A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane.

An improved method for the formation of DNA—RNA hybrids is described. The procedure involves immobilizing denatured DNA on nitrocellulose membrane filters, hybridizing complementary RNA to the membrane-fixed DNA, and eliminating RNA “noise”. Denatured DNA and hybridized RNA remain on the filter throughout the procedure. Unpaired RNA is removed by washing, and RNA complexed over short regions is eliminated by RNase treatment. Many samples can be easily handled, permitting kinetic and saturation studies. Large amounts-of DNA can be loaded on a filter without interfering with the efficiency of the annealing reaction. Moreover, the “noise” can be depressed to a level (0·003% of the input RNA) permitting the identification of small regions of DNA complementary to a given RNA species. The results are quantitatively more certain than annealing in liquid, since the competing DNA renaturation reaction is suppressed.

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