A very difficult 1.1 kb gap to PCR and Clone

Jin Li jlli2000 at yahoo.com
Mon Jul 7 18:35:15 EST 2003

HI, Tom:

Tom Knight <tk at suspiria.ai.mit.edu> wrote in message news:<bdq2ug$420$1 at mercury.hgmp.mrc.ac.uk>...
> It sounds as if the only evidence you have that this gap is real is
> the running position of some (many?) fragments.  What if there were a
> protein, e.g., bound tightly to a region on the chromosome which made
> that piece run abnormally?
This is a very good suggestion.  We suspect that this is origin of
replication region and there is a DNA binding protein that can bind
this region tightly.  We are currently modfiying our DNA extraction
protol to remove possible DNA binding protein now.

> You don't say what happens when you do the many PCRs.  Do they give
> short fragments?

All PCR (with a combination of 6 six primers) produce no bands.
> What happens when you sequence across that region?  Does sequence
> stop?  Or is the problem that you can't clone it?  Or do you see the
> sequence on the far side and reject it "knowing" that it can't be
> right?

So far,  we are unable to clone this region.

> Have you tried sequencing directly from the mitochondrial DNA rather
> than going to clones?  This is often possible with small bacterial
> chromosomes, and is likely possible with a mitochondrial genome.

We tried sequencing directly from mitochondrial DNA and genomiphi
products,  but both of them did not work at all.

> How are you purifying the DNA?  I'd make sure whatever it was included
> one or more steps of phenol/chloroform extraction to get rid of
> whatever proteins were around, followed by several steps of chloroform
> only extractions to get rid of phenol.
> You mention sequence for the gap in a related species -- is there anything
> special about the sequence in that region?  Can you clone that region
> from the other species into your vector?  Have you talked to the people
> who reported the other sequence?

We did other related species and we have problem with only this



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