| Popis: |
Although stutter is a well-known artifact of STR amplification for DNA typing, it is very difficult to reduce. In this study, we determined whether stutter could be reduced by PCR using previously untested high-performance polymerases, or by droplet PCR that reduced the reaction volume to approximately 1 nL. At the D5S818 locus, our results using control DNA 9948 as a template showed that decreasing PCR volume by droplet PCR did not affect the stutter ratio, whereas high-fidelity polymerases, PrimeSTAR HS, PrimeSTAR MAX, iProof HF, and SuperFi, achieved lower stutter ratios than AmpliTaq Gold 360. To confirm this effect of reducing stutter for other STR loci, triplex PCR was performed for D21S11 and D18S51. The results showed that SuperFi had the lowest stutter ratio among the high-fidelity polymerases, followed by PrimeSTAR MAX. PrimeSTAR HS showed higher stutter ratios at D21S11 and D18S51, and iProof HF showed a higher stutter ratio at D18S51, compared with AmpliTaq Gold 360. The stutter ratios of PrimeSTAR MAX and SuperFi at D18S51 were almost the same as that of AmpliTaq Gold 360. Furthermore, we compared the stutter ratio distribution of 63 samples of AmpliTaq Gold 360 and SuperFi, the latter of which had the lowest stutter ratio in this study. The average stutter values at the loci D5S818, D21S11, and D18S51 were 5.56%, 7.47%, and 6.53% for AmpliTaq Gold 360, but 0.86%, 1.64%, and 5.18% for SuperFi, respectively. SuperFi had significantly lower stutter at all three loci (p 300×), but it was considered that slippage was not directly involved in the replication errors. Besides, it was suggested that high processivity, which has been considered to be effective for reducing stutter, was actually ineffective. It is highly possible that the stutter formation mechanism of STR involves slippage resulting from the pausing of polymerase due to conformational changes. Although the details about SuperFi's function as a high-fidelity polymerase have not been published by the manufacturer, it is considered that it relieves the pausing of polymerase to achieve accurate replication. |
