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Chromosomes 2nd Exam > Cytogenetic Methods - Lectures 1 and 2 > Flashcards

Cytogenetic Methods - Lectures 1 and 2 Flashcards

1
Q

FISH

A

Fluorescence in situ hybridization

  • allows for visualization of single nucleic acid sequences in chromosomes with a fluorescent microscope
  • cells are stopped at metaphase
  • must apply a probe for region of interest
  • can identify origin of marker chromosomes
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2
Q

FISH - Indirect labeling

A
  1. Take double stranded DNA and probe for region of interest
    - probe DNA must be complimentary to patient DNA
  2. Label probe with intermediate binding protein biotin
  3. Denature DNA
  4. Hybridize probe and DNA
  5. Primary antibody binding with fluorochrome
  6. Secondary antibody binding with biotin
  7. Signal amplification by further antibody attachment
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3
Q

FISH - Direct labeling

A
  1. Take double stranded DNA and probe for region of interest
  2. Label probe with fluorescent dUTP
  3. Denature DNA
  4. Hybridize probe and DNA
  5. Visualize
    Key difference - no intermediate binding of biotin
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4
Q

Types of molecular probes

A

BAC - Bacterial artificial chromosomes (100-200kb)
YAC - yeast artificial chromosomes
PAC - P1 phage artificial chromosomes (150-300kb)
Cosmids
Fosmids
PCR products - generates tiled labeled probes

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5
Q

Whole Chromosome Paints (hybridizes along entire chromosome)- Advantages

A

Can detect translocations and derivative segments >5Mb

Can detect complex rearrangements

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6
Q

Whole Chromosome Paints (hybridizes along entire chromosome) - Disadvantages

A

Cannot detect inversions or duplications
Cannot detect segments <5Mb
Cannot be used in interphase analysis

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7
Q

Locus-specific probes (hybridizes to a unique sequence) - Advantages

A

Rapid and easy
Detects microdeletions and microduplications >150kb
Can use multiple probes at the same time
Can be used for interphase analysis

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8
Q

Locus-specific probes (hybridizes to a unique sequence) - disadvantages

A

Need to know the locus of interest

Limit to number of probes you can use

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9
Q

Subtelomeric regions

A
  • Located before the telomere cap (TTAGGG) - distal subtelomeric is closest to cap, proximal subtelomeric region is farthest from cap
  • sub-microscopic (cryptic) telomere sequences cannot be detected through traditional banding, but rearrangements in these areas contribute to intellectual disability
  • have highest concentration of genes of any chromosomal area
  • have increased recombination
  • play critical role in pairing at meiosis
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10
Q

Repetitive Sequence Probes (includes subtelomeric regions) - Advantages

A

Rapid
Easy to analyze
Useful in interphase analysis

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11
Q

Repetitive Sequence Probe (includes subtelomeric regions) - Disadvantages

A

Can only identify chromosomes detected by probes
Cannot distinguish whole chromosome aneuploidy from marker chromosomes
Cannot distinguish trisomy from triploidy

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12
Q

Comparative genomic hybridization (CGH)

A

Mixes control (known normal karyotype) and patient (unknown karyotype) DNA
Label patient with Cy3 and control with Cy5
-Ratio of Cy3/Cy5 > 1 - duplication
-Ratio of Cy3/Cy5 < 1 - deletion

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13
Q

Array CGH (aCGH)

A

hybridize DNA to genomic clone microarray

  • analyze fluorescence ratio of patient to control
  • detects deletions, duplications, copy number variants, insertional translocations, mosaicism, and complex rearrangements
  • laser scanner can detect fluorescence
  • sex-matched controls provide greater sensitivity
  • red/green intensity shows deletions or duplications
  • yellow means areas are the same (pt karyotype normal)
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14
Q

aCGH Advantages

A

Combines routine telomere assay and analysis of all disease-specific regions into a single test
Deletions and duplications can be detected simultaneously
Detects CNV in 15-20% of patients with multiple congenital anomalies or intellectual disability

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15
Q

aCGH Disadvantages

A

Does not detect balanced translocations, inversions, and low-level mosaicism

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16
Q

Continguous Gene Deletion Syndrome

A

Common cause of intellectual disability
Syndromes usually sporadic
Involves multiple, functionally unrelated genes
Features of the syndrome may appear as individual mendelian traits
FISH is required for detection

17
Q

Types of Microarray

A

aCGH- array-based comparative genomic hybridization
SNP array - DNA copy number, copy neutral alterations
Combined CGH and SNP

18
Q

Microarray probes

A

BACS (100-200kb)
Fosmids (30-45 kb)
Oligonucleotides (35-60bp)
SNP probes

19
Q

Microarray resolution

A

Total number of probes - 44k (lowest possible), 105k, 180k, 244k, 400k, 1M
Spacing between probes - 7-50kb

20
Q

Alterations in DNA Copy Number - effects

A

Haploinsufficiency / Overexpression of dosage sensitive genes
Unmasked recessive alleles
Remove/rearrange gene regulatory elements
Create fusion gene

21
Q

Microarrays in cancer diagnosis

A

No protein –> Deletions, mutations
Too much protein –> Amplification, mutations
Shortened protein –> Intragenic deletions, mutations
Altered protein –> Fusion genes, mutations

22
Q

aCGH in cancer

A

Detects heterozygous deletions and duplications
Detects homozygous deletions and duplications
Mosaicism

23
Q

Additional info

A

Apparently balanced rearrangements can be unbalanced at the molecular level
Additional genomic imbalances are present in ~50% of apparently normal cases

24
Q

Microarray in cancer - Advantages

A

Genetic classification of disease
Assessment of prognosis and severity
Monitoring of disease and response to treatment
Screening of populations with increased cancer risk
Parental studies (if present in unaffected parent, probably benign in child)

25
Q

Microarray in cancer - Limitations

A

Tumor heterogeneity
Miss low-level mosaicism - only 10-20% mosaicism or higher is detectable
Copy number calculation not always possible
Complexity of molecular changes in cells
Limitations in distinguishing acquired vs inherited copy number variants

26
Q

SNP Array in Clinical Diagnosis

A

Detects polymorphisms within a population, used for association studies
Genotype indentification
If perfect match to probe, hybridized dye detected on probe

27
Q

Uniparental Disomy

A

Parental UPD6 - Transient prenatal diabetes

Maternal UPD15 - Prader-Willi Syndrome

28
Q

Indications for SNP Array

A

Suspected UPD/imprinting disorder
Autosomal recessive disease due to consanguinity
Determine parental origin by trio analysis

29
Q

SNP Array - Advantages

A

Detects copy number variants

Detects copy neutral aberrations

30
Q

SNP Array - Disadvantages

A

No flexibility in probe SNP coverage in genome
More laborious
Lower sensitivity of mosaicism detection

Chromosomes 2nd Exam (10 decks)

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