Rapid DNA Isolation Research Articles

A Simple and Rapid DNA Isolation Method from Dry Pea Seeds Suitable for PCR Analyses

Pea (Pisum sativum L.) is an important legume grown and consumed extensively worldwide. As a rich source of proteins, carbohydrates and vitamins, peas are important in human nutrition. In this study, we compared four different DNA isolation methods from four pea cultivars (F95-927, Specter, Windham and Nicoleta), with some modifications to the original extraction protocols: one SDS extraction buffer without PVP and one SDS extraction buffer with 1% PVP; one CTAB extraction buffer without PVP and one with 1% (1.5%) PVP. For all four extraction methods we used the same quantity of plant material (0.05 g) and an equal quantity of extraction buffer, so the comparison between methods to be very accurate. To establish which is the most efficient extraction method, after DNA isolation and purification, we submitted our samples to PCR analyzes with two markers: ISSR marker 17899A and SSR marker AA175. In my study, based on spectrophotometric measurements and PCR results, I concluded that both CTAB extraction methods were not suitable for DNA extraction from dry pea seeds because they did not show amplification products. The most appropriate DNA extraction method was SDS1 which provided a good quality DNA.

Genome Sequencing of dsDNA-Containing Bacteriophages Directly from a Single Plaque.

The sequencing of phage genomes has become a routine procedure for phage characterization. The protocol presented here allows rapid isolation of DNA from a single phage plaque followed by building ready-to-sequence Illumina-compatible library.

A rapid and reproducible method for isolating genomic DNA from a few crop plants suitable for polymerase chain reaction-based genotyping

As most of the molecular markers in crop molecular breeding programmes are successful based on polymerase chain reaction (PCR), the isolated genomic DNA must be suitable for the same. Though PCR is a robust method and in most of the cases requires only a minute amount of genomic DNA as template, removal of potential PCR-inhibitory factors is quite important. The present work reports the optimization of a rapid genomic DNA isolation method, suitable for PCR-based genotyping of plants. As very minute amount of the genomic DNA isolated in this rapid method was found to be sufficient for PCR, a researcher is capable to go for several hundred independent PCR from single isolation. The method was validated in 4 different crops (wheat, tomato, brinjal and cauliflower) using different PCR-based molecular markers. In case of wheat, genomic DNA isolated in this method was found to be suitable PCR using the specific marker for the detection of the Lr34 gene. For tomato, genomic DNA isolated in this method was successfully used with the molecular markers for the detection of resistance alleles for yellow leaf curl disease and root knot disease. In case of brinjal, the isolated genomic DNA was found to be suitable for simple sequence repeat (SSR) marker assay. In a similar way, genomic DNA isolated in this method from cauliflower leaves was observed to be suitable for amplifying a gene of ~1.5 kb length. Thus, this method will be quite helpful to expedite marker assisted selection of plants in plant molecular breeding programmes.

Rapid and Efficient Isolation of High Quality DNA from Cassava (Manihot esculenta Crantz) Suitable for PCR Based Downstream Applications

Rapid and Efficient Isolation of High Quality DNA from Cassava (Manihot esculenta Crantz) Suitable for PCR Based Downstream Applications

Genomic DNA Isolation from High Polyphenolic Content Grewia asiatica L. Leaf Without Using Liquid Nitrogen

Grewia asiatica (Grewoideae family) is also known as Phalsa, used for the medicinal purpose has a high content of polysaccharides and polyphenols together with flavonoids and other secondary metabolites which obstructs their pure genomic DNA extraction. Although, there are various good quality DNA extraction protocols, extracting DNA from a high phenolic content plant is a challenging task. The present study describes an efficient and rapid DNA isolation protocol by optimizing cetyl trimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP) and sodium dodecylsulphate (SDS) cocktail specifically for the extraction of DNA from polyphenolic leaf of G. asiatica without using liquid nitrogen. Extracted genomic DNA has high purity index (1.95) examined by A260/A280 ratio. The presence of good quality DNA was also evident by gel electrophoresis. The optimized DNA extraction method is efficient for both fresh and old tough leaves with high polyphenolic contents. The achievement of this method in extracting high quality of genomic DNA demonstrated the broad applicability of this method.

Magnetic nanowires for rapid and ultrasensitive isolation of DNA from cervical specimens for the detection of multiple human papillomaviruses genotypes

Detecting human papillomavirus (HPV) is central in diagnosing and monitoring HPV-related disease. However, limited sensitivity and the wide variability of the HPV genome pose challenges in the identification of HPV genes, particularly high-risk types. This study reports the development of polyethyleneimine-conjugated magnetic nanowires (PEI-MNWs) and their use in the isolation, identification, and analysis of multiple genotypes of HPV DNA from cervical cancer specimens. The nanowires are electrochemically doped with a high density of magnetic nanoparticles and biotin moieties during potentiostatic deposition, thereby allowing conjugating cationic branched polymers to direct the attachment of negatively charged DNA molecules with strong magnetic response. For proof of concept, the rapid and ultrasensitive isolation of HPV DNA is performed at concentrations as low as 10pg/mL with an efficiency of >95%. For clinical optimization, the analytical and clinical sensitivity of PEI-MNWs is compared with that of the Roche Cobas 4800 HPV Test and demonstrates excellent correlation for multiple HPV DNA genotypes with superior threshold cycle values. The high sensitivity, specificity, and good reproducibility of PEI-MNWs are particularly well suited for the recovery of DNA and provide significant and clinically meaningful evidence for the early detection and treatment of HPV-associated cancers.

Rapid method for DNA isolation from a tough cell wall green alga Tetraspora sp. CU2551.

Genetic studies are important to understand the complex biological system of various organisms. Some eukaryotic green organisms have tough cell wall which precludes the efficient extraction of the genetic materials. Here, we developed the method for simple and rapid isolation of high quality DNA from a green alga Tetraspora sp. CU2551. The cell homogenization procedures were combined with physical force plus heat treatment to disrupt the cell envelope of Tetraspora sp. CU2551. Without protease treatment, vortexing with glass bead for 30-105s at 70°C led to the isolation of a high purity DNA which was suitable for downstream process. The improved method was successfully developed and could be applied for the rapid isolation of DNA from other unicellular and filamentous green microalgal strains.

Rapid detection of Streptococcus uberis in raw milk by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12ng/assay) and ~60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4×104 cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0% and a diagnostic specificity of 96.0%. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available.

DNA from microdissected tissues may be extracted and stored on microscopic slides.

With regard to complex structure of tissues, laser capture microdissection represents an important step in analytical workflow streaming to proper molecular characterization of different cell types in examined samples. Therefore the simple method for simultaneous processing of higher numbers of microdissected tissues leading not only to rapid and efficient DNA isolation but allowing also the repeated sampling and easy storage may be useful in the practice of histopathological laboratories. We elaborated such a methodology applicable downstream after the microdissection from formalin-fixed paraffin embedded tissues.The tissues for examination are microdissected directly into the circular areas having the diameter 2 mm and marked on the microscopic slide. In this way, one slide is able to accommodate multiple samples. The DNA extraction is performed in low volume of buffer with Proteinase K in a droplet covered by mineral oil just on the slide. Mineral oil in the quality for molecular biology not only avoids evaporation during DNA extraction, but it helps to position the microdisssected tissue, to control the level of cell lysis microscopically and to protect the DNA sample during subsequent manipulations. We provided the evidence that DNA isolated by our methodology remains in the positions on microscopic slide for months without any changes in the lengths of available fragments and that it may be removed from each position repetitively for different kinds of analysis. The new methodological approach presented by us can be practically applied in broad spectrum of laboratories performing routinely genetic analysis on microdissected tissues.

All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

Applying DNA Barcoding to identify species of medicinal plants

According to the World Health Organization (WHO), in recent years, 80% of the population made use of some kind of medical plant in search of relief from any painful or unpleasant symptoms [1]. In 2003, Herbert Paul proposed the use of “DNA Barcoding” as a method to identify species using a small gene sequence from part of the genome of the species [2]. In this way to confirm the species of four compounds of medicinal tea widely consumed by Brazilian population – Melissa officinalis, Mykania laevigata, Vernonia polyanthes and Solanum lycocarpum – we have used the technique of DNA Barcode. For this purpose, the DNA were extracted using the CTAB protocol (cetyl trimethyl ammonium bromide) [3]. The PCR reaction was done using the following primers pairs: rbcLa, psbA – trnH and ITS2. The samples were applied in capillary electrophoresis on ABI 3500 Genetic Analyzer (Applied Biosystems). The table shows the results. The DNA Barcoding results allowed to confirm the identity of M. Laevigata, but not for the other analyzed species. The leaves from S. lycocarpum and V. polyanthes were confirmed by macroscopic and microscopic analyzes when the exsicata were making, which demonstrated that this new methodology helps to improve the studies for the identification using new primers increasing the genetic database of Brazilian species of medicinal plants. Keywords: Brazilian medicinal plants, DNA Barcoding, species identification

English

Advances in DNA technology, such as marker assisted selection, detection of quantitative trait loci and genomic selection also require the isolation of DNA from a large number of samples and the preservation of tissue samples for future use in cacao genome studies. The present study proposes a method for the preservation of sample tissues for DNA extraction and for manual extraction of large number of samples using spheres. The integrity and concentration of the DNA by these methods were assessed and compared with conventional method using mortar. The best parameters in order to obtain a fine powder using spheres was the use of 4 lyophilized leaf disks (50 mg), a single steel ball of 6 mm in diameter, followed by 30 s of manual maceration. The quantity of DNA obtained was four times higher than the conventional method. The purity of the DNA obtained was satisfactory and proved to be amplifiable by PCR using SSR primers. The present approach is a reliable, rapid, simple and consistent DNA isolation method for cacao, compared to the conventional methods. The protocol greatly increases the efficiency of extraction and suggests an inexpensive and practical way of DNA isolation of cacao for large scale.   Key words: DNA extraction, cacao, spheres, lyophilized.

English

Rapid isolation of high-purity microbial genomic DNA is necessary for genome analysis. In this study, the authors compared a one-hour procedure using a microwave with enzymatic and boiling methods of genomic DNA extraction from Gram-negative and Gram-positive bacteria. High DNA concentration and purity were observed for both MRSA and ESBL strains (80.1 and 91.1 µg/ml; OD260/280, 1.82 and 1.70, respectively) when the extraction protocol included microwave pre-heating. DNA quality was further confirmed by PCR detection of mecA and CTX-M. In conclusion, the microwave-based procedure was rapid, efficient, cost-effective, and applicable for both Gram-positive and Gram-negative bacteria. Key words: DNA, MRSA, ESBL, gene, enzymatic, boiling, microwave.

An economical and combined method for rapid and efficient isolation of fungal DNA.

DNA isolation is a crucial step of conducting genetic studies in any organism. However, this process is quite difficult when studying fungi because of the need to damage the fungal cell walls of specific structures. In this study, we developed a method for the rapid and efficient isolation of fungal DNA based on simultaneous mechanical and enzymatic cell wall degradation. There are several typical modifications of the standard phenol-chloroform DNA extraction method. This method can be modified to degrade the fungal cell wall. The first step of the presented DNA extraction included manual homogenization in modified lysis buffer. Next, enzymatic digestion using 2 enzymes was conducted, including lyticase and proteinase K. To carefully select the most favorable conditions, we developed an economical, rapid, and reliable method for fungal DNA extraction that ensures both high efficiency and proper purity, which are essential for further analyses.

Rapid Isolation of DNA from Staphylococcus.

Many methods exist to extract DNA from bacteria. Indeed, there is no shortage of kits available from manufacturers that allow for isolation of highly purified DNA. However, for many applications samples do not need to be extremely pure (i.e., free of contaminating proteins or RNA). Furthermore, for quick genetic screening, it is often useful to have a rapid and inexpensive option for DNA isolation from small samples. For these occasions, the method found in this chapter provides a cost-efficient, yet rapid, isolation of DNA.

Assessment of five soil DNA extraction methods and a rapid laboratory-developed method for quality soil DNA extraction for 16S rDNA-based amplification and library construction

Extraction of DNA from soil samples using standard methods often results in low yield and poor quality making them unsuitable for community analysis through polymerase chain reaction (PCR) due to the formation of chimeric products with smaller template DNAs and the presence of humic substances. The present study focused on the assessment of five different methods for metagenomic DNA isolation from soil samples on the basis of processing time, purity, DNA yield, suitability for PCR, restriction digestion and mDNA library construction. A simple and rapid alkali lysis based on indirect DNA extraction from soil was developed which could remove 90% of humic substances without shearing the DNA and permits the rapid and efficient isolation of high quality DNA without the requirement of hexadecyltrimethylammonium bromide and phenol cleanup. The size of DNA fragment in the crude extracts was >23kb and yield 0.5–5μg/g of soil. mDNA purification using Sephadex G-50 resin yielded high concentration of DNA from soil samples, which has been successfully used for 16S rDNA based amplification of a 1500bp DNA fragment with 27F and 1492R universal primers followed by restriction digestion and mDNA library construction.

Rapid isolation of DNA from Dioscorea species suitable for PCR, restriction digestion and pathogen screening

The methods employed for DNA extraction from many plants is difficult because of the metabolites that interfere with DNA isolation procedures. We have developed a reliable and efficient method for isolating genomic DNA free from polysaccharide, polyphenols and protein contaminants from Dioscorea spp. The method involves inactivation of contaminant proteins by using CTAB/Proteinase K and precipitation of polysaccharides in the presence of high concentration of salt. The purity of genomic DNA was confirmed by A260/280 and A260/230 ratios calculated from the spectrophotometric readings and further by restriction analysis of the isolated DNA using restriction enzymes Eco RI. The total genomic DNA extracted by the new protocol was used for polymerase chain reaction amplification, RAPD analysis, restriction digestion and pathogen screening. The new protocol can be successfully used for both small- and large-scale preparation of genomic DNA from different tissues of Dioscorea spp. The quarantine of seed tubers and use of pathogen-free tubers for planting is a prerequisite for integrated disease management strategy. The protocol can be used for the isolation of genomic DNA from other crop plants too.

Methods for Rapid and Effective PCR-Based Detection of ‘<I>Candidatus</I> Liberibacter solanacearum' From the Insect Vector <I>Bactericera cockerelli</I>: Streamlining the DNA Extraction/Purification Process

This study provides a protocol for rapid DNA isolation from psyllid vectors (Bactericera cockerelli and Diaphorina citri) that can be used directly with DNA-based methods for the detection of 'Candidatus (Ca.) Liberibacter solanacearum,' the bacterial causal agent of potato zebra chip disease and eventually for 'Ca. Liberibacter asiaticus' the causal agent of huanglongbing disease in citrus. The fast DNA extraction protocol was designed to work with conventional polymerase chain reaction (cPCR) DNA amplification as well as Loop mediated PCR DNA amplification. Direct cPCR of the psyllid 28S rDNA gene from samples prepared using the fast DNA extraction method was as reliable as from samples prepared using standard DNA purification (> 97% from live insects) as tested in B. cockerelli. However, samples prepared using the fast DNA extraction method had to be diluted 1:100 in sterile water for reliable amplification, presumably to dilute PCR inhibitors in the crude extract. Similarly, both cPCR and loop mediated PCR DNA amplification detected 'Ca. Liberibacter' in psyllids infected with either the zebra chip or huanglongbing pathogen equally well from diluted samples prepared using the fast DNA extraction method or from samples prepared using a DNA purification step. In addition to being reliable, the time required to complete the fast DNA extraction for 10 samples was on average approximately 5 min and required no special reagents or laboratory equipment. Thus, the fast DNA extraction method shows strong promise as a rapid, reliable, and expedient method when coupled with PCR-based analyses for detection of 'Ca. Liberibacter' pathogens in psyllids.

A rapid and inexpensive one-tube genomic DNA extraction method from Agrobacterium tumefaciens.

Many methods have been used to isolate genomic DNA, but some of them are time-consuming and costly, especially when extracting a large number of samples. Here we described an easy protocol using two simple solutions for DNA extraction from A. tumefaciens cells. Compared with the standard protocol, this protocol allows rapid DNA isolation with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) gDNA extraction was achieved within 15 min; (2) this method was cost-effective, since it only used calcium chloride and lysozyme; SDS, phenol, chloroform and proteinase K were not necessary; (3) the method gave high yield of gDNA (130 ng/loopful culture) compared with standard protocol that was suitable for restriction analysis; (4) the protocol can be carried out in a single test tube and the cells directly from solid media can be used. Thus, this protocol offers an easy, efficient and economical way to extract genomic DNA from A. tumefaciens.

An anion-exchange method to concentrate dissolved DNA from aquifer water

A rapid DNA isolation method was developed to concentrate dissolved DNA (dDNA) in aquifer water for molecular analysis. The aquifer dDNA from the Eastern Snake River Plain Aquifer (ESRPA) was extracted and concentrated using a new method with an anion-exchange Mustang® Q membrane. The concentration of aquifer dDNA in this study ranged from 60 to 264.5ngl−1 in ESRPA aquifer wells. DNA stability in ESRPA aquifer water was also tested in this study. The dDNA extracted from aquifer water samples was used for PCR amplification of bacterial 16S rRNA genes for terminal restriction fragment length polymorphism (T-RFLP) analysis and construction of 16S rRNA gene clone libraries. The ureC gene, IncP, IncQ and IncW plasmid genes were also PCR amplified from dDNA samples. Based on the results, dDNA is relatively stable in aquifer water and can be concentrated by Q membrane method for molecular analysis. The quality of isolated dDNA was suitable as a PCR template.