Q: Inaccuracies in PCR sequencing and genetic variability

[Fred Rickson]

On Thu, 13 Feb 1997, Daniel GAUTHERET wrote:

> I bet this issue has already been debated, but I can't find any relevant
> data
> in the litterature. So here it is:
> Direct sequencing of PCR products often yields sequences with many 
> inaccuracies (Ns). Can we say that the position of these undefined 
> nucleotides is related to actual genetic variation at these particular
> sites?
> 
> Thank you.
> 
> Daniel Gautheret

I suspect that if you are getting more than an acceptable amount of 
misreads, then you are going to have to clone what must be a 
heterogeneous target area.  On a "clean" DNA region I can sequence 5 
times with maybe two changes each time over 300 bases.

Fred Rickson