Element analysis in labeled DNA by x-ray fluorescence

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An x-ray fluorescence method has been developed which allows for the determination of the degree of replacement of thymine in deoxyribonucleic acid (DNA) by the halogenated uracils, 5-bromouracil, 5-iodouracil and 5-chlorouracil, as well as the sulfur-containing analogs, thioguanine (replacing guanine) and 6-mercaptopurine (replacing adenine). In this method the ratio of the number of halogen (or sulfur) to phosphorus atoms is measured for determination of the degree of replacement. This ratio is obtained dispersively without concern for interline interferences due to elemental impurities in the DNA. The more sensitive nondispersive analytical approach is also used in this system for the determination of bromouracil and iodouracil replacement for isolated DNA material of extreme purity. Under our optimum instrumental conditions, the dispersive approach gives an expected relative counting error for the determination of the Br: P ratio in 10 μg samples of DNA containing 0.5% Br by dry weight (equivalent to about 1% replacement) of 8% for a 10 min integration time. An actual precision measurement on 10 μg samples of DNA with a bromine content of 0.8% dry weight gave a relative standard deviation of 3.7% for the Br: P ratio in a 20 min integration time. Precision analysis on 2 μg samples of Br containing DNA shows the experimental error is less than 1.3 times the expected counting statistical error (see “Results”). The precision in the determination of the Br: P ratio on 2 μg samples of DNA was found to remain practically unchanged with the addition of 8 μg of nonphosphorus containing “impurities,” and the measure of the ratio upon the addition of 80% impurities shows a variation of close to 6% for DNA samples containing about 3% Br by dry weight. The expected error in replacement determinations depends on the amount of replacement. For example, maximal replacement in the order of 0.6% of guanine by TG in Escherichia coli B r ( E. coli B r ) has been reported (13). This is about 100 times less than the maximal replacement of thymine by BU reported for E. coli 15T − and HeLa cells. The statistical counting error in the determination of TG replacement would be greater than 10%, for reasonable integration times, if the degree of replacement to be measured is 0.6% or less. It would also be difficult to determine if the analog is actually incorporated or is present as a nonincorporated impurity at these low levels of replacement.