The need for highly specific sequencing primers

Dan Andrews Dan.Andrews at anu.edu.au
Tue Oct 14 09:58:29 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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