154 D. Wright Wilson 



Schmidt-Thannhauser procedure may not separate completely 

 the RNA from the DNA. 



We have usually separated the DNA constituents from 

 those of the RNA by the very effective combination of paper 

 chromatography and ion exchange. The nucleic acids were 

 hydrolysed by heating at 100° with 1 N HCl for 1 hour. 

 This procedure liberated purine bases and the pyrimidine 

 nucleotides. The separation of these compounds by means of 

 paper chromatography was being studied when Smith and 

 Markham (1950) reported that they could be separated by 

 means of a 70 per cent solution of tertiary butyl alcohol in 

 water, the w hole solution made • 8 N with HCl. The com- 

 pounds are now separated by using a combination of paper 

 chromatography and ion exchange and identified by means 

 of the Beckman ultraviolet spectrophotometer. We are now 

 able to identify the four pyrimidine nucleotides, two from 

 RNA and two from DNA. About • 1 ml. of the hydrolysate 

 is placed as a band near the bottom of Whatman No. 1 filter 

 paper, 12 cm. wide and 40 cm. long and allowed to dry. The 

 paper is set up as an ascending column, using Smith and 

 Markham' s tertiary butyl alcohol. After 48 hours the paper 

 is dried at room temperature and examined by the Mineralight 

 lamp. The dark regions are circled with a pencil. From the 

 solvent front down, the bands contain thymidylic acid, 

 uridylic acid, cytidylic acid, adenine, and guanine. Each 

 band is cut out and extracted with water. Each solution is 

 made faintly alkaline and poured on a column containing 

 Dowex-1, 300-400 mesh, which had been previously washed 

 with alkali, acid and water. After thorough washing, the 

 columns are eluted with varying strengths of HCl. Thymidylic 

 acid comes off the column with • 1 N HCl, uridylic acid with 

 the 0-01 N HCl. RNA cytidylic is eluted with -002 N HCl 

 and after it is removed from the column 0-01 N HCl will 

 remove DNA cytidylic acid. The adenine and guanine are 

 not absorbed by the column. 



It has been demonstrated that the added orotic acid does 

 not contaminate the isolated products (Weed and Wilson, 



