Korean Paddy Soil Microbial Community Analysis Method Using Denaturing Gradient Gel Electrophoresis
Myeong-Eun ChoeSung‐Jun HongJong-Hui LimYunyoung KwakChang–Gi BackHee‐Young JungIn-Jung LeeJae‐Ho Shin
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Abstract:
현재 토양 생태에서 토양미생물은 유기물 분해, 질소 순환, 식물의 질소 이용 등 중요한 역할을 하고 있어, 토양 내 미생물 다양성을 분석하기 위한 연구는 지속적으로 진행되어 오고 있다. 본 연구에서는 논 토양의 미생물 생태 다양성을 조사하기 위한 효과적인 방법으로 denaturing gradient gel electrophoresis (DGGE)를 적용하고자 본 연구를 수행하였다. 논 토양 미생물의 DNA를 분리하기 위하여 lysis buffer method, skim milk bead method, sodium phosphate buffer method, Epicentre SoilMaster DNA extraction kit (Epicentre, USA), Mo Bio PowerSoil kit (Mo Bio, USA)를 이용하여 토양 내 gDNA 최적 추출방법을 확인하였다. 그 결과 Mo Bio PowerSoil kit를 사용하였을 때 Shannon 다양성지수가 세균 3.3870, 진균 3.6254으로 미생물 다양성 분석시에 가장 효과적이었다. DGGE 분석을 위한 조건은 세균의 경우 6% polyacylamide gel, 45-60% denaturing gradient였고, 진균의 경우 6% polyacrylamide gel, 45-80% denaturing gradient에서 최적 분석조건을 보였다. 위의 분석법을 적용하여 논 토양내의 미생물 군집의 변화를 살펴보면 시간의 변화 요인에 의해 미생물 변화가 일어나는 것을 알 수 있었다. 본 연구에서 사용된 DGGE 분석법을 통해 논토양 미생물의 분석 가능성을 제시 할 수 있었다. Soil microbes are important integral components of soil ecosystem which have significant and diverse role in organic matter decomposition, nitrogen cycling, and nitrogen fixation. In this study an effective denaturing gradient gel electrophoresis (DGGE) method was employed for paddy soil microbial diversity survey. For optimum paddy soil microbial DNA extraction, different methods such as Lysis buffer, skim milk bead, sodium phosphate buffer, Epicentre Soil Master DNA extraction kit (Epicentre, USA) and Mo Bio Power Soil DNA kit (MO BIO, USA) methods were utilized. Among all the method, using Mo Bio Power Soil kit was most effective. DGGE analysis of Bacteria was carried out at 6% polyacylamide gel and 45-60% denaturing gradient in the optimal conditions. Whereas DGGE analysis of fungi was done at 6% polyacrylamide gel and 45-80% denaturing gradient in the optimal conditions. By applying the above assay, it was found that variation within the microbial community of paddy soil occurs by a factor of time. DGGE assay used in this study through for a variety of soil microbial analysis suggests the potential use of this method.In recent years, denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) are well-established molecular tools in microbial molecular ecology that allows the study of diversity and dynamics of microbial communities. The technique is reliable, reproducible rapid and inexpensive. Here, the principle, the limitations and potentials application of DGGE/TGGE techniques are introduced in this paper.
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To explore an efficient,stable,low-cost DNA extraction method from single nematode for the support of molecular identification of nematodes,using six species of plant-parasitic nematodes as experimental materials, four DNA extraction methods,namely,direct lysis method,freeze-thaw lysis method,cutting lysis method,freeze-thaw cutting lysis method were designed and compared in order to check their efficiency of DNA extraction from single nematode. The results showed that cutting lysis method and freeze-thaw cutting lysis method were the most efficient and stable methods for DNA extraction of single nematode,then the freeze-thaw lysis method,and the direct lysis method was the worst one. Total DNA with high-quality from a single nematode could be gained efficiently and stably through two methods,namely,cutting lysis method and freeze-thaw cutting lysis method. And the extracted DNA meeted requirement of the following PCR amplification.
Alkaline lysis
Lysis buffer
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The AutoMate Express™ Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler™ lysis buffer or PrepFiler BTA™ lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep™ column, the lysate in the sample tubes were loaded onto AutoMate Express™ instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework-type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day-to-day operation.
Lysis buffer
genomic DNA
DNA profiling
STR analysis
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A fast, simple, easy, efficient, and inexpensive method for DNA extraction from malaria parasites collected on filter paper would be very useful for molecular surveillance. The quality and quantity of DNA are critical to molecular diagnosis and analysis. Here, we developed a simple alkali lysis method for DNA extraction from blood samples on filter paper. The results showed that 10–50 mM NaOH and deionized water all effectively isolated parasite DNA at higher parasitemia, as witnessed by successful PCR amplification, while at a parasitemia of 0.01%, the 10 mM NaOH lysis condition generated the best results. Furthermore, DNA extracted by this method was successfully used to amplify a fragment of > 2000 bp. This method successfully extracted DNA from 1 µl of blood at a parasitemia as low as 0.0001% (equivalent to 5 parasites /µl). The DNA isolated by the 10 mM NaOH lysis method was stable to yield PCR products after storage at 4 °C or − 20 °C for 12 months. These results indicate that this alkali lysis method is simple, effective, sensitive, and inexpensive for isolating stable Plasmodium DNA from dried blood spots on filter paper.
Lysis buffer
Alkaline lysis
Filter paper
genomic DNA
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A set of Escherichia coli freshwater isolates was chosen to compare the effectiveness of denaturing gradient gel electrophoresis (DGGE) vs temporal temperature gradient gel electrophoresis (TTGE) for separating homologous amplicons from the respective uidA region differing in one to seven single base substitutions. Both methods revealed congruent results but DGGE showed a five to eight times higher spatial separation of the uidA amplicons as compared with TTGE, although the experiments were performed at comparable denaturing gradients. In contrast to TTGE, DGGE displayed clear and focused bands. The results strongly indicated a significantly higher discrimination efficiency of the spatial chemical denaturing gradient as compared with the temporal temperature denaturing gradient for separating the uidA amplicons. Denaturing gradient gel electrophoresis proved to be highly efficient in the differentiation of E. coli uidA sequence types.
Amplicon
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Denaturing gradient gel electrophoresis (DGGE) has been developed as a technique to screen for mutations in a particular gene. Usually, PCR products of a particular gene in eukaryotic cells, prokaryotic cells, or clones (e.g., wild-type strain and a mutant) are amplified, and separated on a gel in which a gradient of increasing chemical denaturant is established. Under optimal conditions, PCR products that have nucleotide substitutions will migrate to a different position on DGGE in comparison to the original PCR products, and thus allow the detection of mutations in this particular gene.
Strain (injury)
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Lysis buffer
Sample Preparation
Isolation
Solid phase extraction
Alkaline lysis
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Objectives:In order to discuss effect of electrophoresis times on the analysis of microbial community of activated sludge based on targeted sequence of 16S rDNA by double gradient denaturing gradient gel electrophoresis(DG-DGGE).Methods:Genomic DNA was isolated from activated sludge, and 16S rDNA fragments were amplified with there primer sets(338f/534r).PCR products were separated by DG-DGGE,and optimal electrophoresis time was determined by time travel experiment.Results: The results indicated that DGGE profiles were obviously different under different electrophoresis times.While PCR products of V3 region of 16 rDNA(200 bp) were separated in the gel containing 6%-12% acrylamide and 30%-60% denaturant under 200 V,optimal electrophoresis time was 5 hours.
Primer (cosmetics)
Molecular-weight size marker
genomic DNA
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Our previous study revealed that modified MAGSi (magnetic-silica nanopartivel beads) had various sizes and shapes that affected the DNA extraction process. In this study, we combined our MAGSi with several kinds of lysis buffer for DNA extraction to analyze the result. Human saliva DNA was extracted using three kinds of MAGSi. Each of them was made differently. We used ATL buffer, Triton Buffer, and without buffer for cell lysis. The concentration and purity of the extracted DNA were analyzed using spectrophotometry. Extracted DNA also was used as template DNA for DNA amplification using beta globin gene. This study revealed that our MAGSi-1 provided the best result for DNA concentration and purity. This study also revealed that DNA extraction using lysis buffer produced less concentration yield and purity value but it was more consistent. The concentration and purity value were different but consistent. All the extraction can be used as DNA template for amplification of human beta globin gene. Further studies are still needed to determine the effectiveness and consistency of the lysis buffer and our MAGSi.
Lysis buffer
Alkaline lysis
Buffer (optical fiber)
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