DNA Sequence Pile-ups

[Dr. M.D. Jones]

Hi netters.

On the subject of bands across all four lanes in a sequencing gel
(which if they are caused by secondary structure of the DNA
template I call a pile-up) I have the following info.  There is a
paper (DeBorde,DC et al., (1986) Resolution of a common RNA
sequencing ambiguity by terminal deoxynucleotidyl transferase.
Anal. Biochem.157: 275-82) in which such stops caused by reverse
transcriptasewere removed by the addition of TdT, prior to
running the gel, which extended these pile-up bands to the top of
the gel.  Thus, it would seem that this effect results in a
normal base being added to the growing chain and not a dideoxy
terminator base.

If you use Dye-deoxyterminators and an ABI 373A you maybe will
not see this effect?

Mick Jones
Department of Virology
RPMS, London
Email: mjones at rpms.ac.uk