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