AUTOMATED CYCLE SEQUENCING OF PCR TEMPLATES - RELATIONSHIPS BETWEEN FRAGMENT SIZE, CONCENTRATION AND STRAND RENATURATION RATES ON SEQUENCING EFFICIENCY

With the Applied Biosystems 373A automated DNA sequencer, we have syst ematically investigated the amounts of double-stranded PCR fragments o f varying size (200, 564, and 1126 bp) required to give sequence of de fined lengths, up to the maximum possible. Sequencing was performed on purified double-stranded PCR products using the dye terminator chemis try and a thermal cycling procedure. The minimal template concentratio ns allowing determination of short sequences (less than or equal to 16 0 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 ra nge a significant fraction of the template renatured by the mid-point of the sequencing reaction time-course. We conclude that the cyclic se quencing process overcomes competitive strand reannealing of double-st randed 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 s lightly increased for the 564 bp fragment. Excess template is undesira ble 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 sequen cing efficiency.