Extraction of nucleic acids from agarose gels

A rapid, simple and versatile method is described for the extraction from agarose gels of small plasmid molecules and DNA fragments generated by restriction endonucleases. The method may be used also for the extraction of RNA from agarose-urea gels. It is based on the partitioning of nucleic acid molecules into 1-butanol as their quaternary ammonium salts, leaving the neutral agarose in the aqueous phase. The nucleic acid is then recovered as the sodium salt by partition back into an aqueous phase. Nucleic acid samples were found to be unaffected by the treatment, as judged by their ability to be ligated, transformed, nick-translated, and used in an in vitro protein-synthesizing system.     

Hybridization of nucleic acids directly in agarose gels

Nucleic acids, both DNA and RNA, separated on agarose gels can be visualized by direct hybridization of the dried gel with appropriate radioactive probes. This method does not involve the transfer of the nucleic acid from the gel. The method requires less manipulation than other procedures; it is extremely rapid, sensitive, and inexpensive. These attributes make this procedure a valuable alternative or supplement to the commonly used methods for visualization by hybridization of nucleic acids separated on agarose gels.     

The improved method in agarose gel electrophoresis of nucleic acid

In most molecular experiments, nucleic acids are subjected to agarose gel electrophoresis to determine the size of the molecule. The addition of a nucleic acid dye allows the nucleic acid to be detected under the UV image system after running the gel, so the nucleic acid dye is an integral part of the electrophoresis experiment. But when considering the mutagenicity and toxicity of nucleic acid dyes, one must be careful to insure the proper disposal of experimental waste. In this article, a new usage of nucleic acid dye in agarose gel electrophoresis is described where the nucleic acid dyes were added to the loading buffer and nucleic acid marker buffer. The results show that this method has advantages as: a smaller amount of dye can be used, there is less time in contact with the dye, and its operation is easier and reduces toxicity damage. Also the bands showed a much clearer image, having a lower background value. The improved method shows better results with lower toxicity and is superior to the traditional method.     

Isotachophoresis of nucleic acid constituents

Determination methods for purine and pyrimidine bases, nucleosides, nucleotides and related compounds using analytical capillary isotachophoresis are reviewed. First, the isotachophoretic characterization of these compounds, as well as methods for sample preparation prior to analysis, and the different ways of detecting unknown substances in complex biological systems are described. Then applications of isotachophoretic analysis in medical diagnosis and biomedical research are reviewed. In particular, the analysis of purines, pyrimidines, nucleosides and related compounds in blood and serum for the diagnosis and treatment of inherited diseases and cancer is described. Selected applications of nucleotide analysis in biomedical research using different tissue extracts are also reviewed, and some examples of nucleotide-dependent enzymic reactions, which were performed by means of analytical isotachophoresis, are presented.     

Rapid electroelution of nucleic acids from agarose and acrylamide gels

The alkaline/filter DNA elution technique measures single-strand DNA breaks in mammalian cells based on the DNA molecular weight dependent retention of the macromolecule on 2-μm-pore-size filters. Described here is a modification of the technique which uses [³H]thymidine-labeled DNA of γ-irradiated cells as an internal reference. Thus, an increased precision is obtained in the assessment of this type of DNA damage at biologically significant radiation doses (i.e., where cell survival occurs). The measure of DNA damage is based on the actual initial DNA elution rate, i.e., arithmetic ratio of the elution of “test” DNA (i.e., ¹⁴C-labeled DNA) relative to the elution of “reference” DNA (i.e., ³H-labeled DNA). The repair of this damage on postirradiation incubation of the cells is detected as a decrease in the rate of “test” DNA cluted relative to “reference” DNA from unincubated cells. For Chinese hamster V79–171 cells irradiated with 5 Gy (500 rads), repair can be resolved into two first-order processes having rate constants (at 24°C) of ～0.190 and ～0.017 min^?1.     

Electrophoretic elution of nucleic acids from acrylamide and agarose gels

A simple method for electrophoretic elution of nucleic acids from gel slices is described. The procedure utilizes a standard tube gel system and can be completed in as little as one hour. Nucleic acids are recovered in a small volume with almost 100% efficiency. The procedure is applicable equally to acrylamide and agarose gels, and small as well as large RNA and DNA molecules. The eluted nucleic acids are essentially undegraded and are suitable for a variety of structural and biological analyses.     

Two-dimensional agarose gel electrophoresis without gel manipulation

The apparatus and procedure to perform two-dimensional agarose gel electrophoresis without manipulating the gel used for the first electrophoresis (first-dimension gel) have been developed. The procedure is less complex, less damaging to first-dimension gels, and more precise than procedures that require manipulation of the first-dimension gel. When combined with gel-embedding techniques, the procedure presented can be used to perform the second electrophoresis in a gel different from the first-dimension gel. A first-dimension gel too dilute to be manipulated and a more concentrated gel for the second electrophoresis have been used to separate DNA open circles from a mixture of variable-length linear DNAs.     

Extraction of nucleic acids from agarose gel--a quantitative and qualitative comparison of four different methods

Agarose gel electrophoresis is commonly used to separate different species of nucleic acids. We compare four different methods of extraction which are commonly used. These methods include buffer extraction, electroelution, glass bead extraction and extraction of DNA from low-melting agarose. The results show that DNA extracted by these four methods is comparable in their ligability to the PMT 21 vectors and the plasmids with insert can be used for subsequent transfections of competent bacteria. There is a higher yield for buffer extraction and electroelution when compared with glass bead extraction and low melting agarose (p less than 0.05). To conclude, the four commonly used methods for DNA isolation are comparable qualitatively. But the simplest method, namely buffer extraction, has the highest yield.     

The agarose double helix and its function in agarose gel structure

Agarose and eight different derivatives carrying O-methyl, O-sulphate, O-hydroxyethyl or O-carboxyethylidene substituents in various positions were studied by optical rotation, X-ray diffraction and computerised molecular model building methods. All samples showed essentially the same order-disorder transition during gel-sol interconversion. In addition, all the samples that could be made into oriented films or fibres gave X-ray diffraction diagrams corresponding to a common molecular structure. A double helix model for this structure is proposed that has the 0.95 nm axial periodicity observed and a calculated cylindrically averaged Fourier transform in good agreement with the observed (continuous) layer line intensities. Each chain in the double helix forms a lefthanded 3-fold helix of pitch 1.90 nm and is translated axially relative to its partner by exactly half this distance. This model accounts for the sign and magnitude of the optical rotation shift that accompanies the sol-gel transitions and is sterically accessible to each of the various substituted forms. The relationship between agarose gel properties and the double helix is discussed and the structure compared with i-carrageenan.