4 cytogenetic methods discussed in class (in order of resolution)
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
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)
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
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)
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)
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
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
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
Fusion FISH probes
2 probes, 1 for each gene
Normal - both signals are apart
Abnormal - signals are fused/overlapping
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
Method for finding microdeletions?
subtelomeric FISH probes
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
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)
Disadvantages of aCGH
Does not detect balanced translocations
Does not detect small insertions/deletions
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)
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)
What cytogenetic method allows you to look at all levels of resolution?