PHOTOGRAPHIC QUANTIFICATION OF FLUORESCENTLY STAINED DNA IN GELS THROUGH ANALOG MODIFICATION OF SPECTROPHOTOMETER OUTPUT.

All conventional methods for determining the film darkening function rely on external fluorescent standards. Since an equimolar series of molecular weight standards is generally included in gel electrophoresis experiments with DNA, internal standardization is possible. In order to use these standards, assumptions must be made about the distribution of DNA within the band corresponding to a particular fragment. When the Gaussian distribution function and the linear-log darkening function are used to predict the absorbance as a function of position in the band, the absorbance profile describes a parabola. This relationship should be valid except at the 'tails' where the linear-log function fails to describe the film darkening response. D. E. Pulleyblank and others have suggested an approximate method of quantification based on their empirical observation of the quasi-parabolic shape of the absorbance peaks. An analog device was constructed to transform the transmittance output of a Beckman Model 25 spectrophotometer, equipped with a scanning accessory, by the appropriate hyperbolic function. It was found that the analog transfer function is the most accurate method of mass determination examined with significant errors only for the lowest intensity bands. A signal attenuator would be a useful addition to the system since low contrast and high absorbance can drive the recorder off scale.