Lecture 6 - Cytogenetic Techniques

Cytogenetic techniques

  1. Why is conventional G-banding karyotyping (developed in the 1970’s) still the main diagnostic tool for chromosome abnormalities?

    • Easy detection of structural changes → where atypical chromosome events occurred

    • Detect balanced and unbalanced rearrangements

      • as well as more complicated events (Robertsonian, isochromosomes, ring chromosomes

  2. What are some advantages/disadvantages of FISH or SKY compared to G-banding?

    1. What kinds of investigations might FISH be useful for?

      • targets specific regions of interest - design probe to localize

      • disadvantage - only detect changes at specific regions you are testing for

    2. What kinds of investigations might SKY be useful for?

      • paint whole genome (all chromosomes diff colours)

      • complex rearrangements involving multiple chromosomes

        • see many simultaneous alterations in one genome

      • not gene specific

  3. What factors affect the resolution of chromosomal microarray (i.e. how small a change can be detected)?

    • resolution - how small a defect we can see

    • density of array probes in genome, total number of probes

      • if probes are 50kb apart in genome, can’t resolve anything smaller than this

      • some arrays have 44,000 probes vs 5 million, coverage varies between these two

  4. What is hybridized to what in FISH? In microarray?

    • FISH - fluorescent probe to specific genomic region based on sequence similarity

    • Microarray - patient DNA hybridizes to probes that represent regions of the human genome, then fluoresce

      • fluorescence is proportional to concentration

        • more fluorescence = duplication

        • less fluorescence = deletion

  5. What is the difference between an oligo and SNP microarray?

    • Oligo - comparative genomic hybridization

      • probes target patient DNA regardless of SNPs

      • how much DNA is present relative to balanced control sample hybridized with another colour

      • mix equal amounts of labelled DNA, if patient has more DNA in region than control, colour will be different than equal mix and deletion

    • SNP

      • two probes present for every location, one for each allele

      • DNA hybridized, SNP that matches patient fluoresces

        • total fluorescence across genome tells relative copy number

      • also tells genotypes info

        • only one allele fluoresces → homozygous

        • both alleles fluoresce → heterozygous

  6. What are some advantages and disadvantages of chromosomal microarray for diagnosing chromosome abnormalities as compared to G-banding?

    CMA

    G-banding

    advantages:
    - high resolution
    - detect parental origin of variant
    - detect loss of heterozygosity, UPD
    - do not need frozen cells
    - avoid culture failure
    - relatively rapid

    advantages:
    - detect balanced rearrangements
    - positional info
    - cheaper

    disadvantages:
    - cannot detect balanced rearrangements
    - no positional info

    disadvantages:
    - low resolution
    - requires culturing of cells
    - may result in higher burden of maternal contamination

  1. What are some examples of how molecular approaches (array or sequencing) and Gbanding can complement each other and be used together for a more complete understanding of a patient’s phenotype.

    • Detect a large duplication by microarray → tell by G-banding where the duplication occurs (chromosome of origin or elsewhere)

  2. How has diagnosis of microdeletion syndromes (such as Williams syndrome) changed over time and added to our knowledge?

    • G-banding - not reliable, deletions tend to be small

    • FISH - better correlation between clinical features and presence of deletion

    • Array - precise definition of breakpoint, exact determination of genes included in deletion, identification of sequences bordering breakpoints

  3. What is an advantage of long read sequencing over array-based methods or Gbanding?

    • single nucleotide resolution

    • detect smaller structural variants than array-based methods

    • detect parental origin of change, DNA methylation etc.