Columbia University

Donald R Mills

Fred Russell Kramer

Eleanor A Miele

Paul M Lizardi

Vladimir D Axelrod

The present invention provides a replicatable and hybridizable recombinant single-stranded RNA probe molecule comprising: a recognition sequence for the binding of an RNA-directed RNA polymerase; a sequence required for the initiation of product strand synthesis by the polymerase; and a heteroloqus RNA sequence inserted at a specific site in the internal region of the recombinant molecule and complementary to an oligo or polynucleotide of interest. This invention also provides methods for determining the presence of concentration of an oligo- or polynucleotide of interest in a sample and for simultaneously determining the presence or concentration of several different oligo- or polynucleotides of interest in a sample.

Replicatable hybridizable recombinant RNA probes and methods of using same

This invention provides a method for producing a mutant virus or viroid, or a mutant recombinant RNA molecule comprising a single stranded virus genome or a single stranded viroid genome, a circular virus genome or a circular viroid genome, or a segment of a virus genome or a segment of a viroid genome which comprises incubating a recombinant single-stranded RNA molecule comprising a recognition sequence for the binding of an RNA directed RNA polymerase, a sequence for the initiation of product strand synthesis by the polymerase and a heterologous sequence of interest derived from a different RNA molecule inserted at a specific site in the internal region of the recombinant molecule under appropriate selective conditions and for a sufficient period of time permitting the selection of a mutant population, the heterologous sequence of interest comprising the RNA of the single stranded virus genome or the single stranded viroid genome, the circular virus genome or the circular viroid genome or a segment of the virus genome or a segment of the viroid genome.

Autocatalytic replication of recombinant RNA

This invention concerns recombinant RNA molecules comprising a recognition sequence for the binding of an RNA-directed RNA polymerase, a sequence for the initiation of product strand synthesis and a heterologous sequence of interest inserted at a specific site in the internal region of the recombinant molecule. Such recombinant RNA molecules are capable of serving as a template for the synthesis of complementary single-stranded molecules by RNA-directed RNA polymerase. The product molecules so formed are also capable of serving as a template for the synthesis of additional copies of the original recombinant RNA molecule. In a preferred embodiment of the invention Q.beta. replicase is used as the RNA-directed RNA polymerase.

This invention relates to the use of functional reporter molecules in the detection and measurement of targets in a sample. 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.

Replicative RNA-based amplification/detection systems

This invention concerns recombinant RNA molecules comprising a recognition sequence for the binding of an RNA-directed RNA polymerase, a sequence for the initiation of product strand synthesis-and a heterologous sequence of interest inserted at a specific site in the internal region of the recombinant molecule. Such recombinant RNA molecules are capable of serving as a template for the synthesis of complementary single-stranded molecules by RNA-directed RNA polymerase. The product molecules so formed are also capable of serving as a template for the synthesis of additional copies of the original recombinant RNA molecule. In a preferred embodiment of the invention Q.beta. replicase is used as the RNA-directed RNA polymerase. The invention also concerns methods of selectively cleaving RNA molecules at specific sites, methods of constructing recombinant RNA molecules, and methods for the autocatalytic in vitro replication of recombinant RNA molecules. The recombinant RNA molecules of this invention, or fragments thereof, are useful in methods of sequencing nucleic acids, as hybridization probes, as labels, in cell-free methods of protein synthesis, in methods for identifying or characterizing RNA processing enzymes, in methods for producing mutant viruses or viroids and in isolating from mRNA mixtures a desired mRNA in the form of a recombinant mRNA molecule.

Cell-free method for synthesizing a protein

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.

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Englewood, NJ, United States of America

Donald R Mills