A very difficult 1.1 kb gap to PCR and Clone

Jin Li jlli2000 at yahoo.com
Fri Jun 27 17:51:18 EST 2003

   We have shutgun cloned a mitochondrial genome.   However,  by
comparing it with the mitochodrial genome from a close related
species,  we found that a 1.1 kb gap.  We have tried many techniqes,
including PCR with combination of 3 set of primers that surround the
gap with PCR kits from different manufacturers,  amplifing it with
Genomify,  cloning the restriction fragment that contains this gag.
However, none of these techniques worked.  We have even tried to cut
this restriction fragment with different 4 cutters into smaller (20 to
50 bp pieces) and cloned and sequenced these smaller fragments.  We
found the the pieces aligned every where in the restriction fragment
but the 1.1 kb gap.  We have confirmed by 5 or 6 restriction maps that
this restriction fragment that contains the 1.1 kb fragment is real.
But somehow can not be cloned,  PCRed etc.    Does anyone can suggest
what to do?



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