Conditions are described for effecting deoxyribonucleic acid renaturation and deoxyribonucleic acid-ribonucleic acid hybridization in formamide solutions at low temperatures. High specificity and rates of reaction can be achieved by appropriate choice of the formamide and salt conmalian nucleic acids. centrations and the temperature. Reaction conditions can be deduced from the relationship between formamide concentration and reduction in the thermal stability: 1 % formamide reduces the T,,, by 0.72'. These methods are valuable for studies of the prolonged reactions occurring between mamR e n a t u r a t i o n of DNA and hybridization of RNA with DNA are normally studied under reaction conditions involving elevated temperatures. The maximum rate of reaction occurs some O below the T,, or mean thermal denaturation temperature (Marmur and Doty, 1961). However, in studies of nucleic acids of complex organisms where reaction times are prolonged, or where biological activity must be preserved, exposure of nucleic acids to high temperatures presents obvious disadvantages such as chain scission and depurination. These difficulties may be avoided by the use of high concentrations of certain salts, such as 6.2 M NaC104 (Thomas, 1966) or of aqueous solutions of various organic solvents in which the thermal stability of double-stranded polynucleotides is greatly reduced (Herskovits, 1962; Geiduschek, 1962; Levine et al., 1963). A particularly useful organic solvent is formamide in which DNA can be denatured and renatured at room temperature (Helmkamp and Ts'o, 1961 ; Marmur and Ts'o, 1961 ; Subirana and Doty, 1966). This principle has been exploited by Bonner et al. (1967) to allow the formation of DNA-RNA hybrids with filter-bound DNA at 25 in the presence of formamide. The present study explores in detail the relationships among the rate and specificity of reaction, the temperature, and the concentrations of formamide and salt. As a result of the incidence of partially related base sequences (Britten and Kohne, 1968), DNA renaturation or DNA-RNA hybridization reactions involving nucleic acids of mammals or other higher organisms may not display locus specificity. In fact, the extent of cross-reaction among related base sequences is dependent upon the reaction conditions such as temperature and ionic strength (McCarthy and McConaughy, 1968 ; Church and McCarthy, 1968). Thus it is important not only to establish conditions for high rates of reaction and species specificity but also to characterize the nature of the duplexes being formed. Although in aqueous solution cross-reaction among related base sequences is reduced at higher temperatures or lower ionic strengths (McCarthy and McConaughy, 1968), specificity conditions are much more easily adjusted by variation of ionic strength and formamide concentration. High specificity may be obtained at low temperatures, with rates of reaction several times higher than those obtained at 60 or 70 in aqueous solution. Materials and Methods Formamide. The formamide used in these studies was purchased from the Eastman Kodak Co. Optical studies in formamide solution require high solvent purity. The criterion applied was that the optical density of 100% formamide at 270 mp in 1-cm path-length cells not exceed 0.15. All of the experiments described here were carried out with formamide which meets these specifications. Isolation of Nucleic Acids. Bacillus subtilis DNA was prepared as described by Marmur (1961) and 3H-labeled DNA was extracted from cells grown in minimal medium to which 0.5 pCi/ml of thymidine-methyl-H3 was added per hour for 4 hr. * From the Departments of Microbiology and Genetics, University of Washington, Seattle, Washington 98105, and from the Department of DNA was isolated from Drosopl?i/a me/anogaster pupae by a procedure described elsewhere (Laird and McCarthy, 1968). Zoology, University of Texas, Austin, Texas. Received Apri l 18, 1969. Mouse and hamster DNA were extracted from liver as The work was supported in part by a research grant from the. U. S. Public Health Service, Institute of General Medical Sciences (GM 124491, in part by a grant from the National Science Foundation (GB 6099), and in part by National Institutes of Health postdoctoral funds, previously described by Church and McCarthy (1968). Labeled L cells grown in !-4Ci/m1 Of thymidine-methy1-3H added to the medium (McCarthy and McConaughy, 1968). DNA was prepared from with N U C L L I C A C I D R E A S S O C I A T I O N 3280