missing nucleotide close to the primer

Frank O. Fackelmayer Frank at Fackelmayer.de
Wed Feb 13 11:55:56 EST 2002



nsalazar at hgmp.mrc.ac.uk wrote:
> 
> Hi there,
> Is it possible to miss a nucleotide in a stretch of about 60 nucleotides close to the
> primer that was used for the sequencing reaction ?? I got a nucleotide deleted
> (missing) from two different DNA sequences...
> 

it depends on our way of sequencing and your experience in reading
sequencing gels. Reading close to the primer used to be a problem back
in the ages of radioactive sequencing, when we had to use Mn-containing
buffer instead of the Mg-containing one. Today, e.g. with bigdye
chemistry, you can get quite close to the primer, but still the sequence
is worse at the very beginning. 
Did you read the sequencing gel/chromatogram, or did you rely on the
text sequence? In uncertain cases, always consult the gel/chromatogram.
And that´s where your experience comes into play. Often, you can
manually read a gel/chromatogram where the sequencing software has
problems. Especially in "compressed" regions (although they do not
appear often with modern methods), or when the bands run slightly
irregular on the gel.

In case you did check the original gel/chromatogram, and you are
experienced in doing so: No, I don´t feel that the sequencing misses
bases. To be on the safe side, use a different primer and/or sequence
the other strand.

Frank




More information about the Methods mailing list