DNA staining in agarose gels with Zn²+-cyclen-pyrene

A pyrene-labeled Zn2+-cyclen complex for the staining of DNA in agarose gels is reported. The metal chelate coordinates reversibly to the DNA phosphate backbone, which induces the formation of pyrene excimers. The typical pyrene excimer emission is used for the detection of the DNA. Staining is limited to agarose gels and is less sensitive than ethidium bromide, but DNA amounts as low as 10 ng and short DNA strands (～300 b.p.) are detectable. Gel extraction as a standard technique in molecular biology was successfully performed after staining with Zn2+-cyclen-pyrene. Cytotoxicity tests on HeLa and V-79 cells reveal that the zinc-cyclen pyrene probe is significant less toxic compared to ethidium bromide.     

Optimization of PCR protocol in microsatellite analysis with silver and SYBR® stains

Here we present the optimization of PCR conditions for microsatellite analysis of coniferous trees. The use of touchdown protocol for annealing resulted in a high success rate for optimization using fewer temperature profiles. The use of SYBR^￠ Green gel stain to detect PCR products in agarose gels was more sensitive than ethidium bromide. This is valuable for determining the success of PCR reactions and estimating the amount of PCR products formed—which is crucial in determining the dilution required to produce bands of similar intensity upon silver staining of the polyacrylamide gels. The use of SYBR^￠ Gold for staining polyacrylamide gels was not satisfactory in terms of the image quality produced. However, it was comparable to silver staining in terms of sensitivity, and could possibly be used in cases where the products are present as sharp single bands. In those cases, the use of SYBR^￠ Gold gel stain would save time and money for staining polyacrylamide gels.     

Comparison of ethidium bromide and 4′,6-diamidino-2-phenylindole as quantitative fluorescent stains for DNA in agarose gels

Two methods of quantitative, fluorescent detection of DNA bands in agarose gels separated by electrophoresis were evaluated for sensitivity and linearity of response. Comparisons of ethidium bromide staining with a method using 4′,6-diamidino-2-phenylindole (DAPI) developed in this work showed that DAPI is several times more sensitive than ethidium bromide in conditions of comparable background flourescence. Optimum flourescent staining and detection conditions for DNA bands in agarose gels using DAPI are desctribed, and advantages of this method over other fluorescent detection methods are discussed.     

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb¹. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits². During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel''s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight³. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along⁴. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation⁵; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments       

DNA staining in agarose and polyacrylamide gels by methyl green

ABSTRACT

Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G? (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.     

The interaction of platinum complexes with nucleosomes investigated with fluorescent probes

The fluorescent probes, N-(3-pyrene)maleimide, which is specific for histone H3, and terbium (Tb³⁺), which is specific for guanine single-stranded residues in DNA, are used to investigate the interaction of platinum complexes (cis- and trans-dichlorodiammineplatinum(II)) with rat liver and calf thymus nucleosomes. At low concentrations of the drug, lower than most of those reported previously in studies investigating the interaction of the drugs with isolated DNA, N-(3-pyrene)maleimide studies show that profound modifications occur near or in the cysteinyl binding site of histone H3. H3 dimer formation appears to be the cause of the change induced by trans-DDP; however, the effects observed with the cis-isomer do not seem to be correlated with dimer formation. At short incubation times, Tb³⁺ fluorescence shows small changes in DNA conformation, but they are slight when compared to the effect observed with proteins at the same length of incubation. SDS-polyacrylamide gels indicate some changes in protein composition, and agarose gels display a decrease in ethidium bromide staining of the cis-treated DNA. The results suggest that the protein portion, predominantly histone H3, as well as DNA are targets for the platinum derivatives in the nucleosome.     

Interaction of 2‐Chloro‐N10‐Substituted Phenoxazine with DNA and Effect on DNA Melting

Five N¹⁰‐substituted phenoxazines having different R groups and –Cl substitution at C‐2 were found to bind to calf –thymus DNA and plasmid DNA with high affinity as seen from by UV and CD spectroscopy. The effect of phenoxazines on DNA were studied using DNA‐ethidium bromide complexes. Upon addition of phenoxazines, the ethidium bromide dissociated from the complex with DNA. The binding of phenoxazines to plasmid _PUC18 reduced ethidium bromide binding as seen from the agarose gel electrophoresis. Butyl, and propyl substituted phenoxazines were able to release more ethidium bromide compared with that of acetyl substitution. Addition of phenoxazines also enhanced melting temperature of DNA.     

Simultaneous electroelution and concentration of DNA fragments from agarose gels

A rapid and inexpensive method for the electroelution of DNA fragments from agarose gels is described. DNA fragments were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Selected DNA fragments were placed into electroeluter tubes capped with dialysis membrane and electroeluted into a small volume of buffer using a conventional horizontal gel electrophoresis apparatus. The method successfully eluted and concentrated DNA fragments with molecular weights ranging from 2.7 to 13.9 MDa in 3 h.     

Ultrasensitive staining of nucleic acids with silver

A method for ultrasensitive detection of proteins on polyacrylamide gels by staining with silver, recently described by C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert (Science211, 1437–1438 (1981)), was applied with slight modifications to staining nucleic acids. Silver staining of double-stranded DNA was at least 100 times as sensitive as fluorescence staining with ethidium bromide, and at least 20 times as sensitive as staining with ammoniacal silver. The limit of detection of double-stranded DNA was approximately 25–50 pg/band with a cross-sectional area of 5 mm². The intensities of silver staining of double-stranded fragments 271 bp or longer from HaeIII endonuclease digests of φX174 RF DNA were linear over a concentration range of 0.25 to 4 ng DNA/band. RNA and single-stranded DNA species as short as 10 to 20 nucleotides were detected with high sensitivity after electrophoresis on denaturing gels containing urea, suggesting that silver staining may be applicable to the sequencing of a few micrograms of unlabeled DNA. Methods for staining DNA using ammoniacal silver were relatively insensitive for small DNA fragments.     

Characteristics of a PCR-Based Assay for In Planta Detection of Xanthomonas campestris pv. pelargonii

Polymerase chain reaction (PCR) amplification was carried out with a primer pair targeting a sequence in the genome of Xanthomonas campestris pv. pelargonii , the causative agent of bacterial blight in geraniums. PCR amplification with the primer pair XcpMl/XcpM2 using total nucleic acid preparations from 22 geographicallydiverse isolates of X. campestris pv. pelargonii generated a major 197 bp DNA product. In contrast, no major amplification products were consistently generated from 12 other pathovars of X. campestris or from 19 isolates representing 10 different plant pathogenic bacteria, including two other bacterial pathogens of geraniums, Corynebacterium fascians and Pseudomonas cichorii . After PCR using this primer pair, between 1380 and 13800 copies of the X, campestris pv. pelargonii bacterial DNA target as template were detected by ethidium bromide staining of agarose gels, and between 13.8 and 138 copies by blot hybridization to a pathovar-specific biotinylated probe. Similarly, between 630 and 6300 colonyforming units (CFU) of X. campestris pv. pelargonii could be detected after ethidium bromide staining of agarose gels, and between 63 and 630 CFU after blot hybridization. The PCR-based assay was used to identify X. campestris pv. pelargonii in diseased geraniums; whereas discrete amplification products were not obtained with healthy plants.     

Dye titrations of covalently closed supercoiled DNA analyzed by agarose gel electrophoresis.

We have developed a rapid electrophoretic technique for performing ethidium bromide dye titrations in cylindrical 0.7% agarose gels. The technique was used to analyze the extent of supercoiling in circular covalently closed SV40, Co1E1, and pSC101 DNA. We have estimated the superhelical densities of SV40, Co1E1, and pSC101 DNA to be ?0.050, ?0.078, and ?0.085 respectively. The results obtained for native SV40 DNA correlate well with previously published values for the superhelical density of this DNA when these values are corrected to reflect a 26° duplex unwinding angle for ethidium bromide. Ethidium bromide concentrations sufficient to partially relax a supercoiled DNA allow the DNA to be resolved into a series of discrete bands in agarose gels. The distribution of bands represents a natural heterogeneity in the superhelical densities of the DNA molecules in the population.     

A novel procedure for simple and efficient genotyping of single nucleotide polymorphisms by using the Zn2+–cyclen complex

The analysis of single nucleotide polymorphisms (SNPs) is increasingly utilized in the study of various genetic determinants. Here, we introduce a simple, rapid, low-cost and accurate procedure for the detection of SNPs by polyacrylamide gel electrophoresis (PAGE) with a novel additive, the Zn²⁺– cyclen complex (cyclen = 1,4,7,10-tetraazacyclododecane). The method is based on the difference in mobility of mutant DNA (in the same length) in PAGE, which is due to Zn²⁺–cyclen binding to thymine bases accompanying a total charge decrease and a local conformation change of target DNA. Various nucleotide substitutions (e.g. AT to GC) in DNA fragments (up to 150 bp) can be visualized with ethidium bromide staining. Furthermore, heteroduplex and homoduplex DNAs are clearly separated as different bands in the gel. We demonstrate the analysis of single- and multiple-nucleotide substitutions in a voltage-dependent sodium channel gene by using this novel procedure (Zn²⁺–cyclen–PAGE).     

Determination of plasmid copy number by fluorescence densitometry

A simple and reliable method for the determination of plasmid copy numbers by direct fluorescence densitometry of ethidium bromide-stained electrophoretic gels was developed. In developing the method, the following parameters were evaluated and controlled: plasmid DNA trapping in the linear chromosomal DNA, staining-destaining kinetics for ethidium bromide, linearity of the fluorescence response, and the effect of the molecular topology of DNA on ethidium bromide binding to DNA in agarose.     

Behavior of bacteriophage Mu DNA upon infecton of Escherichia coli cells.

The question whether the ends of bacteriophage Mu DNA are fused to form a ring in host cells is critical to the understanding of the mechanism of integrative recombination between Mu DNA and host DNA. We have examined the fate of ³²P-labeled Mu DNA, after infection of sensitive and immune (lysogenic) cells, by sedimentation in sucrose gradients, ethidium bromide/CsCl density centrifugation and by electrophoresis of parental Mu DNA and its fragments in agarose gels. We find that the parental Mu DNA cannot be detected as covalently closed circles at any stage during the Mu life cycle. An interesting form of Mu DNA can be seen after superinfection of immune cells. This form sediments about twice as fast as the mature phage DNA marker in neutral sucrose gradients but yields linear molecules upon phenol extraction. Upon infection of sensitive cells, most of the parental DNA associates with a large complex, presumably containing the host chromosome. When Mu-sensitive cells are infected with unlabeled Mu particles and Mu DNA examined at different times after infection by fractionation in 0.3% agarose gels and hybridization with ³²P-labeled Mu DNA, Mu sequences are found to appear with the bulk host DNA as the phage lytic cycle progresses. However, no distinct replicative or integrative intermediate of Mu, that behaves differently from linear Mu DNA and is separate from the host DNA, can be detected.     

Detection of subnanogram amounts of RNA in polyacrylamide gels in the presence and absence of protein by staining with silver

The new ultrasensitive photochemically derived silver stain described for polypeptides in polyacrylamide gels (Merril et al., Science211, 1437–1438 (1981)) also stains nucleic acid in polyacrylamide gels. Reovirus genome double-stranded (ds) RNA segments were clearly detected in gels at about 0.03 ng/mm² with the silver staining technique when either purified virions or isolated, purified dsRNA was analyzed. The silver stain was about 10 to 30 times more sensitive than ethidium bromide for detecting reovirus dsRNA.     

A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)₃²⁺)-end-labelled primers. In this way, biotin for capture and Ru(bpy)₃²⁺ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)₃²⁺-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.     

A highly sensitive technique for staining DNA and RNA in polyacrylamide gels using silver

A technique is described for staining DNA in polyacrylamide gels with silver. It is rapid, requiring about 30 min for whole staining and development procedure, very simple and at least 20 times more sensitive than ethidium bromide for the staining of double-stranded DNA in polyacrylamide gels. This technique can also be applied for the staining of denatured, single-stranded DNA as well as RNA after their electrophoretic separation on polyacrylamide gels, having the same sensitivity as for double-stranded DNA fragments.     

Covalent binding of ethidium azide analogs to Salmonella DNA in vivo: competition by ethidium bromide.

The photoreactive analogs of ethidium bromide (ethidium mono- and diazide) have been developed as drug probes to determine the actual molecular details of ethidium bromide interactions with DNA. In an effort to demonstrate that the analogs in fact mimic the parent ethidium, competition experiments were designed using ³H thymidine-labeled DNA in intact $\text{Salmonella}$ TA1538, which is reverted by the azide analogs. ¹⁴C-labeled ethidium azide analogs were used in combination with the non-labeled ethidium bromide. The results presented here demonstrate that the parent ethidium competes with the azide analogs as a DNA intercalating drug using CsCl density gradient ultracentrifugation.     

A reappraisal of the role of Zn2+ in TGF-β1-induced apoptosis in primary hepatocytes

There is now a wealth of information regarding the apoptotic mode of cell death and its importance in toxicological studies in many mammalian organs including the liver. In this study, we investigated the modulatory effects of the heavy metal Zn²⁺ on transforming growth factor-β₁ (TGF-β₁)-induced apoptosis in primary rat hepatocytes. Apoptosis induced by TGF-β₁ (1 ng/ml) in hepatocytes was accompanied by nuclear condensation as assessed morphologically by staining with Hoechst 33258 and DNA cleavage as detected biochemically by in situ end-labeling, field inversion and conventional gel electrophoresis. Pretreatment with 100 μmol/L Zn²⁺ abrogated the nuclear condensation, in situ end-labeling, and DNA laddering in TGF-β₁-treated hepatocytes. Surprisingly, Zn²⁺ did not inhibit the formation of high-molecular-weight DNA fragments (30–50 kbp to 250–300 kbp). These data provide evidence that Zn²⁺ exerts its effects on the endonucleases that act downstream in the execution phase of TGF-β₁-induced apoptosis in hepatocytes. This revised version was published online in July 2006 with corrections to the Cover Date.