Electrophoresis paper, nucleotide separation

Other mixtures of oligonucleotides must be fractionated in larger quantities or are too complex to be separated by traditional paper chromatography and electrophoresis. Large quantities (> 1 mg) of material arise in preparative procedures, in nucleotide sequence analysis of nucleic acids which are not radioactively labelled, and in mixtures where the components of interest are present in very small proportions. Column chromatography is widely used in these cases. 

As an example of the use of paper electrophoresis for the fractionation of oligonucleotides consider the fractionation of some decanucleotides obtained from MS2 viral RNA by T1 ribonuclease digestion. The specificity of the enzyme ensured that each oligonucleotide contained only one Gp residue and the decanucleotides were isolated by column chromatography ( 5.2.1.2). The oligonucleotide mixture was electrophoresed for 17 hr at 6 volt/cm in 0.02 M ammonia formate pH 2.7. At this pH the nucleotides, Ap, Cp, Gp, Up have net charges, q, of —0.08, —0.02, —0.66, 0.98 respectively. Thus the mobility of these decanucleotides should depend mostly on the ratio of Ap+Cp to Up in their composition. Good separation in the predicted order has been obtained (Rushizky et al. 1965). 

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 apparatus and practical considerations The mobility of uridylic acid in 0.05 M ammonium formate buffer pH 3.5 is approximately 0.4 cm hr" volt cm". It is commonly necessary for this nucleotide to run about 40 cm along the paper. So, a potential gradient of 10 volt cm" would be needed to complete the run in 10 hr, i.e. a potential difference of 400 volts. In practice very much larger voltages, up to about 5 kV may be used and separations are then achieved in an hour or so. Currents up to about 10 mA per cm width of paper are used. 

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 -... 

Fig. 9. The reaction catalyzed by the sialytransferases acyl is either acetyl or glycolyl. The assay method for these transferases usually involves incubation of acceptor, enzyme, and radioactive CMP-slallc acid followed by high voltage paper electrophoresis in 1% sodium tetraborate (Roseman et

In 1952 Russell Hilmoe and I became interested in ribonucleases and phosphodiesterases. It was clear that new methods would have to be devised in order to study the mechanism of action of such enzymes. At about this time Markham and Smith published their work on cyclic-terminal nucleotides and the separation of nucleotides by paper electrophoresis. I was excited by their work and arranged to spend the year 1953 in Roy Markham s laboratory in Cambridge, England. This turned out to be a very profitable and enjoyable year. I worked not only with Roy Markham and Paul Whitfeld, but also with Dan Brown, who was in Lord Todd s Chemical Laboratory. 

The chief advantages of zone electrophoresis as compared to the boundary procedure are (1) It is possible to obtain complete separation into zones of different migration and thus not only a boundary separation of overlapping zones. (2) The so-called boundary anomalies interfere less in zone electrophoresis, and therefore also substances of low molecular weight (e.g., amino acids, peptides, nucleotides) may be studied. In addition, zone electrophoresis (particularly in filter paper strips) requires only minute quantities of material and can be performed with simple and inexpensive equipment. 

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