Determination of DNA base composition by reversed-phase high-performance liquid chromatography

✍ Scribed by Jin Tamaoka; Kazuo Komagata

• Book ID: 109312708
• Publisher: John Wiley and Sons
• Year: 1984
• Tongue: English
• Weight: 259 KB
• Volume: 25
• Category: Article
• ISSN: 0378-1097
• DOI: 10.1111/j.1574-6968.1984.tb01388.x

✦ Synopsis

DNA base composition was determined by reversed-phase high-performance liquid chromatography (HPLC). DNA was hydrolysed into nucleosides with nuclease P1 and bacterial alkaline phosphatase. The mixture of nucleosides was applied to HPLC without any further purification. One determination by chromatography needed 2 /xg of hydrolysed nucleosides and took only 8 min. The relative standard error of nucleoside analysis was less than 1%. The system described here gives a direct and precise method for determining DNA base composition.

same DNAs, e.g. 50.1 (G + C) mol% was assumed for DNA from Escherichia coli K-12 in [2] and 51.0 (G + C) mol% in [8]. This problem is inevitable for an indirect determination.

Recently, reversed-phase HPLC has been applied to analysis of nucleosides from DNA or RNA [9-11], and complete hydrolysis of tRNA into nucleosides with nuclease P1 and bacterial alkaline phosphatase was studied by Gehrke et al.

[121.

We have developed a direct method to determine DNA base composition by reversed-phase HPLC after complete enzymic hydrolysis of DNA into nucleosides.