I am trying to sequence several alleles consisting of very AT-rich sequences.
I am using an ABI 373 sequencer. The alleles are subcloned into pMos.
I used both T7 dye terminator and cycle sequencing dye terminator sequencing,
but I do not get usable sequences. T7 sequencing failed totally. With cycle
sequencing, A and T peaks are getting smaller soon, C and G peaks are very
high, and the total reading length is about 200.
I do not have these problems with other sequences so I do not believe that
they are due to false plasmid preparation. I suppose that they are related
to the nucleotide composition. I reasoned that I would get better results
if I increased dATP, dye-ddATP, dTTP, dye-ddTTP and decrease dCTP and dGTP and
the respective dye terminators. Therefore, I bought a cycle sequencing dye
terminator core kit that is shipped with dye terminators and dNTP not pooled.
Regrettably, there is no information about the dNTP concentration used in this
kit, and ABI is unwilling to give me that information.
Has anyone any suggestions how to sequence these repeats? Does anybody know
about the dNTP concentrations in the core kit? Has anyone successfully used
other enzymes for this purpose on the ABI 373?