DNase pretreatment of master mix reagents improves the validity of universal 16S rRNA gene PCR results

Abstract

DNase I pretreatment of 16S rRNA gene PCR reagents was tested. The DNase I requirement for the elimination of false-positive results varied between 0.1 and 70 IU per master mix depending on the applied Taq polymerase. PCR sensitivity was mostly maintained when 0.1 IU of DNase I was used.

FIG. 1.

DNase I dosages required to improve universal PCR results. Percentages of PCR runs delivering valid (white bars), false-positive (grey bars), and false-negative (black bars) results depending on DNase I dosages are shown for assays I (A), II (B), and III (C) (for details, see text). Complete elimination of false-positive results and significant increases in valid results were achieved with highly varying amounts of DNase I (0.1 through 70 IU). The asterisks mark the significantly high proportion of valid PCR results obtained with DNase pretreatment compared to the results from PCR runs without DNase pretreatment.

FIG. 2.

Influence of DNase I dosages on the sensitivity of PCR assays. Rate of positive PCR results at the detection limit are given depending on DNase I dosages for assays I (A), II (B), and III (C) (for details, see text). The threshold dosage of DNase I that was required for the complete elimination of false-positive PCR results is underlined. Amounts of DNase I increasing stepwise reduced the sensitivity of the PCRs.