Cytogenetics Flashcards
Karotyping
numerical and large structural abberations
scans whole genome
requires mitotic cells
days to weeks
FISH
metaphase and interphase
targeted cytogenetics
structural abnormalities (aneusomies) < 5Mb, microdeletions, microduplications
cryptic rearrangements
requres knowledge of targets
Array CGH
scan whole genome for copy number changes and small rearrangements
DNA sequencing
now good for detailed views of known gene target, soon useful for detailed scan of whole genome
cytogenetic techniques
karotype
FISH
molecular genetic techniques
array
sequencing
DNA resolution
Karotype Procedure
suspension cultures: blood, bone marrow
monolayer cultures: amniotic fluid, chorionic villi, products of conception, solid tumors
need proliferating cells that live well in a dish
specimens are minced and disaggregated by treatment w collagenase
plated as monolayer cultures
growth
metaplase cells are harvested, swolloe, dropped on glass slides and burst
chromosomes are stained and banded using Giemsa and trypsin (GTG)
structrue of chromosome
dark band - heterochromatin: AT rich, tissues specific genes expressed
Euchromatin: GC-rich, housekeeping genes
acrocentric
“p” short arm is super short - it’s hard to see it but still present
satellite
submetacentric
arm length is unequal
metacentric
2 arms equal in length, balanced translocation = fusion of 2 acrocentric chromosomes
advantages of karotype
numerical abnormalities - too many too few
trisomy 21, 13, 18, Monosomy
large quantitative structural abnormalities (deletions, duplications, amplifications, big translocations)
large qualititave structural abnormalities (translocations, inversions, insertions)
disadvantages of karyotype
requires actively dividing cells
requires time (growth of cells)
resolution varies with tissues
contamination w normal cells (mother and chid, tumor and non tumor)
best for big changes over 5 Mb
usually cannot detect single gene rearrangements (tumors)
FISH procedure
- denature dsDNA into single strands
- add fluoro labled single strand probe (always DNA!) w sequence complentary to chromosomal region of interest
- re-anneal DNA to allow probe binding to target
- wash unbound proble
- evaluate by fluoro microscope - nucleus is counterstained
Metaphase vs Interphase FISH
Metaphase:
requires cell culture
chromosome morphology retained = hybridization signals can be localized
like classic karyotype - stopped them in metaphase,
use knowledge of what chromosome you are looking at - can see how many chromosomes there are (numerical abnormalities!) and molecular abnormalities that are too small to see on karyotye
very good for prenatal! can see number AND microdeletions
Interphase:
fixed tissues
chromosome morphology unavailable
signalys can’t be mapped reliably to chromosomes (FNA, single cells, formalin-fixed paraffin sections)
cells are out of M phase - did not proliferatue - previously fixed tissue, don’t see chromosomes, ony fish signals!
done on a lot of cancer tissues when asking specific questions - don’t really care about karyotype
Break-apart probe
gene specific FISH:
1 gene - 2 probes for 2 diff regions close to each other
normal: both signals together/overlapping
abnormal: signals apart
rearrangement w differnet partners forms fusion protein
Fusion probe
Gene specific FISH
2 genes - 2 probes - one for each gene
normal: both signals apart
abnormal: fused/overlapping
fusion protein is pathologic
disadvantages of FISH
have to know target!
need clinical suspicion
limited number of colors
limited resolution - can’t see small inertions/deletions (have to be able to detect w the eye - if probe is very small can’t see it
aCGH procedure
array + competitive + genomic + hybridization
slide w dots: tons of probes! chunks of DNA printed w areas of interest
pt DNA and controlled DNA - different colors - compete!
if 1:1 yellow - balance!
if more pt dna for region - gain!
if more control dna for a region - loss!
5-15 probes for everything so no mistakes
when aCGH is the right test
looking for small DNA gains/losses that would not be detected by karyotype and can’t target w FISH bc unknown target
developmental delay, autism, growth, recurrent pregnancy loss/unexplained infertility
first tear w many unexplained abnormalities
unbalanced changes!
high resolution, sensitivity, specificity
molecular karyotype
aCGH disadvantages
does not detect balanced translocations, small insertions, deletions
if only a few cells affected - may not see!
mosiacism - array will even out all dna
chimerism - fusion of more than one fertilized zygote in embryonic development
balanced rearrangement
no DNA loss (almost) or gain
array: balanced signal - same amt DNA
reciprocal, inversions
unbalanced rearrangements
DNA loss or gain
Array shows
unbalanced translocations
deletions
duplications
amplifications
Robertsonian Transloaction
whole arm exchange of acrocentric chromosomes - most involve non homologous (13 and 14)
in short arm recombination - bicentric chromosome w 2 long arms and most of short arm material lost
1 centromere is inactive
chromosome is stable
balanced carriers = parent - 45 chromosome
2/3 child is effected w monosomy/trisomy
when sequencing?
mutations, small insertions/deletions
clinical testing:
targeted therapy for tumors
constitutional defects: hearing loss, cardiomyopathy, syndromes (risk for next preg>
genotyping - hla testing paternity forensics
pharmacogenetics - drug metabolism
