Metin Colpan

High-performance liquid chromatography of high-molecular-weight nucleic acids on the macroporous ion exchanger, nucleogen

Abstract The application of Nucleogen★, a series of novel silica gel-based anion exchangers of different pore size in the range 60–4000 A, to analytical and large-scale high-performance liquid chromatography of a wide variety of nucleic acids is described. The influence of pore size, pH, temperature, eluting salts, bivalent metal ions, denaturing reagents and flow-rate on resolution, purity and capacity was studied. High resolution was obtained for oligonucleotides up to a chainlength of 37, natural RNSs from tRNS up to phage RNA (molecular weight 1,000,000), double-stranded RNA and DNA fragments, and supercoiled plasmids from a crude cell lysate. Recovery was over 95%; the isolated nucleic acids were pure according to gel electrophoresis, physico-chemical criteria, and activity in enzymatic tests.

Large-scale purification of viroid RNA using Cs2SO4 gradient centrifugation and high-performance liquid chromatography

A procedure for the purification of viroid RNA from tomato plants is described which yields up to a milligram of viroid RNA of gel electrophoretic homogeneity within 2 days. This technique is at least three times as fast as previous methods and is generally applicable to other RNA species. Plant material was homogenized and phenol extracted. In a Cs2SO4 density gradient, viroid RNA together with low-molecular-weight RNA, was separated from large single-stranded RNA, DNA, polysaccharides, polyphenols, and other compounds. The separation is based on the differences in the buoyant density and on the selective precipitation of large single-stranded RNA in Cs2SO4. Further purification of viroid RNA was achieved by HPLC over a weak anion exchanger linked to silica gel of optimized pore size. The elution was carried out by a salt gradient with complete exclusion of divalent metal ions. The procedures were applied to whole plants, leaves, stems, roots, cells, and protoplasts. The yields of nucleic acids at the different steps of purification are given for leaves, stems, and roots.

High-performance liquid chromatography of dna restriction fragments

High-performance liquid chromatography on Nucleogen-DEAE 4000-10 has been applied to several problems of the isolation of DNA restriction fragments. Large amounts of DNA fragments of high purity are necessary for biophysical studies and for molecular hybridization in basic research, as well as in medical diagnosis. The influence of various parameters, such as buffer, pH, eluting salt, gradient slope, flow-rate and the addition of urea on the resolution of fragments by high-performance liquid chromatography were studied on an analytical scale, and the optimal conditions were then used for the large-scale preparation of milligram amounts. The best resolution of fragments between 25 and 1500 base pairs was obtained with a linear gradient from 500 mM to 1200 mM sodium chloride in 6 M urea -30 mM sodium phosphate (pH 6.0). Quantitative data are given for the purity and recovery of the sample, and the capacity and lifetime of the column. The following applications of high-performance liquid chromatography of restriction fragments are described: preparation of 2 mg of fragments, separation of 1 mg of DNA insert from 7 mg of its plasmid vector, and analysis of DNA-RNA hybrids.

Dynamics and Interactions of Viroids

Viroids are single stranded circular RNA molecules of 120,000 daltons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure. The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.

A microcell for the temperature-jump technique☆

Abstract The volume of the sample in the temperature-jump technique was reduced from 1.2 ml to 80 μl. This was achieved by a simple modification of a commercially available cell. The sample is placed in a small chamber which is enclosed within a larger chamber containing buffered agarose gel. Sample and surrounding gel are heated simultaneously by discharge. Experimental examples in which the denaturation of tRNA and of viroid-RNA was measured are given. The signal-to-noise ratio is as high as in the conventional cell, problems of high-voltage arcing and solution cavitation have not been observed, and the optical signal can be recorded longer than a second without being influenced by recooling.

Chapter 4 High Performance Liquid Chromatography of Nucleic Acids

Publisher Summary Isolation of altogether different but well-defined nucleic acids is of major interest in present-day biochemistry, biophysics, molecular biology and gene technology. For example, in biophysical chemistry of nucleic acids large amounts of homogeneous nucleic acid material are needed. The requirements range from synthetic oligonucleotides with defined sequences which are used to determine the elementary parameters of nucleic acid structure and stability, up to large natural nucleic acids such as plasmids which are the subject of studies about supercoiled structures and nucleic acid topology. For biochemical studies on RNA, e.g. tRNA, viroid RNA or viral RNA, highly purified fractions of these RNAs from natural sources have to be prepared. Often, one is confronted with the difficulty of those RNAs being present in the cell together with a vast excess of other, sometimes quite similar, nucleic acids. Other new applications of HPLC have emerged from recent developments in molecular biology and gene technology. Routinely, for the different steps of the cloning and sequencing procedures synthetic oligonucleotides, DNA fragments and plasmids must be prepared in great purity. Purity alone, however, does not suffice as a specification; in addition, they are supposed to be highly active in a variety of enzymatic reactions and biological transformations. Another new field of HPLC of nucleic acids arises in diagnostic applications for medicine and phytopathology. In these new areas of application too, highly purified probes of recombinant DNA or RNA will be required, e.g. for testing for infectious diseases or for genetically determined disorders.