Since the Phusion and K APAHiFi polymerases have been designed for reduced cycling times, we used increased denaturation and annealing temperatures and shortened thermal cycling times (per the manufacturer's recommendations) as follows: an initial denaturation at 98°C for 1 min; 30 cycles of 98°C for 20 s, 64°C for 15 s, and 72°C for 20 s; hold at 72°C for 5 min; and an indefinite hold at 4°C. To ensure that any improvements in sequence quality were not a result of changing the thermal cycling parameters alone, we also performed PCR with AmpliTaq Gold polymerase using the adjusted thermal conditions.
Reactions containing Herculase II Fusion, or Topo Taq HF were amplified with conditions similar to those for AmpliTaq Gold, as follows: an initial denaturation at 95°C for 3 min; 30 cycles of 95°C for 30 s, 58°C for 30 s, and 68°C for 30 s; hold at 72°C for 5 min; and an indefinite hold at 4°C. Amplification products were sequenced using the methods outlined for generating the baseline quality scores.
To ensure that any improvements in sequence quality were not a result of changing the thermal cycling parameters alone, we also performed PCR with Taq DNA polymerase using the adjusted thermal conditions used for the other enzymes.Data analysis
Sequence trace files were inspected and edited using Sequencher 4.8 (Gene Codes Corporation, Ann Arbor, MI, USA). Most sequence chromatograms were qualitatively inspected by visual means to assess whether there was any improvement over standard reaction conditions. For most trials, this was sufficient to determine that there was no significant improvement in quality. PCR products that were amplified more than once under the same PCR conditions showed little variation in sequence quality (data not shown).
In order to obtain a quantitative assessment of quality improvement for improved chromatograms, Sequencher was used to generate quality scores for sequences generated under standard PCR conditions with AmpliTaq Gold, and compared with sequences using the other four enzymes. Poor sequence quality as a result of mononucleotide repeats are typically restricted to nucleotides that are after the 3′ end of the repeat. In order to provide an estimate of quality unbiased by the relative position of the mononucleotide repeat in the sequence, we restricted the quality assessment to the portion of the sequence that was between the 3′ end of the mononucleotide repeat (the first repeat in cases where there were two) and the reverse priming site. For each sequence, we determined the percentage of bases within this region with quality scores >20.Results and discussion
Despite the importance of accurate sequencing and genotyping of microsatellites to various biological and forensic applications, limited progress has been made toward the reduction of in vitro frameshift mutations. To date, manufacturers have focused primarily on improving polymerase error rate, performance with difficult GC-rich templates, and processivity. While significant improvements have been made in these particular areas, the options for sequencing through long mononucleotide repeats in PCR products are few.
The most effective method of alleviating frameshift error is still the use of thermolabile polymerases at low extension temperatures (3). Hite et al. (3) hypothesized that the low extension temperature (37°C) used with thermolabile polymerases decreases the likelihood of dissociation of the 3′ end of the nascent strand, thereby reducing the occurrence of slipped-strand mispairings. Although the use of thermolabile DNA polymerases may be effective in this regard, the application to PCR and cycle sequencing reactions is not generally practical due to the need to add enzyme after each denaturation step. Computational methods have been developed to model and interpret stutter patterns for automated genotyping applications (4,28) but the optimal solution would be to reduce the degree of stutter product formation in the first place.
All of our attempts to improve the quality of sequence data from AmpliTaq Gold–generated PCR products by altering various parameters of the PCR constituents or the thermal profile (including the same thermal conditions as used for the other polymerases) failed to yield any improvements in quality.
The use of different polymerases yielded varying results. The sequence quality of samples amplified using Topo Taq HF was similar to that generated using AmpliTaq Gold. Improvement in sequence quality scores was demonstrated with the use of KAPAHiFi relative to AmpliTaq Gold for sequences containing runs of 10 or 11 bp, but sequences from samples with longer runs showed no consistent improvement.