Quantitation of submicrogram concentrations of DNA by a modified Laurell electrophoresis method

Charles R. Steinman

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Quantitation of DNA at concentrations below 1 μg/ml is difficult to achieve reliably, especially in the presence of interfering substances that are commonly present in complex biological mixtures such as human plasma. In addition, non-specific binding of DNA, presumably by protein, becomes a significant problem at low DNA concentrations. Rocket immunoelectrophoresis, as described by Laurell (1966), is limited in its sensitivity by the inability to visualize the immune precipitates at the very low antibody concentrations necessary to provide a linear assay in the submicrogram range of DNA concentrations. The present report is of a modification of the Laurell technique, using ethidium bromide staining with fluorescence enhancement, in combination with photography using a highly sensitive film and an appropriate light filter to allow measurement of the otherwise invisible rocket-shaped precipitates. In order to apply this assay to a complex biological fluid such as plasma, it was found necessary to add guanidine hydrochloride to the plasma which probably serves to minimize interfering protein-DNA interactions. The result is a reproducible, linear assay for DNA applicable to plasma or serum and presumably also to other biological fluids in the range of 0.125-20 μg of double stranded DNA per ml. To achieve this sensitivity, sample sizes of 3 μl were used so that, in absolute terms, the minimum quantitatible amount of DNA was 0.4 ng.

Original languageEnglish
Pages (from-to)373-378
Number of pages6
JournalJournal of Immunological Methods
Volume31
Issue number3-4
DOIs
StatePublished - 27 Dec 1979

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