Fluorometric determination of DNA in cartilage of various species

Abstract

A sensitive, modified 3,5-diaminobenzoic acid (DABA), fluorometric DNA assay was developed and compared to mithramycin and ethidium bromide assays in determining the DNA content of dense connective tissues including: Swarm rat chondrosarcoma, rabbit, dog, monkey, and most importantly, adult human articular cartilage. In the more cellular cartilages, the three methods gave equivalent results. However, in the relatively acellular human cartilage, the DABA method was shown to be superior. Both the mithramycin and ethidium bromide gave falsely high values compared to the DABA method, which by subtraction after DNase digestion approached the DABA value. The latter was completely DNase sensitive. With the DABA method, the DNA content of human cartilage can be obtained on less than 5 mg wet weight of fresh, alcohol-fixed, or lyophilized material. While the DNA can also be released by digestion with papain or protease from Streptomyces griseus, proteinase K was preferable. The comparison of literature values for other fluorometric and spectrophotometric assays of human cartilage suggest these methods overestimate human articular cartilage DNA concentrations, whereas the DABA values were in line with those predicted from previous morphometric analysis. Thus, the modified method represents an improvement in DNA analysis of dense connective tissues.