scholarly journals Endophytic Microbial Community DNA Extraction from the Plant Phyllosphere

BIO-PROTOCOL ◽  
2017 ◽  
Vol 7 (4) ◽  
Author(s):  
Carlos Ruiz-Pérez ◽  
María Zambrano
Keyword(s):  
Microbial Community ◽  
Dna Extraction

scholarly journals Validating DNA Extraction Protocols for Bentonite Clay

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Katja Engel ◽  
Sara Coyotzi ◽  
Melody A. Vachon ◽  
Jennifer R. McKelvie ◽  
Josh D. Neufeld
Keyword(s):  
Microbial Community ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Bentonite Clay ◽  
Dna Recovery ◽  
Blocking Agents ◽  
Clay Matrix ◽  
Microbial Dna

ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.

scholarly journals Impact of Enhanced Staphylococcus DNA Extraction on Microbial Community Measures in Cystic Fibrosis Sputum

PLoS ONE ◽  
2012 ◽  
Vol 7 (3) ◽  
pp. e33127 ◽  
Author(s):  
Jiangchao Zhao ◽  
Lisa A. Carmody ◽  
Linda M. Kalikin ◽  
Jun Li ◽  
Joseph F. Petrosino ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Microbial Community ◽  
Dna Extraction ◽  
Cystic Fibrosis Sputum

scholarly journals Development of Simple Methods of DNA Extraction from Environmental Samples for Monitoring Microbial Community Based on PCR.

2000 ◽  
Vol 36 (4) ◽  
pp. 193-204 ◽  
Author(s):  
KAZUNARI SEI ◽  
KEN-ICHIRO ASANO ◽  
NAOHIRO TATEISHI ◽  
KAZUHIRO MORI ◽  
MICHIHIKO IKE ◽  
...  
Keyword(s):  
Microbial Community ◽  
Dna Extraction ◽  
Environmental Samples ◽  
Community Based

scholarly journals Effect of DNA Extraction Methods on the Apparent Structure of Yak Rumen Microbial Communities as Revealed by 16S rDNA Sequencing

2015 ◽  
Vol 64 (1) ◽  
pp. 29-36 ◽  
Author(s):  
YA-BING CHEN ◽  
DAO-LIANG LAN ◽  
CHENG TANG ◽  
XIAO-NONG YANG ◽  
JIAN LI
Keyword(s):  
Microbial Community ◽  
Bacterial Community ◽  
Microbial Communities ◽  
16S Rdna ◽  
Dna Extraction ◽  
Extraction Methods ◽  
16S Rdna Sequencing ◽  
Bead Beating ◽  
Rdna Sequencing ◽  
Dna Extraction Methods

To more efficiently identify the microbial community of the yak rumen, the standardization of DNA extraction is key to ensure fidelity while studying environmental microbial communities. In this study, we systematically compared the efficiency of several extraction methods based on DNA yield, purity, and 16S rDNA sequencing to determine the optimal DNA extraction methods whose DNA products reflect complete bacterial communities. The results indicate that method 6 (hexadecyltrimethylammomium bromide-lysozyme-physical lysis by bead beating) is recommended for the DNA isolation of the rumen microbial community due to its high yield, operational taxonomic unit, bacterial diversity, and excellent cell-breaking capability. The results also indicate that the bead-beating step is necessary to effectively break down the cell walls of all of the microbes, especially Gram-positive bacteria. Another aim of this study was to preliminarily analyze the bacterial community via 16S rDNA sequencing. The microbial community spanned approximately 21 phyla, 35 classes, 75 families, and 112 genera. A comparative analysis showed some variations in the microbial community between yaks and cattle that may be attributed to diet and environmental differences. Interestingly, numerous uncultured or unclassified bacteria were found in yak rumen, suggesting that further research is required to determine the specific functional and ecological roles of these bacteria in yak rumen. In summary, the investigation of the optimal DNA extraction methods and the preliminary evaluation of the bacterial community composition of yak rumen support further identification of the specificity of the rumen microbial community in yak and the discovery of distinct gene resources.

scholarly journals Releasing the microbes from old bones: the effect of different DNA extraction protocols on microbial community profiling

2020 ◽  
Vol 6 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Anne Marie Høier Eriksen ◽  
Lara Puetz ◽  
Carolina Rocha ◽  
Tue K. Nielsen ◽  
Lars H. Hansen ◽  
...  
Keyword(s):  
Microbial Community ◽  
Dna Extraction ◽  
Microbial Community Profiling ◽  
Community Profiling

scholarly journals Evaluating DNA Extraction Methods for Community Profiling of Pig Hindgut Microbial Community

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0142720 ◽  
Author(s):  
Yang Lu ◽  
Philip Hugenholtz ◽  
Damien John Batstone
Keyword(s):  
Microbial Community ◽  
Dna Extraction ◽  
Extraction Methods ◽  
Community Profiling ◽  
Dna Extraction Methods

scholarly journals Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method

AMB Express ◽  
2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Ayomi Dilhari ◽  
Asanga Sampath ◽  
Chinthika Gunasekara ◽  
Neluka Fernando ◽  
Deepaka Weerasekara ◽  
...  
Keyword(s):  
Microbial Community ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Chronic Wounds ◽  
Chronic Wound ◽  
Extraction Methods ◽  
Wound Infections ◽  
Dna Extraction Methods ◽  
The Impact ◽  
Chronic Wound Infections

AbstractInfected chronic wounds are polymicrobial in nature which include a diverse group of aerobic and anaerobic microorganisms. Majority of these communal microorganisms are difficult to grow in vitro. DNA fingerprinting methods such as polymerase chain reaction-denaturation gradient gel electrophoresis (PCR-DGGE) facilitate the microbial profiling of complex ecosystems including infected chronic wounds. Six different DNA extraction methods were compared for profiling of the microbial community associated with chronic wound infections using PCR-DGGE. Tissue debris obtained from chronic wound ulcers of ten patients were used for DNA extraction. Total nucleic acid was extracted from each specimen using six DNA extraction methods. The yield, purity and quality of DNA was measured and used for PCR amplification targeting V2–V3 region of eubacterial 16S rRNA gene. QIAGEN DNeasy Blood and Tissue Kit (K method) produced good quality genomic DNA compared to the other five DNA extraction methods and gave a broad diversity of bacterial communities in chronic wounds. Among the five conventional methods, bead beater/phenol–chloroform based DNA extraction method with STES buffer (BP1 method) gave a yield of DNA with a high purity and resulted in a higher DGGE band diversity. Although DNA extraction using heat and NaOH had the lowest purity, DGGE revealed a higher bacterial diversity. The findings suggest that the quality and the yield of genomic DNA are influenced by the DNA extraction protocol, thus a method should be carefully selected in profiling a complex microbial community.

scholarly journals Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

2014 ◽  
Vol 80 (6) ◽  
pp. 1985-1994 ◽  
Author(s):  
Yuki Morono ◽  
Takeshi Terada ◽  
Tatsuhiko Hoshino ◽  
Fumio Inagaki
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Dna Extraction ◽  
Extraction Method ◽  
Cell Disruption ◽  
Rrna Genes ◽  
Dna Integrity ◽  
Microbial Cells ◽  
Sediment Samples ◽  
Dna Extraction Method

ABSTRACTA prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods.

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