Automated cycle sequencing of PCR templates: Relationships between fragment size, concentration and strand renaturation rates on sequencing efficiency

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Abstract
With the Applied Biosystems 373A automated DNA sequencer, we have systematically investigated the amounts of double-stranded PCR fragments of varying size (200, 564, and 1126 bp) required to give sequence of defined lengths, up to the maximum possible. Sequencing was performed on purified double-stranded PCR products using the dye-terminator chemistry and a thermal cycling procedure. The minimal template concentrations allowing determination of short sequences (160 bases) were essentially identical for the fragments studied. Maximal possible sequence determination from the 200 bp fragment was achieved over a wide concentration range, despite the fact that within this range a significant fraction of the template renatured by the mid-point of the sequencing reaction time-course. We conclude that the cyclic sequencing process overcomes competitive strand reannealing of double-stranded PCR products. The sequencing concentration-response curves for the 564 bp and 1126 bp fragments were similar to each other, although the minimal template concentrations required to read >300 bases were slightly increased for the 564 bp fragment. Excess template is undesirable for optimal sequence length determination, but this is unlikely to be solely due to strand reannealing as single-stranded M13 templates in super-optimal concentrations also showed marked reduction in sequencing efficiency.