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.Key words: extracellular DNA, real-time long PCR, DNA uptake, in situ RCA Extracellular DNA is able to provide new genetic traits and/or genetic diversity through its integration into the genome or its replication as part of the genome following its uptake into bacteria. This process, known as natural genetic transformation, is one of the major mechanisms of lateral gene transfer. As little as 153 bp and 311 bp can facilitate the integration of foreign DNA into the Streptococcus pneumoniae and Pseudomonas stutzeri genomes through natural transformation, respectively 30,35) . In addition, Acinetobacter calcoaceticus can be transformed by 294-bp PCR fragments 33) . Because the extracellular DNA in natural environments comprises 0.15 to 35.2 kbp, it is long enough to provide genetic diversity to a recipient genome even if it is not long enough to encode a complete gene 24) . One of the most important requirements for transformation to occur in natural environments is the persistence of intact extracellular DNA 43) . Previous studies suggest that the fate and persistence of extracellular DNA differs greatly depending on the particular environment 40) . The fate of extracellular DNA in the environment should be accurately and sensitively monitored in order to appreciate the potential for genetic transformation for prolonged periods.The persistence and state of donor DNA molecules have been investigated using electrophoresis after DNA extraction and concentration, conventional PCR, or genetic transformation with competent cells or with selective medium 6,8,29) . These techniques are limited in their ability to monitor the fate of donor DNA due to reproducibility and sensitivity of quantification. In the case of genetic transformation for examination of the transformability of target DNA, it is difficult to distinguish donor DNA from DNA indigenous to environmental samples because t...

Particle size distribution and optimal capture of aqueous macrobial eDNA

“…We sequentially filtered water from a lake and pond inhabited by Common Carp (Cyprinus carpio Linnaeus, 1758, hereafter Carp) to create a series of size fractions containing the particles retained at each size, from ≥180 lm to <0Á2 lm. In aquatic microbiology, 0Á2 lm is the filter nominal pore size below which aqueous eDNA is considered to exist as extracellular molecules free in solution (Matsui et al 2004;Maruyama et al 2008), and the vertebrate mitochondrion measures 0Á2-8 lm (Flindt 2006 p. 254). Thus, our size fractions spanned from extracellular and extraorganellar DNA free in solution (hereafter free eDNA; <0Á2 lm) to particles larger than most vertebrate cells (≥180 lm).…”

Inter-comparison of marine microbiome sampling protocols

“…Another study, however, highlighted that different filter materials and DNA extraction protocols can introduce false negative detection of microeukaryotic operational taxonomic units (OTUs) and underestimate diversity [ 35 ]. Size fractionation is used to separate microbial cells by size [ 36 , 37 ], thus selecting prokaryotes from larger microeukaryotes and discriminating free-living cells from particle attached ones. Classically, samples are divided into picoplankton (0.2–3 µm), nanoplankton (3–20 µm) and microplankton (20–200 µm) size fractions, based on the historic division of planktonic size fractions [ 38 ].…”