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Flashcards in 09/26 - 10/01 (Svetlana's lectures) Deck (40)
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1

FISH represents a cytochemical technique which allows what?

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

2

What does FISH stand for?

Fluorescence in situ Hybridization (FISH)

3

Principal of FISH (steps of INDIRECT LABELING)

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

4

Principal of FISH (Steps of DIRECT LABELING)

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

5

Molecular probes (list them)

- 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)

6

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

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

7

Specific kinds of molecular probes (by region)

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

8

What are the advantages and disadvantages of whole chromosome paints?

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

9

Locus-Specific Probes

- Disease locus - gene specific
- Subtelomere - specific

10

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

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

11

Describe the telomere structure

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)

12

Subtelomeres

- 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

13

Subtelomere Region-specific Probes

p-arm probes fluoresce green
q-arm probes fluoresce red

14

Locus-specific probes

- Deletion
- Duplication
- Amplification
- Translocation
- Gene break/rearrangement
- Gene fusion

15

Advantages and Disadvantages of Repetitive Sequence Probes

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.

16

Clinical indications for Rapid Prenatal Diagnosis by FISH

- 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

17

Functions of the telomeres

- 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

Comparative Genomic Hybridization (CGH)

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

19

Alterations in DNA Copy Number

- 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

Types of arrays for copy number studies:

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

21

Chromosomal aberrations detected by aCGH

- Deletions
- Duplications
- Insertional translocations
- Mosaicism (whole chromosome trisomy or segment)
- Complex rearrangements

22

Array CGH advantages and disadvantages

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

Genomic Resolution

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

24

Contiguous Gene Deletion Syndromes

- 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

Contiguous Gene Deletion/Duplication Syndromes

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

26

Submicroscopic chromosome rearrangements

- microdeletions & microduplications
- Contiguous gene deletion syndromes
- Interstitial
- Terminal
- Monogenic genomic syndromes

27

Microarray Types

- 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

Microarray Resolution

- 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

Clinical microarray results interpretation

- 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

Alterations in DNA Copy Number

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