DNase I Activity Retained after Heat Inactivation in Standard Buffer

For the detection of RNA transcripts by RT-PCR, prior removal of genomic DNA must be performed. To remove genomic DNA, RNA is often prepared by DNase I digestion following phenol extraction. Recently, one-tube or onebuffer systems of RT-PCR were developed to prevent loss of RNA and to reduce the risk of contamination (2,6). In these methods, DNase I is added before RT. Taq DNA polymerase preparations could be a source of genomic DNA contamination in PCR (9). DNase I treatment of Taq DNA polymerase is recommended particularly when the primers for the highly conserved 16S rRNA gene are used (7,8). Digestion of Taq enzyme with DNase I (4 U DNase I/10 U Taq DNA polymerase in a 10-μL volume) was recommended by Rochelle et al. (8) to remove DNA contamination of the enzyme preparation. In each of these methods, DNase I in the reaction mixture was inactivated by heating at 75°C–95°C for 5–10 min. The heated DNase I solution was then carried over to the next PCR step (3,4). We show here that the buffers used for the heat inactivation are crucial. Heating in PCR buffer alone is inadequate to inactivate DNase I. Taq DNA polymerase (AmpliTaq; PΕ Biosystems, Urayasu, Chiba, Japan) was treated with DNase I (Nippon Gene, Toyama, Toyama, Japan) (4 U DNase I/10 U Taq DNA polymerase in 40 μL PCR buffer) at 37°C for 30 min. The mixture was heated at 95°C for 10 min to inactivate DNase I and then used for PCR. The Taq DNA polymerase treated with DNase I required 100-fold more template DNA than the untreated Taq DNA polymerase for producing the same intensity of the band (data not shown). We suspect this was due to the activity of the DNase I, which remained active after the heat inactivation. To test this possibility, 50 ng EcoRI-cleaved pBR322 was treated with 0.2 U/50 μL or 1 U/50 μL DNase I that had been heated for various periods. The treatment condition was either incubation at 37°C for 60 min or a thermal cycling program consisting of 30 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 30 s. Using 0.2 U/50 μL DNase I, which was recommended by Rochelle et al. (8), degradation of DNA was observed within 20 min in both assay conditions (Figure 1A). With 1 U/50 μL DNase I, Fiorenza and Mangia (3) and Huang et al. (4) used 1 U/50 μL and 2.5 U/50 μL DNase I in RT conditions, respectively. DNA degradation was observed even in 90-min heated samples in the former assay condition and with 40 min of heating in the latter condition (data not shown). These experiments showed that DNase I in PCR buffer could not be completely inactivated by heating at 95°C for 10 min. Heating at 95°C for 10 min in the Tris-HCl buffer (pH 8.3) included in the Taq DNA polymerase kit failed to inactivate DNase I, but heating in the sodium acetate buffer (pH 5.2) included in the DNase I kit or in the Tris-HCl buffer (pH decreased to 5.2) did so. As the pH was different between these buffers, we examined the effect of pH on heat treatment of DNase I in PCR buffer. In the pH range of 7.0–8.5, 20 U/40 μL DNase I were active after heating at 95°C for 10 min. However, DNase I was inactivated after heating in pH lower than 5 or higher than 9 (Figure 1B). The efficient heat inactivation of DNase I in acidic conditions agreed with both Kunitz’s earlier observation (5) and the observaBenchmarks