Nucleic Acids and Proteins in Soil: 8 (Soil Biology)

Part of the Soil Biology book series (SOILBIOL, volume 8) Stabilization of Extracellular DNA and Proteins by Transient Binding to Various Soil Components .
Table of contents

Yet, the course of biology had been nudged in a new direction. Nucleic acids are involved in the most important processes occurring in living cells and possess an enormous potential for specific interactions with different biopolymers. Specificity of recognition of DNA and RNA species by complementary nucleic acids is unique in the world of biopolymers. A great number of the proteins involved in processes of replication, transcription, and translation are also capable of highly specific recognition of certain nucleotide sequences and specific structural motifs in folded nucleic acids.

Therefore, nucleic acids have always attracted the attention of pharmacologists dreaming of highly specific therapeutics, i. Nucleic acids can be extracted directly from soil to analyze the microbial community. Alternatively cells can first be separated from the soil then the nucleic acids extracted. The latter approach decreases contamination by soil components but may also be biased towards cells that are separated more easily from soil.

All methods for nucleic acid extraction and purification follow the same principles. Cell lysis is the first step, followed by separation of the nucleic acids from the other cellular components and finally concentrating the nucleic acids into a working volume. There are many papers that outline these methods and commercial kits are marketed by a number of companies. An important factor when extracting nucleic acids from soil is to ensure the approach achieves equivalent lysis of all cell types so that downstream analysis is representative of the community.

This is the main source of bias in direct analyses methods of soil-extracted nucleic acids. Many laboratories have chosen to use mechanical methods, such as bead beating, to ensure cell lysis. Lysis efficiency can be determined by microscopic examination of the sample before and after the extraction procedure.

The other factor that must be considered is soil composition because nucleic acids are charged and will bind to components, particularly clay. After the nucleic acids are extracted a number of methods can be considered that examine nucleic acids directly or indirectly after PCR amplification. The studies of nucleic acids have also paved the way for the development of biochemistry, molecular biology, biotechnology and modern medicine.

Highlights

Moreover, the genetic codons were decoded in by Marshall Nirenberg and Heinrich J. Reflecting upon the history of biomedical research, it is obvious that discoveries in nucleic acids have played indispensable roles in biomedical advances. In recent years, with the rapid progress in genetics and genomic sciences, scientists have identified that many diseases including certain devastating cancers are actually caused by genetic mutations.

Theoretically, if the genetic mutation in diseased cells can be corrected or replaced by directly delivering nucleic acids to the target cells, the disease can be cured at the genetic level. Moreover, many previously undruggable targets such as microRNA and long non-coding RNA can be modulated by nucleic acids with high specificity and low toxicity. However, despite their spectacular therapeutic potential, many nucleic-acid-based drugs also have poor drug-like properties such as low bioavailability, sensitivity to enzyme-mediated degradation, short half-life in vivo, and low permeability to cross the cell membrane.

In addition, for the successful management of various chronic diseases, long-term stable and controllable gene expression or interference will be needed. Moreover, the safety of the nucleic-acid drugs is also critically important. Similar to small molecule and protein drugs, the toxicities and adverse side effects of nucleic acid drugs must be controlled within an acceptable range when they are used in the clinic. To overcome these challenges, various delivery approaches and formulations were used to optimize the efficacy and safety profiles of nucleic-acid-based drugs such as virus-mediated delivery, lipid-mediated delivery, polymer-mediated delivery, and physical delivery methods.

Among these, polymer-based nanocarriers have emerged as one of the most promising approaches for delivering therapeutic nucleic acids both in vitro and in vivo. In this chapter, we will first discuss the challenges and barriers for therapeutic nucleic acids delivery, and then we will elaborate on the advantages, uniqueness, and recent progress of polymer-based nanocarriers for therapeutic nucleic acids delivery. Moreover, we will also discuss the considerations for manufacturing, safety issues, and regulatory requirements for these novel nanocarriers.

Nucleic acids are polymers of nucleotides: Deoxyribonucleic acid DNA encodes the genetic information of the cell. DNA is composed of a deoxyribose ring and one of four bases: These four bases comprise the genetic code. It is RNA that directs protein synthesis. Nucleic acids can become degraded during transport or storage.

Nucleic acids

RNA is especially prone to degradation, and special procedures must be implemented in the laboratory when working with RNA. Degradation of target nucleic acids can result in false-negative results or in inaccurate quantification in quantitative assays [44]. Nucleases may be present in the specimens, and nucleic acid may be degraded by nucleases. Nucleases may be removed by using a nucleic acid extraction procedure that degrades proteins. RNA is especially prone to degradation by RNases, enzymes that are ubiquitous in the environment.

Target degradation can be prevented or minimized by maintaining the samples in the correct environmental conditions prior to use in the assay. All samples should be handled using procedures that are optimized to prevent degradation of nucleic acids. If degradation of the target is suspected, the quality of the extracted nucleic acid should be checked with UV spectroscopy or some other method. Nielsen, in Methods in Enzymology , The solution should be incubated on ice ethanol or at room temperature isopropanol to allow the DNA to precipitate.

Bacterial communities were sufficiently well preserved at the rDNA and rRNA level although storage effects showed as slightly decreased alpha diversity indices for the prolonged storage of freeze-dried samples for 7 days. Our results suggest that proper sampling design followed by immediate freeze-drying of soil samples enables short-term transportation of soil samples across continents. Biodiversity research on soil microorganisms is conducted throughout the world Ramette and Tiedje ; Tedersoo et al.

New PDF release: Nucleic Acids and Proteins in Soil: 8 (Soil Biology)

In depth analysis of microbial communities is realized by high-throughput sequencing generating millions of nucleic acid reads using next generation sequencing NGS platforms Caporaso et al. However, nucleic acids are prone to degradation Wackernagel and optimal sampling and sample processing methods include the immediate freezing of soil samples until processing. Whenever sampling location and processing laboratories are distantly apart, the reliable freezing of samples in liquid nitrogen tank or dry ice during transportation is challenging, costly and not always realizable.

Though the advance in NGS and the possibility to analyze large number of samples lead to large scale and integrated biodiversity studies at a global scale, soil sample storage and transportation across continents still remain a big challenge. Storage of samples at elevated temperatures presumably after chemical preservation, air-drying or freeze-drying are potential alternatives. In several molecular studies, storage of untreated soil samples at ambient temperatures resulted in only minor changes of microbial communities Rubin et al. Nevertheless sample- and microbial type dependent changes were observed Cui et al.

Chemical preservatives directly interact with the sampled materials, and discrepancies in preservation efficiencies for variable sample characteristics Rissanen et al. Freeze-drying is the process where water is removed via sublimation from the frozen sample due to the application of vacuum Adams Nucleic acids in soils are liable to degradation by microbial nucleolytic enzymes Antheunisse ; Greaves and Wilson ; Wackernagel Water removal by freeze-drying prohibits diffusion of molecules in the soil matrix and withdraws the protein hydrate shell synced diminishing enzyme activity Ball ; Kurkal et al.

The freeze-drying process is non-toxic.

DNA Structure and Replication: Crash Course Biology #10

Dried samples do not require temperature control during transportation, are reduced in weight, harbor no risk of solution leakage and can be declared as inactivated samples Adams Freeze-dryers have a wide application in industry and science. In the vicinity of the specific sampling site they could be accessible via collaborations or bought in variable configurations. To our knowledge only two studies evaluated freeze-drying of soil samples in relation to investigations of bacterial communities. Both studies indicate promising potential of freeze-drying for soil sample preservation.

However, their investigations were not comprehensive as restricted to only one microbial target bacteria and one soil sample in the RNA study. Furthermore, the effect of storage conditions of freeze-dried samples for sample transportation was not investigated. In the present study we assessed the application of freeze-drying as soil storage and safe sample transportation method.

We investigated the DNA and RNA based bacterial and arbuscular mycorrhizal AM fungal communities on two freeze-dried grassland soils using pyrosequencing. Sample transportation across continents is fastest by airplane. However, an airport might be several hours or days apart from the sampling site. We assumed that transportation from field site to processing lab could be accomplished in a minimum of 1 and a maximum of 7 days. But even across temperate regions cooling could be required during very hot summer weathers. We hypothesized that freeze-drying is a suitable soil sample treatment prior to short-term storage and transportation to i preserve both microbial DNA and RNA and ii enable unbiased detection of fungal and bacterial communities using NGS approaches.

Two grassland plots of different soil and land use types Table 1 , online supplementary Fig. HEG01 was a fertilized meadow, mown twice a year, and HEG08 an unfertilized pasture grazed by cattle. In total, five soil cores with a diameter of 5cm were collected in the edges and the center of each subplot in a depth of 0—10cm. The rooted surface layer was removed and the five soil cores of one subplot were combined to a composite sample.

The soil was sieved through a 2mm mesh and mixed with a sterilized spoon. Samples were snap frozen in liquid nitrogen and transported on dry ice to the laboratory. Land-use intensity LUI category was assigned according to Wiesner et al. Freeze-dried samples were stored in the presence of blue silica gel within sealed plastic bags. For each temperature treatment three replicates were stored for either 1 day or 7 days online supplementary Fig. Nucleic acids of all samples were extracted in the same run. Sample material could be gained from control samples without prior thawing.

Summing up the number of nucleic acid extracts of control samples and freeze-dried samples, we analyzed 28 DNA and 28 RNA extracts, in the following referred to as a total of 56 samples. For each experimental treatment nucleic acid extracts of one sample replicate were subjected to quality analysis by gel electrophoresis. DNA extracts were loaded on an 1. Amplicon libraries were prepared with pyrosequencing fusion primers.

The bacterial 16S rRNA gene was amplified with the reverse primer R coupled to a barcode and the pyrosequencing adapter B.

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The forward primer F was coupled to pyrosequencing adapter A. The forward primer NS31 was fused to the barcode and the pyrosequencing adapter B while both PCR reverse primers were coupled with the adapter A. The sequencing plate was divided into four lanes. Quality filtering of raw sequences was done with the Mothur software v. Sequences were trimmed to nt length v4—v5 region after removal of reads with an average quality value below 20, occurrence of ambiguous nucleotides or if barcodes exceeded more than one mismatch. As the bacterial rRNA gene was sequenced starting with the gene reverse primer, bacterial sequences were flipped.

In case the alignment still showed end gaps as for the bacterial dataset, uniform start and end positions were explicitly set for a second screening step. Chimera check was done with uchime Edgar et al. Rare OTUs with less than four reads were removed from both datasets. Statistical analyses were done with R version 3. Nucleic acid yields were log transformed. The outlier function of the outliers package was applied to identify datapoints that potentially needed removal from the dataset prior to alpha diversity analysis of variances ANOVA.

Identified outlying datapoints were only removed if a visible deviation appeared in diversity index plotting and Non-metric multidimensional scaling NMDS ordination plotting or if the violation of test assumptions normality of model residuals and homogeneity of variances could be avoided. Specific formulation of treatment contrasts is shown in online supplementary Table S2. Linear regression models included the plot as fixed factor and the treatment with defined contrasts as fixed factor while interaction terms were only included if the model fit was much better as determined by a lower Akaike Information Criterion AIC value.

Homogeneity of variance was assessed by Levene test, while normal distribution of model residuals was inspected by Shapiro tests. Permanova analysis was carried out by the adonis function vegan package to determine the significance of the factors sampling plot, freeze-drying, storage duration and storage temperature on the bacterial and AM fungal community.


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High-molecular weight DNA was recovered from frozen and freeze-dried soil samples online supplementary Fig. RNA yields were neither affected by freeze-drying nor by storage time or temperature. Nucleic acid yields were log transformed prior to analysis. From the total of 56 nucleic acid samples, bacterial 16S raw sequences were obtained.

After quality filtering, the number of bacterial sequences was normalized to the minimum number of sequences per sample resulting in bacterial reads per sample, which clustered into bacterial abundant OTUs containing at least three reads. The true bacterial diversity still exceeded the recovered OTUs as indicated by rarefaction curves online supplementary Fig. The bacterial community comprised 14 phyla and six candidate divisions online supplementary Table S3.

The AM fungal dataset of 18S reads comprised 83 sequences. After quality filtering, the number of AM fungal sequences was normalized to the minimum number of sequences per sample resulting in AM fungal reads per sample, which clustered into 66 abundant OTUs. Most AM fungal rarefaction curves online supplementary Fig.

S4b did not reach saturation but came closer to saturation level than the bacterial samples. Bacterial diversity was not affected by freeze-drying of soil samples itself but by a prolonged storage duration of 7 days Table 5 , online supplementary Fig. At the DNA level, the observed species number and Shannon diversity were significantly lower for freeze-dried samples stored for 7 days than for freeze-dried samples stored only for 1 day.


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At the RNA level the mean OTU richness of freeze-dried samples compared to control samples and the number of shared OTUs with the control varied strongly between sampling plots and treatments. Nevertheless, neither freeze-drying nor tested storage conditions were found to significantly affect the detected alpha diversity of AM fungi in the soil samples Table 6 , online supplementary Fig. NMDS ordination plots showed a clear clustering of bacterial communities in respect to plot and nucleic acid origin Fig. In the RNA based analysis bacterial communities were enriched for Deltaproteobacteria online supplementary Fig.