1. Field of the Invention
Recent advances in the general field of molecular biology have made it possible to detect specific genes of clinical and commercial importance. For example, the structures of various genes and gene sequences associated with specific human diseases are known, as are various techniques for detecting the presence of such genes. It is therefore possible to diagnose human disease at the genetic level.
The most common technique for detecting a particular gene sequence is hybridization. A particular nucleotide sequence or "probe" is marked with a detectable label, typically a radioactive label or chemical modification, and combined with the nucleic acid sample of interest, either in situ as part of intact cells or as isolated DNA or RNA fragments. The sample can be either free in solution or immobilized on a solid substrate. If the probe molecule and nucleic acid sample hybridize by forming a strong non-covalent bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. The probe's detectable label provides a means for determining in a known manner whether hybridization has occurred and for measuring the amount of DNA/RNA sample present. The hybridization technique is of prime importance in basic research directed at understanding the relationship between nucleotide sequences and their function, as well as in diagnostic use to detect known aberrant genes or disease agents such as viruses or bacteria.
The main limitation of present gene detecting methods is that they are not sensitive enough and therefore require a relatively large amount of sample to accurately verify the existence of a particular gene sequence. This is not surprising since the detection of a single gene in the entire genetic repertoire of a human being requires locating one part in one to ten million. In fact, most hybridization methods require at least one to ten micrograms of purified DNA, representing a substantial sample of cells, to perform a reliable analysis. This limitation is particularly significant in pre-natal diagnosis of genetic disorders where only a small cell sample can be taken or in identifying infectious agents such as viruses in small tissue samples. Consequently, there is a substantial need for gene detecting methods which will increase the sensitivity of the hybridization assay without sacrificing its specificity.
2. Description of the Prior Art
The hybridization procedure typically includes the initial steps of isolating the DNA sample of interest and purifying it chemically. The DNA sample is then cut into pieces with an appropriate restriction enzyme. The pieces are separated by size through electrophoresis in a gel, usually agarose or acrylamide. The pieces of interest are transferred to an immobilizing membrane in a manner that retains the geometry of the pieces. The membrane is then dried and prehybridized to equilibrate it for later immersion in a hybridization solution.
A probe labeled with a radioactive isotope is constructed from a nucleotide sequence complementary to the DNA sample by a conventional nick translation reaction, using a DNase and DNA polymerase, although other types of labels can be used. The probe and sample are then combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules supported on the membrane. The signal of the bound probe molecules is typically detected and quantified by autoradiography and/or liquid scintillation counting.
Southern, J. Mol. Biol. 98:503 (1975) teaches the transfer of DNA fragments to strips of nitrocellulose after the fragments have been resolved by electrophoresis in agarose gels. Immobilization of DNA on diazobenzyloxymethyl cellulose is taught by Noyes and Stark, Cell 5:301 (1975). Alwine et al., PNAS USA 74:5350 (1977) teach a method for detecting specific RNA molecules that are resolved in agarose gels, transferred to diazobenzyloxymethyl paper and then hybridized with DNA probes. Similarly, Reiser et al., Biochem. Biophys. Res. Comm. 85:1104 (1978), teach the detection of small DNA fragments resolved in polyacrylamide gels by immobilizing the same on diazobenzyloxymethyl paper and then hybridizing them to DNA probes.
Both Bittner et al., Anal. Biochem. 102:459 (1980) and Stellwag and Dahlberg, Nucl. Acid Res. 8:299 (1980) teach the use of an electrical current to transfer DNA and RNA fragments from either agarose or acrylamide gels to diazobenzyloxymethyl paper. Similarly, electrophoretic transfer of nucleic acids to diazophenylthioether paper is taught by Reiser and Wardale, Eur. J. Biochem. 114:569 (1981).
A New England Nuclear (Boston, Mass.) technical bulletin entitled "Gene Screen Hybridization Transfer Membrane Instruction Manual", by D. J. Green and D. R. Rittenbach (1982), teaches a modification of two different techniques ("electrophoretic transfer" and "capillary wicking") for immobilizing DNA and RNA samples on a porous substrate, specifically nylon-base membranes.
Nucleic acid probes have heretofore been labeled separately with either tritium or radioactive phosphorus [.sup.32 P] by nick translation, Rigby et al., J. Mol. Biol. 113:237 (1977) or with biotinylated uridine, Narayanswami, Hutchison and Ward, J. Cell Biol. 95:74a (1982). Other references, besides Rigby et al., identifying radioactive labels for DNA probes include Wahl et al., U.S. Pat. No. 4,302,204 (.sup.32 P); Falkow et al., U.S. Pat. No. 4,358,535 (.sup.32 P, .sup.3 H, .sup.14 C); and Axel, et al., U.S. Pat. No. 4,399,216 (.sup.32 P) A New England Nuclear technical bulletin, Vincent et al., "Preparation of DNA Labeled With High Specific Activity [.sup.35 S]-Deoxyadenosine 5' - [.alpha.-Thio] Triphosphate; the Use of .sup.35 S-Labeled Nucleic Acids as Molecular Hybridization Probes" (1982), teaches that a hybridization probe labeled with [.sup.35 S]-deoxyadenosine 5' [.alpha.-thio] triphosphate is qualitatively indistinguishable from a conventional .sup.32 P labeled probe.
Wahl et al., U.S. Pat. No. 4,302,204 teach the use of ionic polymers, particularly dextran sulfate, to increase the local concentration of nucleic acids in hybridization reactions and in turn increase the signal of the DNA sample of interest. It also describes the use of a depurination step to increase the efficiency with which very large nucleic acid segments are transferred from a gel to a solid membrane.
A method of incorporating a phosphorothioate analog of deoxynucleotides, namely .sup.32 S, into DNA polymers, using known DNA polymerases (E. coli DNA polymerase I and E. coli DNA polymerase III) is described by Kunkel et al., PNAS USA 78:6734 (1981). This reference mentions the use of thionucleotides to induce site-specific mutations as an application of the method. Putney et al., PNAS USA 78:7350-7354 (1981) teach that a protective effect against enzymatic degradation occurs following the incorporation of a thionucleotide (.sup.32 S) into DNA polymers.
The use of a polyfunctional disulfide compound to cross-link protein molecules is taught by Kotani et al., U.S. Pat. No. 4,287,345.
The present invention improves prior methods of detecting DNA genes and gene sequences by amplifying the detectable signal generated by bound probe molecules, thereby providing an accurate and reliable method for detecting and quantifying particular nucleotide sequences, even in relatively small samples. Accordingly, the present invention includes as its objects the following:
(1) to increase the mass of complementary DNA bound to specific gene sequences immobilized on a membrane; PA0 (2) to increase the signal-to-noise ratio produced in hybridization reactions involving immobilized DNA; PA0 (3) to improve the resolution of bands visualized by autoradiography of radioisotopically-labeled DNA; PA0 (4) to produce radioisotopic probes which will provide the same intensity of autoradiographic signal as obtained with .sup.32 P-labeled probes, but with less danger of radiation exposure to the worker due to the decreased emission energy of the probe; PA0 (5) to improve existing protocols for hybridizations, especially filter hybridizations, with .sup.35 S-labeled probes and reduce both the direct cost of the reaction mixture and the labor cost of the procedure; and PA0 (6) to produce a system of signal amplification which is not dependent upon the use of radioisotopes and which is compatible with a variety of probe-labeling systems.
Other objects will be apparent from the Description of a Specific Embodiments.