DNA ladders can be used to size polyphosphate resolved by polyacrylamide gel electrophoresis

PAGE is often used to resolve inorganic polyphosphates (polyP), but unfortunately polyP size ladders are not commercially available. Since several dyes that are commonly used to detect nucleic acids in gels also stain polyP, we examined the utility of commercially available DNA size ladders for estimating polyP polymer lengths by gel electrophoresis. Narrow size fractions of polyP were prepared and their polymer lengths were quantified using NMR. Commercially available DNA ladders and these polyP fractions were then subjected to PAGE to determine the relationship between migration of DNA vs polyP, which was found to be: log₁₀(dsDNA length in bp) = 1.66 × log₁₀(polyP length in phosphate units) − 1.97. This relationship between DNA and polyP size held for a variety of different polyacrylamide concentrations, indicating that DNA size ladders can readily be employed to estimate polyP polymer lengths by PAGE.     

Properties of nucleic acid staining dyes used in gel electrophoresis

Nucleic acid staining dyes are used for detecting nucleic acids in electrophoresis gels. Historically, the most common dye used for gel staining is ethidium bromide, however due to its toxicity and mutagenicity other dyes that are safer to the user and the environment are preferred. This Short Communication details the properties of dyes now available and their sensitivity for detection of DNA and their ability to permeate the cell membrane. It was found that GelRed? was the most sensitive and safest dye to use with UV light excitation, and both GelGreen? and Diamond? Nucleic Acid Dye were sensitive and the safer dyes using blue light excitation.     

A color‐code for glycosaminoglycans identification by means of polyacrylamide gel electrophoresis stained with the cationic carbocyanine dye Stains‐all

Cationic dyes such as toluidin blue are commonly employed to visualize glycosaminoglycans (GAGs) on electrophoresis gels; however, the carbocyanine‐based dye Stains‐all have been increasingly used to stain the non‐sulfated hyaluronic acid and other GAGs in submicrogram quantities. In this short communication, we demonstrate that Stains‐all is able to stain the most common GAGs on polyacrylamide gels with distinct and contrasting colors in a reproducible manner. We also show that this staining method is useful to identify GAGs present both in mixtures and in submicrogram quantities. Therefore, Stains‐all has shown to be useful in identifying GAGs on polyacrylamide gels with basis on their specific colors, at least on screening level.     

Two-dimensional coordination polymer-based nanosensor for sensitive and reliable nucleic acids detection in living cells

A reliable and sensitive strategy which can assess nucleic acid levels in living cells would be essential for fundamental research of biomedical applications. Some nanomaterial-based fluorescence biosensors recently developed for detecting nucleic acids, however, are often with expensive, complicated and timeconsuming preparation process. Here, by using a facile bottom-up synthesis method, a two-dimensional(2 D) coordination polymer(CP) nanosheet, [Cu(tz)](Htz = 1,2,4-triazole), was successfully...     

A simple system for staining protein and nucleic acid electrophoresis gels

Researchers in molecular biology spend a significant amount of time tending to the staining and destaining of electrophoresis gels. Here we describe a simple system, costing approximately $100 and taking approximately 1 h to assemble, that automates standard nucleic acid and protein gel staining protocols. Staining is done in a tray or, with DNA gels, in the electrophoresis chamber itself following automatic detection of the voltage drop. Miniature pumps controlled by a microcontroller chip exchange the necessary solutions at programmed time intervals. We demonstrate efficient and highly reproducible ethidium bromide and methylene blue staining of DNA in agarose gels and Coomassie blue and silver staining of proteins in polyacrylamide gels.     

Characterization of the selective staining of DNA on gels using two fluorochromes

Ethidium bromide (EB) and 4'-6-diamidino-2-phenylindole (DAPI) are both well-known fluorochromes for detecting DNA fragments. EB binds to DNA by intercalation and DAPI binds in the DNA minor groove. We previously developed a staining method using both EB and DAPI that is selective for AT-rich DNA fragments. Using this double-staining method, AT-rich DNA fragments are visualized as bluish-white fluorescent bands. To further characterize this method, a series of synthetic DNA fragments were designed with systematic variations in the length, AT content, and DNA sequence pattern. The staining properties of these fragments were determined in the presence of DAPI and EB, and the following results were obtained. (i) In a series of fragments with three AT base pairs followed by one GC base pair, the stained DNA fragments exhibited different fluorescent colors and varied from bluish (more DAPI staining) to pinkish (less DAPI staining) in the order 5'-AAA-3', 5'-AAT-3', 5'-ATA-3', 5'-TTA-3'. (ii) In fragments with constant AT content, the blue fluorescent color increased with increasing number of A (or T) nucleotides, due to increased DAPI binding. The blue color was saturated when the number of A (or T) nucleotides was 12 or greater. (iii) The fluorescent color of the stained DNA fragments changed in the order of red-orange, pink, pinkish-white, white, bluish-white, blue as the AT content increased from 0 to 100%. Thus, the fluorescent color of DNA fragments stained with DAPI and EB depends on base composition and nucleotide sequence, suggesting that individual stained DNA fragments may have characteristic and specific fluorescent colors. The fluorescent color emitted by specific stained DNA fragments in the presence of EB and DAPI can be analyzed with a high degree of sensitivity and resolution using the XYZ colorimetric system.     

Application of silver staining to the rapid typing of the polymorphism of HLA-DQ alleles by enzymatic amplification and allele-specific restriction fragment length polymorphism.

A rapid and highly sensitive silver staining method, originally developed for the detection of proteins, was slightly modified to detect nucleic acids in polyacrylamide gels. The second exons of the histocompatibility antigen HLA-DQA 1 and DQB 1 genes were selectively amplified from genomic DNA by the polymerase chain reaction (PCR). Digestion of the PCR products by endonucleases, followed by their size-separation on polyacrylamide gels and visualization by silver staining, allowed us to define the HLA-DQ alleles of the genomic DNA. The intensity of staining of digested PCR-amplified DNA is linear from at least 8 to 18 ng for fragments of lengths ranging from approximately 40 to 200 bp. Thus, silver staining in combination with PCR and allele-specific restriction fragment length polymorphism provides a simple, safe, and rapid method for accurate definition of HLA-DQ alleles at the nucleotide level in the clinical typing laboratory.     

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1-0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc-imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.     

Simultaneous binding of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin and 4',6-diamidino-2-phenylindole at the minor grooves of poly(dA).poly(dT) and poly[d(A-T)(2)]: fluorescence resonance energy transfer between DNA bound drugs

The spectral properties of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) bound to poly(dA).poly(dT) and poly[d(A-T)(2)] in the presence and in the absence of 4',6-diamidino-2-phenylindole (DAPI) have been studied. DAPI fits deeply into the minor groove of both poly(dA).poly(dT) and poly[d(A-T)(2)], and TMPyP is also situated at the minor groove. The nature of the absorption, circular dichroism (CD), and flow linear dichroism (LD) spectra of the TMPyP-poly(dA).poly(dT) and -poly[d(A-T)(2)] complexes in the Soret band is essentially unaffected whether the minor groove is blocked by DAPI or not, although small variations been noticed in the presence of DAPI. Furthermore, a close analysis of the reduced LD spectrum in the Soret band results in angles of approximately 80 degrees and 55 degrees between transition moments of the TMPyP and DNA helix axes in the absence of DAPI. All these observations indicate that the side of TMPyP whose structure resembles that of classical minor groove binding drugs does not fit deeply into the minor groove. This suggests that TMPyP binds across the minor groove: two positively charged pyridiniumyl rings interact electrostatically with negatively charged phosphate groups of DNA. When DAPI and TMPyP are simultaneously bound to poly(dA).poly(dT) or poly[d(A-T)(2)], the fluorescence intensity of DAPI decreases as TMPyP concentration increases, indicating that the excited energy of DAPI is transferred to TMPyP.     

Interaction of nucleic acids with the glycocalyx

Mammalian cells resist the uptake of nucleic acids. The lipid bilayer of the plasma membrane presents one barrier. Here, we report on a second physicochemical barrier for uptake. To create a sensitive probe for nucleic acid-cell interactions, we synthesized fluorescent conjugates in which lipids are linked to DNA oligonucleotides. We found that these conjugates incorporate readily into the plasma membrane but are not retained there. Expulsion of lipid-oligonucleotide conjugates from the plasma membrane increases with oligonucleotide length. Conversely, the incorporation of conjugates increases markedly in cells that lack the major anionic components of the glycocalyx, sialic acid and glycosaminoglycans, and in cells that had incorporated highly cationic lipids into their plasma membrane. We conclude that anionic oligosaccharides provide a formidable barrier to the uptake of nucleic acids by mammalian cells. This conclusion has implications for genomic stability, as well as the delivery of genes and siRNAs into mammalian cells.     

Structural analysis of metal ion ligation to nucleotides and nucleic acids using pulsed EPR spectroscopy

Metal ions play key structural and functional roles in many nucleic acid systems, particularly as required cofactors for many catalytic RNA molecules (ribozymes). We apply the pulsed EPR technologies of electron spin-echo envelope modulation and electron spin-echo-electron nuclear double resonance to the structural analysis of the paramagnetic metal ion Mn(II) bound to nucleotides and nucleic acids. We demonstrate that pulsed EPR, supplemented with specific isotope labeling, can characterize ligation to nucleotide base nitrogens, outer-sphere interactions with phosphate groups, distances to sites of specific (2)H atom labels, and the hydration level of the metal ion. These techniques allow a comprehensive structural analysis of the mononucleotide model system MnGMP. Spectra of phenylalanine-specific transfer RNA from budding yeast and of the hammerhead ribozyme demonstrate the applicability of the methods to larger, structured RNA systems. This suite of experiments opens the way to detailed structural characterization of specifically bound metal ions in a variety of ribozymes and other nucleic acids of biological interest.     

Extraction processes reduce polyphosphate ion migration,dispersion and diffusion as detected with gel electrophoresis and 31P DOSY NMR

Inorganic polyphosphates (polyPs) have been identified in eukaryotic and prokaryotic cells alike. Various extraction methods have been optimized as a necessary step before identification and measurement of these polymers. Three commercially available sodium polyP glasses were either dissolved or dissolved and extracted by two commonly used polyP extraction techniques – perchloric acid or buffered phenol–chloroform. The products were separated by polyacrylamide gel electrophoresis (PAGE), stained with toluidine blue O, and the migration results quantitatively compared. Both extraction processes reduced the relative migration distances of the peak and leading edges, and the stained band lengths, suggesting reduced polyP migration and dispersion. ³¹P diffusion-ordered spectroscopy nuclear magnetic resonance confirmed that polyP extraction by perchloric acid or phenol–chloroform processes reduced polyP diffusion coefficients and suggested hydrolytic degradation with stronger end-chain signals. Reduced polyP diffusivity after extraction makes possible an overestimation of synthetic polyP chain length assignment when compared to unextracted polyP ladders with PAGE. The mechanism(s) for reduced synthetic polyP diffusion after extraction and intracellular chemical environment effects on migration are not known.     

Polyacrylamide gel electrophoresis of fluorophore-labeled hyaluronan and chondroitin sulfate disaccharides: application to the analysis in cells and tissues

This report describes a new formulation of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides (PAGEFS) for the analysis of hyaluronan (HA) and chondroitin sulfate (CS) Delta-disaccharides. PAGEFS relies on derivatization of reducing ends of HA- and the variously sulfated CS-derived Delta-disaccharides with 2-aminoacridone (AMAC), followed by electrophoresis under optimized buffer conditions (Tris-borate and Tris-HCl) and on polyacrylamide gels (25% T/3.75% C). The method was applied to the analysis of glycosaminoglycans (GAGs) from the human umbilical cord tissue and GAGs isolated from human aortic smooth muscle cell cultures. The obtained results were in agreement with those obtained after an analysis with high-performance liquid chromatography (HPLC). On the basis of these results, PAGEFS is a rapid and sensitive method for the analysis of the total amount of HA- and CS-derived disaccharides, as it allows analyzing 20 samples in minigels in one run and provides quantitation with relatively high sensitivity (less than 25 pmol per disaccharide). In addition, PAGEFS overcomes the lack of commercial gels described previously for the separation of AMAC-labeled disaccharides. Therefore, the method proposed here is an economic and useful tool for a fast screening of GAGs in biological samples, particularly when a high number of samples should be analyzed.     

Counterion-dye staining for DNA in electrophoresed gels using indoine blue and methyl orange

In this study, we describe a sensitive staining method for DNA in agarose and polyacrylamide gels using organic visible dyes, indoine blue (IB) and methyl orange (MO). The counterion-dye staining method uses two oppositely charged dyes to form a hydrophobic ion pair complex in the staining solution. A decrease in the number of free forms of dyes in staining solution can enhance the selectivity of binding between the dye and DNA, and can reduce nonspecific background staining. As a result, the sensitivity of counterion-dye staining was significantly improved compared with other dye-based staining. This method uses a staining solution consisting of 0.008% IB, 0.002% MO, 10% ethanol and 0.2 M sodium acetate at pH 4.7, and can detect 5 ng of lambda DNA/HindIII within 60 min in agarose gels and 10 ng of PhiX174 DNA/HaeIII within 20 min in polyacrylamide gels.     

Dimeric calixarenes: a new family of major-groove binders

A new class of potent DNA binding agents is presented. Dimeric calix[4]arenes with cationic groups at their upper rims and flexible alkyl bridges can be synthesized from triply acyl-protected calix[4]arene tetramines in relatively short synthetic sequences (3-5 steps). The compounds attach themselves to double-stranded nucleic acids in a noncovalent fashion, with micro- to nanomolar affinities. Guanidinium headgroups with their extended hydrogen-bonding "fingers" are more powerful than ammonium groups, and the benzylamine series is superior to the anilinium series (see below). The new ligands easily distinguish between RNA and various DNA types, and produce characteristic changes in UV/Vis, fluorescence, CD, as well as NMR spectra. Especially extended oligonucleotides of more than 100 base pairs are bound with affinities increasing from RNA (10?μM K(d))相似文献   

Investigation of the interaction between curcumin and nucleic acids in the presence of CTAB

It is found that nucleic acid can enhance the resonance light scattering (RLS) enhancement effect of curcumin (CU) in the presence of cationic surfactant cetyltrimethylammonium bromide (CTAB). The investigation indicates that in BR (pH 4.3) buffer, both the positive CTAB and negative yeast RNA (yRNA) combine and form a positive large association, then which is bound on the two carbon atoms of the carbonyls of CU through hydrogen bond and hydrophobic force and form CU-CTAB-yRNA ternary complex, resulting in the RLS enhancement of this system. Based on it, a sensitive method for determination of nucleic acids at ngml(-1) is established.     

Increased sensitivity for Coomassie staining of sodium dodecyl sulfate-polyacrylamide gels using PhastSystem Development Unit

Staining of proteins in PhastGel gradient media with Coomassie Blue R 350 was considerably improved using a lower concentration of methanol (10% v/v) and 2% ammonium sulfate in the staining solution and 10% acetic acid for destaining. The detection limit in sodium dodecyl sulfate-polyacrylamide gels was lowered by a factor of 10 to about 2 ng per protein band. The Coomassie staining method was adapted to the newly developed silver staining procedure so that both can be used in parallel in PhastSystem.     

Ultrasensitive polychromatic silver staining of sodium dodecyl sulfate-polyacrylamide gels with the Gelcode system using the PhastSystem Development Unit

The Gelcode color-based silver staining system, an improved formulation based on the original publication by Sammons et al. (Electrophoresis 1981, 2, 141-147) has been adapted to automated rapid staining in the PhastSystem Development Unit. The use of elevated temperatures in the fixation, washing, staining, and stabilization steps of the protocol reduces the total time of the process from 18 h to 1 h. The limit of detection, which is at least tenfold more sensitive than the silver staining protocol recommended for the PhastSystem, corresponds to 0.05-0.1 ng of protein per band. The method is applicable to both one- and two-dimensional polyacrylamide gels.     

A 2,7-carbazole-based dicationic salt for fluorescence detection of nucleic acids and two-photon fluorescence imaging of RNA in nucleoli and cytoplasm

A new carbazole-derived dicationic compound, namely 2,7-bis(1-hydroxyethyl-4-vinylpyridinium iodine)-N-ethylcarbazole (2,7-9E-BHVC), with a large two-photon action absorption cross section in nucleic acids has been obtained. Moreover, it possesses the potential of imaging RNA in nucleoli and cytoplasm in two-photon fluorescence microscopy and exhibits good counterstain compatibility with the commercial fluorescent nucleic dye DAPI.