Overcoming Sequencing Secondary Structure

Seth Findley findley at u.washington.edu
Fri Jun 10 16:48:25 EST 1994


Here's a technical tip for those of you trying to overcome secondary 
structure problems on sequencing gels. In particular, compression and 
abberent migration of bands:

USB sells c7dGTP mixes in the kit, but a much more effective mix replaces 
*both* dGTP *and* dATP with their c7 (7-deaza) analogs. You will need to 
replace the dGTP on a per-mole basis with c7dGTP; and also replace 
the dATP with c7dATP on a per-mole basis. You will need to do this in both 
the Labelling and Termination mixes. You will also only be able to use a 
"c-minus" labelling mix (requiring hot dCTP).

You will also need to reduce the ddGTP and ddATP concentraions by one-half.

It is sometimes necessary to follow the Termination reactions with the 
Terminal Transferase/dNTP  chaseto get rid of polymerase stops (bands 
across all four lanes).

The above reagents do an incredibly good job at decompression. I was able 
to pull twelve well-spaced bands out of a secondary structure gob that 
looked like it had maybe 5 bands in it. Aberrant spacing is a pretty good 
clue that there are more bands than you might guess.


Really, this stuff works great!




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