancers
- Antibiotics
- Antifungals
- Maximise numbers of cells in division:
- The best time to harvest is when the cells are in their most active phase
- Bloods (for constitutional studies) are usually in culture for three days (72-hour cultures)
- Bone marrows and leukaemic bloods are harvested after 1 to 4 days
- Tissues grown on a substrate may take 7 – 30 days to produce enough cells
- Prenatal tissues will usually stop growing after ~50 divisions
Non-allelic Homologous Recombination (NAHR)
- DNA recombination process that occurs between non-identical but similar DNA sequences
- Results in genetic imbalances or syndromes due to changes in chromosome structure
U-type Exchange
- Process through which chromosomes can undergo rearrangement, leading to genetic disorders
- Two highly similar regions on the same chromosome incorrectly line up and form a loop
- Loop leads to the inversion of a specific segment of DNA, resulting in a genetic disorder
Learn about the differences between constitutional and acquired cells. Constitutional cells come from normal tissues or are passed down from parents, while acquired cells have undergone genetic changes due to factors like environmental exposure or disease. Explore examples like blood, skin biopsies, and tumour biopsies.
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