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.     

A refined method for silver staining of nucleic acids fixed on nitrocellulose

A refined silver staining method was developed to stain nucleic acids fixed onto nitrocellulose membranes and nylon-based membranes. Approximately 4 ng RNA or DNA can be stained with this method with no protein interference. This method involves simple repetition of immersions of membranes in three solutions prepared from common chemicals. The total staining time is less than 30 min.     

A dumbbell molecular beacon for the specific recognition of nucleic acids

A dumbbell molecular beacon (DMB) was designed and synthesized with the attachment of a fluorophore and a quencher at two ends. This DMB probe can be used to detect single mismatch of a 20mer oligodeoxynucleotide in two different buffers and discrimination factors were as high as 60 at 37°C. Statistics of single substitutions of analytes showed that both substituted positions and substituted nucleotides have important contributions for this probe to efficiently distinguish the true analyte from mismatched ones. Hybridization kinetics of DMB with the target oligonucleotide was also studied.     

A silver staining procedure for nucleic acids in polyacrylamide gels without fixation and pretreatment

Silver staining of nucleic acid has been widely used in molecular marker analysis such as simple sequence repeat (SSR), single-strand conformation polymorphism (SSCP), and amplified fragment-length polymorphism (AFLP). Many alternatives to silver staining methods have been described, but these methods are not efficient or cost-effective. Here we report a silver staining method that requires less than 10 min for one gel and can save chemicals as well. It has a detection limit of approximately 5.6 pg of DNA/mm² in nondenaturing polyacrylamide gels and 12.8 pg/mm² in denaturing polyacrylamide gels.     

Clip-phen conjugates for the specific cleavage of nucleic acids

For the first time Clip-Phen (1) was conjugated to oligonucleotides to provide very efficient tools for the cleavage of nucleic acids at specific positions. The synthesis of the conjugates as well as the cleavage experiments are reported.     

Cytochemical differentiation of nucleic acids with a Schiff-methylene blue sequence.

A method is described whereby a Feulgen type of hydrolysis of deoxyribonucleic acid is carried out on paraffin sections of routinely fixed tissues by controlled exposure of the sections to Bouin's fluid. Subsequent staining with Schiff reagent followed by methylene blue distinguishes red-to purple-stained deoxyribonucleic acid from blue-stained ribonucleic acid. This Schiff-methylene blue sequence visualizes ribonucleic acid in nucleoli and the chromidial substance of various normal and neoplastic cells and provides an assessment of their protein synthetic activity. The method has proved valuable in demonstrating normal immunocytes and immunoglobulin-forming tumor cells in pathologic specimens.     

Red-blue staining of hydrolysed nucleic acids in paraffin sections

A previous treatment with 10% HC1 in tetrahydrofuran for 2-3 min at 37° C hydrolyses DNA while substantially preserving RNA in formol-fixed paraffin sections. If this treatment is followed by dyeing with basic fuchsin-thiazine or oxazine mixtures, the basic fuchsin stains DNA, the blue dye cytoplasmic RNA, though nucleolar RNA is not well preserved. A specimen sequence is to treat the hydrolysed section with a mixture of 1% aqueous trimethylthionin (Chroma), 15 ml; 0.1% basic fuchsin (G. T. Gurr), 4 ml; and glacial acetic acid, 1 ml. Stain for 15-30 min, dehydrate in acetone, then pass sections through xylene to polystyrene. The specificity of this stain for cytoplasmic RNA is sharper than that of methyl green-pyronin; hence the technic given can be a useful addition to the standard Unna-Pappenheim procedure.     

A simplified method for staining mast cells with astra blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however; the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl2-6H2O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.     

Preparation of radioiodinated peptide nucleic acids with high specific activity

Peptide nucleic acids (PNAs) have stronger affinity and greater specificity than do oligonucleotides for binding to DNA and RNA and, as such, have potential utility as probes in molecular biology applications. In this study, a novel approach for labeling the PNA with radioiodine that avoided solubility issues and poor labeling encountered when trying to radioiodinate PNAs directly in solution was developed. For this approach, a purpose-designed prosthetic group that incorporated both a radioiodinatable tyrosine and a triphenylphosphonium (TPP) moiety was synthesized. The latter is an organic cation that combines the properties of good solubility in both aqueous and organic solvents with a strong retention by reverse phase HPLC. Following radioiodination of the TPP-based prosthetic group in phosphate buffer, the prosthetic group was purified and coupled to the terminal amine of 15-mer PNA on the solid phase resin. After cleavage and deprotection of the PNA from the resin, the presence of the TPP group resulted in a clean separation of radioiodinated PNA from unlabeled PNA, yielding a high-specific activity probe in a single HPLC run. As an example of a potential molecular biology application of the resultant (125)I-labeled PNA probe, it was used to detect mRNA for the Lcn2 gene in Northern blotting.     

Methyl green-pyronin; basis of selective staining of nucleic acids

1. Methyl green stains selectively highly polymerized desoxyribonucleic acid, and fails to stain, to any significant extent, depolymerized desoxyribonucleic acid and ribonucleic acid. 2. Pyronin stains preferentially low polymers of nucleic acid. 3. Triphenylmethane dyes with two amino groups appear to share the selectivity of methyl green. Those with three amino groups are not selective. 4. A stereochemical hypothesis is offered to account for these observations.     

A specific effect of copper on methylene blue sensitized photodegradation of nucleic acid derivatives

Among several metal ions tested, Cu²⁺ was unique in slowing down methylene blue sensitized photodynamic breakdown of some nucleic acid bases and nucleosides. The t_1/2 values were increased in the case of xanthine and uric acid by Cu²⁺, but without any alteration in the nature or amounts of photoproducts formed. Xanthine was degraded quantitatively to allantoin and urea. The breakdown of the sugar moiety of nucleosides was more drastically retarded than that of the purine ring. The decomposition rate and its magnitude was dependent on the concentration of Cu²⁺ as well as the nucleoside. The most profound increase in t_1/2 values was found with xanthosine—7 min for the purine ring and 65 min for the ribose moiety, at 0.6 mM Cu²⁺ Hg²⁺ in the case of xanthine, and some paramagnetic metal ions in the case of the nucleosides, slowed down the photobreakdown to a small extent; however, differential effects were not observed unlike with Cu²⁺. None of the other metal ions tested significantly influenced the process.