Abstract: Compositions of transposome complexes for generating DNA fragments with specific 5?- and 3?-tags. Kits for generating libraries for sequencing, with transposome complexes, enzymes, oligonucleotides or other components.

Abstract: The present invention provides methods, compositions, and kits for generating rRNA-depleted samples and for isolating rRNA from samples. In particular, the present invention provides compositions comprising affinity-tagged antisense rRNA molecules corresponding to substantially all of at least one rRNA molecule (e.g., 28S, 26S, 25S, 18S, 5.8S and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S and 5S prokaryotic rRNA molecules) and methods for using such compositions to generate rRNA-depleted samples or to isolate rRNA molecules from samples.

Abstract: The present invention provides compositions and methods for controlling the copy number for a broad range of plasmids and uses thereof. Disclosed is a host cell for conditional control of copy number of a plasmid, which host cell comprises a poly(A) polymerase gene that is operably joined to a conditionally inducible promoter, and a method for cloning and stably maintaining a DNA sequence encoding a heterologous polypeptide in the host cell.

Abstract: The present invention provides novel compositions, kits and methods employing RNA 5? polyphosphatases, RNA 5? monophosphatases, capping enzymes, decapping enzymes, nucleic acid pyrophosphatases and RNA ligases, as well as other enzymes, for selective 5? ligation tagging of desired classes of RNA molecules that differ with respect to particular chemical moieties on their 5? ends. The 5?tagged RNA molecules can be used for synthesis of tagged first-stand cDNA, double-stranded cDNA, and sense or antisense RNA for a variety of uses.

Abstract: Methods, compositions and kits are disclosed for obtaining directionally truncated polypeptides by inserting a transposon. Preferably the transposon comprises a selectable marker and an ori, and optionally a promoter, a ribosome binding site and a translation start codon, into a target sequence in vitro or in vivo. Amplification products, varying in length depending on the transposon insertion site, are obtained using one primer that anneals to the target sequence and a second primer that anneals to the transposon. Amplification products are ligated to circular dsDNA, transformed into host cells, and individual colonies, each containing a directionally truncated clone of the target sequence, are obtained by plating on medium for which the selectable marker encodes resistance. Directionally truncated polypeptides encoded by the target sequence are obtained in vivo by inducing an RNAP in the host cells that uses the promoter or, in vitro by cell-free transcription and translation.

Abstract: The present invention provides methods, compositions and kits for using a transposase and a transposon end for generating extensive fragmentation and 5?-tagging of double-stranded target DNA in vitro, then using a DNA polymerase for generating 5?- and 3?-tagged single-stranded DNA fragments without performing a PCR amplification reaction, wherein the first tag on the 5?-ends exhibits the sequence of the transferred transposon end and optionally, an additional arbitrary sequence, and the second tag on the 3?-ends exhibits a different sequence from the sequence exhibited by the first tag. The method is useful for generating 5?- and 3?-tagged DNA fragments for use in a variety of processes, including processes for metagenomic analysis of DNA in environmental samples, copy number variation (CNV) analysis of DNA, and comparative genomic sequencing (CGS), including massively parallel DNA sequencing (so-called “next-generation sequencing.

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: The present invention relates generally to methods, compositions and kits for synthesizing sense RNA molecules from one or more RNA molecules of interest in a sample. In exemplary embodiments, the methods use a terminal tagging oligoribonucleotide (rTTO) to join a DNA sequence tag to the 3?-termini of first-strand cDNA molecules. The use of an rTTO comprising ribonucleotides results in decreased oligonucleotide-derived background synthesis of RNA in the absence of sample RNA and, surprisingly and unexpectedly, also results in significantly increased yields of sense RNA molecules that exhibit sequences that are substantially identical to those of the RNA molecules of interest in the sample. The sense RNA molecules also have an RNA sequence tag on their 5?-termini that is useful for fixing the lengths of sense RNA molecules that are synthesized in a second or subsequent round.

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: 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 and compositions are provided for assaying for a target analyte in a sample by formation of a substrate for a replicase. The target analyte, if present in the sample, is first bound to a reporter probe. The reporter probe comprises a first portion and a second portion. The first portion comprises a polynucleotide that encodes at least part of a sequence for an RNA that is a substrate for replication. The second portion comprises a molecule that has affinity for an analyte. The reporter probe itself is not a substrate for a replicase. However, a replicase substrate is generated by treating reporter probe which is bound to analyte with a composition having nuclease activity in order to release the parts of the first portion, and then the released parts and one or more mononucleotides or oligonucleotides comprising missing parts of the substrate sequence are joined with a composition having ligase activity.

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.

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 analyzing a DNA molecule is disclosed. In one embodiment the method comprises the steps of exposing a DNA molecule to an effective amount of a chemical modification reagent wherein the reagent converts guanine to 8-hydroxyguanine. The oxidized product is then exposed to a DNA glycosylase enzyme and the DNA molecule is cleaved at the site of the 8-hydroxyguanine. The fragments are then resolved by electrophoresis and the position of guanine residues within the DNA molecule is determined. In a preferred embodiment of the present invention, the modification reagent is a thiazine dye and the enzyme is FPG protein.

Abstract: A method of improving the synthesis of full-length cDNA transcripts by Mn.sup.++ -dependent reverse transcriptases, preferably DNA-dependent DNA polymerases, is disclosed.

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: An essentially pure preparation of thermostable RNase H isolated from Thermus flavus is disclosed. In one preferable form of the invention, the RNase H is capable of biological activity after incubation at temperatures equal to or greater than 70.degree. C. for at least ten minutes. The present invention is also a method of digesting RNA polymers that are in duplex form with a DNA molecule. The method comprises exposing the duplex to the isolated thermostable RNase H. The present invention is also a genetic construct capable of expressing a thermostable RNase H.