The detection of specific nucleic acid sequences is an important tool for diagnostic medicine and molecular biology research. Gene probe assays currently play roles in identifying infectious organisms such as bacteria and viruses, in probing the expression of normal genes and identifying mutant genes such as oncogenes, in typing tissue for compatibility preceding tissue transplantation, in matching tissue or blood samples for forensic medicine, and for exploring homology among genes from different species.
Ideally, a gene probe assay should be sensitive, specific and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis (for a review, see Abramson et al., Current Opinion in Biotechnology, 4:41-47 (1993)).
In contrast, specificity remains a problem in many currently available gene probe assays. The extent of molecular complementarity between probe and target defines the specificity of the interaction. Variations in the concentrations of probes, of targets and of salts in the hybridization medium, in the reaction temperature, and in the length of the probe may alter or influence the specificity of the probe/target interaction.
It may be possible under some limited circumstances to distinguish targets with perfect complementarity from targets with mismatches, although this is generally very difficult using traditional technology, since small variations in the reaction conditions will alter the hybridization. New experimental techniques for mismatch detection with standard probes include DNA ligation assays where single point mismatches prevent ligation and probe digestion assays in which mismatches create sites for probe cleavage.
Finally, the automation of gene probe assays is an area of high interest. Such assays generally rely on the hybridization of a labelled probe to a target sequence followed by the separation of the unhybridized free probe. This separation is generally achieved by gel electrophoresis or solid phase capture and washing of the target DNA, and is generally quite difficult to automate easily.
Immobilization of biomolecules on solid phases is widely used. Of particular interest is the immobilization of nucleic acids on solid surfaces for use in nucleic acid detection systems. There are a number of known techniques for the immobilization of nucleic acid on solid supports, including Hegner et al., FEBS Letters 336(3):452 (1993); Millan et al., Anal. Chem. 65:2317 (1993); Southern et al., Nucleic Acids Res. 22(8):1368 (1994); Maskos et al., Nucleic Acids Res. 20(7):1679 (1992); Palecek, Electroanalysis 8:7 (1996); Hashimoto et al., Anal. Chem. 66:3830 (1994); Su et al., Anal. Chem. 66(6):769 (1994); Chrisey et al., Nucleic Acids Res. 24(15):3031 (1996); Williams et al., Nucleic Acids Res. 22(8):1365 (1994); Xu et al., J. Am. Chem. Soc. 117:2627 (1995); Millan et al., Electroanalysis 4:929 (1994); Lee et al., Science 266:771 (1994); Millan et al., Anal. Chem. 66:2943 (1994); and Xu et al., J. Am. Chem. Soc. 116:8386 (1994).
It is an object of the present invention to provide novel compositions for the detection of nucleic acids, and methods of using the compositions.
USPTO Backgrounds
USPTO Backgrounds