A Rapid and Economical Method for Efficient DNA Extraction from Diverse Soils Suitable for Metagenomic Applications

Selvaraju, Gayathri Devi; Fathima, Anwar Aliya; Radha, Sudhakar; Arunraj, Rex; Curtis, Wayne R.; Ramya, Mohandass

doi:10.1371/journal.pone.0132441

Cited by 48 publications

(33 citation statements)

References 40 publications

(53 reference statements)

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“…As a final added advantage, the overall processing time for the ISB method was shorter (1:30 h) than 5 to 7 h of the earlier reported methods (Sagar et al 2014). The commercial kit (CK) took the least time for DNA extraction (Table 3); however, the processing cost of 1 g of soil or tissue for a single reaction is about US$7 which is quite high if large numbers of samples are needed to be processed (Devi et al 2015).…”

Section: Discussionmentioning

confidence: 95%

“…6 and 7). A potential reason for low performance of cell disruption in soil samples by bead beating of power soil kit may be due to the increase of viscosity and the presence of a high concentration of insoluble materials during the beating process (Devi et al 2015). High losses of input DNA during the application of commercial kits have been reported (Roossinck et al 2010;Vo and Jedlicka 2014;Sadeghi et al 2013).…”

Section: Discussionmentioning

confidence: 99%

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Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Ahmadi

Kowsari

Azadfar

et al. 2018

Annals of Forest Science

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& Key message Two new efficient, fast and low-cost metagenomic DNA extraction methods were developed for different Persian oak tissues (leaf, stem, root, and rhizospheric) and soil samples. & Context The new "omics" studies on the genus Quercus are of importance to help finding efficient strategies for overcoming environmental challenges, and to do this, presence of efficient DNA extraction protocols for different Quercus species are very critical. & Aims The objective of the present study was to develop new efficient methods for extraction of metagenomic DNA (mDNA) from of Persian oak (Quercus brantii Lindl.) tissues. & Methods The efficiency of two newly developed mDNA extraction methods, including indirect SDS-based (ISB or concentrate method) and one spin column-based method (SCB) were compared to that of two classical direct methods, including CTABbased and SDS-based methods, and two commercial mDNA extraction kits. & Results The maximum average yield of mDNA for all samples (leaf, stem, root, bulk, and rhizospheric soils) was obtained by SCB (258 ng/μl) and ISB (189 ng/μl) methods, respectively. Successful PCR amplification for 16SrRNA and ITS sequence was consistently observed for ISB, SCB, and kit-extracted mDNAs, which confirmed the high purity of mDNA extracted by these methods. The new methods showed more than 96% quantitative PCR efficiency, and partial restriction digestion and metagenomic library construction confirmed the high efficiency of the newly developed methods. & Conclusion It could be concluded that two new protocols enhanced efficiency (yield, purity, and cost) of mDNA extraction from different tissues of Persian oak.

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Ahmadi

Kowsari

Azadfar

et al. 2018

Annals of Forest Science

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show abstract

“…Successful DNA isolation would be one that could extract the high yield consistently across the samples from a large number of individuals with high quantity, purity and the less time period. Different kinds of methods are available for gDNA extraction based on chemical and mechanical lysis of cells (Devi et al, 2015). There are an ambiguity and problems in choosing the best methods that not only are able to recover a high amount of nucleic acids but also give a higher yield of amplifiable copies.…”

Section: Discussionmentioning

confidence: 99%

Novel extraction of high quality genomic DNA from frozen bovine blood samples by using detergent method

Goud¹,

Upadhyay²,

Kumar³

et al. 2018

Open Vet J.

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DNA is the prerequisite for life’s inception that transfers hereditary information, past several years; various types of commercial kits are made available which vary depending on the type of the biological sample being used. The present study is focused on developing an improvised methodology for the isolation of genomic DNA from stored bovine blood samples. DNA was isolated by using the conventional Phenol: Chloroform: Isoamyl alcohol (PCI) method and Detergent method. The aim of the study was to make a comparative analysis and evaluation of these two methods to identify the one that gives a superior quality and quantity of genomic DNA. Total (n=48) each duplicate blood samples from three different buffalo(Bubalus bubalis) breeds Banni, Surti, Murrah, three zebu cattle (Bos indicus) breeds Kankerj, Gir, Sahiwal were collected from the jugular vein. The quantity, purity of the genomic DNA was assessed based on the total DNA yield, purity ratios, spectral profile, agarose gel electrophoresis analysis and polymerase chain reaction amplification of MC1R gene product without any inhibitors. The results of our study suggest that detergent method is also suitable for extraction of genomic DNA from the bovine blood and results were significant (*P>0.05). The total mean yield was found to be 329.05±11 μg/5ml for all six breeds while the PCI method was employed. The total mean yield of the gDNA for all six breeds was 406.6±43 μg/5ml of blood when the detergent method was used. One way ANOVA test showed that the total DNA yield varied depending on the isolation method used. The DNA yield obtained from the DG method was (***P< 0.001) significant as compared to the PCI method (**P<0.01).

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“…The concentration and purity of genomic DNA were determined by measuring the A 260/ A 280 ratio. Genomic DNA was extracted from the soil samples as previously described by Devi .…”

Section: Methodsmentioning

confidence: 99%

Metagenome complexity and template length are the main causes of bias in PCR‐based bacteria community analysis

Peng

Wang

et al. 2018

J Basic Microbiol

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Multitemplate PCR is used widely for the study of microbial community diversity. Although such studies have established the abundance of different groups within many natural ecosystems, these reports are limited by uncertainties such as bias and artifacts in the PCR. Bias which is introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. In this study, factors leading to the bias of the multitemplate PCR in bacterial communities were examined and optimized. Comparisons between PCR cycle parameters, DNA polymerases, PCR primer degeneracy, and 16S rRNA gene fragments GC content, revealed that annealing temperatures and DNA structure are predominant factors contributing to the observed bias. Pre-digestion of metagenomic DNA with the restriction enzyme Sau3A I and decreased annealing temperature reduced the bias significantly. The application of these optimized conditions to the ten-species model community in a soil sample verified the validity of these treatments.

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A Rapid and Economical Method for Efficient DNA Extraction from Diverse Soils Suitable for Metagenomic Applications

Cited by 48 publications

References 40 publications

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Novel extraction of high quality genomic DNA from frozen bovine blood samples by using detergent method

Metagenome complexity and template length are the main causes of bias in PCR‐based bacteria community analysis

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