Standard DNA Isolation Research Articles

Environmental DNA Isolation, Validation, and Preservation Methods.

Environmental DNA (eDNA) workflows contain many familiar molecular-lab techniques, but also employ several unique methodologies. When working with eDNA, it is essential to avoid contamination from the point of collection through preservation and select a meaningful negative control. As eDNA can be obtained from a variety of samples and habitats (e.g., soil, water, air, or tissue), protocols will vary depending on usage. Samples may require additional steps to dilute, block, or remove inhibitors or physically break up samples or filters. Thereafter, standard DNA isolation techniques (kit-based or phenol:chloroform:isoamyl [PCI]) are employed. Once DNA is extracted, it is typically quantified using a fluorometer. Yields vary greatly, but are important to know prior to amplification of the gene(s) of interest. Long-term storage of both the sampled material and the extracted DNA is encouraged, as it provides a backup for spilled/contaminated samples, lost data, reanalysis, and future studies using newer technology. Storage in a freezer is often ideal; however, some storage buffers (e.g., Longmires) require that filters or swabs are kept at room temperature to prevent precipitation of buffer-related solutes. These baseline methods for eDNA isolation, validation, and preservation are detailed in this protocol chapter. In addition, we outline a cost-effective, homebrew extraction protocol optimized to extract eDNA.

RELATIONSHIP BETWEEN GRK4 POLYMORPHISMS AND ESSENTIAL HYPERTENSION. A STUDY IN A GROUP OF TURKISH SUBJECTS

Background/Purpose:
 G protein-coupled receptor kinase (GRK4) is associated with essential hypertension (EHT). GRK4 regulates sodium balance in the proximal tubules of the kidney. In this study, the possible roles of A142V and A486V polymorphisms in the development of hypertension were investigated in a Turkish population.
 Methods: Genomic DNA was obtained from white blood cells using the standard DNA isolation kit (Roche). Genotypes of variants of GRK4 were determined by Polymerase chain reaction (PCR) - Restriction fragment length polymorphism (RFLP) analysis. 
 Results: In this study, GRKA142V and A486V polymorphisms were found to be associated with EHT in the Turkish population (P

Drop-to-drop liquid-liquid extraction of DNA in an electrowetting-on-dielectric digital microfluidics.

Recent advancements in microfluidics and lab-on-a-chip technologies enabled miniaturization and automation of many downstream nucleic acid analysis steps such as PCR. However, DNA extraction/isolation protocol remains a stand-alone sample preparation step. For a quick sample-to-result solution, downstream protocols and sample preparation protocols need to be seamlessly integrated into a single lab-on-a-chip platform. As a step toward such integration, this paper introduces microfluidic DNA isolation using the liquid-liquid extraction (LLE) method in the drop-to-drop (DTD) format. The electrowetting-on-dielectric digital microfluidic platform is capable of handling a two-phase liquid system easily, which enables DTD LLE. In this study, the extraction of plasmid DNA (pDNA) from an aqueous sample to an ionic liquid is demonstrated. Prior to pDNA extraction study, the DTD LLE protocol was developed and optimized using organic dyes as solutes. The selective extraction of pDNA in the presence of proteins as interfering molecules is also demonstrated. This work implies that DTD LLE can substitute for magnetic beads steps in standard DNA isolation protocols.

Isolation and Quantification of Extracellular DNA from Biofluids.

Extracellular DNA is studied as a diagnostic biomarker, but also as a factor involved in the pathophysiology of several diseases due to its pro-inflammatory properties. Extracellular DNA can be extracted from plasma, urine, saliva or other biofluids using standard DNA isolation procedures and specialized commercial kits. Sample preparation for isolation is important, freezing and thawing may affect the amount of extracellular DNA extracted. Subsequent centrifugations remove cells and cell debris from the samples to obtain true extracellular DNA. Small volume of samples especially from animal experiments is often an issue and it affects the DNA yield. Very short fragments ( 100 bp) can be lost during isolation and are difficult to quantify using PCR. Fluorometric methods asses all stained DNA fragments. Selecting the method for quantification of extracellular DNA is crucial and combination of at least two methods is ideal. Standardization of procedures or at least their reporting in research papers is of utmost importance for comparison of results.

The hidden ‘mycobacteriome’ of the human healthy oral cavity and upper respiratory tract

The incidence of opportunistic non-tuberculous mycobacteria (NTM) infections has increased considerably in the past decades causing an array of infections, including respiratory and soft-tissue infections. NTM are ubiquitous and can be found in numerous environments, including households and water plants. However, NTM have not been reported to be associated with the healthy human oral microbiome. Since the oral cavity and upper respiratory track are the main ports of entry of microorganisms into the human body, elucidating NTM diversity and prevalence will assist in the assessment of the potential risks of infection elicited by these opportunistic pathogens. Here, we report the identification of a ‘non-tuberculous mycobacteriome’ in healthy individuals. We employed a modified DNA extraction procedure in conjunction with mycobacterial-specific primers to screen niches in the oral cavity (buccal mucosa and dental plaque) and upper respiratory tract (nostrils and oropharynx) of 10 healthy subjects. A total of 50 prevalent operational taxonomic units sequenced on MiSeq (Illumina) using 16S rRNA V3–V4 region were detected across all screened niches, showing the presence of diverse NTM communities. NTM DNA was detected in the nostrils of all 10 subjects, in buccal mucosa of 8 subjects, in the oropharynx of 7 subjects, and in the dental plaques of 5 subjects. Results from quantitative PCR showed each individual harbored 103–104 predicted NTM per each screened niche. The modification of standard DNA isolation methods to increase sensitivity toward mycobacterial species represents an important step to advance the knowledge of the oral as well as the overall human microbiome. These findings clearly reveal for the first time that healthy individuals harbor a ‘non-tuberculous mycobacteriome’ in their oral cavity and upper respiratory tract and may have important implications in our understanding of infections caused by NTM.

The effect of age on DNA concentration from whole saliva: implications for the standard isolation method.

Adequate quantity and quality of the DNA isolated from saliva samples are crucial for ensuring successful genotyping rates in genetic studies. However, there is little information about these issues when saliva samples are collected from children. The objectives of this study were to assess whether there are differences in DNA quality or quantity isolated from saliva samples of children at different ages and adolescents compared to adults and, if so, to establish a modified protocol to improve and standardize DNA isolation from saliva samples of children. Saliva samples were collected with Oragene DNA Sample Collection Kit from 41 healthy subjects including children of different ages, adolescents, and adults. Quantity and quality of isolated DNA were determined spectrophotometrically. DNA concentration and age were positively correlated (r = 0.676, P < 0.001). A high percentage of samples from children below 12 years yielded DNA concentrations <100 ng/µL and DNA quality a260/a280 ratios of <1.8. Modifying the standard DNA isolation method raised DNA quantity and quality in these critical samples. Age determines, at least in part, the high variability observed in the concentration of DNA isolated from saliva samples. This fact should be taken into account for a better standardization of the DNA isolation to ensure DNA banking in large-scale genetic studies involving children.

Sensitive life detection strategies for low-biomass environments: optimizing extraction of nucleic acids adsorbing to terrestrial and Mars analogue minerals

The adsorption of nucleic acids to mineral matrixes can result in low extraction yields and negatively influences molecular microbial ecology studies, in particular for low-biomass environments on Earth and Mars. We determined the recovery of nucleic acids from a range of minerals relevant to Earth and Mars. Clay minerals, but also other silicates and nonsilicates, showed very low recovery (< 1%). Consequently, optimization of DNA extraction was directed towards clays. The high temperatures and acidic conditions used in some methods to dissolve mineral matrices proved to destruct DNA. The most efficient method comprised a high phosphate solution (P/EtOH; 1 M phosphate, 15% ethanol buffer at pH 8) introduced at the cell-lysing step in DNA extraction, to promote chemical competition with DNA for adsorption sites. This solution increased DNA yield from clay samples spiked with known quantities of cells up to nearly 100-fold. DNA recovery was also enhanced from several mineral samples retrieved from an aquifer, while maintaining reproducible DGGE profiles. DGGE profiles were obtained for a clay sample for which no profile could be generated with the standard DNA isolation protocol. Mineralogy influenced microbial community composition. The method also proved suitable for the recovery of low molecular weight DNA (< 1.5 kb).

RT-qPCR detection ofAspergillus fumigatusRNAin vitroand in a murine model of invasive aspergillosis utilizing the PAXgene®and Tempus™ RNA stabilization systems

Molecular diagnosis of invasive aspergillosis (IA) is a potentially life-saving tool in the care of at-risk individuals. To date, the development of PCR-based diagnostic tests has been hampered by the lack of standardization in the methods for such critical activities. In this study, we used both spiked volunteer blood samples and a murine model of IA to test the utility of the PAXgene and Tempus systems for stabilization and isolation of fungal RNA from blood as part of an evaluation of a new diagnostic strategy. In spiking experiments, RNA isolation followed by RT-qPCR that targeted the 18S gene was compared to a standard DNA isolation and qPCR assay that targeted the ITS ribosomal region. We demonstrated that both PAXgene and Tempus RNA stabilization and extraction systems followed by RT-qPCR had similar performance in detecting fungal RNA in blood samples from Aspergillus fumigates-infected mice. In spiked samples, the Tempus system performed better than the PAXgene system as it detected 100% of all samples spiked with 10 or 20 germinated Aspergillus conidia/ml blood sample as compared to the PAXgene system which detected 33% and 56% of the samples spiked with 10 or 20 conidia/ml, respectively. The stabilization of fungal nucleic acids in blood samples and its efficient isolation by a commercial method is an important step in the development of standardized molecular diagnostic tools that are needed to improve the outcomes for individuals with IA.

Changes in the Structure of DNA Molecules and the Amount of DNA Per Plastid During Chloroplast Development in Maize

We examined the DNA from chloroplasts obtained from different tissues of juvenile maize seedlings (from eight to 16 days old) and adult plants (50-58 days old). During plastid development, we found a striking progression from complex multigenomic DNA molecules to simple subgenomic molecules. The decrease in molecular size and complexity of the DNA paralleled a progressive decrease in DNA content per plastid. Most surprising, we were unable to detect DNA of any size in most chloroplasts from mature leaves, long before the onset of leaf senescence. Thus, the DNA content per plastid is not constant but varies during development from hundreds of genome copies in the proplastid to undetectable levels in the mature chloroplast. This loss of DNA from isolated, mature chloroplasts was monitored by three independent methods: staining intact chloroplasts with 4',6-diamidino-2-phenylindole (DAPI); staining at the single-molecule level with ethidium bromide after exhaustive deproteinization of lysed chloroplasts; and blot-hybridization after standard DNA isolation procedures. We propose a mechanism for the production of multigenomic chloroplast chromosomes that begins at paired DNA replication origins on linear molecules to generate a head-to-tail linear concatemer, followed by recombination-dependent replication.

One-step isolation of plant DNA suitable for PCR amplification

We report a one-step extraction technique for the isolation of plant DNA, DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm2 in less than 20 min, with no tube changes. The method was tested on several plant specA00AK020ies. The described method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated by the novel method to PCR products generated using standard DNA isolation techniques.

PCR detection of Fusarium oxysporum f.sp. gladioli race 1, causal agent of Gladiolus yellows disease, from infected corms

Fusarium oxysporum f.sp. gladioli (FOG) race 1 infects both large‐ and small‐flowered Gladiolus cultivars. Race 2 isolates infect only small‐flowered cultivars but can be present as epiphytes on large‐flowered plants. When 160 arbitrary 10‐mer oligonucleotide primers were tested on FOG by PCR to find RAPD markers specific for race 1, the RAPD primer G12 amplified two discriminating DNA fragments, AB (609 bp) and EF (1196 bp), in race 1 isolates only. Both fragments were cloned and sequenced. Two pairs of race 1‐specific primers for multiplex PCR were designed. Tests of 112 F. oxysporum isolates by PCR showed that, in almost all cases, race 1 isolates of vegetative compatibility group 0340 could be distinguished with these primers. Seven putative race 1 isolates did not react in multiplex PCR; hybridization studies with labelled AB and EF DNA fragments showed that these isolates belong to separate groups. A bioassay was developed to detect corms that were latently infected with FOG race 1. Gladiolus corms were homogenized and incubated for 5 days at 28°C in a semiselective medium to induce growth of Fusarium. Cultivated mycelium was isolated and subjected to the developed multiplex PCR after standard DNA isolation or disruption by microwave treatment.

Rapid extraction of DNA from archival clinical specimens: our experiences.

The analysis of DNA extracted from archival clinical specimens using polymerase chain reaction represents the basis of a variety of research and diagnostic protocols in medicine. However, the selection of optimal DNA extraction method is critical if such an analysis is to be successful. Recently, we have evaluated a number of rapid DNA extraction protocols in order to find the most suitable method for routine processing of the most common archival materials in pathological and cytological laboratories: paraffin-embedded tissues and Papanicolaou- or Giemsa-stained smears. Our results demonstrate that rapid DNA extraction methods have comparable DNA extraction efficiencies with standard DNA isolation protocols on archival clinical specimens with the exception of Giemsa-stained smears.

Rapid and effective processing of blood specimens for diagnostic PCR using filter paper and Chelex-100.

AIM: To provide a more efficient method for isolating DNA from peripheral blood for use in diagnostic DNA mutation analysis. METHODS: The use of blood impregnated filter paper and Chelex-100...

Bacterial Lipopolysaccharide Copurifies with Plasmid DNA: Implications for Animal Models and Human Gene Therapy

During the course of gene therapy experiments in rodents, using intramuscular injections of plasmid DNA derived from Escherichia coli, we noted dose-related toxicity. This observation prompted a search for possible contaminants of DNA samples. We used the highly specific and sensitive limulus amoebocyte lysate assay (LAL), to monitor endotoxin bioactivity in DNA samples, and found plasmid DNA derived from standard E. coli bacterial strains, using traditional DNA isolation protocols, to be heavily contaminated with endotoxin, or lipopolysaccharide (LPA). Standard DNA isolation procedures resulted in the copurification of up to 500 micrograms/ml of LPS. LPS is a potent inducer of cytokines and other inflammatory mediators, and may complicate the use of naked DNA in gene therapy. The copurification of endotoxin with plasmid DNA also has important implications for in vitro transfection studies and microinjection of DNA into embryos. A simple and efficient protocol to reduce LPS contamination of plasmid DNA was developed. The conversion of intact bacteria to spheroplasts prior to the isolation of plasmid DNA, incubation with lysozyme, treatment with the detergent n-octyl-beta-D-thioglucopyranoside (OSPG) and polymyxin-B (PMB) chromatography, allowed the isolation of plasmid DNA containing less than 50 ng/ml LPS. This represents a 10,000-fold reduction in LPS contamination, compared to conventional methods of plasmid DNA purification, avoids potentially toxic reagents such as ethidium bromide, and produces a higher yield of plasmid DNA.

The use of herbarium specimens in DNA phylogenetics: Evaluation and improvement

During the last few years we have been confronted with the need to use herbarium specimens in the molecular phylogeny studies, since it is generally difficult to obtain living material of some rare species. Ancient DNA has been sequenced, and there are also reports on successful DNA amplification from herbarium specimens. However, it is not easy to obtain amplified DNA from the first herbarium sample tested. In this paper, experiments are described about trials of DNA amplification from two to 151-year-old herbarium specimens of plant species we needed for our projects. Of the 17 herbarium samples tested only two allowed DNA amplification under standard DNA isolation conditions. Different types of PCR inhibiting activities were demonstrated in DNA extracts. In some of the extracts there was extremely low concentration of template with satisfactory quality. In some instances, PCR inhibiting activities were successfully removed by treating them either with insoluble polyvinylpyrrolidone or by adding bovine serum albumin (BSA) to the amplification mixture. However, some PCR-inhibiting activities were resistant to the treatments described above. When the concentration of template was very low, a second PCR amplification with internal primers was necessary to increase the amount of DNA for sequencing. Nevertheless, contamination of either DNA extract or amplification mixture were sometimes observed, and consequently precautions were taken to minimize them. Finally, successful amplification was obtained in eight samples out of the 17 examined.