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sequencing riddle...

taguebw at wfu.edu taguebw at wfu.edu
Sat Apr 5 12:34:01 EST 1997

In article <5i4afv$9k1 at net.bio.net>, Hiranya Roychowdhury
<hroychow at NMSU.Edu> wrote:

One of our
> colleagues has amplified a piece of DNA from a plant using a single primer
> (i.e. amplified region bordered by inverted repeats...). Now the teaser
> (may not be one for some of you): How can this piece of DNA be sequenced
> WITHOUT having to CLONE it? In other words, the investigator wants to use
> the same primers to sequence the pcr products directly after purifying
> them!

> Humbly,
> Hiranya.
> Dr. Hiranya Sankar Roychowdhury
> Plant Genetic Engineering Lab.
> New Mexico State University
> Las Cruces, NM 88003
> Ph. (505) 646-5785
> hroychow at nmsu.edu

Run a strand-separating gel to separate the two strands and isolate the
two strands. Check out Molecular Cloning, Maniatis et al., 1982 pp. 179ff.

This was a fairly standard technique when I was doing Maxam-Gilbert
sequencing. We use to do this with 32P end-labelled fragments to generate
assymetrically label templates. The presence of 32P might be a problem if
you doing chain-termination sequencing but Maniatis et al., indicates that
the strand separation gel can be done with cold DNA followed by EtBr

My 2 electrons...

Brian Tague
Department of Biology
Wake Forest University
Winston-Salem NC 27109

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