A very difficult 1.1 kb gap to PCR and Clone

Tom Knight tk at suspiria.ai.mit.edu
Sat Jun 28 09:33:07 EST 2003


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?

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

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?

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.

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?




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