FLUORESECENT IN SITU HYBRIDISATION (FISH)

The reporter fluorescent molecule is tagged on the cDNA molecule by using the Nick translation technique developed by Rigby and Paul Berg in 1977. In this method, the enzyme DNase I cuts DNA and DNA polymerase I makes DNA. These two are combined in a reaction along with dNTPs with the dUTP labeled with a red or green fluorescence. The DNA pol I adds dNMP’s to the 3’OH terminus in the nick that has already been created by DNases thereby incorporating the DNA with fluorescent nucleotides. This technique known as Fluorescence in situ Hybridization (FISH) allows knowing the status in the interphase unlike in karyotyping where you need a metaphase chromosome. The application of FISH technique was illustrated by taking the example of chronic mylogenous leukemia (CML) in which the karyotype analysis revealed that there was a   9-22 translocation in the chromosome (Philadelphia chromosome)