Principles and methods for the analysis and purification of synthetic deoxyribonucleotides by high-performance liquid chromatography.

The need for high-purity oligodeoxyribonucleotides for various applications has resulted in the development of novel synthesis, purification, and analytical techniques. A diversity of methods, including polyacrylamide slab gel electrophoresis, capillary gel electrophoresis, as well as high-performance liquid chromatography (HPLC), have been successfully used to aid in the characterization and isolation of these synthetic compounds. The information contained in this review article primarily details both the theoretical and practical aspects related to the use of HPLC for the analysis and purification of synthetic DNA. In addition, a variety of postsynthesis sample preparation protocols, commonly employed prior to and after HPLC, are described.

HPLC analysis of synthetic oligonucleotides using spectral contrast techniques.

The need for high-purity oligodeoxyribonucleotides for various applications has resulted in the development of novel synthesis, purification and analytical techniques. A diversity of methods, including polyacrylamide slab gel electrophoresis, capillary gel electrophoresis, as well as HPLC, have been successfully used to analyze material throughout the purification process. This study demonstrates how the application of spectral comparison techniques to synthetic products resolved by anion-exchange HPLC can distinguish deletion fragments (i.e., "N-1" sequences) from full-length products. Such analysis techniques can also differentiate partial from fully phosphorothioated DNA sequences. In combination, HPLC separation and spectral analysis technology provide information not obtainable with any other single analytical method.

Analysis of synthetic oligodeoxyribonucleotides by capillary gel electrophoresis and anion-exchange HPLC.

Polyacrylamide slab gel electrophoresis has been traditionally used for the analysis of synthetic oligodeoxyribonucleotides. As the demand for oligonucleotides of high purity has increased, a need for additional high resolution analytical techniques has arisen. This report describes the principles and application of capillary gel electrophoresis and high-performance liquid chromatography for the analysis of synthetic DNA varying in base composition and length. From the results of a series of comparative experiments, the relative advantages and disadvantages of each technique are discussed.

Growth of Legionnaires disease bacterium (Legionella pneumophila) in chemically defined medium.

A chemically defined medium containing 21 amino acids and inorganic salts was developed which supported the growth of four isolates of Legionnaires disease bacterium (Legionella pneumophila). Growth in liquid defined medium at 37 degrees C with shaking approximated the generation time and growth kinetics observed for growth in complex media. After a 3-h lag, the culture grew exponentially with a generation time of 6 h and reached a maximum optical density of 230 Klett units (170 Klett units corrected for pigment). A soluble brown pigment was first observed as the culture entered late exponential to early stationary phase of growth. Morphologically, L. pneumophila grew in the liquid defined medium with extensive filamentation and numerous intracellular lipid granuoles. L-Serine, L-methionine, and L-cysteine were required for optimum growth. The latter amino acid could be replaced by L-cystine or reduced glutathione but not by D-cysteine, thiomalate, thioglycollate, or 2-mercaptoethanol. Ferric iron was needed for maximum growth, but supplemental iron was not an essential growth requirement. Carbohydrates (i.e., glucose) or organic acids did not stimulate growth. In fact, pyruvate, acetate, and citrate all gave varying degrees of inhibition (69, 37, and 0% of control growth, respectively).

Magnitude of pollution indicator organisms in rural potable water.

A total of 460 water samples were randomly drawn from the potable water supply sources of rural communities in three counties of South Carolina. About 10% of the population, not incorporated in municipalities, was sampled. The samples were tested for total coliforms, Escherichia coli, and fecal streptococci. Significant levels of these pollution indicator organisms were detected in almost all the water supplies. Total coliforms were the most common, and only 7.5% of the water supplies were uncontaminated. E. coli, considered a reliable indicator of recent and dangerous pollution, was observed in 43% of the water supplies. Statistical analyses indicated that the bacterial populations, especially E. coli, were associated with the supply source depth and its distance from the septic tank. Total coliform counts were also weakly correlated to the pH of the water.

Binding of cholesterol by Neisseria gonorrhoeae.

The binding of [1,2-3H]cholesterol to Neisseria gonorrhoeae CS-7, Pseudomonas aeruginosa, and Salmonella typhimurium (smooth and rough strains) was investigated. The kinetics of cholesterol binding to N. gonorrhoeae CS-7 demonstrated that binding occurred slowly with maximum binding by 10 h. Under optimum conditions, a large percentage (65%) of the added cholesterol was associated with the cells. Chemical fractionation revealed that ca. 98% of the labeled cholesterol was associated with the cell membrane(s). The bound cholesterol was not esterified and was associated primarily with the cytoplasmic membrane. Intact gonococci bound 4 to 30 times more cholesterol than the deep rough mutant S. typhimurium TA1535, the wild-type S. typhimurium DB-21, and P. aeruginosa. In contrast, isolated cell membranes from all organisms rapidly bound cholesterol to the same extent. Therefore, the outer membrane can function as a permeability barrier to cholesterol. Cholesterol binding to both whole cells and isolated cell membranes was influenced by the incubation temperature. The rate of cholesterol binding by whole cells of N. gonorrhoeae decreased markedly at lower temperatures, with almost complete cessation of binding at 0 degrees C. A similar temperature effect on the binding of cholesterol to isolated membranes was not observed. Thus, the effect of temperature on the binding of cholesterol to whole cells was an effect not on the actual binding process but rather on the ability of the cholesterol molecule to penetrate the lipid domain of the gonococcal outer membrane.

Cortical potentials associated with the detection of visual events.

A positive-going potential, which reaches a maximum at the vertex and midline parietal scalp electrodes, occurs in the human being when an infrequent, significant event occurs in a continuously observed visual display. It is not time locked to eye movements or operant response and appears to be generated when the observer recognizes an event that he has been instructed to detect.

Potable water quality in rural Georgetown County.

Drinking water supplies of 161 rural communities, in Georgetown County, South Carolina, were randomly selected for sample collection. The analysis showed that most of the waters were slightly acidic. Low, but acceptable concentrations of chloride, copper, fluoride, sodium, cadmium, nitrate and phosphate were found. A few water samples showed higher then recommended levels of arsenic, mercury, zinc and lead. Although only 2% of the samples exceeded the mandatory limit of 0.05 ppm for arsenic, 72% exceeded the recommended level of 0.01 ppm. The mandatory limit for manganese was exceeded in 37% of the waters while 88% exceeded the limit for iron. The high iron content was generally responsible for the high turbidity found in 45% of the samples. The well depth and the consumer income had some bearing on water quality. Statistical evidence suggested that septic tank seepage was partially responsible for nitrate, phosphate, iron and arsenic contamination of shallow water supplies. Lead concentrations appear to vary according to the plumbing used and the pH of the waters.