Electrophoresis DEAE paper

A variety of powerful methods is available for fractionating short oligonucleotides. Work on nucleotide sequence analysis of small RNA molecules (n=80 or 120) and more recently viral RNA molecules (n= 3000) and even ribosomal precursor RNAs (up to 45 s) (Holley et al. 1965a Brownlee 1972 Maden et al. 1972) has stimulated the development of these procedures. They can also be used for synthetic oligonucleotides (e.g. Hachman and Khorana, 1969). The methods depend largely on chromatography and electrophoresis on filter papers, diethylaminoethyl(DEAE) paper, cellulose acetate, thin layers of cellulose or polyethyleneimine (PEI)-cellulose, or columns. 

Many of the fractionation problems can be overcome by the careful use of cellulose acetate and DEAE-paper electrophoresis (Brownlee 1972) but the methods are limited to radio-chemical quantities. 

Other useful pH values are pH 1.9 where fractionation depends mostly on the number of Up residues pH 3.5 where the four main ribonucleotides may be separated and higher pH values where differences between Ap and Cp can be exploited, although Rushizky et al. (1965) did not have much success at pH 4.0-4.4 with penta-to heptanucleotides. Degradation of purine nucleotides may occur at pH 1.9, although this is not observed on DEAE-paper electrophoresis and deamination of cytosine to uridine may occur at very high pH values. 

Electrophoresis is a possible procedure for fractionating sequence isomers since the resistance to motion will be affected by secondary structure and charge distribution as well as by any effect due to differences in total charge. This can be seen on cellulose acetate at pH 3.5 by the separation of the tetranucleotides Gp(Gp, Ap)Up for example (Sanger et al. 1965). Electrophoresis on DEAE-paper in 7% formic acid is particularly powerful, and admirably complements electrophoresis on cellulose acetate at pH 3.5. For example, ApGpGpUp is separated from Gp(Gp, Ap)Up (Brownlee 1972). 

Another way of dealing with an excised zone is described by Adesnik (1970), and has been adapted to strips of gel cut from broad slabs on which are run as much as 5 mg of RNA (Adams et al. 1969). The gel slice is coarsely chopped up and placed into a cylindrical tube 10 x 1 cm, one end of which is covered with DEAE paper, and over this a layer of cellophane attached to the tube with a rubber band. The tube is filled with buffer (0.04 M Tris acetate, pH 8.3) and placed in a disc electrophoresis apparatus. After electrophoresis the RNA is found trapped in the ion-exchange paper, from which it is eluted as before. 

Nucleobases and nucleosides [19] and mixtures of mononucleotides [15] can be separated by paper electrophoresis and oligonucleotides by two-dimensional paper electrophoresis-chromatography [80] or by two-dimensional electrophoresis on cellulose acetate and DEAE-paper [84]. Chromatographic and electrophoretic techniques are complementary in that compounds which cannot be fractionated suitably by the former can usually be completely separated by the latter. 

Fig. 4. — Monitoring of the Multiple Molecular Forms of Tomato Pectinesterase by Starch-gel Electrophoresis.98 [ENZ, detection of pectinesterase activity by paper print with pectin and Bromothymol Blue PROT, protein staining with nigrosin O, origin. Key A, 1 crude tomato extract after ammonium sulfate salting-out, and dialysis 2 pectinesterase fraction from column of DEAE-Sephadex A-50 3 and 4 pectinesterase fractions from column of Sephadex G-75. B, Two parts of the same gel after horizontal slicing 1, 500 fig of the isolated form of pectinesterase from a column of CM-Seph-adex C-50 with 175 mM phosphate-sodium chloride buffer 2, active fraction at 150 mM buffer 4 and 5, 250 fig and 1 mg of the isolated form of pectinesterase, respectively.]...

The purification of glutamine cyclotransferase from papaya latex has been carried out by Messer and Ottesen (116). A batch procedure was used for the removal of impurities by passage of a papaya latex extract through a thin layer of carboxymethyl-Sephadex the active protein was separated by selective elution. Additional purification was achieved by chromatography on a column of carboxymethyl-Sephadex and by gel filtration on Sephadex G-100. The purified enzyme was homogeneous by the criteria of paper electrophoresis, ultracentrifugation, and gel filtration on Sephadex G-100 columns and chromatography on carboxy-methyl- or DEAE-Sephadex. The electrophoretic behavior of the enzyme indicates that it is a basic protein with an isoelectric point near... 

Another modification in which form thin sheet electrophoresis is employed is known as ionophoresis. In this method, devised by Sanger and his co-workers, high voltage electrophoresis is carried out on ion-exchange paper. It is a rapid method of great resolution and sensitivity and is used in Biochemistry for the separation of constituents of a highly complex mixture e.g. a mixture of oligoribonucleotides produced by partial enzymic hydrolysis of RNA. For separation of the constituents of this hydrolysate mixture, a two-dimensional technique is used in which the mixture is subjected to electrophoresis on cellulose acetate and then the partially separated mixture is transferred to a DEAE-cellulose... 

Fig 2. Effect of template type and concentration on yield from an ECL random prime reaction. Yields given are total amounts ofDNA present (labeled DNA plus template) following each labeling reaction. The reactions were carried out for 1 h at 37 C. The N-ras, abl, and K-ras proto-oncogene inserts were excised from pSP65 plasmid by coRI, separated from the plasmid by gel electrophoresis, and purified using DEAE-cellulose paper. 

To study sequence, a sample of the RNA was divided into two batches, one treated with RNase and the other with taka-diastase. At the end of the incubation, in both cases the RNA is split into mononucleotides and oligonucleotides. Various mono- and oligonucleotides can be separated by chromatography on DEAE-cellulose columns. In the case of dinucleotides, the two nucleotides are split by alkaline treatment, and the mononucleotides are separated by paper chromatography or electrophoresis. The position of the 3 -... 

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