[109] Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA

Abstract The GC content of highly purified DNA samples can be estimated from their thermal denaturation temperatures, employing small amounts of DNA (10–40 μg). The method is simple, convenient and utilizes readily available laboratory equipment; the thermal denaturation profile also gives a good picture of the quality of the DNA preparation. Denatured DNA present in native preparations, or single stranded DNA obtained by denaturing native DNA, or isolated from natural sources such as bacteriophage φX174, give broad thermal transition profiles. Denatured DNA which has been renatured by annealing, or which renatures spontaneously because it is cross-linked, yields thermal denaturation profiles which are almost similar to that of native DNA. A recent interesting observation 38 is that the supercoiled component of polyoma DNA has a T m which is 17° higher (in SSC) than the circular, nonsupercoiled or linear polyoma components. The authors would like to emphasize a point which is troublesome to many investigators, namely, the degree of hyperchromicity resulting from the denaturation of DNA. When the denaturing agent (e.g., heat, alkali) is still present, the increase in absorbance in approximately 40% at 260 mμ. Upon removal of the denaturing agent the difference in absorbance of native and denatured DNA is approximately 12%, reflecting the re-formation of intra- and intermolecular short helical structures. It should also be pointed out that irreversible denaturation is accomplished only after the last hydrogen bonds present in the GC rich regions of the DNA have been disassociated. Until this point is reached, the melting of the double-helical molecule is completely reversible, even if the heated solution is rapidly cooled. When equipment is available, analytical CsCl density gradient centrifugation is the preferred technique for estimating the GC content of DNA, especially if only small quantities (in the range of 1 μg) and/or only partially purified DNA are available. 39 If possible, base compositions should be determined by two independent methods. Discrepancies in the base composition by the two methods are indicative of the presence of unusual bases. 36 When large numbers of samples are to be compared, melting profiles and buoyant density determinations used together provide a great deal of information as to the composition and quality of the samples.