Randolph L. Rill

Ultrasensitive staining of nucleic acids with silver.

Abstract 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 ( Science 211 , 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 mm 2 . The intensities of silver staining of double-stranded fragments 271 bp or longer from Hae III 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.

Electron microscopy of liquid crystalline DNA: direct evidence for cholesteric-like organization of DNA in dinoflagellate chromosomes

Freeze-fracture-etch replicas of concentrated DNA solutions which appeared, by polarized light microscopy, to be in a cholesteric-like liquid crystalline state were examined by high resolution transmission electron microscopy (TEM). Individual DNA molecules were resolvable, and the microscopic morphologies observed for such replicas confirmed the cholesteric organization of DNA molecules in this liquid crystalline state. Furthermore, replica morphologies were strikingly similar to TEM images of dinoflagellate chromosomes in both thin section and freeze-etch replicas, providing strong support for the cholesteric DNA packing model proposed for the organization of DNA in these chromosomes by Bouligand and Livolant.

Ordered phases in concentrated DNA solutions

Aqueous solutions of concentrated DNA, a strong polyelectrolyte, in 1:1 electrolyte form liquid-crystalline phases analogous to those observed for other semi-rigid neutral polymers and weak polyelectrolytes. Phase transitions were examined in detail for DNA fragments with a contour length (500 A) approximating the persistence length. These fragments form at least three lyotropic phases. The lowest density or “precholesteric” phase appears to be a nematic with a slight and easily variable twist. The intermediate density phase is a true cholesteric with a pitch of ≈2.1 μm. With increasing concentration the cholesteric phase unwinds prior to formation of the third, high density, columnar phase. Phase transition boundaries were determined as functions of DNA concentration from 10 to 300 mg/ml solvent, supporting electrolyte concentration from 0.01 to 1.0 M, and temperature from 20°C to 60°C. Critical concentrations for formation of anisotropic phase (Ci) and disappearance of isotropic phase (Ca) were only moderately dependent on temperature. Ci, Ca, and the pitch of the cholesteric phase were surprisingly insensitive to the supporting electrolyte concentration. The insensitivity can be most simply related to the high concentrations required for anisotropic phase formation by these rather short, highly charged rod-like DNA fragments. At high concentrations the DNA counterions contribute significantly to the effective ionic strength, hence overall charge screening, and the counterion atmosphere as monitored by 23Na NMR, appears to be perturbed by inter-rod interactions.

Chapter 6 Isolation and Characterization of Chromatin Subunits

Publisher Summary This chapter discusses methods used for isolation and characterization of chromatin subunits. Most reported physical studies of chromatin subunits have used more or less refined preparations of core particles. The particles are easily prepared in quantity because of their nuclease resistance and can be resolved to a fairly high degree of homogeneity. Studies of the entire repeating unit containing 200 base pairs of DNA have been inhibited by the fact that these structures are observed as intact entities only in the initial stages of digestion, and even then exhibit a rather broad heterogeneity in DNA size. This heterogeneity presumably results from the possibility that the first cleavage within spacer regions can occur at random over a range of sites and may be compounded by heterogeneity of spacer lengths. The techniques described in the chapter are employed for the isolation of core particles. If digestion conditions are carefully controlled, they can also be used for the preparation of small quantities of repeating units. Nuclease digestion of nuclei or chromatin with staphylococcal nuclease is discussed and isolation of core particle subunits and other nucleoprotein fragments is illustrated in the chapter.

DNA electrophoresis in agarose gels: A simple relation describing the length dependence of mobility

Electrophoretic mobilities of DNA molecules ranging in length from 100 to 10 000 base pairs (bp) were measured in gels of eleven concentrations of agarose from 0.5 to 1.5%. Excellent fits of the dependence of mobility on DNA length were obtained with the relationship

DNA electrophoresis in agarose gels: Effects of field and gel concentration on the exponential dependence of reciprocal mobility on DNA length

{1 \over {\mu \left( {L} \right)}} = {1 \over {\mu _l }} - \left( {{1 \over {\mu _l }} - {1 \over {\mu _s }}} \right)e^{ - L/\gamma }

Terraces in the cholesteric phase of DNA liquid crystals

showing an e–L/γ crossover, where L is the length of a DNA fragment and γ is a crossover length ranging from 8000 to 12 000 bp. The other parameters in the fit are νs the mobility of short DNA with unit charge in the limit as length is extrapolated to zero, and νl, the mobility of long DNA as length is extrapolated to infinity. This exponential relationship should be a useful interpolation function for determining DNA lengths over a wide range. The simplicity of this relationship may be of more fundamental significance and suggests that some common feature dominates the electrophoresis of double stranded DNA fragments in agarose gels, regardless of length.

Capillary gel electrophoresis of nucleic acids in pluronic F127 copolymer liquid crystals

Electrophoretic mobilities of DNA molecules ranging in length from 200 to 48 502 base pairs (bp) were measured in agarose gels with concentrations T = 0.5% to 1.3% at electric fields from E = 0.71 to 5.0 V/cm. This broad data set determines a range of conditions over which the new interpolation equation ν(L) = (β+α(1+exp(–L/γ))–1 can be used to relate mobility to length with high accuracy. Mobility data were fit with χ2 > 0.999 for all gel concentrations and fields ranging from 2.5 to 5 V/cm, and for lower fields at low gel concentrations. Analyses using so‐called reptation plots (Rousseau, J., Drouin, G., Slater, G. W., Phys. Rev. Lett. 1997, 79, 1945–1948) indicate that this simple exponential relation is obeyed well when there is a smooth transition from the Ogston sieving regime to the reptation regime with increasing DNA length. Deviations from this equation occur when DNA migration is hindered, apparently by entropic‐trapping, which is favored at low fields and high gel concentrations in the ranges examined.

Enrichment of transcribed and newly replicated DNA in soluble chromatin released from nuclei by mild micrococcal nuclease digestion

Near the transition to the columnar phase, the cholesteric liquid crystal phase in an aqueous solution of DNA fragments with contour lengths approximating the persistence length undergoes an unwinding of the cholesteric pitch. Unwinding of the cholesteric with planar alignment of the fragments was studied by polarized light microscopy. Terraces or ‘‘Grandjean planes’’ of cholesteric are seen as uniformly birefringent fields of distinct hues (typically blue), bounded by lines which moved as the local concentration of DNA increased. These lines are interpreted as disclination lines, bounding regions of different total twist, which move as the intrinsic pitch of the cholesteric varies with concentration.

Non-histone proteins of soluble nucleoproteins released from mouse myeloma nuclei by mild micrococcal nuclease digestion.

SummaryThe liquid crystalline gel phases of solutions of Pluronic F127, a triblock copolymer, were recently introduced as an alternative to disordered solutions of random coil polymers as replaceable media for capillary gel electrophoresis (CGE). Pluronic F127, from BASF, is a copolymer of poly(ethylene oxide) and poly(propylene oxide) with the approximate formula (EO)106 (PO)70 (EO)106. Polymer chains aggregate into spherical micelles in aqueous solutions, with poly(propylene oxide) chains creating a hydrophobic core surrounded by brushes of hydrated poly(ethylene oxide) tails. Crowding at high concentrations promotes ordering of micelles. Solutions in the range of about 14–24 % polymer are self-supporting, gel-like cubic liquid crystals at 25–30°C, but when cooled they become low viscosity liquids that are easily loaded into capillaries. This article reviews applications of Pluronic F127 media for capillary gel electrophoresis separations of nucleic acids of several types including oligonucleotides, double stranded DNA fragments, and supercoiled plasmid DNAs.