Abstract: This invention relates to the use of functional reporter molecules in the detection and measurement of RNA sequences in a sample, as a determination, for example, of pathogenic disease existence or potential. The invention is predicated on the utilization of nucleotide sequences, one having a probe sequence linked to a sequence capable of initiating replication by an RNA-dependent RNA polymerase. The other is capable of hybridizing to a strand separated from the extension product of the first nucleotide sequence after hybridization to a specific target sequence. The extension product of the second hybridized nucleotide sequence serves as a template source for autocatalytic replication by the RNA-dependent RNA polymerase. The replication products are detected as a means for detection of nucleic acid sequences.

Abstract: This invention relates to the use of functional reporter molecules in the detection and measurement of nucleic acid sequences in a sample, as a determination, for example, of pathogenic disease existence or potential. The invention is predicated on the utilization of a transcription step between the production of an appropriate reporter molecule and replication based amplification in order to increase the number of detectable species as an indirect reference to target nucleic acid sequence.

Abstract: Highly sensitive methods for assaying for biopolymers, such as by immunoassay or nucleic acid probe hybridization assay, and compositions for carrying out the methods, are provided. The methods employ as reporter group a RNA capable of being autocatalytically replicated by an RNA-dependent RNA polymerase, such as the replicase of bacteriophage Q.beta.. The high sensitivity of the assay methods is due to the rapid, exponential increase in concentration of such a replicative RNA, associated specifically with biopolymer analyte, that can be achieved by autocatalytic RNA replication.

Abstract: A probe for the detection of a nucleic acid target sequence containing a molecular switch comprising three essential elements: a probe sequence of 20-60 nucleotides surrounded by switch sequences of 10-40 nucleotides which are complementary to each other, wherein the state of the switch is useful for selectively generating a detectable signal if the probe is hybridized to a target; also, assays and kits utilizing such probes.

Abstract: Fluorescent linker moieties are provided which comprise a fluorescent compound such as fluorescein attached to a linker moiety such that a functional group of the linker is available for attachment to an affinity molecule such as a nucleic acid which has an N.sup.4 (substituted amino) cytosine moiety. Probes tagged with fluorescent derivatives such as fluorescein, tetramethyrhodamine or tetraethylrhodamine may be detected by fluorescence spectroscopic methods.

Abstract: A first mixture is prepared that contains labeled chain fragments which each has a common end adjacent to a primary nucleotide and a termination at a position from the primary through an nth nucleotide, the first mixture containing nucleotide chain fragments of each length from termination at the primary through termination of the nth nucleotide. A second mixture is prepared that contains labeled chain fragments beginning at the common end and terminating at positions from the first through the nth nucleotide, the second mixture containing chain fragments of each length terminating wherever either a first or a second of the four nucleotides occurs. A third mixture is prepared that contains labeled chain fragments beginning at the common end and terminating at a position from the first through the nth nucleotide, the third mixture containing chain fragments of each length terminating wherever the first or a third of the four nucleotide sequences occurs.

Abstract: Nucleic acid hybridization probes are provided which comprise an N.sup.4 -(substituted amino)cytosine moiety, wherein the substituted amino group comprises a tag moiety, whereby the probe is detected. Methods of preparing probes of the invention, intermediates used in such methods, and methods of using the probes of the invention in hybridization assays are also provided. Typical tag moieties employed with the probes of the invention are biotinyl, aminothiadiazole and fluorescein derivatives, connected to N.sup.4 -amino groups of modified cytosines of the probe through linker moieties. Probes tagged with biotin are typically detected by binding to the biotinyl moieties, through a streptavidin or avidin molecule, a reporter group which includes streptavidin or avidin and then detecting a signal due to the reporter group.

Abstract: Nucleic acid hybridization probes are provided which comprise nucleoside bases or terminal nucleotide phosphates chemically linked to aromatic sulfonamide inhibitors of carbonic anhydrase. Methods of preparing probes of the invention, intermediates used in such methods, and methods of using the probes of the invention in hybridization assays are also provided. A probe of the invention is detected by binding to it a reporter group, such as a homopolymer or heteropolymer of enzymes, which includes a carbonic anhydrase which binds to the inhibitor linked to the probe, and then detecting the bound reporter group, as by production of a fluorescent or colored product in a reaction catalyzed by an enzyme component of the reporter group. Also provided are enzyme immunoassays wherein detection of antibody is by a process which comprises a chemical reaction catalyzed by a carbonic anhydrase.