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Journal ArticleDOI

Reexamination of the Association Between Melting Point, Buoyant Density, and Chemical Base Composition of Deoxyribonucleic Acid

01 Mar 1970-Journal of Bacteriology (American Society for Microbiology (ASM))-Vol. 101, Iss: 3, pp 738-754
TL;DR: In this paper, the base composition of deoxyribonucleic acid (DNA) was calculated by regression and correlation analysis and treated statistically by using only sets of data on DNA determined with the same strains.
Abstract: The equations currently used for the calculation of the chemical base composition of deoxyribonucleic acid (DNA), expressed as moles per cent guanine plus cytosine (% GC), from either buoyant density (ρ) or midpoint of thermal denaturation (Tm) were recalculated by using only sets of data on DNA determined with the same strains. All available information from the literature was screened and supplemented by unpublished data. The results were calculated by regression and correlation analysis and treated statistically. From the data on 96 strains of bacteria, it was calculated that% GC = 2.44 (Tm – 69.4). Tm appears to be unaffected by the substitution of cytosine by hydroxymethylcytosine. This equation is also valid for nonbacterial DNA. From the data on 84 strains of bacteria, the relation% GC = 1038.47 (–1.6616) was calculated. The constants in this equation are slightly modified when data on nonbacterial DNA are included. Both correlations differ only slightly from those currently used, but now they lean on a statistically sound basis. As a control, the relation between ρ and Tm was calculated from data of 197 strains; it agrees excellently with the above two equations.
Citations
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Journal ArticleDOI
TL;DR: High-performance liquid chromatography is a promising alternative for determining the G+C content of bacterial deoxyribonucleic acid (DNA) and may also be more accurate than indirect methods, such as the buoyant density and thermal denaturation methods.
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 content. 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.

4,685 citations

Journal ArticleDOI
TL;DR: It is concluded that ANI can accurately replace DDH values for strains for which genome sequences are available and reveal extensive gene diversity within the current concept of "species".
Abstract: DNA-DNA hybridization (DDH) values have been used by bacterial taxonomists since the 1960s to determine relatedness between strains and are still the most important criterion in the delineation of bacterial species. Since the extent of hybridization between a pair of strains is ultimately governed by their respective genomic sequences, we examined the quantitative relationship between DDH values and genome sequence-derived parameters, such as the average nucleotide identity (ANI) of common genes and the percentage of conserved DNA. A total of 124 DDH values were determined for 28 strains for which genome sequences were available. The strains belong to six important and diverse groups of bacteria for which the intra-group 16S rRNA gene sequence identity was greater than 94 %. The results revealed a close relationship between DDH values and ANI and between DNA-DNA hybridization and the percentage of conserved DNA for each pair of strains. The recommended cut-off point of 70 % DDH for species delineation corresponded to 95 % ANI and 69 % conserved DNA. When the analysis was restricted to the protein-coding portion of the genome, 70 % DDH corresponded to 85 % conserved genes for a pair of strains. These results reveal extensive gene diversity within the current concept of "species". Examination of reciprocal values indicated that the level of experimental error associated with the DDH method is too high to reveal the subtle differences in genome size among the strains sampled. It is concluded that ANI can accurately replace DDH values for strains for which genome sequences are available.

3,471 citations


Cites background from "Reexamination of the Association Be..."

  • ...+ 47] 2 36 uC, where 36 uC is the correction for the presence of 50 % formamide (De Ley, 1970; McConaughy et al., 1969)....

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Journal ArticleDOI
TL;DR: The system described here gives a direct and precise method for determining DNA base composition by reversed-phase high-performance liquid chromatography (HPLC).
Abstract: 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 μg 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.

2,468 citations

Journal ArticleDOI
TL;DR: The optical method of De Ley et al. (1970) for determining DNA/DNA homologies was reexamined and the agreement is excellent above a degree of binding of 25-30%.

1,695 citations

Journal ArticleDOI
TL;DR: The results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 value corresponding to about 4,000 completely different genomes of standard soil bacteria.
Abstract: Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS) Images

1,688 citations

References
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Journal ArticleDOI
J. Marmur1, Paul Doty1
TL;DR: The previously discovered linear relation between the base composition of DNA, expressed in percentage of guanine plus cytosine bases, and the denaturation temperature, T m, has been further investigated and it appears that the measurement of the T m is a satisfactory means of determining base composition in DNA.

4,154 citations

Journal ArticleDOI
TL;DR: A comprehensive study of the buoyant density of DNA as a function of composition has been made and the linear relation previously reported has been confirmed.

1,549 citations

Journal ArticleDOI
TL;DR: The DNA homopolymers dA and dT have been prepared enzymically using Escherichia coli DNA polymerase, and their properties have been studied, but the homopolymer pair dA:rU is not stable under any conditions of temperature and salt concentration tested.

466 citations