Abstract: An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Abstract: An RNA ligase derived from bacteriophage TS2126 which infects Thermus scotoductus, nucleic acids comprising nucleotide sequences of open reading frame (ORF) and polypeptides encoded by the nucleic acids, are described.

Abstract: The present invention relates to a method of detecting a base at a pre-determined position in a nucleic acid molecule by performing enzymatic detection reactions using base-specific detection oligomers, where each oligomer is specific for a particular base at the predetermined position, and then comparing the enzymatic detection reactions to determine which base is present at the position, with an enzyme-disabling agent being present during the enzymatic detection reaction. In preferred embodiments the enzymatic detection reaction is an oligomer elongation extension reaction, catalysed by, among others, polymerase or ligase. Also disclosed are methods of performing the assay in a liquid phase and on microarrays.

Abstract: The present invention relates to the design and use of nucleic acid molecules to create novel materials. The present invention further relates to the use of DNA as a bulding block for DNA-materials that are of high yield and purity and that can be incorporated into larger structures.

Abstract: The presently claimed invention provides methods and kits for amplifying a target sequence from within a nucleic acid population. The presently claimed invention provides selection probes which are complementary to at least a portion of said target sequence and mechanisms for adding a probe sequence to the 3? end of a target sequence that is hybridized to a selection probe. The added 3? probe sequence and a probe sequence added at the 5? end of the target by adaptor ligation allow for selective amplification of the target sequence.

Abstract: The present invention provides random cDNA expression vector libraries, comprising expression vectors which comprise random cDNAs positioned in sense and antisense orientation, which are useful for the delivery and expression of a combination of genetic effector types to host cells. Methods for producing these libraries through bi-directional cloning of random cDNAs are also provided. Also provided herein are methods of using these libraries to screen for agents capable of modulating cell phenotype in desirable ways.

Abstract: The present invention is a method for detecting DNA sequence differences including single nucleotide mutations or polymorphisms, one or more nucleotide insertions, and one or more nucleotide deletions. Labeled heteroduplex PCR fragments containing base mismatches are prepared. Endonuclease cleaves the heteroduplex PCR fragments both at the position containing the variation (one or more mismatched bases) and to a lesser extent, at non-variant (perfectly matched) positions. Ligation of the cleavage products with a DNA ligase corrects non-variant cleavages and thus substantially reduces background. This is then followed by a detection step in which the reaction products are detected, and the position of the sequence variations are determined.

Abstract: The present invention is drawn to a method for characterising nucleic acid molecules, which comprises the steps of: i) introducing a modified base which is a substrate for a DNA glycosylase into a DNA molecule; ii) excising the modified base with the DNA glycosylase to generate an abasic site; iii) cleaving the DNA at the abasic site to generate and release an extendible upstream DNA fragment having a 3? hydroxyl terminus; and iv) incubating the released extendible upstream DNA fragment in the presence of an enzyme allowing for extension thereof and an additional template nucleic acid and analysing resultant fragment(s).

Abstract: Recombinant DNA vectors and methods for cloning and expressing nucleic acid molecules by using a combination of site-specific recombination and end-to-end joining or linking of nucleic acid molecules, such as endonuclease restriction digestion and ligation. The DNAs, vectors and methods can be used for inserting, exchanging, transferring a variety of DNA segment(s) both in vitro and in vivo. Also disclosed are linker molecules and methods using these linkers the can be used for cloning a gene of interest into an expression vector in one-step. The linker sequences comprise adapter sequences for cloning purposes, as well as eukaryotic and prokaryotic ribosome binding sites for increase translation efficiency.

Abstract: The present invention provides novel polynucleotides encoding BGS-42 polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel BGS-42 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

Abstract: The present invention provides efficient transfer of genes into host cells or embryos to transform the cells or embryos by transposition vectors using the minimal amount of nucleotide sequences in the transposon piggyBac required for gene transfer. The transformed cells or embryos may also be developed into transgenic organisms.

Abstract: A method of making very long, double-stranded synthetic poly-nucleotides. A multiplicity of short oligonucleotides is provided. The short oligonucleotides are sequentially hybridized to each other. Enzymatic ligation of the oligonucleotides provides a contiguous piece of PCR-ready DNA of predetermined sequence.

Abstract: The present invention describes a method for identifying one or more of a plurality of sequences differing by one or more single base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences. The method includes a ligation phase, a capture phase, and a detection phase. The ligation phase utilizes a ligation detection reaction between one oligonucleotide probe, which has a target sequence-specific portion and an addressable array-specific portion, and a second oligonucleotide probe, having a target sequence-specific portion and a detectable label. After the ligation phase, the capture phase is carried out by hybridizing the ligated oligonucleotide probes to a solid support with an array of immobilized capture oligonucleotides at least some of which are complementary to the addressable array-specific portion. Following completion of the capture phase, a detection phase is carried out to detect the labels of ligated oligonucleotide probes hybridized to the solid support.

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: A method of analyzing intestinal bacteria flora which comprises: extracting a sample from a subject; extracting bacteria to prepare a bacteria suspension; extracting DNAs of bacteria from the bacteria suspension to prepare a DNA extract liquid; amplifying a specific region such as 16S rDNA using the DNA extract liquid; fractionating the amplified fragments by electrophoresis to obtain a fractional pattern; and comparing the fractional pattern with preliminarily obtained electrophoretic patterns of amplified fragments of intestinal bacteria flora, thereby analyzing the intestinal bacteria flora of the subject.

Abstract: Methods for discriminating between fully complementary hybrids and those that differ by one or more base pairs and libraries of unimolecular, double-stranded oligonucleotides on a solid support. In one embodiment, the present invention provides methods of using nuclease treatment to improve the quality of hybridization signals on high density oligonucleotide arrays. In another embodiment, the present invention provides methods of using ligation reactions to improve the quality of hybridization signals on high density oligonucleotide arrays. In yet another embodiment, the present invention provides libraries of unimolecular or intermolecular, double-stranded oligonucleotides on a solid support. These libraries are useful in pharmaceutical discovery for the screening of numerous biological samples for specific interactions between the double-stranded oligonucleotides, and peptides, proteins, drugs and RNA.

Abstract: The increased use of nucleotide sequence data mining techniques has amplified the demand for efficient methods of producing recombinant proteins in eukaryotic cells. A strategy is provided for enhancing the synthesis of recombinant amino acid sequences by polymerizing expression cassettes in vitro before producing recombinant hosts.

Abstract: The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of nucleic acid from various viruses in a sample.

Abstract: The present invention relates to genetically engineered attenuated viruses and methods for their production. In particular, the present invention relates to engineering live attenuated viruses which contain a modified NS gene segment. Recombinant DNA techniques can be utilized to engineer site specific mutations into one or more noncoding regions of the viral genome which result in the down-regulation of one or more viral genes. Alternatively, recombinant DNA techniques can be used to engineer a mutation, including but not limited to an insertion, deletion, or substitution of an amino acid residue(s) or an epitope(s) into a coding region of the viral genome so that altered or chimeric viral proteins are expressed by the engineered virus.

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: The present invention describes a method for identifying one or more of a plurality of sequences differing by one or more single base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences. The method includes a ligation phase, a capture phase, and a detection phase. The ligation phase utilizes a ligation detection reaction between one oligonucleotide probe, which has a target sequence-specific portion and an addressable array-specific portion, and a second oligonucleotide probe, having a target sequence-specific portion and a detectable label. After the ligation phase, the capture phase is carried out by hybridizing the ligated oligonucleotide probes to a solid support with an array of immobilized capture oligonucleotides at least some of which are complementary to the addressable array-specific portion. Following completion of the capture phase, a detection phase is carried out to detect the labels of ligated oligonucleotide probes hybridized to the solid support.

Abstract: The present invention provides vectors and methods which improve the efficiency of nucleic acid insertion into circular vectors, which generally facilitate nucleic acid cloning and specifically facilitate the preparation of DNA libraries. In general, the present invention involves separation of the cloning process into two distinct steps: (a) insertion which is done at a high nucleic acid concentration favoring intermolecular joining, and (b) circularization which is performed at a low nucleic acid concentration favoring intramolecular circularization. The present vectors generally have distinct insertion ends and circularization ends which are blocked from covalent joining during the insertion step. Circularization ends contemplated by the present invention include complementary cohesive ends and topoisomerase-linked ends. The present vectors and methods allow minute amounts of nucleic acid inserts to be efficiently cloned.

Abstract: The present invention provides methods for the amplification of nucleic acid molecules. Methods for amplifying target polynucleotides, including mRNA, using oligonucleotides, DNA and RNA polymerases are provided. The invention further provides compositions and kits for practicing the methods, as well as methods which use the amplification products.

Abstract: Provided are compositions and methods useful for modulating the activity of autoinducer synthase catalysts. A method for identifing modulators of the autoinducer synthesis reaction is also provided. Such modulators are useful for controlling bacterial growth and can be used for therapeutic treatment of bacterial infections particularly in immunocompromised subjects. They are also useful in treating disease states associated with autoinducer synthesis and biofilm development.

Abstract: This invention relates to a method wherein special adaptors and primers are used for single stranded cDNA amplification. The method comprises contacting RNA with an anchored cDNA synthesis primer which can anneal to RNA, a suitable enzyme which possesses reverse transcriptase activity, and a special designed adaptor which can efficiently be ligated to the 3′ end of single stranded cDNA by T4 DNA ligase. Processes are provided for using specific primers annealing to the adaptors in the 5′ and 3′-end of the single stranded cDNA for PCR amplifying the cDNA. The invention describes the use of this approach for generation of probes starting from low amounts of RNA to be used e.g. in hybridization towards micro-arrays.

Abstract: The invention relates to methods for the qualitative and quantitative determination of differentially expressed mRNA molecules. Said methods are used especially to determine all possible mRNA molecules present in a cell or a tissue, and to compare them with other cells or tissues, with other conditions (stages of disease or development), or with stages of treatment for these conditions. The method provided for in the invention therefore makes it possible, for example, to establish a comprehensive map of the different mRNA molecules present in a defined mRNA population and subsequently to use the preferably digital information obtained in this way in data base analyses.

Abstract: The present invention provides a method for covalently joining a DNA strand to an RNA strand using a topoisomerase enzyme. This invention also provides a method for obtaining a cDNA corresponding to a gene using a topoisomerase enzyme.

Abstract: The present invention relates to a process for the preparation of full-length complementary DNA (cDNA).

Abstract: Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The method is based on stand displacement replication of the nucleic acid sequences of interest by multiple primers. In one preferred form of the method, referred to as multiple strand displacement amplification, two sets of primers are used, a right set and a left set. The primers in the right set are complementary to one strand of the nucleic acid molecule to be amplified and the primers in the left set are complementary to the opposite strand. The 5′ end of primers in both sets are distal to the nucleic acid sequence of interest when the primers have hybridized to the nucleic acid sequence molecule to be amplified. Amplification proceeds by replication initiated at each primer and continuing through the nucleic acid sequence of interest. A key feature of this method is the displacement of intervening primers during replication by the polymerase.

Abstract: The invention provides a method and related kits and reagents for producing, purifying, isolating, or enriching desired nucleic acids from a library. The desired nucleic acids are produced from polymerase enzyme extension products, generated from specific primers or sets of primers, in a form that is immediately replicable in a host cell. The invention can be practiced in solution phase, thus eliminating the need for solid phase filter hybridizations, column hybridizations, or gel electrophoresis purification when enriching for or isolating a target sequence or vector from one or more libraries.

Abstract: The present invention relates to a method for the determination of the presence and amount of DNA in a sample. The method is based on the use of a nucleic acid template dependent enzyme in combination with a random primer to generate an enzymatic product which incorporates a binding species and a detectable species covalently linked.

Abstract: Method for the detection of the antibiotic resistance spectrum of Mycobacterium species present in a sample, possibly caused to the identification of the Mycobacterium species involved, comprising the steps of: (i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample; (ii) if need be amplifying the relevant part of the antibiotic resistance genes present in said sample with at least one suitable primer pair; (iii) hybridizing the polynucleic acids of step (i) or (ii) with at least one of the rpoB gene probes, as specified in table 2, under the appropriate hybridization and wash conditions; (iv) detecting the hybrids formed in step (iii); (v) inferring the Mycobacterium antibiotic resistance spectrum, and possibly the Mycobacterium species involved from the differential hybridization signal(s) obtained in step (iv).

Abstract: The presently claimed invention provides methods and kits for amplifying a target sequence from within a nucleic acid population. The presently claimed invention provides selection probes which are complementary to at least a portion of said target sequence and mechanisms for adding a probe sequence to the 3′ end of a target sequence that is hybridized to a selection probe. The added 3′ probe sequence and a probe sequence added at the 5′ end of the target by adaptor ligation allow for selective amplification of the target sequence.

Abstract: Nucleic acid sequences, oligonucleotides and a method for detection of SRSV, in particular, a virus which belongs to Genotype II (GII), in clinical examinations, public health examinations, food evaluations and food poisoning examinations are provided.

Abstract: Methods of manipulation of nucleic acid, in particular amplification by means of the polymerase chain reaction (PCR), including use of oligonucleotides and combinations and kits comprising such oligonucleotides, also methods comprising use of nested PCR, allowing for improved results in methods wherein large numbers of nucleic acid fragments are manipulated by means of PCR and electrophoresis. Oligonucleotides are provided for use a size standards in electrophoresis, and internal controls allowing for calculation of relative amounts of material present. Improved results can be achieved in methods of profiling mRNA transcribed in a system under investigation.

Abstract: A method for generating and amplifying closed circular DNA having a specific sequence in vitro in a cell-free system is disclosed. Prior to the invention of this method, closed circular DNA could only be amplified in vivo in appropriate host cells. The essence of the method is the inclusion of a thermostable DNA ligase in a PCR reaction. This procedure is referred to as ligation-during-amplification (LDA), in which the fully extended DNA strands are ligated by the DNA ligase and used as templates for subsequent amplification. Closed circular DNA having a specific sequence can be selectively amplified exponentially by the use of two sequence-specific primers in the LDA reaction. In addition, one or more site-specific mutations can be introduced into a closed circular DNA by the use of one or more mutagenic primers in the LDA reaction. Various thermostable DNA polymerases and thermostable ligases can be used for LDA amplification.

Abstract: A process for generating multiple linear complements of a single strand, circular nucleic acid template containing at least one cleavage site is described. The process consists of combining the single strand, circular nucleic acid template with polynucleotide primers under conditions sufficient for hybridization; extending the polynucleotide primer more than once around the circle to generate a complementary displacement of more than one continguous complement of the single strand, circular nucleic acid template. Also described is a process of synthesizing novel single strand, circular nucleic acids between 30 an 2200 nucleotides. The process consist of synthesizing a linear polynucleotide; combining the linear polynucleotide with a complementary linking oligonucleotide under conditions sufficient for hybridization; and ligating the linear polynucleotide pto produce a single strand, circular nucleic acid.

Abstract: A method of evaluating a risk of a subject to develop vascular complications is disclosed. The method is effected by determining a haptoglobin phenotype of the subject and thereby evaluating the risk of the subject to develop vascular complications. The risk is decreased in patients with haptoglobin 1-1 phenotype as compared to patients with haptoglobin 1-2 or haptoglobin 2-2 phenotypes.

Abstract: cDNA including the 5′-terminal sequence of full-length mRNA with a cap structure is synthesized from a mRNA sample containing the full-length mRNA with the cap structure and non-full-length mRNA without any cap structure in mixture. At the first step, the phosphate group at 5′-terminus of the non-full-length mRNA in the mRNA sample is removed. At the second step, the cap structure at the 5′-terminus of the full-length mRNA in the mRNA sample is removed. At the third step, an oligoribonucleotide is ligated to the phosphate group at 5′-terminus of mRNA generated through the first and second steps. At the fourth step, mRNA with the oligoribonucleotide ligated at the 5′-terminus thereof at the third step is subjected to a reverse transcriptase process using a short-chain oligonucleotide capable of being annealed to an intermediate sequence within the mRNA as primer, to synthesize a first-strand cDNA.

Abstract: Novel solution-based methods and materials, including apparatus, for sequence analysis by hybridization are provided.

Abstract: The present invention provides a method for the rapid simultaneous production of a plurality of single-stranded DNA circles having a predetermined size and nucleotide sequence using pre-designed hairpin oligonucleotides containing complementary sequences for directing ligation to form dumbbell-shaped monomers followed by heat denaturation to yield single-stranded DNA circles.

Abstract: A plurality of oligonucleotides are caused to bind specifically to target nucleic acid which is predictive of a disease state or a biological condition within cells containing the target nucleic acid. The respective oligonucleotides, which may be functionilized or may be present in the form of oligonucleotide analogs, carry with them a plurality of synthons. Such synthons, which may be identiphores, toxiphores, or other precursors to biologically effective molecules, interact when specific binding of the respective oligonucleotides occurs at sites adjacent to each other on the target nucleic acid. The resulting interaction gives rise to the synthesis, generation or release of highly active biological molecules in situ in the cell in which the specific binding takes place. This permits the use of extraordinarily toxic molecules for use in killing cells containing the target nucleic acids. Imaging and other uses are also provided by the present invention.

Abstract: A method for the detection of telomerase activity, for the detection of cancer cells and for the diagnosis of cancer comprising amplifying an oligonucleotide sequence extended by a DNA extension reaction with a telomerase and hybridizing the resulting amplified product with a probe labelled with a non-radioactive material to detect the telomerase activity, as well as a diagnositic kit for use in said method for the detection and diagnosis.

Abstract: Detection of phenols using engineered bacteria. A biosensor can be created by placing a reporter gene under control of an inducible promoter. The reporter gene produces a signal when a cognate transcriptional activator senses the inducing chemical. Creation of bacterial biosensors is currently restricted by limited knowledge of the genetic systems of bacteria that catabolize xenobiotics. By using mutagenic PCR to change the chemical specificity of the Pseudomonas species CF600 DmpR protein, the potential for engineering novel biosensors for detection of phenols has been demonstrated. DmpR, a well-characterized transcriptional activator of the P. CF600's dmp operon mediates growth on simple phenols. Transcription from Po, the promoter heading the dmp operon, is activated when the sensor domain of DmpR interacts with phenol and mono-substituted phenols.

Abstract: The present invention provides a dual selection cassette (DSC) comprising first and second DNA segments having homology to a eukaryotic viral vector, positive and negative selection genes, each operably linked to their own promoter, and one or more unique restriction enzyme sites (URES) or sitey-directed homologous recombination sites. The present invention also provides a plasmid, pN/P, comprising an independent positive selection marker gene, an origin of replication, and a dual selection cassette. The dual selection cassette and pN/P plasmid can be used to produce eukaryotic gene transfer vectors without requiring temporally-linked double recombination events or the use of specialized bacterial strains that allow the replication of plasmids comprising defective origins of replication. This method usefully increases the ratio of desired to undesired plasmid and vector constructs. Additionally, this invention provides a method for the creation of eukaryotic viral vector libraries.

Abstract: A process to obtain linear double-stranded covalently closed DNA “dumbbell” constructs from plasmids by restriction digest, subsequent ligation with hairpin oligodesoxyribonucleotides, optionally in the presence of restriction enzyme, and a final digestion with endo- and exonucleolytic enzymes that degrade all contaminating polymeric DNA molecules but the desired construct. The invention also provides a process to obtain said dumbbell constructs employing endonuclease class II enzymes. Furthermore, the invention provides a process to obtain linear, covalently closed DNA molecules, such as plasmids, free from contamination by genomic DNA, by submitting the DNA preparation to a facultative endonucleolytic degradation step and an obligatory exonucleolytic degradation step.

Abstract: This invention relates to a pair of probes for the detection of a target nucleic acid, said pair of probes comprising (1) probe 1 having base sequence B1 complementary to A1 between two specific sequential base sequences A1 and A2 of the nucleic acid, wherein (a) the 5′-terminus of base sequence B1 is phosphorylated and (b) a first solid phase immobilizing part is bound to the 3′-terminus of base sequence B1, and (2) probe 2 having base sequence B2 complementary to A2 between base sequences A1 and A2, wherein (a) the 5′-terminus of base sequence B2 is bound to one end of a cleavage part and (b) a second solid phase immobilizing part is bound to the other part of the cleavage part. When the target nucleic acid is hybridized above the solid phase of the invention where said probes are immobilized on its surface, the probes on the solid phase occupy spatial positions beneficial to the formation of a hybrid and thus forms the hybrid efficiently.

Abstract: The present invention concerns a process for the amplification of nucleic acids, wherein the tailing of the nucleic acid to be amplified is effected by extending the nucleic acid with ribonucleotides at its 3′ end with the aid of terminal transferase. Further, a kit for the amplification of a nucleic acid, which includes at least one ribonucleotide and terminal transferase, is provided.

Abstract: The present invention is directed to an approach to identify changes in gene expression by selective amplification of 3′ fragments of double stranded cDNAs.

Abstract: The base sequence data of a gene is analyzed. A set of degenerate probes are hybridized to a single-stranded nucleic acid analyte derived from a gene, the nucleic acid analyte is used as a template and the probes are used as primers for a thermo-cycled polymerase reaction, the reaction products obtained from the respective probes are separated by gel electrophoresis and the electrophoresis patterns for the probes are compared, to allow for feature extraction of the base sequence of the nucleic acid analyte, including its squencing.