A DNA cycle sequencing reaction that minimizes compressions on automated fluorescent sequencers

Abstract

We recently demonstrated that most band compressions (> 70%) on DNA sequencing gels result from the presence of a single sequence motif, 5'-YGN1-2AR-3', where Y and R indicate base-pairing pyrimidine and purine residues, respectively. This finding raised the possibility that the use of 7-deaza-dATP instead of dATP in chain termination sequencing reactions would resolve most of the band compressions. Thus, we examined the effects of 7-deaza-dATP on DNA sequencing using thermostable DNA polymerases. The results indicate that the replacement of dATP with 7-deaza-dATP in conventional cycle sequencing reactions can successfully eliminate most band compressions without sacrificing sequencing performance.