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摘要
An improving knowledge of bacterial community within natural environments including forest soils and leaf litters requires extraction of nucleic acids directly from environmental samples since molecular approaches provide less biased access to a larger portion of uncultivable microorganisms. However, when DNA was extracted successfully from these samples, it might still have been difficult to apply it as a template for polymerase chain reaction (PCR) amplifications due to the effect of PCR inhibitors. Various compounds from plant tissues including polysaccharides, phenolic compounds and especially humic acids can inhibit PCR amplification. Some of these inhibitors could inhibit PCR amplification by chelating the Mg(2 ) (cofactor for Taq polymerase), or by binding to target DNA, and PCR amplification would consequently be interfered with. Therefore, eliminating the effects of these PCR inhibitors is one of the most important steps for PCR-based molecular techniques. Four different methods were assessed in this study to purify the genomic DNA extracted from F, L layer leaf litters and forest soil in an exotic pine plantation of southeast Queensland, Australia. Three samples including two leaf litters and one forest soil were collected with a core (25 x 40 cm) from a 22-year-old slash pine plantation in southeast Queensland, Australia. The DNA fragments were extracted directly using the Ultra Clean (TM) Mega Prep Soil DNA kit (Mo Bio Labs, Solana Beach, CA). Then, four different purification methods were applied and compared to purify the DNA for PCR amplification, which include PVPP, Sephadex (TM) spin column, low-melting agarose gel and a new modified gel purification method. The purified DNA from these four purification methods was detected by agarose gel electrophoresis, and the purity and usefulness of DNA samples were ultimately determined by successful PCR amplifications. The DNA was extracted from each sample using the Ultra Clean (TM) Mega Prep Soil DNA kit, and the DNA eluents were dark in colour and sometimes formed compact aggregates. Subsequently, PCR amplification from such samples failed, although a series of dilutions had been made from neat to 1:10(3). The DNA purification step could not, therefore, be avoided. It was observed that both the colour of eluent and the DNA concentration decreased gradually after elution. Considering the difficulties of removing PCR inhibitors and the possibility of high DNA losses, 50-200 A mu l of sample DNA was used for purification. Four DNA purification methods (the PVPP spin column, Sephadex (TM) spin column, low-melting agarose gel and the modified gel purification method) were applied and compared on leaf litter and soil samples. The DNA purified by the modified gel purification method provided the best PCR products for 16S rRNA gene amplification, but the other methods, PVPP, Sephadex (TM) spin column and low-melting agarose gel, produced very weak or no products. Thus, in this study, DNA fragments which were purified by the modified gel purification method were amplified efficiently. This may be attributed to running the low-melting agrose gel for a longer time, which could remove substantial humic substances and also some other compounds from the samples and, thus, prevent them from being involved in PCR amplification. A new modified gel purification method which can improve DNA purification and PCR amplification of environmental DNA is first introduced in this study. Comparing PVPP, Sephadex (TM) spin column, low-melting agarose gel and modified gel purification method for the effect of DNA purification, the modified gel purification method is more successful in removing the PCR amplification inhibitors and obtaining the highly purified PCR amplifiable high-molecular-weight DNA. The method described here is cheap, fast and easy to operate. It suggests in this study that the method containing less and easier following steps should be widely used to relieve the heavy working load of molecular-biological researchers. This study introduces a new modified DNA purification method, and it is found that this modified gel purification method is effective in removing the PCR inhibitors and obtains highly purified DNA from leaf litters for PCR amplification. The modified gel purification method may have wider applications, although it was only assessed on leaf litter and soil samples. The effect of the modified gel purification method on the DNA purification would need to be further investigated on a variety of samples which suffered from PCR inhibitors, such as clinical samples, plant tissues and environmental samples.
- 出版日期2009-6
