In a large polymer molecule (such as a DNA chain) which is made from multiple component monomers containing many atoms, how was it determined that the chain was made up from the four groups of specific combinations of monomers of elements (in this case, adenine, thymine, guanine and cytosine)? In other words how were they able to determine that the DNA contained the four specific bases before they knew the structure of the DNA molecule? In general, doesn't breaking up a large molecule into its constituent atoms also break up the monomers?


1 Answer 1


In the early experiments performed by Erwin Chargaff and co-workers to determine the composition of DNA, leading to the deduction of the Chargaff's rules, the total DNA was extracted, chemically digested and the products were analysed using paper chromatography. The DNA was not broken down to the level of atoms which is certainly impossible using the standard chemical treatment procedures.

This is an excerpt from one of the earliest papers by Chargaff [1]:

Two procedures were compared. In one, which will be referred to here as Procedure 1, the nitrogenous components were liberated and determined by the methods described previously from this laboratory (5, 8, 12, 13); i.e., the purines were set free by N H2S04 (1 hour, 100o) and chromatographed in aqueous butanol-diethylene glycol (NH3 atmosphere); the pyrimidines were liberated with concentrated formic acid (2 hours, 175o) after removal of the purines as the hydrochlorides by treatment with methanolic HCl, and chromatographed in aqueous butanol.

The second method, Procedure 2, is considerably simpler and permits the use of even smaller quantities of DNA (2 to 6 mg. for a complete analysis) than was possible heretofore; its use was briefly mentioned in a recent paper (14). It is based on the simultaneous liberation of all purines and pyrimidines by the hydrolysis of the DNA with concentrated formic acid and on their separation, on the same chromatogram, by means of ammoniacal butanol. When it is desired to establish complete balances, i.e. to express the molar distribution of individual nitrogenous constituents not only in terms of P present in the hydrolysate, but also in relation to the total N and P of the nucleic acid, about 6 mg. of DNA are used in the analysis. The sample (dried in a high vacuum at 60o for 3 hours) was heated in a sealed small bomb tube (about 220 × 6.5 mm.) with 0.8 cc. of concentrated formic acid (98 to 100 per cent) at 175o for 2 hours. The hydrolysate was transferred quantitatively to a 1 cc. volumetric flask and adjusted to volume by means of small portions of distilled water which served to rinse the hydrolysis vessel. Aliquots, measured with an accurate microburette in the usual manner (12), were employed for the calorimetric P determination (0.04 cc. of hydrolysate) and for the chromatographic separation (0.01 cc.). When very small quantities of DNA were to be analyzed, 2 mg. of the substance were heated with 0.2 cc. of concentrated formic acid and the hydrolysate was, without volume adjustment, analyzed for P and for individual purines and pyrimidines. In this case, all computations were based on the ratio of moles of nitrogenous constituent to moles of phosphorus present in the hydrolysate (13, 14). The chromatographic and spectroscopic techniques were, in general, similar to those described previously (8, 12). The paper sheets (16 × 46 cm.), carrying five lanes 3 cm. wide, were left for 20 minutes in an ammonia atmosphere in order to neutralize the acid. The solvent used for the chromatographic separation consisted of a mixture of 6 volumes of n-butanol and 1 volume of 0.6 N aqueous ammonia. Two beakers, one containing the solvent mixture, the other 0.6 N ammonia, were placed along- side each paper sheet at the bottom of the jar used for chromatography; the paper sheets were suspended, the solvent trough being left empty, and allowed to equilibrate with the solvent atmosphere for 1 hour in the closed vessel. Then the trough was filled with the solvent mixture and the separation permitted to proceed for 18 to 20 hours, at which time the solvent front had passed over the lower edge of the paper. After the papers had dried in air, the position of the separated components was marked under a Mineralight lamp or a similar ultraviolet lamp.4 The components, in the order of increasing distance from the starting point, were aligned as follows (the distance of thymine arbitrarily taken as 100) : guanine (26), cytosine (47), adenine (71), thymine. The quantitative determinations were carried out as described before (12).

Chargaff, Erwin, et al. "The composition of the desoxyribonucleic acid of salmon sperm." Journal of Biological Chemistry 192.1 (1951): 223-230.


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