VNTR sequencing

Launce Gouw launce at apollo.med.utah.edu
Thu Oct 4 12:16:34 EST 1990


Hi, 
We are trying to sequence the VNTR in the minisatellite region
adjacent to the ApoB gene.  So far, in order to avoid subcloning
(lazy, I know) we've been PCRing up the region and trying to sequence
it.  The region PCRs up beautifully, but ss asymmetric amplification
is the pits.  We've also tried lambda exo degradation of ds PCR
product (amplified with a phosphorylated primer/nonphosphorylated
primer) with no better results.  It appears the polymerase is
"slipping" on the ss VNTR DNA during dideoxy synthesis giving us bands
on all four lanes everywhere.  This might even occur if we end up
subcloning.  Any suggestions? Comments? Questions?  HELP!
--
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