The Fixation and Retention of Viral and Mammalian Deoxyribonucleic Acids on Nitrocellulose Filters

Abstract

A study has been made of the factors involved in the ability of denatured deoxyribonucleic acid to bind irreversibly to three different brands of nitrocellulose filters. The DNA was obtained from Polyoma virus: from the mouse line of L-929 cells in monolayer culture, and from mouse embryos. It was found that, although smaller size DNA could be fixed well to nitrocellulose filters, considerable loss of such DNA occurred during the incubation procedures used for standard hybridization tests. In addition, loss of fixed DNA was enhanced by increasing the temperature, or decreasing the salt concentration, of the incubation medium. Thus DNA of molecular weight 3 × 106 daltons could be retained quantitatively on the filters during incubation in 0.9 M NaCl – 0.09 M sodium citrate, at a temperature of 65° for not more than 24 h. A decrease in ionic strength or an increase of temperature or time of incubation encouraged loss of DNA. The DNA of smaller molecular size was correspondingly more susceptible to loss. For DNA smaller than 3.0 × 106 mol wt, hybridization should be carried out at lower temperature in the presence of formamide.The three commonly used brands of nitrocellulose filters were equivalent in their ability to adsorb and irreversibly fix each of the DNA preparations tested, and in the capacity of the fixed DNA to hybridize with complementary RNA.