Separation of Nucleic Acids

Advances in the application of pulsed electric fields for the separation and analysis of large macromolecules use of multidimensional electric fields for enhanced separation of nucleic acids... 

A wide variety of bases, nucleosides and nucleotides have been separated using porous layer bead ion exchangers. A representative chromatogram of the separation of ribonucleoside mono-phosphoric acids from the work of Smukler ( ) is shown in Figure 4. Recently, ion exchangers chemically bonded to small particle diameter (> 10 ym) silica have been successfully applied to the separation of nucleic acid constitutents (37). The rapid separations using such supports undoubtedly mean that they will find increasing use in the future. 

Kasai, K., Size-dependent chromatographic separation of nucleic acids, /. Chromatogr., 618, 203, 1993. 

Separation of proteins Separation of nucleosides Separation of nucleic acid bases and their nucleosides... 

Cadet J, Voituriez L, Berger M (1983) Separation of nucleic acid components and their radiation-induced degradation products on chemically bonded C12 reversed-phase thin-layer plates. J Chromatogr 259 111-119... 

In summary the deleterious effects of alkali on the proteins and the large-scale impracticality of the heat-shock (endogeneous ribonuclease) process, plus the accompanying proteolysis and denaturation of proteins, clearly indicate the need for better methods to facilitate the large-scale separation of nucleic acids from yeast proteins. 

As mentioned before, a number of these methods are discussed at length in this book (see Chapters 2-6). The methodologies for separation of nucleic acids, oligonucleotides, and monoclonal antibodies are covered in Chapters 8, 14, and 15. The following chromatographic methods, as they relate to separations of monoclonal antibodies, are discussed in Chapter 15 ... 

Table 4-5 The Effective Separation Range of Agarose Gels of Various Composition for Separation of Nucleic Acids ...

The separation of nucleic acids by CE has become a steadily growing area of interest, especially since the inception of the Human Genome Initiative. This interest originally stemmed from the use of polyacrylamide (PA) or agarose slab gel electrophoresis as the accepted standard for nucleic acid separation (Stellwagen, 1987). 

Capillary electrophoretic separation of nucleic acids has been reviewed (Cohen et al., 1987a Kuhr, 1990 Gebauer and Thormann, 1991). In nucleotide and nucleoside analysis, MEKC has been the method of choice, using SDS (Row et al. 1987 Cohen et al., 1987b Kasper et al., 1988), dodecyltrimethylammonium bromide, or hexadecyltriethylammonium bromide (Liu et al., 1989). Other applications concerning chemically modified nucleotides, nucleosides, and nucleobases can be found in papers by Lecoq et al. (1991) and Thormann et al. (1992). 

IPC on non-porous alkylated PS-DVB particles provided high resolution separation of nucleic acids with very short analysis times [32]. Due to its intrinsic pH stability, a PS-DVB monolith was used to bring pH into play for selectivity adjustment in separation of the peptide mixture. Since selectivities were significantly different with acidic and alkaline eluents, a two-dimensional IPC at high pH and at low pH was successfully performed [33]. 

Nesterenko, E.P. et al. Separation of nucleic acid precursors on an amphoteric surfactant modified monolith using combined eluent flow, pH and concentration gradient. J. Sep. Set 2007, 30, 2910-2916. 

Dickman, M.J. Effects of sequence and structure in the separation of nucleic acids using ion pair reverse phase liquid chromatography. J. Chromatogr. A 1076, 83-89 (2005)... 

Gel electrophoresis can be used in semi-preparative separations of nucleic acids, with either agarose or acrylamide as matrix. The DNA or RNA band of interest is first located on the gel, usually by staining, and a piece of gel containing the band is excised. DNA can be extracted from the gel by various methods, either by electroelution in a dialysis bag, or by transfer of the DNA to a DEAE cellulose membrane. Alternatively, if the gel separation is carried out using low melting agarose, the piece of gel can be warmed to melt it, and the freed DNA can be adsorbed onto matrices such as DEAE cellulose, or fine glass beads, from which it can be released after... 

Scott et al. [4] applied the technique to the separation of nucleic acid bases and an example of their results is shown in figure 8. A solution of uracil, hypoxanthine, guanine and cytosine was used as the mobile phase and the upper chromatogram shows a peak for cytosine that was present 0.4 mg/ml in excess of the concentration in the mobile phase. As the charge was 600 pi the peak represents a total excess mass of 0.24 pg. No other peaks are shown, as the other solutes are present in the sample in the same concentration as the mobile phase. 

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