The need for highly specific sequencing primers

Dan Andrews Dan.Andrews at anu.edu.au
Mon Nov 17 06:59:52 EST 1997


Hi Weblanders,

I'm sequencing a mitochondrial gene from a number of primate species
and am discovering a higher than normal degree of divergence between
these genes, but I am also finding that this sequencing really only
works when I'm using primers 100% homologous to their target sequence.
This is OK, but I really am churing through the forms for getting new
primers synthesised.  Is this unusual to need such high homology
between primer and target sequence to achieve a successful sequencing
reaction?  Does anybody know if there are any tricks for getting
around this?  I've tried dropping the annealing temperature of the
sequencing reaction a bit with some success.  I'm sequencing directly
from a PCR product rather than from a clone.

Thanks,  Dan Andrews

Human Genetics Group, John Curtin School of Medical Research,
Australian National University






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