sequencing post-lineraization

Bruce A.Roe broe at ou.edu
Sat Oct 13 07:54:40 EST 2001


Alex,

    Sequencing directly off BACs can be difficult to say the least.
I suggest you PCR the region of interest (or in this case difficulty)
and sequence the PCR product.  However, use two different PCR
reaction mixes, one containing everything including all 4 dNTPs and the
other containing everything except replacing the dGTP with the same
amount of 7-deaza-GTP (7-Deaza-2'-deoxy-guanosine-5'-triphosphate),
(Roche Molecular Biochemicals, formally Boehringer Mannheim,
Cat. No. 0988537).

    The PCR product containing 7-deaza-G instead of dG, will not
form the secondary structure that may be present in this region and
be quite easy to sequence through with normal BigDye3 or BigDye1
reaction conditions either with or without 5% DMSO.

    Give it a try as we have had some remarkable success with sequencing
realllllly difficult G-rich templates when the G is replaced by 7-deaza-G.

Cheers,
--bruce



Alex Laufer wrote:

> Hi all,
>
> Perhaps one or more of you have thoughts on the subject.  I'm attempting to
> sequence internally in a BAC.  The read goes out ~200 bps into a nasty
> repeat region (mostly GT, w/an occasional A) and then signal dies off,
> probably due to secondary structure.  I've tried Big Dye (v. 2 & 3), dGTP,
> dGTP & Big Dye 2 combined, & dRhodamine terminator chemistries, with varying
> amounts of DMSO & cycling conditions.  The best result has always been with
> Big Dye 3, but its still not good enough.  The latest advice from ABI was to
> use a rare cutter or topoisomerase to linearize the template prior to
> sequencing.  Has anyone tried either of these methods, and specifically, is
> there any cleanup necessary prior to sequencing?  Is there any thing else I
> should attempt (aside from shotgunning the BAC)?
>
> Thanks in advance for your advice, Alex
>
> ---

--
*************************************************************************
Bruce A. Roe, Ph.D  George Lynn Cross Research Professor
                    Department of Chemistry and Biochemistry
                    University of Oklahoma, Norman, OK 73019-0370, U.S.A.
Phone: (405) 325-4912 or 7610;  FAX: (405) 325-7762;  e-mail: broe at ou.edu
********************** http://www.genome.ou.edu/ ************************


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