1. Field of the Invention
This invention relates to compositions and assay methods for the hybridization and extraction of nucleic acids. In particular, this invention relates to compositions and methods to release nucleic acids from cells in complex biological samples or specimens while simultaneously hybridizing complementary nucleic acids released during lysis. The methods are based on the use of the chemical family of compounds termed lactams, preferably pyrrolidones, which promote nucleic acid base pairing, and which are effective in the extraction and purification of nucleic acids, and, alternatively, highly sensitive assays for ribonucleic acid are also described that employ a heating step.
2. Brief Description of the Relevant Art
Organic solvents such as phenol and chloroform are traditionally used in techniques employed to isolate nucleic acid from procaryotic and eucaryotic cells or from complex biological samples. Nucleic acid isolations typically begin with an enzymatic digest performed with proteases followed by cell lysis using ionic detergents and then extraction with phenol or a phenol/chloroform combination. The organic and aqueous phases are separated and nucleic acid which has partitioned into the aqueous phase is recovered by precipitation with alcohol. However, phenol or a phenol/chloroform mixture is corrosive to human skin and is considered as hazardous waste which must be carefully handled and properly discarded. Further, the extraction method is time consuming and laborious. Marmur, J. Mol. Biol., 3:208-218 (1961), describes the standard preparative procedure for extraction and purification of intact high molecular weight DNA from procaryotic organisms using enzymatic treatment, addition of a detergent, and the use of an organic solvent such as phenol or phenol/chloroform. Chirgwin et al., Biochemistry, 18:5294-5299 (1979) described the isolation of intact RNA from tissues enriched in ribonuclease by homogenization in GnSCN and 2-mercaptoethanol followed by ethanol precipitation or by sedimentation through cesium chloride.
Further, the use of chaotropic agents such as guanidine thiocyanate (GnSCN) are widely used to lyse and release nucleic acid from cells into solution, largely due to the fact that the chaotropic salts inhibit nucleases and proteases. However, it has proved difficult to isolate the nucleic acids from these chaotropic salt solutions due to the incompatibility of the chaotropes with ionic detergents and the inability to easily partition the nucleic acid into an aqueous phase, given such high molar concentrations of salt used and the chaotropic behavior of the salts.
Nucleic acid hybridization is a known and documented method for identifying nucleic acids. Hybridization is based on base pairing of complementary nucleic acid strands. When single stranded nucleic acids are incubated in appropriate buffer solutions, complementary base sequences pair to form double stranded stable molecules. The presence or absence of such pairing may be detected by several different methods well known in the art.
Most hybridization assays previously described involve multiple steps such as the hybridization technique described by Dunn & Hassell in Cell, Vol. 12, pages 23-36 (1977). Their assay is of the sandwich-type whereby a first hybridization occurs between a "target" nucleic acid and a "capture" nucleic acid probe which has been immobilized on a solid support. A second hybridization then follows where a "signal" nucleic acid probe, typically labelled with a radioactive isotope, hybridizes to a different region of the immobilized target nucleic acid. The hybridization of the signal probe may then be detected by, for example, autoradiography.
Ranki et al. in U.S. Pat. Nos. 4,486,539 and 4,563,419 describe sandwich-type assays which first require steps to render nucleic acids single stranded and then the single stranded nucleic acids are allowed to hybridize with a nucleic acid affixed to a solid carrier and with a nucleic acid labelled with a radioisotope. Thus, the Ranki et al. assay requires the nucleic acid to be identified or targeted in the assay to be first rendered single stranded.
One approach to dissolving a biological sample in a chaotrope solution and performing molecular hybridization directly upon the dissolved sample is described by Thompson and Gillespie, "Analytical Biochemistry," 163:281-291 (1987). See also PCT Application US87/01023, Feb. 5, 1986. Cox et al. have also described the use of GnSCN in methods for conducting nucleic acid hybridization assays and for isolating nucleic acid from cells (European Application No. 84302865.5, May 12, 1984).
Bresser, Doering and Gillespie, "DNA," 2:243-254 (1983), reported the use of NaI, and Manser and Gefter, Proc. Nat'l. Acad. Sci. USA, 81:2470-2474 (1984) reported the use of NaSCN to make DNA or mRNA in biological sources available for trapping and immobilization on nitrocellulose membranes in a state which was suitable for molecular hybridization with DNA or RNA probes.
Hybridization media and extraction solutions which avoid the problems encountered with the use of chaotropic agents and toxic organic solvents, but which also yield sensitive assays would be desirable. Further, procedures which minimize the handling of reagents and samples would be advantageous.
USPTO Backgrounds
USPTO Backgrounds