CFM 2: Molecular Diagnostics and Cytogenetics

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CFM 2: Molecular Diagnostics and Cytogenetics
2015-09-08 21:27:02
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  1. 4 cytogenetic methods discussed in class (in order of resolution)
    • Karyotyping
    • FISH
    • Array CGH
    • DNA sequencing
  2. 4 types of chromosomes based on centromere location
    • Telocentric: centromere on end
    • Acrocentric: p-arm is so short it is hard to see
    • Submetacentric: centromere not quite in the middle
    • Metacentric: centromere right in the middle
  3. What tissues can you analyze with karyotyping?
    Cells that proliferate in a culture (e.g. amniotic fluid, chorionic villi, umbilical blood, preimplantation blastomeres, blood, fibroblasts, hematologic or solid tumors)
  4. Pros and cons of karyotyping
    • Pros: good for looking at numerical abnormalities (trisomy, monosomy); large quantitative structural abnormalities (deletions, duplications, amplifications); large qualitative structural abnormalities (translocations, inversions, insertions)
    • Cons: Requires actively dividing cells, requires time to grow the cells, resolution varies with tissues, low resolution (best for changes >5Mb), contamination with normal cells
  5. FISH is good for?
    • Identification of complex or cryptic rearrangements
    • Identification of segmental aneusomies
    • Identification of marker or ring chromosome origin
    • Aneuploidy (extra or missing copies of single chromosomes)
  6. How does FISH work?
    • Take a cell
    • Denature dsDNA into ssDNA strands
    • Add fluorescence labeled ss probes to bind to complementary sequences on chromosomal region of interest
    • Re-anneal DNA to allow probe to bind
    • Wash off unbound probe
    • Evaluate by fluorescence microscope (nucleus counterstained with DAPI)
  7. Metaphase FISH vs. Interphase FISH
    • Metaphase FISH: FISH done on karyotyped cells, so you can see which chromosome the problem happens on; requires cell culture
    • Interphase FISH: cannot tell which chromosome the genetic change happens on
  8. How to check for trisomy 21 quicker than karyotyping?
    Use centromeric FISH probe specific to chromosome 21 centromere and see how many chromosome 21s you have
  9. Break-apart FISH probes
    • 2 probes for 2 different regions of a single gene close to each other
    • Normal - both signals are together and overlapping
    • Abnormal - the signals are apart
  10. Fusion FISH probes
    • 2 genes
    • 2 probes, 1 for each gene
    • Normal - both signals are apart
    • Abnormal - signals are fused/overlapping
  11. Disadvantages of FISH?
    • You have to know something about your target
    • Clinical suspicion is crucial
    • Limited number of colors
    • Limited resolution; cannot visualize small insertions/deletions because you need to find them by eye
  12. Method for finding microdeletions?
    subtelomeric FISH probes
  13. aCGH
    • Array competitive genomic hybridization
    • Patient DNA probed one color; control DNA probed another
    • Mix pt and control DNA in equal amounts
    • Add to an array with probes for specific regions of genome
    • If there is a gain in pt's DNA, it will be more the pt's color than the control's
    • If there is a loss in pt's DNA, it will be more control's color than pt's
  14. When to use aCGH?
    Looking for small DNA gains/losses that would not be detected by karyotype and we cannot target them with FISH (unknown target)
  15. Disadvantages of aCGH
    • Does not detect balanced translocations
    • Does not detect small insertions/deletions
    • Detection limit
    • Mosaicism (presence of two or more populations of cells with different genotypes in one individual who has developed from a single fertilized egg)
    • Chimerism (two or more genotypes occur from fusion of more than one fertilized zygote in early stages of embryonic development)
  16. Robertsonian translocation
    • Whole arm exchange in acrocentric chromosomes (most often non-homologous chromosomes) that leads to viable fetus
    • Long arms from acrocentric chromosomes fuse to form one chromosome with all long arms, and one with all short arms (which gets lost)
  17. What cytogenetic method allows you to look at all levels of resolution?
    NextGen sequencing