Abstract: Provided is kit for and a method of amplifying a nucleic acid using rolling cyclic amplification (RCA), including amplifying a nucleic acid together with formation of a single-strand circular DNA template using RCA by reacting a reaction solution including: (a) two hairpin oligos, (b) a target nucleic acid, (c) a DNA ligase,(d) an endonuclease, (e) a DNA polymerase, and (f) a primer.

Abstract: Methods, devices, and systems for performing intermittent detection during analytical reactions are provided. Such methods facilitate collection of reaction data from disparate reaction times. Further, such methods are useful for reducing photo-induced damage of one or more reactants in an illuminated analytical reaction at a given reaction time. In preferred embodiments, the reaction mixture is subjected to at least one illuminated and non-illuminated period and allowed to proceed such that the time in which the reaction mixture is illuminated is less than a photo-induced damage threshold period.

Abstract: An active ubiquitin E3 ligase, GRAIL, is crucial in the induction of anergy in cells of the immune system, and in the regulation of cellular proliferation. GRAIL is shown to associate with, and be regulated by Otubain isoforms, including OTUBAIN-1 (DOG, the Destabilizer of GRAIL) and an alternative reading frame splice variant of OTUBAIN-1 (SOG, the Stabilizer of GRAIL). These proteins play opposing roles in the regulation of GRAIL auto-ubiquitination and consequently on its ability to induce anergy and regulate cellular proliferation. DOG serves as an adaptor protein, recruiting the DUB USP8. One major substrate for USP8 is the Ras exchange factor Ras-GRF1, and this protein can be found in a complex with USP8 and GRAIL, which complex is ubiquitinated by GRAIL.

Abstract: A method of modulation of synthesis capacity on and cleavage properties of synthetic oligomers from solid support is described. The method utilizes linker molecules attached to a solid surface and co-coupling agents that have similar reactivities to the coupling compounds with the surface functional groups. The preferred linker molecules provide an increased density of polymers and more resistance to cleavage from the support surface. The method is particularly useful for synthesis of oligonucleotides, oligonucleotides microarrays, peptides, and peptide microarrays. The stable linkers are also coupled to anchor molecules for synthesis of DNA oligonucleotides using on support purification, eliminating time-consuming chromatography and metal cation presence. Oligonucleotides thus obtained can be directly used for mass analysis, DNA amplification and ligation, hybridization, and many other applications.

Abstract: Provided is a method for analysis of DNA methylation. A first method comprises: (1) a step in which analyte DNA is digested with methylation-insensitive restriction enzyme(s) that produce overhanging end(s) and that contain methylated cytosine or methylatable cytosine in recognition sequence(s) thereof; (2) a step in which adaptor(s) capable of regenerating recognition sequence(s) of the aforesaid methylation-insensitive restriction enzyme(s) is/are ligated to both ends of DNA fragment(s) obtained at the aforesaid step (1); and (3) a step in which DNA construct(s) obtained at the aforesaid step (2) is/are digested with methylation-sensitive restriction enzyme(s) that recognize the same recognition sequence(s) as the aforesaid methylation-insensitive restriction enzyme(s).

Abstract: The invention is based upon the discovery that small nucleic acids from non-viral pathogens are able to cross the kidney and are present in urine of a subject when the subject is infected with the non-viral pathogen. These transrenal DNAs are especially prevalent at smaller sizes under about 300 bp. Thus the invention provides compositions and methods for the diagnosis of infection of a subject with non-viral pathogens through the detection of transrenal nucleic acids from those pathogens in the urine of the subject.

Abstract: The present invention relates to a reaction mixture for the amplification of nucleic acids, the non-methylated cytosine bases of which have been converted to uracil bases by means of a bisulfition reaction. The invention also discloses methods for amplifying bisulfited nucleic acid and for determining the nucleic acid methylation state, and also kits based on the reaction mixture according to the invention.

Abstract: The present invention relates to a polymorphic CYP2C8-polynucleotide. Moreover, the invention relates to genes or vectors comprising the polynucleotides of the invention and to a host cell genetically engineered with the polynucleotide or gene of the invention. Further, the invention relates to methods for producing molecular variant polypeptides or fragments thereof, methods for producing cells capable of expressing a molecular variant polypeptide and to a polypeptide or fragment thereof encoded by the polynucleotide or the gene of the invention or which is obtainable by the method or from the cells produced by the method of the invention. Furthermore, the invention relates to an antibody which binds specifically the polypeptide of the invention. Moreover, the invention relates to a transgenic non-human animal. The invention also relates to a solid support comprising one or a plurality of the above mentioned polynucleotides, genes, vectors, polypeptides, antibodies or host cells.

Abstract: Provided herein are methods for ligation of polynucleotides containing modified ligation components, particularly modified ligase cofactors, modified acceptors and modified donors. The methods readily applied to ligation-based assays for detection of a nucleic acid sequence where the use of the modified cofactor improves discrimination between matched and mismatched templates. Furthermore, the use of the modified ligation components reduces or eliminates the ligation in the absence of nucleic acid template.

Abstract: The invention provides compositions and methods relating to delivery of agents in vivo or in vitro. More specifically, the invention provides nanoparticles synthesized from crosslinked nucleic acids, optionally having a lipid shell or coating, and may further comprise for example small molecule or high molecular weight compounds as therapeutic or diagnostic agents.

Abstract: The invention provides compositions and methods relating to delivery of agents in vivo or in vitro. More specifically, the invention provides nanoparticles synthesized from crosslinked nucleic acids, optionally having a lipid shell or coating, and may further comprise for example small molecule or high molecular weight compounds as therapeutic or diagnostic agents.

Abstract: The present invention provides a method of covalently joining a DNA strand to an RNA strand comprising (a) forming a topoisomerase-DNA intermediate by incubating a DNA cleavage substrate comprising a topoisomerase cleavage site with a topoisomerase specific for that site, wherein the topoisomerase-DNA intermediate has one or more 5? single-strand tails; and (b) adding to the topoisomerase-DNA intermediate an acceptor RNA strand complementary to the 5? single-strand tail under conditions permitting a ligation of the covalently bound DNA strand of the topoisomerase-DNA intermediate to the RNA acceptor strand and dissociation of the topoisomerase, thereby covalently joining the DNA strand to the RNA strand. The present invention also provides a method of tagging a 5? end of an RNA molecule. The present invention further provides a DNA-RNA molecule which has been joined in vitro by the use of a topoisomerase. The present invention also provides a method of tagging a 5? end of an mRNA.

Abstract: The invention is directed to a variety of multiplexing methods used to amplify and/or genotype a variety of samples simultaneously.

Abstract: Provided herein are methods, compositions and kits to extract and relatively enrich by physical separation or amplification short base pair nucleic acid in the presence of a high background of genomic material (e.g., host or maternal nucleic acids).

Abstract: Described herein are methods and reagents for the ligation of a peptide acceptor to an RNA, as well as the RNA-peptide acceptor products.

Abstract: An object of the present invention is to provide a simple and safe method for synthesis of single-stranded or double-stranded DNA and a kit for performing the synthesis.

Abstract: Methods and compositions are provided for repairing a polynucleotide so that it can be copied with improved fidelity and/o yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a DNA ligase and an effective amount of at least one endonuclease as well as a cofactor selected from NAD+ or ATP.

Abstract: The present invention relates to a method for producing a DNA chip, which comprises the steps of: (a) cloning a probe, where a linker is coupled to one or both ends of an oligonucleotide to be integrated on a slide, into a vector; (b) transforming host cells with the vector; (c) culturing the transformed host cells, to recover the probe where the linker is coupled to one or both ends of the oligonucleotides; and (d) integrating the recovered double-helical probes on a slide. Also, the present invention relates to a DNA chip for HPV diagnosis produced by the method, and a method for diagnosing the presence or genotype of HPV using the DNA chip.

Abstract: The present invention is directed generally to methods facilitating the cloning of nucleic acid molecules. In particular, the invention relates to the use of polymerase inhibitors, including but not limited to anti-polymerase antibodies (such as anti-Taq antibodies) and fragments thereof, to inactivate residual polymerase activity remaining after the amplification (particularly via PCR) of a target nucleic acid molecule. The invention further provides compositions, particularly storage-stable compositions, comprising one or more components, such as one or more restriction endonucleases and one or more polymerase inhibitors, that are useful in cloning amplified or synthesized nucleic acid molecules by the above-described methods. The invention also relates to nucleic acid molecules produced by these methods, and to genetic constructs (such as vectors) and host cells comprising these nucleic acid molecules.

Abstract: The present invention relates to a single-chain circular RNA having a sustained or slow-releasing RNA interference effect, characterized in that the single-chain circular RNA comprises a sense strand sequence, an antisense strand sequence complementary to the sense strand sequence, identical or different two loop sequences between the sense strand and the antisense strand, connecting both strands, wherein the sense strand and the antisense strand are paired to form a stem.

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: The invention provides nucleoside and nucleotide molecules containing cleavable linkers linking a label such as a dye. The invention also provides nucleosides and nucleotide molecules containing a blocking group, either removable or non-removable. The invention additionally provides methods of using the nucleoside and nucleotide molecules containing a cleavable linker and/or a blocking group.

Abstract: Compositions and methods for chemical ligation are provided. Methods for nucleic acid sequencing, nucleic acid assembly and nucleic acid synthesis are also provided.

Abstract: The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5?-CMeCpGG-3?. Methods for making and using the rMcrA protein, and derivatives thereof are provided.

Abstract: Methods and compositions for making and using pre-adenylated oligonucleotide sequences are provided.

Abstract: The present invention provides a method of covalently joining a DNA strand to an RNA strand comprising (a) forming a topoisomerase-DNA intermediate by incubating a DNA cleavage substrate comprising a topoisomerase cleavage site with a topoisomerase specific for that site, wherein the topoisomerase-DNA intermediate has one or more 5? single-strand tails; and (b) adding to the topoisomerase-DNA intermediate an acceptor RNA strand complementary to the 5? single-strand tail under conditions permitting a ligation of the covalently bound DNA strand of the topoisomerase-DNA intermediate to the RNA acceptor strand and dissociation of the topoisomerase, thereby covalently joining the DNA strand to the RNA strand. The present invention also provides a method of tagging a 5? end of an RNA molecule. The present invention further provides a DNA-RNA molecule which has been joined in vitro by the use of a topoisomerase. The present invention also provides a method of tagging a 5? end of an mRNA.

Abstract: A method of synthesizing a cDNA chain using an insoluble carrier having on the surface thereof a polymer substance containing a first unit having a group derived from a phosphoric ester composing the hydrophilic portion of phospholipid, and a second unit having a group derived from carboxylic acid having an electron-attractive substituent bound to a carbonyl group, which includes immobilizing a polynucleotide for DNA elongation; bringing a solution containing an RNA fragment, nucleotide monomers, and a reverse transcriptase or an enzyme having polymerase activity into contact with the surface of the insoluble carrier; and allowing the polynucleotide for DNA elongation immobilized on the surface of the carrier to elongate using the RNA fragment contained in the solution as a template, to thereby form a single-strand cDNA.

Abstract: Methods, reagents, and kits for (mis)ligating oligonucleotide probes or for identifying at least one target nucleotide are disclosed. One can enhance the generation of misligation product using a ligase under reaction conditions and with reagents where that particular ligase is prone to misligation. Alternatively, one can decrease or avoid generating misligation products using a particular ligase under reaction conditions and using reagents where that ligase is at least less prone to misligation. In certain embodiments, the recombinant ligase from Archaeoglobus fulgidus (Afu) is employed due to its unique misligation properties.

Abstract: Methods are provided for making a circular duplex polynucleotide. Such methods can include providing a mixture of sequence specific linear duplex polynucleotides and denaturing and reannealing the polynucleotide mixture under conditions such that some of the polynucleotides form circular duplexes that comprise the desired polynucleotide.

Abstract: The present invention is related to a method for the manufacture of a nucleic acid molecule comprising the following steps: a) providing a first at least partially double-stranded oligonucleotide, whereby the oligonucleotide comprises a first and a second single-stranded overhang, b) providing a second at least partially double-stranded oligonucleotide, whereby the oligonucleotide comprises a recognition site for a first type IIS restriction enzyme which cuts outside its recognition site, a modification allowing the oligonucleotide to be coupled to a surface and a single-stranded overhang, c) ligating the first oligonucleotide and the second oligonucleotide via the first single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide, generating a first ligation product, whereby the first ligation product comprises a single-stranded overhang essentially corresponding to the second single-stranded overhang of the first oligonucleotide, d) cutting the first l

Abstract: Methods for measuring environmental parameters using chemical recording are provided. In some embodiments, the methods include generating a polymer comprising an ordered series of chemical units, wherein the position and number of each chemical unit in the polymer is indicative of a reading of the environmental state variable at a given point in time. The presently disclosed subject matter also provides compositions that can be employed in and/or that employ the disclosed methods for recording environmental state variables.

Abstract: Transposase proteins that are modified relative to and have higher transposase activities than the wild-type Tn5 transposase are disclosed. A transposase protein of the present invention differs from the wild-type Tn5 transposase at amino acid position 41, 42, 450, or 454 and has greater avidity than the wild-type Tn5 transposase for at least one of a Tn5 outside end sequence as defined by SEQ ID NO:3, a Tn5 inside end sequence as defined by SEQ ID NO:4, and a modified Tn5 outside end sequence as defined by SEQ ID NO:5. Also disclosed are various systems and methods of using the transposase proteins of the present invention for in vitro or in vivo transposition.

Abstract: The invention is directed to a variety of multiplexing methods used to amplify and/or genotype a variety of samples simultaneously.

Abstract: The invention provides methods for sequencing by hybridization (SBH) using pools of probes that allow greater efficiency in conducting SBH by reducing the number of separate measurements of hybridization signals required to identify each particular nucleotide in a target nucleic acid sequence. The invention also provides pools and sets of pools of probes, as well as methods of generating pools of probes.

Abstract: Methods and systems for removing masking agents from test samples, e.g., DNA-containing samples obtained from living subjects, when they are submitted for or subjected to molecular assays. The present invention allows molecular assays of nucleic acids in bodily fluids and excretions, such as urine, blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid, salivary fluid, vaginal fluid, stool, seminal fluid, and sweat to be carried out with greater sensitivity. The masking agents are suppressed by contacting a test sample with an amount of one or more divalent metal chelators and an amount of one or more chelator enhancing components. The amounts of the divalent metal chelator(s) and the chelator enhancing component(s) are selected such that interference of a masking agent on a molecular assay of a nucleic acid-containing test sample are suppressed, and upon contact with the divalent metal chelator(s)/chelator enhancing component(s), the masking agents are suppressed.

Abstract: The present invention provides at least one isolated linear composite nucleic acid molecule comprising at least one first tag from at least one first nucleic acid molecule and at least one second tag from at least one second nucleic acid molecule, wherein the first and second nucleic acids interact in a nucleic acid mixture; and wherein the first and second tags are from different nucleic acid molecules. The invention also provides a method of producing at least one isolated linear composite nucleic acid and to a method of detecting and/or identifying nucleic acid interactions.

Abstract: The invention relates to a method for producing polymers, in particular synthetic nucleic acid double strands of optional sequence, comprising the steps: (a) provision of a support having a surface area which contains a plurality of individual reaction areas, (b) location-resolved synthesis of nucleic acid fragments having in each case different base sequences in several of the individual reaction areas, and (c) detachment of the nucleic acid fragments from individual reaction areas.

Abstract: Compositions that describe a thermostable DNA ligase isolated from Thermococcus sp. (strain 9° N-7) and methods for making und using the same are described. The thermostable DMA ligase depends on ATP and not NAD+ as a cofactor during ligation, and retains activity at 100° C.

Abstract: The invention features methods of analyzing the kinetics properties of transfection reactions. Also featured are methods for creating structural promoters which are effectively unregulated by enhancers and repressors. The structural promoters are significantly more active than the native promoter sequences upon which they are based.

Abstract: Methods, systems and kits are provided for detecting molecules expressing a selected epitope in a sample through use of an epitope detector containing a single chain Fv for the selected epitope or a constrained epitope specific CDR, CDR mimetic or engineered CDR structure attached to an oligonucleotide.

Abstract: We describe a method of selecting an enzyme having replicase activity, the method comprising the steps of: (a) providing a pool of nucleic acids comprising members each encoding a replicase or a variant of the replicase; (b) subdividing the pool of nucleic acids into compartments, such that each compartment comprises a nucleic acid member of the pool together with the replicase or variant encoded by the nucleic acid member; (c) allowing nucleic acid replication to occur; and (d) detecting amplification of the nucleic acid member by the replicase. Methods for selecting agents capable of modulating replicase activity, and for selecting interacting polypeptides are also disclosed.

Abstract: Disclosed is a modified hyperthermophilic DNA ligase having improved DNA binding ability and reactivity. The modified hyperthermophilic DNA ligase has an amino acid sequence corresponding to the amino acid sequence of a heat-resistant DNA ligase derived from a thermophilic bacterium, a hyperthermophilic bacterium, a thermophilic archaebacterium, or a hyperthermophilic archaebacterium, except with at least two of charged amino acids in the C-terminal helix region each being substituted by alanine, threonine, or serine residues.

Abstract: The present invention relates to a method of producing single-stranded nucleic acid molecules from oligo- or polynucleotides wherein each of said oligo- or polynucleotides has a predefined 5? or 3? terminus, comprising the steps of (a) annealing an adaptor oligonucleotide simultaneously or step by step to (aa) a first oligo- or polynucleotide; and (ab) a second oligo- or polynucleotide wherein the 5?-terminus of said adaptor oligonucleotide is complementary in sequence to the 5? terminus of said first oligo- or polynucleotide and the 3?terminus of said adaptor molecule is complementary in sequence to the 3? terminus of said second oligo- or polynucleotide; and optionally (a?) simultaneously with or subsequently to step (a) annealing at least one further adaptor oligonucleotide to free termini of said first or second oligonucleotides and to free termini of further oligo- or polynucleotides; (b) optionally filling in gaps between the neighbouring ends of said oligo- or polynucleotides; (c) ligating said oligo-

Abstract: The present invention features methods and compositions for the renaturation, hybridization, association, or reassociation of nucleic acids that combines both acceleration of the reaction rate and improved specificity.

Abstract: Nucleic acid sequences encoding at least a portion of a polypeptide are directly incorporated into a plasmid by DNA polymerization or reverse transcription of a nucleic acid template. In particularly preferred embodiments, nucleic acid sequences encoding at least a portion of an antibody are directly incorporated into a plasmid by reverse transcription of messenger RNA (mRNA).

Abstract: The invention provides methods for evolving a polynucleotide toward acquisition of a desired property. Such methods entail incubating a population of parental polynucleotide variants under conditions to generate annealed polynucleotides comprising heteroduplexes. The heteroduplexes are then exposed to a cellular DNA repair system to convert the heteroduplexes to parental polynucleotide variants or recombined polynucleotide variants. The resulting polynucleotides are then screened or selected for the desired property.

Abstract: The method of the invention improves the specificity of a ligation reaction carried out between a first double-stranded polynucleotide having a single-stranded portion and a second polynucleotide having a complementary single-stranded portion, the second polynucleotide being present in a sample comprising a mixture of different polynucleotides. The method comprises contacting the sample, under hybridising conditions, with the first polynucleotide and one or more third polynucleotide(s) wherein the third polynucleotide(s) comprises a single-stranded portion that differs from the single-stranded portion of the first polynucleotide by at least one base substitution.

Abstract: The present invention is the development of a simple and specific quantitative method for the determination of P. falciparum DNA in malaria that involves the direct detection of the highly 42-kDa conserved C-terminal region of P. falciparum merozoite surface protein (MSP1) gene. This procedure entails the amplification of the 42-kDa C-terminal region of the MSP1 gene by using the PCR technique in the presence of digoxigenin-11-dUTP and the synthesis of the specific biotin labeled nucleotide probes directed to the 42-kDa C-terminal region of the MSP1 gene. These specific probes are then used in the Enzyme Linked Immunosorbent Assay (ELISA) for the quantitative determination of the 42-kDa C-terminal region of the MSP1 gene which leads to the quantitative determination of P. falciparum DNA in malaria for quantitative diagnostic purpose as well as for monitoring the efficacy of antimalarial treatment.

Abstract: The present invention is related generally to analysis of polynucleotides, particularly polynucleotides derived from genomic DNA. The invention provides methods, compositions and systems for such analysis. Encompassed by the invention are constructs that include pairs of target sequences which are separated by a known distance in the polynucleotide from which they are derived.

Abstract: Methods, systems and kits are provided for detecting, molecules expressing a selected epitope in a sample through use of an epitope detector containing a single chain Fv for the selected epitope or a constrained epitope specific CDR attached to an oligonucleotide.