Abstract: The present invention relates to the discovery of RNA 5? polyphosphatase enzymes not previously described in the art, methods for discovery of said enzymes, compositions of said enzymes, methods for making said enzymes, and various methods and kits for using said enzymes for biomedical research, for human and non-human diagnostics, for production of therapeutic products, and for other applications. In particular, some embodiments provide compositions, kits and methods for employing RNA polyphosphatases for isolation, purification, production, and assay of capped RNA using a biological sample or a sample from an in vitro capping reaction wherein the sample also contains RNA that is not capped. Other embodiments provide compositions, kits and methods wherein RNA polyphosphatases comprise signal-amplifying enzymes for analyte-specific assays.

Abstract: The present invention relates to the discovery of RNA 5? polyphosphatase enzymes not previously described in the art, methods for discovery of said enzymes, compositions of said enzymes, methods for making said enzymes, and various methods and kits for using said enzymes for biomedical research, for human and non-human diagnostics, for production of therapeutic products, and for other applications. In particular, some embodiments provide compositions, kits and methods for employing RNA polyphosphatases for isolation, purification, production, and assay of capped RNA using a biological sample or a sample from an in vitro capping reaction wherein the sample also contains RNA that is not capped. Other embodiments provide compositions, kits and methods wherein RNA polyphosphatases comprise signal-amplifying enzymes for analyte-specific assays.

Abstract: The present invention provides methods, compositions and kits for using an RNA polymerase for making transcription products corresponding to a target sequence by obtaining circular single-stranded DNA transcription substrates using a promoter primer that encodes one strand of a double-stranded promoter. The invention has broad applicability for research, diagnostic and therapeutic applications, such as preparing cDNA corresponding to mRNA, making sense or anti-sense probes, detecting gene- or organism-specific sequences, or making RNAi.

Abstract: The present invention provides methods, compositions and kits for using an RNA polymerase for making transcription products corresponding to a target sequence by obtaining single-stranded DNA transcription substrates that comprise a single-stranded promoter that is operably joined to a single-stranded target sequence. The invention has broad applicability for research, diagnostic and therapeutic applications, such as preparing cDNA corresponding to full-length mRNA, making sense or anti-sense probes, detecting gene- or organism-specific sequences, cloning, cell signaling or making RNA for use in RNAi.

Abstract: Methods, compositions and kits for amplifying a target sequence by strand displacement replication using strand-displacing primers. The method uses primers that have only ribonucleotides or purine ribonucleotides and at least one 2?-substituted pyrimidine-2?-deoxyribonucleotide.

Abstract: A method for synthesizing a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate using a mutant polymerase having a reduced discrimination between canonical and non-canonical substrates is disclosed. The method comprises incubating a template nucleic acid in a reaction mixture comprising the mutant nucleic acid polymerase and the appropriate canonical and non-canonical nucleoside triphosphates which are desired substrates for the mutant nucleic acid polymerase. The present invention is also a method of determining the sequence of a nucleic acid molecule using the mutant polymerase to create a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate.

Abstract: Methods, compositions and kits for amplifying a target sequence by strand displacement replication using strand-displacing primers. The method uses primers that have only ribonucleotides or purine ribonucleotides and at least one 2?-substituted pyrimidine-2?-deoxyribonucleotide.

Abstract: Methods, compositions and kits for amplifying a target sequence by strand displacement replication using strand-displacing primers. The method uses primers that have only ribonucleotides or purine ribonucleotides and at least one 2?-substituted pyrimidine-2?-deoxyribonucleotide.

Abstract: A method for synthesizing a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate using a mutant polymerase having a reduced discrimination between canonical and non-canonical substrates is disclosed. The method comprises incubating a template nucleic acid in a reaction mixture comprising the mutant nucleic acid polymerase and the appropriate canonical and non-canonical nucleoside triphosphates which are desired substrates for the mutant nucleic acid polymerase. The present invention is also a method of determining the sequence of a nucleic acid molecule using the mutant polymerase to create a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate.

Abstract: A method for retrofitting DNA in a single-copy or high-copy vector, such as a fosmid or BAC, whereby an artificial transposon is used to introduce a conditional multi-copy origin of replication (“ori”) into the DNA in said vector. Following random in vitro or in vivo transposition of the ori-containing transposon into DNA in the single-copy or low-copy vector, the resulting insertion clones are introduced into a special host strain that contains a gene which encodes a polypeptide required for replication from the multi-copy ori. However, since the gene for this polypeptide is expressed from a tightly-regulated inducible promoter, the polypeptide is not expressed in the absence of inducer. On addition of inducer to the culture medium, the host cell synthesizes the polypeptide, which in turn activates replication from the multi-copy ori, thereby increasing the amount of clone DNA synthesized by the cell.

Abstract: It can be difficult to achieve efficient transformation of many strains of bacterial cells due in part to the presence of one or more restriction and modification (R-M) systems in the cells that restricts unmodified transforming DNA. Phage T7 OCR protein is a potent inhibitor of Type I R-M systems. Methods are disclosed for improving transformation efficiency of Eubacterial and Archaebacterial cells having an R-M system by introducing into the cells an inhibitor of the restriction activity. For example, addition of 1–5 micrograms of T7 OCR protein to 50 microliters of electrocompetent cells having a Type I R-M system prior to electroporation significantly increased transformation efficiency by unmodified plasmids, fosmid clones, and artificial transposons comprising synaptic complexes.

Abstract: The present invention provides methods, compositions and kits for using an RNA polymerase for making transcription products corresponding to a target sequence by obtaining circular single-stranded DNA transcription substrates using a promoter primer that encodes one strand of a double-stranded promoter. The invention has broad applicability for research, diagnostic and therapeutic applications, such as preparing cDNA corresponding to mRNA, making sense or anti-sense probes, detecting gene- or organism-specific sequences, or making RNAi.

Abstract: The present invention comprises novel methods, compositions and kits that use N4 vRNAP deletion mutants to detect and quantify analytes comprising one or multiple target nucleic acid sequences, including target sequences that differ by as little as one nucleotide or non-nucleic acid analytes, by detecting a target sequence tag that is joined to an analyte-binding substance.

Abstract: Methods, compositions and kits kits for amplifying a target sequence by strand displacement replication using strand-displacing primers. The method uses primers that have only ribonucleotides or purine ribonucleotides and at least one 2′-substituted pyrimidine-2′-deoxyribonucleotide.

Abstract: The present invention comprises novel methods, compositions and kits comprising monopartite or bipartite target probes and an RNA polymerase to detect and quantify analytes comprising one or multiple target nucleic acid sequences, including target sequences that differ by as little as one nucleotide, or to detect and quantify non-nucleic acid analytes by detecting a target sequence tag that is joined to an analyte-binding substance. The method, called “target-dependent transcription,” consists of an annealing process, a DNA ligation process, a transcription process, and a detection process. The invention also comprises novel methods, compositions and kits for amplifying RNA, including strand-displacement reverse transcription and rolling circle reverse transcription.

Abstract: The present invention provides methods, compositions and kits for using an RNA polymerase for making transcription products corresponding to a target sequence by obtaining single-stranded DNA transcription substrates that comprise a single-stranded promoter that is operably joined to a single-stranded target sequence. The invention has broad applicability for research, diagnostic and therapeutic applications, such as preparing cDNA corresponding to full-length mRNA, making sense or anti-sense probes, detecting gene- or organism-specific sequences, cloning, cell signaling or making RNA for use in RNAi.

Abstract: A method for synthesizing a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate using a mutant polymerase having a reduced discrimination between canonical and non-canonical substrates is disclosed. The method comprises incubating a template nucleic acid in a reaction mixture comprising the mutant nucleic acid polymerase and the appropriate canonical and non-canonical nucleoside triphosphates which are desired substrates for the mutant nucleic acid polymerase. The present invention is also a method of determining the sequence of a nucleic acid molecule using the mutant polymerase to create a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate.

Abstract: A method for synthesizing a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate using a mutant polymerase having a reduced discrimination between canonical and non-canonical substrates is disclosed. The method comprises incubating a template nucleic acid in a reaction mixture comprising the mutant nucleic acid polymerase and the appropriate canonical and non-canonical nucleoside triphosphates which are desired substrates for the mutant nucleic acid polymerase. The present invention is also a method of determining the sequence of a nucleic acid molecule using the mutant polymerase to create a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate.

Abstract: A method for synthesizing a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate using a mutant polymerase having a reduced discrimination between canonical and non-canonical substrates is disclosed. The method comprises incubating a template nucleic acid in a reaction mixture comprising the mutant nucleic acid polymerase and the appropriate canonical and non-canonical nucleoside triphosphates which are desired substrates for the mutant nucleic acid polymerase. The present invention is also a method of determining the sequence of a nucleic acid molecule using the mutant polymerase to create a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate.

Abstract: A method of characterizing a nucleic acid molecule is disclosed. The method comprises synthesizing DNA in the presence of a reaction mixture comprising a nucleic acid template, a primer molecule, an enzyme that extends the primer so that a DNA molecule may be synthesized, four canonical deoxynucleoside triphosphates and at least one non-canonical deoxynucleoside triphosphate. The non-canonical deoxynucleoside triphosphate is incorporated into the synthesized DNA in place of a portion of only one canonical deoxynucleoside triphosphate. The synthesized DNA is treated with an N-glycosylase that excises a base portion of the non-canonical deoxynucleoside triphosphate from the synthesized DNA. The DNA is then treated in such a manner that the phosphodiester backbone of the DNA is broken at the abasic site, thus creating at least two DNA fragments. The fragments are separated according to size.