Rapid cleanup of bacterial DNA from field samples

Polymerase chain reaction (PCR) is an in vitro enzymatic, synthetic method used to amplify specific DNA sequences from organisms. Detection of target DNA using gene probes allows for absolute identification not only of specif ic organisms, but also of genetic material in recombinant organisms. A majo r challenge facing detection of DNA from field samples is that they are alm ost sure to contain impurities, especially impurities that inhibit amplific ation through PCR. For example, humic material even in quantities as small as 1 ng have been shown to inhibit PCR. DNA has been extracted from sewage/ stool samples, food, sputum, water and sediment, and human DNA likewise is being extracted from sources as varied as forensic samples of blood, cigare tte butts and human remains. However, multi-step, time consuming methods ar e required to isolate the DNA from the surrounding contamination. This rese arch focuses on developing a method for rapid cleanup of DNA which combines extraction and purification of DNA while, at the same time, removing inhib itors from 'dirty samples' to produce purified, PCR-ready DNA. GeneReleaser (TM) produces PCR-ready DNA in a rapid 5-min protocol. We report here the r apid extraction/purification of plasmid DNA from recombinant Escherichia co li. GeneReleaser(TM), inhibitor and similar to 10(3) cfu recombinant E. col i containing a plasmid insert were added to PCR tubes, vortexed for 30 s an d microwaved for 5 min. DNA was PCR-amplified and identified by gel electro phoresis. GeneReleaser(TM) (GR) resin was able to cleanup samples containin g typical aerosol and water/soil contaminants (dust-60 mu g, pollen-100 mu g, soot-250 mu g, humic acid-75 ng). While these inhibitors were easily rem oved prior to PCR amplification, other complex inhibitors found in soil and food samples remain a major challenge and detection of DNA in these materi als typically requires multi-step procedures that may take up to a day. The advantages of using GR are that it is rapid and inexpensive. (C) 1999 Else vier Science B.V. All rights reserved.