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Chromosomes Exam 2 > 09/26 - 10/01 (Svetlana's lectures) > Flashcards

09/26 - 10/01 (Svetlana's lectures) Flashcards

1
Q

FISH represents a cytochemical technique which allows what?

A

Which allows the visualization of single nucleic acid sequences in chromosomes in the fluorescence microscope.

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2
Q

What does FISH stand for?

A

Fluorescence in situ Hybridization (FISH)

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3
Q

Principal of FISH (steps of INDIRECT LABELING)

A

1a) Double Stranded chromosome DNA
1b) Probe for region to be investigated
2) Probe labeling with biotin
3) Denaturation
4) HYBRIDIZATION
5) Primary antibody with fluorochrome
6) Secondary antibody with biotin
7) Amplification of signal by attachment of further primary antibody

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4
Q

Principal of FISH (Steps of DIRECT LABELING)

A

1a) Double stranded chromosome DNA
1b) Probe for region to be investigated
2) Probe labeling with fluorescent dUTP
3) Denaturation
4) HYBRIDIZATION
5) VISUALISATION

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5
Q

Molecular probes (list them)

A
  • BAC (Bacterial artificial chromosomes) (100-200 kb)
  • PAC (P1 phage artificial chromosomes) (150-300 kb)
  • YAC (yeast artificial chromosomes) (300-1000 kb)
  • Cosmids (30-100 kb)
  • Fosmids (35-45 kb)
  • PCR products (1-30 kb)
    (Sure FISH) (set of 200 bp)
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6
Q

What are SureFISH oligonucleotide probes able to do that BAC probe FISH are not?

A
  • SureFISH has the ability to target unique, non-repetitive sequences, where BAC is not able to do this.
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7
Q

Specific kinds of molecular probes (by region)

A
  1. Whole chromosome paint (wcp)
  2. Locus-specific (unique sequence) probe
  3. Repeat sequence probes (telomeric or centromeric)
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8
Q

What are the advantages and disadvantages of whole chromosome paints?

A

Advantages:

  • Detect translocations and derivative segments >5Mb
  • Detect complex rearrangements

Disadvantages:

  • Cannot detect inversions or duplications
  • Cannot detect segments less than 5 Mb
  • Cannot be used in interphase analysis
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9
Q

Locus-Specific Probes

A
  • Disease locus - gene specific

- Subtelomere - specific

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10
Q

What are the advantages and disadvantages of locus-specific probes?

A

Advantages:

  • Rapid and easy
  • Detect microdeletions and duplications >150 Kb (or size of probe)
  • Can use multiple probes at the same time
  • Can be used for interphase analysis

Disadvantages:

  • Need to know the locus of interest
  • Limitation to the number of probes used
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11
Q

Describe the telomere structure

A

Chromosome “Cap” (telemore repeats TTAGGG)

Distal Telomere Associated Repeats (TAR) shared by many chromosomes (distal subtelomeric sequence)

Proximal TARs - shared by fewer chromosomes (Proximal subtelomeric sequence)

Location of Subtelomere FISH probes (Chromosome unique sequence)

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12
Q

Subtelomeres

A
  • Sub-microscopic (cryptic) telomeric rearrangements cannot be seen by conventional chromosomes banding
  • Cryptic rearrangements have been implicated in up to 6% of unexplained mental retardation.
  • Method exists for assaying all 41 unique telomeres (no p arm for 13, 14, 15, 21, and 22; Xp same as Yp) simultaneously on one slide.
  • Highest concentration of genes of any chormosomal region - therefore submicroscopic deletions and duplications would have significant impact
  • Increased genetic recombination at telomeres - with male rate being higher than females for most chromosomes.
  • Telomeres play a critical role in chromosomes pairing at meiosis
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13
Q

Subtelomere Region-specific Probes

A

p-arm probes fluoresce green

q-arm probes fluoresce red

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14
Q

Locus-specific probes

A
  • Deletion
  • Duplication
  • Amplification
  • Translocation
  • Gene break/rearrangement
  • Gene fusion
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15
Q

Advantages and Disadvantages of Repetitive Sequence Probes

A

Advantages:

  • Rapid
  • Easy to analyze
  • Useful in interphase analysis

Disadvantages:

  • Identify only chromosomes detected by probes
  • Cannot distinguish whole chromosomes aneuploid from marker chromosomes
  • Cannot distinguish trisomy from triploidy.
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16
Q

Clinical indications for Rapid Prenatal Diagnosis by FISH

A
  • Rapid FISH is done when there is a high-index of suspicion of a chromosome disorder and/or pregnancy is at an advanced stage (20 weeks)
  • Abnormal ultrasound scan
  • Advanced maternal age
  • Biochemical indication of possible aneuploid fetus
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17
Q

Functions of the telomeres

A
  • Protect chromosomes from degradation
  • Prevent end-to-end fusion
  • Facilitate the interaction between the chromosome ends and the nuclear envelope
  • Assist in chromosome pairing, recombination, and proper segregation
  • Control the cell aging
18
Q

Comparative Genomic Hybridization (CGH)

A

Control genomic DNA (known normal karyotype) + Test genomic DNA (unknown karyotype) and put mix through normal metaphase

19
Q

Alterations in DNA Copy Number

A
  • Size: single gene - whole chromosome
  • Abnormality; deletion - amplification
  • Some variations among normal individuals
  • Can cause defects in human development
  • Contributions to cancer
  • Can effect function and gene expression
20
Q

Types of arrays for copy number studies:

A

1) Clone arrays
2) Whole genome oligonucleotide arrays
3) Oligo-based BAC emulation arrays
4) High resolution targeted arrays

21
Q

Chromosomal aberrations detected by aCGH

A
  • Deletions
  • Duplications
  • Insertional translocations
  • Mosaicism (whole chromosome trisomy or segment)
  • Complex rearrangements
22
Q

Array CGH advantages and disadvantages

A

Advantages:

  • Combined routine telomere assay and analysis of all disease-specific regions in a single test
  • Duplications and deletions can be detected simultaneously

Disadvantage:
- Does not detect balanced translocations, inversions, or low level mosaicism

23
Q

Genomic Resolution

A
  • Karyotype: 5-10 Mb
  • FISH (40-250 Kb per clone, single site)
  • CMA (average resolution ~30 kb, whole genome)
24
Q

Contiguous Gene Deletion Syndromes

A
  • Common cause of MR and developmental defects
  • Syndromes are usually sporadic (rare familial cases known)
  • Involves multiple, functionally unrelated genes, each independently contributing to the phenotype (often single gene, transcription factor)
  • Features of the syndrome may occur as individual Mendelian traits
25
Q

Contiguous Gene Deletion/Duplication Syndromes

A 
- Same as contiguous gene deletion syndromes list with addition of:
Molecular cytogenetics (FISH) required for detection (interphase FISH for microduplication)
26
Q

Submicroscopic chromosome rearrangements

A
  • microdeletions & microduplications
  • Contiguous gene deletion syndromes
  • Interstitial
  • Terminal
  • Monogenic genomic syndromes
27
Q

Microarray Types

A
  • Array-based Comparative Genomic Hybridization (aCGH) -
    DNA COPY NUMBER
  • SNP-based microarray - DNA COPY NUMBER, COPY NEUTRAL DNA ALTERATIONS (UPD,LOH,AOH)
  • Combined CGH + SNP
28
Q

Microarray Resolution

A
  • Total number of probes on microarray (44K; 105K; 180K; 244K; 400K; 1 Mill)
  • Spacing between probes (7-50 kb)
  • Minimal number of probes incorporated into software algorithm (5-50 probes)
  • Coverage of the particular region of genome
29
Q

Clinical microarray results interpretation

A
  • Number of probes: minimum 5 consecutive
  • Threshold: deletion (-0.5 non-mosaic); duplication (+0.3)
  • CNV Size: depends from array resolution
  • Coordinates: hg18, hg19
30
Q

Alterations in DNA Copy Number

A
  • Haploinsufficiency/overexpression of dosage sensitive genes
  • Unmask recessive allele - remaining copy has a mutation
  • Remove/rearrange regulatory gene elements
  • Create fusion gene
31
Q

Protein levels and molecular changes for cancer development

A
  • No protein - deletions, mutations
  • Too much protein - amplifications, mutations
  • Shortened protein - intragenic deletions, mutations
  • Abnormal protein - gene fusions, mutations
32
Q

Array CGH in cancer diagnosis

A
  • Significant number of clinically relevant genomic alterations is missed by currently available cytogenetics techniques
  • Apparently balanced rearrangements are frequently unbalanced at the molecular level
  • Additional genomic imbalanced are present in about 50% of cases
  • Does not require cell culture
  • Array CGH unravel novel recurrent genomic alterations
33
Q

Array CGH in cancer can detect

A
  • Heterozygous deletion - single copy loss (1 copy number)
  • Homozygous deletion - loss of 2 copies of DNA (0 copy number)
  • Heterozygous duplication - gain of 1 copy (3 copy number)
  • Homozygous duplication - gain of 2 copies (4 copy number)
  • Mosaicism (calculation of copy number is difficult)
34
Q

Advantages of microarray in cancer diagnosis

A
  • Genetic classification
  • Assessment of prognosis and severity
  • Monitoring the course of the disease and response to the therapy
  • Screening of populations with increased cancer risk (cancer predisposition syndromes)
35
Q

aCGH

A
  • aCGH: high resolution genome analysis technique to detect DNA copy number alterations (deletion, duplication)
  • aCGH: detects CNV in 15-20% of patients with MCA and intellectual disabilities
  • Apparently balanced rearrangements are frequently unbalanced at the molecular level
  • Array CGH unravel novel recurrent genomic alterations
  • Genomic imbalances are frequent findings in cancer samples
  • Gene dosage alteration is a common mechanism of genetic disease
36
Q

Limitations of microarray in cancer diagnosis

A
  • Tumor heterogeneity
  • Low level of abnormal cells (10% mosaicism detectable)
  • Copy number calculation is not always possible
  • Complexity of molecular changes in cancer cells
  • Limitations in distinguishing constitutional vs. acquired CNV
37
Q

SNPs in the human genome

A
  • a SNP is a variation at a single site in DNA< is the most frequent type of variation in the genome
  • Human genome contains more than 5 million common SNPs with minor allele frequencies (MAF) > 10%
  • SNP array is a type of DNA microarray which is used to detect polymorphisms within a population, used for association studies
  • Haplotype - a sequence of consecutive allels on a particular chromosome
  • HapMap - haplotype map describes the common patterns of human genetic variation
38
Q

SNP arrays

A
  • DNA copy number changes
  • Uniparental disomy (UPD)
  • Consanguinity
  • Identity by descent (IBD)
  • Absence of heterozygosity (AOH)
  • Loss of heterozygosity (LOH) in cancer cells
39
Q

Indications for SNP array

A
  • Suspected UPD/conditions associated with imprinting
  • Autosomal recessive condition due to (suspected common ancestry, consanguineous family)
  • Determine parental origin of the chromosomal abnormality by trio analysis
40
Q

SNP array Advantages and disadvantages

A
  • Advantages: detects DNA copy variations and detects DNA copy neutral alterations
  • Disadvantages: No flexibility in probe SNP coverage in genome, lower sensitivity for mosaicism detection, more laborious

Chromosomes Exam 2 (11 decks)

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