Molecular cytogenetics

FISH (fluorescent in situ hybridization)

• detection of target DNA sequences directly on chromosome preparation (in situ) using specific fluorescently labeled DNA probe, the probe is hybridized with complementary segments of the chromosomal DNA
• different modifications according sequences of interest:
  • centromeric probes – rapid counting of particular chromosome number (e.g. sex chromosomes, anuploidies)
  • locus-specific probes – microdeletion syndromes, subtelomeric regions...
  • whole-chromosome (painting) probes – translocations, insertions...
  • M-FISH (multicolor) - all chromosomes labeled by different combination of five fluorochromes to differ from each other, detection of complex rearrangements (e.g. in leukemia cells)
  • M-banding – multicolor combinations of fluorochromes along the chromosome enable to detect breakpoints in specific chromosome rearrangements; method is used especially in haematooncologic patients to help with assessment of prognosis and individual therapy indication according to their tumor subtype

CGH (comparative genomic hybridization)

• detection of quantitative - unbalanced genomic changes (gain or loss), not able to detect balanced rearrangements
• comparison of tested and control DNA (labeled with different fluorochromes, applied as probes on normal chromosomal preparation) used in ratio 1:1
• primarily developed for analysis of solid tumors

Microarrays

• molecular cytogenetic method with much higher resolution level (10-100 kb) then routine cytogenetic methods (karyotyping, 5-10 Mb)
• whole-genome analysis
• main disadvantage – method is targeted only on unbalanced changes, not able to detect balanced rearrangements
• great for detection of submicroscopic microdeletions or microduplications in patients with unexplained mental retardation and/or developmental delay
• reaction is performed on special slides („chips“) with small target region of thousands of pits with short specific chromosomal fragment in each; after reaction the slide is scanned and result from every pit is demonstrated on the chromosome map with the precise location
• data are compared with international databases and genotype-phenotype correlation with prognosis assessment should be commented in clinical report
• two basic modifications: array-CGH x SNP-array
  • Array-CGH: based on CGH method (above) but with higher resolution
  • SNP-array: based on detection of single nucleotide polymorphisms (SNPs) in the genome