Peattie DEPC sequencing and sequencing gels

[Erich Chapman]

Hi,

For about a month now I've been trying to sequence a 40mer RNA using direct chemical modification as described by Peattie.  I am trying for the A specific reaction in which DEPC (diethyl pyrocarbonate) is used to first modify the N7 of A>G and subsequent aniline induced cleavage.  I realize that RNAse U2 cleaves a A residues but I need the chemical method to comment on the N7 of the A's and as far as I can tell there's only 1 RNAse U2 supplier (pierce).

When I run the sequencing gels I see specific cleavage occuring at A's, however the bands containing DEPC reactions migrate slower and cause -OH ladders and a T1 ladder to merge into them.  I had thought that electric fields, salt concentrations, dye concentration had caused this merging but am now convinced it is the influence of the carrier tRNA added to the DEPC reactions.  I can't find documentation of this anywhere but my lab notices that a sample with significantly more RNA can cause neighboring lanes to distort or simply cause the highly loaded lane to move slower.  I was wondering if anyone can confirm this.

Also if anyone had a modernized procedure for Peattie A specific sequencing it'd be much appreciated.

One more thing too... which I phosphorimage the resulting gels I see the expected smiling of the bands but the smiling extends into lanes where I didn't load sample, as if the radioactive RNA has move laterally in the gel.  I am curious if this could be due to the water added around the edges of the gel in order to pull it onto Whatman paper or if the phenomenon has another basis.

I'd greatly appreciate any advice.

Erich Chapman
Ph.D. Student
University of Oregon