34. THE RIBONUCLEIC ACIDS OF VIRUSES 257 



(1) Phenol Method. Westphal et al. b9 treated bacteria with a warm homo- 

 geneous phenol-water mixture followed by separation of the two phases. 

 Bacterial protein was dissolved in the phenol phase, and nucleic acid and 

 polysaccharide in the aqueous phase. This method proved well-suited, with 

 slight modification, for splitting TMV and isolating an infectious RNA of 

 high molecular weight. The detailed procedure is as follows. 



A 3-6% TMV solution in 0.01-0.05 M phosphate buffer, pH 6-7, is shaken vigor- 

 ously for 10-15 minutes in the presence of an equal volume of 80% phenol. After a 

 short centrif ligation at low speed, the emulsion is separated into a lower phenol phase 

 containing all of the protein and an upper aqueous phase containing all of the RNA. 

 The aqueous phase is treated with phenol at least twice, in this case by shaking for 

 5-10 minutes in the presence of a small volume of phenol. The phenol dissolved in 

 the aqueous phase is removed by repeated extraction with peroxide-free ether, and 

 the ether is removed with N> gas. The RNA dissolved in the aqueous phase can be 

 used direct!}' for biological tests, or for other chemical and physical studies, or may 

 be precipitated with 2 volumes of alcohol (with the addition of several drops of 1 M 

 acetate buffer to facilitate the precipitation), or with 2 M NaCl (1 part to 1 part of 

 the RNA solution) prior to use. RNA precipitated by these methods and then redis- 

 solved shows no loss in activity. 



If no effort is made to have a quantitative yield, the entire procedure can be per- 

 formed in about an hour. For quantitative extractions, it is sufficient to remove, 

 quantitatively, the aqueous phase immediately after separation of the two phases. 

 This may be accomplished by washing the phenol phase with buffer or water. 



TMV-RNA prepared in this manner is biologically active with an infec- 

 tivity some 0.1-0.5% of that of an equivalent amount of RNA in the 

 intact virus. 3 The advantages of the phenol method are quite obvious. 

 The splitting of nucleoprotein can be performed at low temperatures with- 

 out employing extreme pH values. The direct contact with phenol leads to 

 an immediate denaturation of any enzymes which may be present, in 

 particular ribonuclease. The denatured enzymes are dissolved in the phenol 

 phase. Thus, the phenol method is generally applicable for the isolation of 

 RNA and especially in those cases where the presence of enzymes, which 

 may degrade RNA, is unavoidable. 



If it is necessary to separate nucleic acid from polysaccharides which are 

 simultaneously extracted, as is the case for extraction from complex animal 

 viruses or from tissues, one may precipitate the RNA with NaCl. According 

 to Kirby 60 it is also possible to separate RNA from polysaccharides by ex- 

 traction with 2-methoxy-ethanol from a solution containing potassium 

 phosphate. 



(2) Detergent Methods. 4 Four volumes of a 1% solution of TMV containing 10" 4 XI 

 Versene is warmed to 50°C, adjusted to pH 8.5 at that temperature, mixed with 1 



59 O. Westphal, O. Liideritz, and F. Bister, Z. Naturforsch. 7b, 148 (1952). 



60 K. S. Kirby, Biochem. J. 64, 405 (1956). 



