Abstract: The present invention provides methods for isolating a defined quantity of DNA target material from other substances in a medium. The method may be carried out using a known quantity of a silica-containing solid support, such as silica magnetic particles, having a definable capacity for reversibly binding DNA target material, and DNA target material in excess of the binding capacity of the particles. The methods of the present invention involve forming a complex of the silica magnetic particles and the DNA target material in a mixture of the medium and particles, and separating the complex from the mixture using external magnetic force. The DNA target material may then be eluted from the complex. The quantity of DNA target material eluted may be determined based on a calibration model. The methods of the present invention permit isolation of DNA target material which is within a known quantity range.

Abstract: The present invention entails methods and kits for carrying them out based on the discovery that an RNA replicase, such as Q&bgr; replicase, has DNA-dependent RNA polymerase (“DDRP”) activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2′-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase.

Abstract: The present invention provides methods for isolating a defined quantity of DNA target material from other substances in a medium. The method may be carried out using a known quantity of a silica-containing solid support, such as silica magnetic particles, having a definable capacity for reversibly binding DNA target material, and DNA target material in excess of the binding capacity of the particles. The methods of the present invention involve forming a complex of the silica magnetic particles and the DNA target material in a mixture of the medium and particles, and separating the complex from the mixture using external magnetic force. The DNA target material may then be eluted from the complex. The quantity of DNA target material eluted may be determined based on a calibration model. The methods of the present invention permit isolation of DNA target material which is within a known quantity range.

Abstract: The present invention entails methods, and kits for carrying them out, based on the discovery that an RNA replicase, such as Q&bgr; replicase, has DNA-dependent RNA polymerase (“DDRP”) activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2′-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase. The discovery of this DDRP activity provides methods of the invention for nucleic acid amplification wherein a nucleic acid, with a DNA segment with the sequence of an RNA that is autocatalytically replicatable by an RNA replicase, is provided as a substrate for the replicase. The replicase catalyzes synthesis, from the DNA segment, of the RNA, which the replicase then autocatalytically replicates. The invention entails use of the amplification methods in detecting nucleic acid analytes, as in nucleic acid probe hybridization assays.

Abstract: The present invention entails methods, and kits for carrying them out, based on the discovery that an RNA replicase, such as Q&bgr; replicase, has DNA-dependent RNA polymerase (“DDRP”) activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2′-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase. The discovery of this DDRP activity provides methods of the invention for nucleic acid amplification wherein a nucleic acid, with a DNA segment with the sequence of an RNA that is autocatalytically replicatable by an RNA replicase, is provided as a substrate for the replicase. The replicase catalyzes synthesis, from the DNA segment, of the RNA, which the replicase then autocatalytically replicates. The invention entails use of the amplification methods in detecting nucleic acid analytes, as in nucleic acid probe hybridization assays.

Abstract: The present invention provides a method for isolating RNA from a biological material comprising RNA and contaminants, wherein: the biological material is disrupted in the presence of a chaotropic agent, the resulting lysate is diluted to precipitate out contaminants, and the precipitate is removed from the lysate. RNA is preferably isolated from the resulting cleared lysate, using a silica matrix to bind and then release RNA bound thereto under particular conditions. The present invention also provides a method for isolating RNA from a solution comprising RNA and DNA, wherein: the RNA and DNA are bound to a silica matrix in the presence of at least one binding enhancer, the DNA is digested with DNase, and the RNA fluted therefrom.

Abstract: The present invention entails methods, and kits for carrying them out, based on the discovery that an RNA replicase, such as Q.beta. replicase, has DNA-dependent RNA polymerase ("DDRP") activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2'-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase. The discovery of this DDRP activity provides methods of the invention for nucleic acid amplification wherein a nucleic acid, with a DNA segment with the sequence of an RNA that is autocatalytically replicatable by an RNA replicase, is provided as a substrate for the replicase.

Abstract: Methods for controllably creating agglomerates from particles of colloidal dimensions. Creation of agglomerates permits physical isolation or separation of, e.g., unwanted cell debris from cleared cell lysate comprising target particles, by the deposition or creation of a blocking or masking layer. Chaotropic agents are utilized to enhance colloidal particle agglomeration. The resulting agglomerant or "floc" can be deposited, as needed, in a separation/isolation process to create a blocking or masking layer on an "as needed" basis.

Abstract: Methods for separating target particles, from a test medium in which the particles are suspended. The method contemplates utilization of blocking or masking particles which block or mask unwanted biological debris which tends to interfere with formation of complexes by which the target particles can be isolated. After centrifugation, the masking particles create a barrier between unwanted debris and test medium containing the target particles which permits the particles to be manipulated or modified without removing them from the debris. In a preferred aspect, magnetic separation techniques are used. In this aspect, non-magnetic 0.04 micron carboxylated polystyrene blocking particles are employed.

Abstract: Apparatus and methods for separating magnetically responsive particles from a nonmagnetic test medium in which the magnetically responsive particles are suspended. The separator comprises a container comprising, e.g., plurality of multiwell chambers, which hold the nonmagnetic test medium, one or more pins disposed substantially within the test medium, an external magnet for producing a magnetic field within the test medium. The external magnet is proximate the pins so as to create magnetic flux lines, magnetizing the pins and creating a magnetic field substantially parallel to the longitudinal axis of the pins thereof in the test medium. According to the method of the invention, the container holding the test medium is positioned in the separator, producing a magnetic field operative to cause the magnetically responsive particles to be attracted to and adhere to the pins and which is substantially parallel thereto.