Lecture 6 - Cytogenetic Techniques

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

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

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

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

• 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

What is hybridized in FISH and 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 fluoresces (fluorescence proportional to concentration of sequence)

What is Oligo microarray

comparative genomic hybridization which probes target patient DNA regardless of SNPs

mix equal amounts of labelled DNA, if patient has more/less DNA in region than control, colour will be different than equal mix

What is SNP microarray

Two probes present for each location, one for each allele
DNA hybridizes and SNP that matches patient fluoresces, total fluorescence across genome tells relative copy number and genotype info
- only one allele = homozygous
- both alleles fluoresce = heterozygous

How can molecular approaches and Gbanding complement each other for a more complete understanding of a phenotype

Detect large duplication by mirroarray

G-banding indicates where duplication occurs

Advantages of long read sequencing

Single nucleotide resolution, detect smaller structural variants, detect parental origin of change, DNA methylation etc.

How have methods of diagnosis 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

Advantages and disadvantages of chromosomal microarray (CMA)

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

Disadvantages:
- cannot detect balanced chromosomal rearrangements
- no positional info

Advantages and disadvantages of G-banding

Advantages:
- detect balanced rearrangements
- positional info
- cheaper

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