Nucleic acids, fractionation oligonucleotides

For very dilute nucleic acid solutions, precipitation with polyethylene glycol (PEG) is preferred. PEG 6000 is added to a concentration of 10% (w/v), and the solution is allowed to stand on ice for 2 h. The precipitate is removed by centrifugation and washed with 70% (v/v) ethanol. PEG can be used for fractional precipitation, since high molecular weight DNA is precipitated at lower PEG concentrations than low molecular weight DNA. It should be emphasised that oligonucleotides of chain length less than 20 cannot be precipitated effectively with either alcohol or PEG. 

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. 

Gel electrophoresis is a powerful and versatile method to resolve mixtures of different nucleic acid molecules and allows the fractionated molecules (i) to be viewed directly, (ii) to be recovered in pure form or (iii) to be characterized directly by hybridization. Hybridization of the probes to fractionated DNA ( Southern technique ) (Southern, 1975) or fractionated RNA ( Northern technique ) (Al-wine et al., 1979) can be achieved after the transfer of the resolved molecules to a membrane, but in some cases also directly in the gel using oligonucleotide probes ( unblot ) (Purrello and Balazs, 1983 Tsao et al., 1983). The steps in these protocols are summarized in Table 9.1. Simultaneous extraction of DNA and RNA (Section 3.4.3) (Chan et al., 1988) may be advantageous when the mass of tissue available is small. 

Immobilized nucleic acid bases, nucleosides or oligonucleotides may be used for separation, fractionation and structure determination of various nucleic acids and enzymes participating in their synthesis and degradation. Schott et al. [139,229] made use of immobilized defined oligonucleotides for the selective separation of free nucleotides on the basis of a base-pairing mechanism. Complementary oligonucleotides in the mobile phase are selectively adsorbed on the immobilized template if... 

Analytical Techniques and Physical Methods.— The mapping of oligonucleotides and nucleic acid digests on cellulose or cellulose-polyethyleneimine has been described recently, and columns of mercurated dextran or dihydroxyborylcellulose have been used to fractionate nucleotide mixtures. Electrophoresis on polyacrylamide gels has been advocated as a rapid method for desalting and fractionating mixtures of oligonucleotides. ... 

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