Application of real-time long and short polymerase chain reaction for sensitive monitoring of the fate of extracellular plasmid DNA introduced into river waters.

ABSTRACT

The precise estimation of extracellular DNA, long enough to encode a gene, is valuable for determining its potential involvement in genetic transformation. Here, the applicability of real-time long PCR was examined by using target DNA of different lengths and transformation with competent cells to monitor the fate of plasmid DNA released into rivers. Detection limits of the PCR were 7 and 30 copies reaction(-1) for a plasmid (4.1 kbp), and 30 and 3×10(4) copies reaction(-1) for lambda DNA (8.6 kbp and 15.5 kbp). The copy numbers of the plasmid obtained by the real-time long PCR were highly correlated with those determined by the transformation metod (R(2)=0.98). Real-time PCRs targeting a short fragment and full-length plasmid DNA were carried out to monitor fragmentation during 506 h of incubation. After 75 h, more than 100-fold larger amounts of the short fragments persisted compared to the full-length plasmid and the values remained constant in the following days. Real-time long PCR revealed that long DNA persisted in river water for prolonged periods of incubation and is thus useful to assess the fate of target DNA in natural water systems.