Precise Measurement of the G+C Content of Deoxyribonucleic Acid by High-Performance Liquid Chromatography

Abstract

High-performance liquid chromatography is a promising alternative for determining the G+ C content of bacterial deoxyribonucleic acid (DNA). The method which we evaluated involves enzymatic degradation of the DNA to nucleosides by P1 nuclease and bovine intestinal mucosa alkaline phosphatase, separation of the nucleosides by high-performance liquid chromatography, and calculation of the G + C content from the apparent ratios of deoxyguanosine and thymidine. Because the nucleosides are released from the DNA at different rates, incomplete degradation produces large errors in the apparent G+ C content. For partially purified DNA, salts are a major source of interference in degradation. However, when the salts are carefully removed, the preparation and degradation of DNA contribute little error to the determination of G+C cmtent. This method also requires careful selection of the chromatographic conditions to ensure separation of the major nucleosides from the nucleosides of modified bases and precise control of the flow rates. Both of these conditions are achievable with standard equipment and C18 reversed-phase columns. Then the method is precise, and the relative standard deviations of replicate measurements are close to 0.1%. It is also rapid, and a single measurement requires about 15 min. It requires small amounts of sample, and the G+C content can be determined from DNA isolated from a single bacterial colony. It is not affected by contamination with ribonucleic acid. Because this method yields a direct measurement, it may also be more accurate than indirect methods, such as the buoyant density and thermal denaturation methods. In addition, for highly purified DNA, the extent of modification can be determined.The measurement of G + C content is one of the most common measurements performed on bacteria. Its greatest values are probably its usefulness as a taxonomic marker and its usefulness in distinguishing phenotypically similar microorganisms. G + C content is also a fundamental property of cellular deoxyribonucleic acid (DNA) and is correlated with the amino acid composition of proteins, codon usage in messenger ribonucleic acid, auxotrophy for specific bases, and other properties of general biological interest (1, 8, 9, 19,25,26,29,36). Therefore, interest in this parameter is not limited to microbial taxonomists.Although the G + C content of DNA has a precise analytical meaning, the techniques for determining this parameter are often indirect and lack a high degree of precision. For instance, the standard deviations of the most widely used methods, the buoyant density centrifugation and thermal denaturation methods, are usually about k1.0 and k0.4 mol% G+C, respectively (5,20,21,23,28,34). The accuracy of both of these methods is also affected by the purity of the DNA and the presence of modified bases (20, 28). The other methods which are occasionally used, the chemical modification and ultraviolet absorption methods, also present difficulties under certain experimental conditions (3, Recently, high-pe...

Paenibacillus yonginensis sp. nov., a potential plant growth promoting bacterium isolated from humus soil of Yongin forest

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