Rapid cleanup of bacterial DNA from field samples

摘要

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 specific organisms, but also of genetic material in recombinant organisms. A major challenge facing detection of DNA from field samples is that they are almost sure to contain impurities, especially impurities that inhibit amplification 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, cigarette butts and human remains. However, multi-step, time consuming methods are required to isolate the DNA from the surrounding contamination. This research focuses on developing a method for rapid cleanup of DNA which combines extraction and purification of DNA while, at the same time, removing inhibitors from 'dirty samples' to produce purified, PCR-ready DNA. GeneReleaser super(TM) produces PCR-ready DNA in a rapid 5-min protocol. We report here the rapid extraction/purification of plasmid DNA from recombinant Escherichia coli. GeneReleaser super(TM), inhibitor and similar to 10 super(3) cfu recombinant E. coli containing a plasmid insert were added to PCR tubes, vortexed for 30 s and microwaved for 5 min. DNA was PCR-amplified and identified by gel electrophoresis. GeneReleaser super(TM) (GR) resin was able to cleanup samples containing 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 removed prior to PCR amplification, other complex inhibitors found in soil and food samples remain a major challenge and detection of DNA in these materials typically requires multi-step procedures that may take up to a day. The advantages of using GR are that it is rapid and inexpensive.