differential molecular screening
lelyc at u.washington.edu
Sun Oct 1 23:14:12 EST 1995
I have been doing DD for the past year or so, with some success. I am
now in the process of screening bands of interest, and I am coming up
with some interesting results.
The main criticism of the method is reproducibility. ie. If you do the
differential display in 3 different experiments, you will get a different
banding pattern. This could be due to differences due to slight
variability between experiments, or just to the inherent problems of
using PCR in a quantitative way. I have just isolated bands from *one*
differential display experiment, and then screen the bands on dot blots
after reamplifying. It turns out that many of the bands do *not*
correspond to the DD, but some do, and this is reproducible on more than
one dot blot.
Therefore, if you use DD, make you you have an effective way of screening
your bands, and don't give because of low reproducibility in the early
stages of the screen.
In terms of sensitivity for low abundancy genes, I can't say very much
except for the fact that there is a possibility that I may have found a
putative transcription factor.
I have never tried subtractive hybridization, but I can say that DD *is*
technically easy...I have so far cloned over 30 bands in the past year.
However, I will say that others have had problems reamplifying, which is
the hardest part of the whole technique. Here, I would recommend playing
around with Mg conc.
Good luck! Feel free to ask further questions if you'd like. Looks like
we are just around the corner from each other.
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