During DNA FISH, you are labeling a specific sequence of DNA with a fluorescently labeled complementary strand of DNA.
To do this, you have to denature the DNA and then introduce your probe for overnight hybridization.
What, thermodynamically, is happening? Is the fluorescent probe competing with the other (naturally complementary strand) of DNA?
If the probe hybridization is effective, what happens with the “left over” (natural) complementary strand of DNA? is it just hanging out elsewhere or is there some sort of triple stranded DNA forming?
Hope this makes sense. Any papers on the thermodynamics/theory of FISH are appreciated. I am currently searching myself and will update this thread if I find any that help me answer my question.
Thank you in advance.
Hi Ian,
The time needed for half of the probe to be hybridized is called Cot1/2 or Cot half. Cot1/2 is related to the inverse of the molar concentration. Probes are usually much shorter than the genome. Molar concentration of probe is several order of magnitude higher than the genome. Accordingly, the genome DNA stay denatured when probe is hybridized.
Thank you Akihiko Yamagishi
So will the "final product" (after hybridization at 37C and post-hybridization washes) look like how I have drawn here? (see attached drawing, rough drawing, of course, but appreciate any insight)
Where the genomic "AGA" sequence is just 'free-floating' and the fluorescent probe (AGA) has now hybridized with the genomic "TCT", thanks to better kinetics due to being smaller and in excess concentration compared to the genomic AGA single strand?
Or will it look like something different?
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