help with DNAse footprinting...

Richard Converse Richard.Converse at UC.EDU
Thu Apr 23 22:11:46 EST 1998


Hi everyone!

    I am looking for help with DNAse footprinting. We are trying to run
footprinting experiments on a tissue specific promoter we are interested
in, and we are having little luck--the main problem has to do with the
starting material:

   We have tried end-labelling and recessed-end fill ins to label our
DNA fragment for analysis--the problem is that when we run this product
on a sequencing gel we see at least 2 major products separated by about
100 bp. Our product is 400bp, and we have tried to label a single strand
using several strategies (which I'll describe next). What I want to find
out, is whether or not these products are the 2 strands of the starter
DNA, or whether we have some other problem. Our different labelling
strategies have been as follows:

1. end-label one of the 2 PCR primers with PNK, and heat inactivate the
PNK @ 95degrees C for 1 hour--use the labelled primer in a PCR reaction
to generate an asymmetrically labelled product.

2. run PCR on the templated DNA and end-label both ends of the final
product with PNK followed by heat inactivation of the PNK @ 95degrees C
for 1 hour. Follow this with dialysis, and cut one of the 2 ends of this
product with 1 of 2 unique enzyme sites which we engineered into each of
the primers (one primer contains a NotI site, and one contains an AscI
site). Use this product in footprinting analysis.

3. run PCR on the templated DNA and end-label both ends of the final
product with PNK followed by heat inactivation of the PNK @ 95degrees C
for 1 hour. Follow this with dialysis, and cut one of the 2 ends of this
product with 1 of 2 unique enzyme sites which we engineered into each of
the primers (one primer contains a NotI site, and one contains an AscI
site). Agarose gel-Purify this product and use it for analysis.

4. Cut the product out of a plasmid clone which we have, end-label both
ends of this product with PNK followed by phenol extraction of the PNK,
and cut one of the 2 ends of this product with 1 of 2 unique enzyme
sites  which occur in the fragment at one of the 2 ends. Agarose
gel-Purify this product and use it for analysis.

5. Same as #4, except use a Klenow fill-in to label the DNA fragment.


    The problem with all of these methods in our hands is that they
produce 2 major bands on a sequencing gel--one at 400bp and one at
300bp. My questions are the following:

1. What are these 2 products? Are  they the 2 strands of the parent
fragment, separated via the denaturing conditions of the sequencing gel?

2. How can we get a totally pure, strand- specific starting product?

3. Are there commercial services for this?


       Please e-mail me back at Richard.Converse at uc.edu with any
suggestions!!


                                    Many thanks,

                                    Richard--






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