An improved method for purifying genomic DNA from forest leaf litters and soil suitable for PCR |
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Authors: | Li Zhang Zhihong Xu Bharat K C Patel |
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Institution: | (1) Microbial Discovery Research Unit, School of Biomolecular and Physical Sciences, Griffith University, Brisbane, Queensland, 4111, Australia;(2) Centre for Forestry and Horticultural Research, School of Biomolecular and Physical Sciences, Griffith University, Brisbane, Queensland, 4111, Australia |
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Abstract: | Background, aim and scope 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 Mg2+ (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.
Materials and methods Three samples including two leaf litters and one forest soil were collected with a core (25 × 40 cm) from a 22-year-old slash
pine plantation in southeast Queensland, Australia. The DNA fragments were extracted directly using the Ultra Clean™ 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.
Results and discussion The DNA was extracted from each sample using the Ultra Clean™ 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 1:103. 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 μl of sample DNA was used for purification. Four DNA purification methods (the PVPP spin column, Sephadex™
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™ 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.
Conclusions 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 ™ 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.
Recommendations and perspectives 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. |
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Keywords: | DNA DNA purification Forest leaf litter PCR amplification PCR inhibitors |
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