FISH Flashcards
What is FISH?
Fluorescent probes bind to only those parts of a nucleic acid sequence with a high degree of sequence complementarity
Fluorescence microscopi is then used to find out where to probe is bound to the chromosomes
Purpose of FISH?
Used to identify specific DNA sequences to identify chromosomal abnormalities
What is a probe?
A single strand of DNA or RNA that is complementary to a nucleotide sequence of interest (i.e. attracts similar chromosomes)
What are the 3 types of FISH probes?
Type 1 - Repetitive sequences including those at the centromere of a chromosome
Type 2 - DNA segments that will bind to and cover the entire length of a chromosome
Type 3 - DNA segments from specific genes or regions on a chromosome that have been previously mapped or identified
Process of FISH?
- Make a probe complementary to a known sequence labelled with fluorescent marker
- Denature known chromosome sequence
- Denature probe - allow to hybridise to complementary sequence
- Wash off excess probe
- Observe chromosomes under fluorescent microsome
What happens if micro deletion is present?
Won’t be DNA present during DNA denaturing so specific tagged probe won’t be able to recognised complementary DNA and bind
What type of cells can FISH be performed on?
Interphase (non-dividing) and metaphase (dividing) cells
What is comparative genome hybridisation (CGH) used for?
Reveals loss or gain of chromosomal regions in test samples compared to normal controls - recall any major disruptions in the duplication process
What is cytogenetic localisation?
FISH produces fluorescent red signal at site of specific DNA sequence
Several probes, each corresponding to a defined genomic segment, can be simultaneously analysed and ordered with respect to each other using multicoloured FISH
What is chromosome painting / spectral karyotyping (SKY)?
An image of coloured chromosomes - SKY and multicolour FISH (m-FISH) paint each chromosome in one of 24 colours
