A rapid and mild procedure for the isolation of DNA from mammalian cells

A procedure is described for the isolation of DNA from mammalian cells using polycarbonate filters. This method results in high yields of large-molecular-weight DNA, which is essentially free from protein and RNA. The procedure is rapid (approximately 2 h), does not require organic solvents, and can isolate 1.5–140 μg DNA per filter without the addition of carrier. The isolation of DNA from aflatoxin B₁-treated cells by the filter method is described in order to illustrate its advantages for the preparation of DNA containing lesions of low chemical stability.     

Rapid isolation of DNA from Staphylococcus aureus.

We describe a Staphylococcus aureus bulk DNA isolation procedure which uses detergent and guanidine hydrochloride to free the nucleic acid from contaminants. The procedure is rapid and yields high-molecular-weight DNA suitable for molecular biological procedures.     

Rapid isolation of DNA from Staphylococcus aureus

We describe a Staphylococcus aureus bulk DNA isolation procedure which uses detergent and guanidine hydrochloride to free the nucleic acid from contaminants. The procedure is rapid and yields high-molecular-weight DNA suitable for molecular biological procedures.     

A robust universal method for extraction of genomic DNA from bacterial species

The intactness of DNA is the keystone of genome-based clinical investigations, where rapid molecular detection of life-threatening bacteria is largely dependent on the isolation of high-quality DNA. Various protocols have been so far developed for genomic DNA isolation from bacteria, most of which have been claimed to be reproducible with relatively good yields of high-quality DNA. Nonetheless, they are not fully applicable to various types of bacteria, their processing cost is relatively high, and some toxic reagents are used. The routine protocols for DNA extraction appear to be sensitive to species diversity, and may fail to produce high-quality DNA from different species. Such protocols remain time-consuming and tedious, thus to resolve some of these impediments, we report development of a very simple, rapid, and high-throughput protocol for extracting of high-quality DNA from different bacterial species. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone (PVP) and RNA contamination was precipitated using LiCl. The UV spectrophotometry and gel electrophoresis analysis resulted in high A ₂₆₀/A ₂₈₀ ratio (>1.8) with high intactness of DNA. Subsequent evaluations were performed using some quality-dependent techniques (e.g., RAPD marker and restriction digestions). The isolated DNA from 9 different bacterial species confirmed the accuracy of this protocol which requires no enzymatic processing and accordingly its low-cost making it an appropriate method for large-scale DNA isolation from various bacterial species.     

Rapid Isolation of DNA from Dry and Fresh Samples of Plants Producing Large Amounts of Secondary Metabolites and Essential Oils

The presence of certain metabolites has been observed to interfere with DNA isolation procedures and downstream reactions such as DNA restriction, amplification and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yields with a single protocol, and thus, even closely related species may require different isolation protocols. Here we describe the essential steps of a rapid DNA isolation protocol that can be used for diverse medicinal and aromatic plants, which produce essential oils and secondary metabolites such as alkaloids, flavanoids, phenols, gummy polysaccharides, terpenes and quinones. The procedure is applicable to dry as well as fresh plant tissues. This protocol, in our experiments, permitted isolation of DNA from tissues of diverse plant species and produced fairly good yields. The isolated DNA proved amenable to PCR amplification and restriction digestion.     

Rapid isolation of eukaryotic DNA

Guanidine hydrochloride (GHCl) is frequently used for the isolation of RNA from eukaryotic cells. However, it appears that its use for the rapid isolation of chromosomal DNA has been little appreciated. Intact and readily digestible DNA was prepared from a range of murine tissues and cell lines which had been dispersed in GHCl and ethanol precipitated, and then rinsed in 70% ethanol and resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. This simple technique is ideally suited to the preparation of DNA from large numbers of tissue samples.     

Simultaneous purification of DNA and RNA from small numbers of eukaryotic cells

An extraction procedure for the simultaneous isolation of RNA and DNA from tissue culture cells is described. The procedure is a variation of the guanidium/lithium chloride method for RNA isolation which is rapid, simple, and avoids costly ultracentrifugation equipment. The genomic DNA yielded by this procedure is greater than 50 kb in length and may be readily cleaved by restriction endonucleases. Sufficient DNA for Southern blot analysis, and RNA for Northern blot or nuclease protection analysis, can be obtained from as few as 2 x 10(6) cells, making this method particularly suitable for the genetic screening of large numbers of individual, stably transfected cell clones.     

Isolation of DNA using magnetic nanoparticles coated with dimercaptosuccinic acid

Lately, the isolation of DNA using magnetic nanoparticles has received increased attention owing to their facile manipulation and low costs. Although methods involving their magnetic separation have been extensively studied, there is currently a need for an efficient technique to isolate DNA for highly sensitive diagnostic applications. We describe herein a method to isolate and purify DNA using biofunctionalized superparamagnetic nanoparticles synthesized by a modified polyol method to obtain the desired monodispersity, followed by surface modification with meso-2,3-dimercaptosuccinic acid (DMSA) containing carboxyl groups for DNA absorption. The DMSA-coated magnetic nanoparticles (DMSA-MNPs) were used for the isolation of DNA, with a maximum yield of 86.16%. In particular, we found that the isolation of DNA using small quantities of DMSA-MNPs was much more efficient than that using commercial microbeads (NucliSENS-easyMAG, BioMérieux). Moreover, the DMSA-MNPs were successfully employed in the isolation of genomic DNA from human blood. In addition, the resulting DNA–nanoparticle complex was directly subjected to PCR amplification without prior elution, which could eventually lead to simple, rapid, sensitive and integrated diagnostic systems.     

一种适于PCR扩增的小麦基因组DNA快速提取法

许多小麦分子生物学研究需要对大量的小麦样品进行PCR检测,因此,建立一种快速提取小麦基因组DNA的方法十分必要。根据国外报道的一种快速提取水稻和玉米基因组DNA的方法,我们对部分提取步骤进行变动后,在小麦上进行了尝试,长度为1．5kb的片段能得到稳定的扩增。该方法样品研磨在1.5ml的离心管内进行,后续操作不用酚、氯仿、CTAB、SDS和巯基乙醇,整个提取过程不需要使用通风橱,操作步骤简单,花费时间少,而且提取的小麦基因组DNA完整性好,量也较可观。一个DNA样品可供50～100次PCR反应使用,适用于小麦遗传多样性、分子标记辅助选择、转基因后代检测以及引物筛选、分子标记定位等多种研究。     

Rapid small-scale DNA isolation from filamentous cyanobacteria

Abstract A rapid small-scale DNA isolation procedure is described for the filamentous cyanobacteria, which yields enough chromosomal and plasmid DNA for restriction endonuclease digestions, Souther hybridizations, and electroelution from gels for further manipulation. DNA from seven strains of cyanobacteria were isolated and analyzed on agarose gels.     

A new method for the purification of DNA-binding proteins with sequence specificity

We describe a technique for a rapid and efficient isolation and purification of proteins binding to defined DNA sequences. Cloned double-stranded DNA was covalently coupled to m-aminobenzyloximethylcellulose in order to purify proteins which recognize and bind to specific sequences on the DNA. The purification of two DNA-binding proteins from Drosophila melanogaster is demonstrated using the respective cloned DNA sequences.     

Extraction of DNA from soil using nanoparticles by magnetic bioseparation

Aims: To develop a simple, rapid and inexpensive soil DNA extraction protocol. Methods and Results: The protocol relies on the use of superparamagnetic silica‐magnetite nanoparticles for the isolation and purification of DNA from soil samples. DNA suitable for use in molecular biology applications was obtained from a number of soil samples. Conclusions: The DNA extracted using the tested method successfully permitted the PCR amplification of a fragment of the bacterial 16S rDNA gene. The extracted DNA could also be restriction endonuclease digested. Significance and Impact of the Study: The protocol reported here is simple and permits rapid isolation of PCR‐ready soil DNA. The method requires only small quantities of soil sample, is scalable and suitable for automation.     

Co-precipitation of protein and polyester as a method to isolate high molecular weight DNA

DNA isolation is often the limiting step in genetic analysis using PCR and automated fragment analysis due to low quality or purity of DNA, the need to determine and adjust DNA concentrations after isolation etc. Several protocols have been developed which are either safe and provide good quality DNA or hazardous and provide excellent quality DNA. In this brief communication I describe a new and rapid method of DNA isolation which employs the co-precipitation of protein and polyester, in the presence of acetone, to remove contaminating proteins from a lysed-tissue sample, thus leaving high quality pure DNA. The advantages of this method are increased safety over the phenol:chloroform and the chaotrophic salt methods and increased purity over the salting-out method. Since the concentrations of DNA isolated using this method are relatively consistent regardless of the amount of starting tissue (within limits), adjustments of the DNA concentrations before use as templates in PCR's are not necessary.     

Rapid method for DNA isolation from a tough cell wall green alga <Emphasis Type="Italic">Tetraspora</Emphasis> 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 30–105 s 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.     

Isolation of high-molecular-weight DNA from insects.

A simple and rapid method for the isolation of high-molecular-weight DNA from insects is described. The method does not require CsCl ultracentrifugation or extensive dialysis. High-molecular-weight DNA was obtained within 24 h. Since the entire insect was used for DNA isolation, an initial nuclei-enriched fraction was required. Genomic DNA was extracted from lysed nuclei by organic phase separation (liquid/liquid extraction). This method has been successfully applied to the isolation and purification of DNA from eight different adult insects (Heliothis zea, Musca autumnalis, M. domestica, Blatta orientalis, Tenebrio molitor, Lymantria dispar, Ostrinia nubilalis, and Manduca sexta). The recovered DNA can be cleaved with restriction endonucleases, ligated efficiently using standard cloning vectors, and hybridized to synthetic oligonucleotides.     

A rapid and easy method for the DNA extraction from <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

DNA isolation from C. neoformans is difficult due to a thick and resistant capsule. We have optimized a new and rapid DNA isolation method for Cryptococcus using a short urea treatment followed by a rapid method using a chelex resin suspension. This procedure is simpler than previously reported methods.     

Comparison between modified DNA extraction protocols and commercial isolation kits in grapevine (Vitis vinifera L.)

Various protocols have been developed and used for DNA extraction in grapevine. However, owing to the long duration of the isolation steps in previously developed protocols, researchers have preferred to use isolation kits for studies in recent years. In our study, the DNA yield and purity obtained using six methods - namely three DNA isolation protocols and three commercial DNA isolation kits - were compared. Modifications were made and the isolation steps were shortened in the previously developed DNA isolation protocols to achieve more rapid and practical protocols. The samples were taken from plants grown under vineyard and greenhouse conditions in two periods during spring and autumn. The best results among the six DNA isolation methods were discussed. The results were also supported with polymerase chain reaction analyses conducted with isolated DNAs.     

Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.     

High Throughput BAC DNA Isolation for Physical Map Construction of Sorghum (Sorghum bicolor)

With the aim of constructing a physical map of sorghum, we developed a rapid, high throughput approach for isolating BAC DNA suitable for restriction endonuclease digestion fingerprinting, PCR- based STS-content mapping, and BAC-end sequencing. The system utilizes a programmable 96 channel liquid handling system and associated accessories that permit bacterial cultivation and DNA isolation in 96-well plate format. This protocol details culture conditions that optimize bacterial growth in deep-well plates and criteria for BAC DNA isolation to obtain high yields of quality BAC DNA. The system is robust, accurate, and relatively cost-effective. The BAC DNA isolation system has been tested during efforts to construct a physical map of sorghum.     

Isolation of DNA from blood.

A rapid, economical method of DNA isolation from blood was developed that yields DNA suitable for Southern analysis and polymerase chain reactions without organic solvent extractions. Bovine DNA was prepared from peripheral leukocytes and nuclei using pronase E digestion and ethanol precipitation. This isolation method readily adapts to multiple samples. The DNA is characterized by high yield, solubility, lack of protein contamination, and ease of restriction endonuclease digestion.