Sequencing mRNA from variable regions

Patrick HJ Falckh p.falckh at rmit.edu.au
Tue Jan 9 20:05:10 EST 1996


Peggy,

   More of a comment than any real "insights" so I don't know if this 
will help but here it is anyway.
   You haven't mentioned how big the product is, or how much sequence 
you can obtain. One approach would be to use the "single" band product 
as a probe on the mRNA from both patients who display the single band 
and those that are "normal".  This would indicate whether it is unique 
enough to the single band patients or not.
   The faint background ladder can come from too much target in your 
sequencing reaction.
   There is always competition in the PCR reaction and the target with 
the higher expression will tend to "over-shadow" lower expressed 
sequences, so it may be that the "single-band" patients have a target 
sequence that is differentially expressed.  Whether this is a "real" 
product related to the receptor directly or a protein that may interact 
with the variable region is what I would consider to be interesting.

   Good Luck

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