Abstract: Methods and apparatus of some aspects of the invention relate to the synthesis of high fidelity polynucleotides. In particular, aspects of the invention relate to concurrent enzymatic removal of amplification sequences and ligation of processed oligonucleotides into nucleic acid assemblies. According to some embodiments, the invention provides a method for producing a target nucleic acid having a predefined sequence. In some embodiments, the method comprises the step of providing a plurality of oligonucleotides, wherein each oligonucleotides comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) a 5? sequence flanking the 5? end of the internal sequence and a 3? flanking sequence flanking the 3? end of the internal sequence, each of the flanking sequence comprising a primer recognition site for a primer pair and a restriction enzyme recognition site.

Abstract: Disclosed herein are methods, compositions and systems that facilitate accurate phasing of sequence data such as genomic sequence data through the segmentation and rearrangement of nucleic acid molecules in such a way as to preserve individual molecules phase or physical linkage information. This is variously accomplished by binding molecules independent of their phosphodiester backbones, cleaving the molecules, ligating, and sequencing the molecules through long-read sequencing technology to recover segment sequence information spanning at least more than one segment.

Abstract: A microfluidic chip, device, system, the use thereof and method for the generation of aqueous droplets in emulsion oil for nucleic acid amplification.

Abstract: The present invention relates to methods for generating a labelled nucleic acid from an RNA comprising a 5? protecting group, said method comprises the steps of obtaining a mixture of template strands of nucleic acids, said mixture comprising said RNA and further potentially other nucleic acids without a 5? protecting group, annealing at least one oligonucleotide primer to the template strand of said RNA and potentially other nucleic acids, and template sequence dependent extending said primer, thereby obtaining a complementary nucleic acid strand annealed to its template strand, or providing the RNA in duplex with a complementary nucleic acid strand annealed to its template strand, and optionally modifying the extension product of said nucleic acids without 5? protecting group either on the 5? end of the template strand or on the 3? end of the complementary strand, or both, and labelling a complementary nucleic acid of a double stranded nucleic acid not modified, wherein therefore the labelled nucleic acid d

Abstract: The present invention is directed to methods to prepare a DNA molecule or a plurality of DNA molecules by random fragmentation. In some embodiments, the present invention regards preparing a template for DNA sequencing by random fragmentation. In specific embodiments, the random fragmentation comprises chemical fragmentation, mechanical fragmentation, or enzymatic fragmentation. In further specific embodiments, a universal sequence is attached to the 3? end of the DNA fragments, such as by ligation of an adaptor sequence or by homopolymeric tailing with terminal deoxynucleotidyltransferase. In other embodiments, a library is prepared with methods of the present invention.

Abstract: The present invention is directed to methods and compositions for adding tails of specific lengths to a substrate polynucleotide. The invention also contemplates methods and compositions for immobilization of tailed substrates to a solid support. The disclosure contemplates that the attenuator molecule is any biomolecule that associates with a tail sequence added to a substrate polynucleotide and controls the addition of a tail sequence to the 3? end of the substrate polynucleotide. The sequence that is added to the substrate polynucleotide is referred to herein as a tail sequence, or simply a tail, and the process of adding a nucleotide to a substrate polynucleotide is referred to herein as tailing.

Abstract: Methods are provided for ligating a 3? adapter and a 5? adapter to a target polynucleotide so as to avoid adapter dimer formation. Embodiments of the methods include adding a blocking oligonucleotide after the first ligation in which a 3? adapter is ligated to the target polynucleotide so that the blocking oligonucleotide is capable of hybridizing to excess 3? adapter and the ligated 3? adapter. Subsequently, a 5? adapter is ligated to the target polynucleotide thus avoiding adapter dimer formation.

Abstract: Select embodiments of the present invention employ biological means to direct assemble CNT-based nanostructures, allowing for scaling to macrostructures for manufacture. In select embodiments of the present invention, a method is provided for assembling DNA-functionalized SWNTs by phosphodiester bonding catalyzed by ssDNA-ligase to form macroscopic CNT aggregates.

Abstract: Provided herein are methods for generation and amplification of a single-stranded DNA circle in a single reaction vessel from a linear DNA without any intervening purification steps. The single-stranded DNA circle is generated via a template-independent single-stranded DNA ligation. Whole-genome amplification of circulating nucleic acids extracted from blood is provided. Kits for performing the disclosed methods are also provided.

Abstract: This disclosure provides, among other things, a method of combining nucleic acid fragments, comprising: (a) providing two double-stranded DNA molecules with a common sequence, wherein the common sequence is at the end of each molecule; (b) nicking one strand in the common sequence of both molecules at a respective nicked site; (c) moderately denaturing both molecules to remove a single-stranded fragment from the nicked site to one end of each molecule, wherein the single-stranded fragment includes the common sequence in part or in whole, resulting in an overhanging sequence in each molecule, and the overhanging sequences in both molecules are complementary to each other; (d) allowing the overhanging sequences of both molecules to anneal to each other, and ligating the molecules. Alternative ways for performing the method are also provided.

Abstract: In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of at least one agent that generates a ligatable terminal 5? phosphate group by removing an adenylate group from a terminal 5? phosphate of a nucleic acid. In some embodiments, an aprataxin enzyme can catalyze removal of an adenylate group from a terminal 5? phosphate of a nucleic acid. In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of an aprataxin enzyme under conditions suitable for ligating nucleic acid ends.

Abstract: Provided herein is a method for producing a population of oligonucleotides that has reduced synthesis errors. In certain embodiments, the method comprises: a) obtaining an initial population of hairpin oligonucleotide molecules that each comprise a double-stranded stem region and a loop region; b) contacting the double-stranded region of the hairpin oligonucleotide molecules with a mismatch binding protein; and c) eliminating any molecules that bind to the mismatch binding protein, thereby producing a population of oligonucleotides that has reduced synthesis errors. A kit and a composition for performing the method are also provided.

Abstract: Methods for developing a binding-element are provided. A mixture comprising a target molecule, a plurality of oligonucleotides and a ligase is provided, followed by binding the oligonucleotides to the target molecule to form an oligonucleotides-target molecule complex. The oligonucleotides bound to the target molecule are ligated to form the binding-element. The binding-elements are separated from the mixture.

Abstract: The present disclosure relates to systems and methods for nucleic acid ligation. In particular, the present disclosure provides oligonucleotide adaptors for use in nucleic acid ligation reactions.

Abstract: Methods, assays, compositions and kits for the ligation of short polynucleotides are presented herein. The short polynucleotides are optionally no more than 7 nucleotides in length, and can be as short as 3 or 4 nucleotides in length. The ligation is optionally performed by CV ligase.

Abstract: A method for detecting nucleic acids by (a) providing a sample having target nucleic acids, each nucleic acid having contiguous first, second, and third domains; (b) contacting the sample with probe sets to form hybridization complexes, wherein each probe set includes (i) a first probe having a sequence that is complementary to the first domain; and (ii) a second probe having a sequence substantially complementary to the third domain; (c) extending the first probes along the second domains of the complexes while the complexes are immobilized on a solid support; (d) ligating the extended first probes to the second probes to form templates; (e) amplifying the templates with primers that are complementary to the first and second priming sequences to produce amplicons; and (f) detecting the amplicons on the surface of a nucleic acid array.

Abstract: The invention provides compositions and methods for making closed nucleic acid structures in which one or both strands are continuous. The closed nucleic acid structures can be used as sequencing templates among other applications.

Abstract: Methods and kits for joining two or more polynucleotides to form a product polynucleotide are provided. A mixture contains a first polynucleotide comprising a selectable marker. The mixture further contains a second polynucleotide comprising a first typeIIs recognition sequence and a second typeIIs recognition sequence. The second polynucleotide is other than the first polynucleotide. The mixture further contains a first typeIIs restriction endonuclease that cleaves the first typeIIs recognition sequence to produce a first end, a second typeIIs restriction endonuclease that cleaves the second typeIIs recognition sequence to produce a second end, and a DNA ligase. The first end is not compatible with the second end. The combined actions of the enzymes in the mixture join the first polynucleotide to the second polynucleotide forming a product polynucleotide, which is obtained by transforming the mixture into a host cell.

Abstract: This invention is intended to enhance and improve the resistance of a single- or double-stranded nucleic acid fragment comprising a base sequence of a functional nucleic acid to degradation by nucleolytic enzymes in a simple and cost-effective manner. The single- or double-stranded nucleic acid fragment comprises, ligated to at least one 3? end thereof, a hairpin-shaped DNA comprising: (A) a nucleic acid region consisting of 2 to 5 arbitrary nucleotides; (B) a nucleic acid region consisting of a “gna” or “gnna” base sequence, wherein each “n” represents “g”, “t”, “a”, or “c”, a base analogue, or a modified base; and (C) a nucleic acid region consisting of a base sequence complementary to the nucleic acid region (A), sequentially ligated from the 5? end toward the 3? end, wherein at least one of two 3? terminal nucleotides from at least one 3? end of the single-stranded nucleic acid fragment or the double-stranded nucleic acid fragment is modified.

Abstract: Multiple properties of plasmonic assemblies are determined by their geometrical organization. This patent focuses on the formation of Janus structure of the asymmetric assembly structure of the gold nanorods and gold nanoparticles. Chiral structure of gold nanorods and gold nanoparticles can be obtained through the characterization of optical spectra of the Janus structure. And it opens the door for the explanation of the mechanism of the chirality, plays a strong guiding role in the negative refractive material above and has good application prospects.

Abstract: Disclosed are methods and compositions for detection and amplification of nucleic acids, wherein two DNA strands hybridized to an RNA strand are ligated. In one aspect, the disclosed methods include removal of an energy source, such as ATP, upon charging a ligase to form an enzyme-AMP intermediate, and then adding substrate, which results in one complete round of RNA-templated DNA ligation. In another aspect, the ligation reaction is accomplished by use of a mixture of at least two different ligase enzymes. The disclosed methods and compositions for RNA-templated DNA ligation may be particularly useful for detection of RNA sequence variants, for example RNA splice variants, and for quantitative expression analysis.

Abstract: Disclosed are methods, compositions and kits related to making double-tagged DNA libraries from RNA/DNA samples. A double-tagged oligonucleotide (DTO) is employed to efficiently add two different tags to ends of DNAs to make a double-tagged DNA libraries. Also disclosed are methods to make mate pair libraries using the double-tagged oligonucleotide, and methods to make double-tagged single stranded DNA. The double-tagged DNA libraries of the invention are ready to be used on next generation sequencing machines.

Abstract: The invention provides compositions and methods for ligating single stranded nucleic acids wherein the ligation is based on fast, efficient, and low-sequence bias hybridization of an acceptor molecule with a donor molecule. In one embodiment, the structure of the donor molecule comprises a stem-loop intramolecular nucleotide base pairing (i.e., hairpin) and a 3?-overhang region such that the overhang is able to hybridize to nucleotides present in the 3? end of the acceptor molecule.

Abstract: Compositions and methods are provided for selection and enrichment of a target gene from a library of polynucleotide sequences such as might be formed from a genome or by random mutagenesis of a genetic sequence. The selection and enrichment occurs in aqueous droplets formed in an emulsion that compartmentalize individual polynucleotides from the library or a plurality of polynucleotides that may include polynucleotides not derived from the library, transcription and translation reagents and optionally additional chemical and enzyme reagents. The selection and enrichment method utilizes a polynucleotide adaptor which when ligated to the polynucleotide fragment enables amplification to occur in the presence of an adaptor specific primer.

Abstract: This disclosure relates to methods for creating engineered templates that are useful for amplification of one or more antibody genes without the use of gene-specific primers. More specifically, templates engineered using these methods in a polymerase chain reaction setting which allows for the specific amplification of one or more antibody genes.

Abstract: Disclosed are methods, compositions and kits related to making double-tagged DNA libraries from RNA/DNA samples. A double-tagged oligonucleotide (DTO) is employed to efficiently add two different tags to ends of DNAs to make a double-tagged DNA libraries. Also disclosed are methods to make mate pair libraries using the double-tagged oligonucleotide, and methods to make double-tagged single stranded DNA. The double-tagged DNA libraries of the invention are ready to be used on next generation sequencing machines.

Abstract: This disclosure provides method for adding an adaptor to a genomic sequence by invasive cleavage, as well as a kit for performing the method. In some embodiments, the method comprises: a) hybridizing genomic DNA to an adaptor comprising a double stranded region and a single stranded region comprising a 5? overhang to produce a substrate for a flap endonuclease; b) cleaving the substrate using the flap endonuclease; c) ligating the recessed end of the double stranded region to the fragment to produce an adaptor-ligated DNA; d) intramolecularly ligating the ends of the adaptor-ligated DNA to produce a circular DNA molecule; and e) enzymatically processing the circular DNA molecule using an oligonucleotide that hybridizes to the adaptor and an enzyme. A kit for performing the method is also provided.

Abstract: Compositions and methods are provided for enhancing enzymatic ligation between nucleic acid fragments that relies on one or more small molecule enhancers having a size of less than 1000 daltons. For example, enhancement of ligation efficiencies are observed for double-stranded nucleic acid fragments that are blunt-ended, have a single nucleotide overhang at the ligation end, or have staggered ends compared to ligation under similar conditions in the absence of the one or more small molecule ligation enhancer. The use of small molecule enhancers for ligating nucleic acids results in an increased number of transformed host cells after transformation with the ligated molecules. This enhancement can be observed with chemically transformed host cells and with host cells transformed by electroporation.

Abstract: Described herein are techniques for assembling a polynucleotide encoding a transcription activator-like effector nucleases (TALEN). The techniques ligate and digest necessary modules for a TALEN assembly in one reactor or system. Methods and Kits for generating a TALEN are also described.

Abstract: Described herein are techniques for assembling a polynucleotide encoding a transcription activator-like effector nucleases (TALEN). The techniques ligate and digest necessary modules for a TALEN assembly in one reactor or system. Methods and Kits for generating a TALEN are also described.

Abstract: Methods are described for determining the pattern of cytosine methylation in a DNA specimen, where the methods involve comparing the amount of DNA fragments generated by a methylation-sensitive restriction enzyme with the amount of DNA fragments generated by a methylation-insensitive isoschizomer of the methylation-sensitive restriction enzyme.

Abstract: The present invention provides a method for the assembly of a polynucleic acid sequence from a plurality of nucleic acid sequences in which the polynucleic acid sequence is of a formula Nn+1, in which N represents a nucleic acid sequence and where n is 1 or greater than 1 and each N may be the same or a different nucleic acid sequence, in which the method comprises: (i) providing a first nucleic acid sequence N1 which has an oligonucleotide linker sequence L13? at the 3?-end of the nucleic acid sequence; (ii) providing a second nucleic acid sequence N2 which optionally has an oligonucleotide linker sequence L23? at the 3?-end of the nucleic acid sequence and which has an oligonucleotide linker sequence L25? at the 5?-end of the nucleic acid sequence, wherein the 5?-end linker sequence L25? of nucleic acid sequence N2 is complementary to the 3?-end linker sequence L13? of nucleic acid sequence N1; (iii) optionally providing one or more additional nucleic acid sequences N, wherein nucleic acid sequence N2

Abstract: The present invention is directed to a method for performing an RT-PCR for amplifying a target RNA including the steps of (i) cultivation of a population of adherent cells in a cell culture vessel (ii) lysis of the population of adherent cells which is supposed to contain the target RNA in the sample vessel with a lysis buffer comprising between 0.05 M and 1 M of a chaotropic agent (iii) adding reagents to the sample vessel which are necessary to perform a reverse transcription reaction such that the the chaotropic agent is present in a concentration of about 10 to 60 mM in the sample vessel, and reverse transcribing the target RNA and (iv) amplifying the first strand cDNA by means of subjecting the sample to multiple cycles of a thermocycling protocol.

Abstract: In a method for generating a long nucleic acid molecule, nucleic acids immobilized on a surface and having overlapping complementary sequences is released into solution. The overlapping complementary sequences are hybridized to form hybridized nucleic acids, followed by extension or ligation of the hybridized nucleic acids to synthesize the long nucleic acid molecule. The nucleic acids may comprise first and second series of nucleic acids having redundant overlapping sequences, wherein nucleic acids from the first and second series are complementary to each other. The complementary nucleic acids are hybridized to form the hybridized nucleic acids. The generated long nucleic acid molecule may have a predetermined sequence element, and it may be introduced into a system wherein the predetermined sequence element is required for replication, such that replication of the synthesized long nucleic acid molecule is indicative of the presence of the predetermined sequence element in the long nucleic acid molecule.

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: The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome.

Abstract: The present invention relates to methods of assembling a plurality of genetic units to form synthetic genetic constructs. This method involves appending universal adapter oligonucleotides and flexible adapter oligonucleotides to the 5? and 3? ends of separate genetic units to be assembled to form separate dual extended genetic units. The dual extended genetic units are assembled together via homologous recombination between the flexible adapter oligonucleotide portions of the dual extended units to form synthetic genetic constructs. The present invention further relates to synthetic genetic constructs formed using the methods of the present invention, and vectors, cells, and organisms containing such synthetic genetic constructs.

Abstract: Disclosed, for example, are methods comprising cleaving an uncleaved probe to form a cleaved oligonucleotide flap, forming a hybridization complex between the cleaved oligonucleotide flap, a bridging oligonucleotide, and a capture oligonucleotide that is immobilized on a surface, such that the oligonucleotide flap and the capture oligonucleotide are hybridized to immediately adjacent, complementary regions of the bridging oligonucleotide, ligating the oligonucleotide flap to the capture oligonucleotide to form an immobilized ligation product, and detecting the ligation product.

Abstract: Compositions and methods are provided for selection and enrichment of a target gene from a library of polynucleotide sequences such as might be formed from a genome or by random mutagenesis of a genetic sequence. The selection and enrichment occurs in aqueous droplets formed in an emulsion that compartmentalize individual polynucleotides from the library or a plurality of polynucleotides that may include polynucleotides not derived from the library, transcription and translation reagents and optionally additional chemical and enzyme reagents. The selection and enrichment method utilizes a polynucleotide adaptor which when ligated to the polynucleotide fragment enables amplification to occur in the presence of an adaptor specific primer.

Abstract: The present invention is directed to compositions and methods for nucleic acid identification and detection. Compositions and methods of the present invention include extracting and fragmenting target nucleic acids from a sample, using the fragmented target nucleic acids to produce target nucleic acid templates and subjecting those target nucleic acid templates to amplification methods to form nucleic acid nanoballs. The invention also includes methods of detecting and identifying sequences using various sequencing applications, including sequencing by ligation methods.

Abstract: The invention provides compositions and methods for making closed nucleic acid structures in which one or both strands are continuous. The closed nucleic acid structures can be used as sequencing templates among other applications.

Abstract: Methods and kits for covalently joining a 3? nucleic acid fragment having a 5?-hydroxyl terminus to a 5? nucleic acid fragment having a 3?-phosphate terminus are disclosed. The methods include the step of contacting the 3?-phosphate terminus of a first nucleic acid molecule and the 5?-hydroxyl terminus of a second nucleic acid molecule with an isolated 2?,3?-cyclic phosphate RNA ligase (RtcB) and a purine triphosphate in the presence of manganese (II) ion, whereby the 3?-phosphate terminus of the first nucleic acid molecule and the 5?-hydroxyl terminus of the second nucleic acid molecule are covalently joined. Although the purine triphosphate used is generally GTP or dGTP, if the method is performed in the presence of an Archease, any purine triphosphate may be used. Accordingly, the disclosed kits include isolated RtcB, along with a purine triphosphate and/or an isolated Archease.

Abstract: The present invention is directed to methods and compositions for the use of microsphere arrays to detect and quantify a number of nucleic acid reactions. The invention finds use in genotyping, i.e. the determination of the sequence of nucleic acids, particularly alterations such as nucleotide substitutions (mismatches) and single nucleotide polymorphisms (SNPs). Similarly, the invention finds use in the detection and quantification of a nucleic acid target using a variety of amplification techniques, including both signal amplification and target amplification. The methods and compositions of the invention can be used in nucleic acid sequencing reactions as well. All applications can include the use of adapter sequences to allow for universal arrays.

Abstract: Biosensors, compositions comprising biosensors, methods of producing biosensors, and methods of using biosensors are disclosed. The biosensors comprise a fluorogen-activating peptide and a blocking peptide. The fluorogen-activating peptide and blocking peptide are covalently linked through a peptide linker. The blocking peptide associates with the fluorogen-activating peptide thereby blocking an active domain of the fluorogen-activating peptide when the linker is in an unmodified state. The peptide linker may contain an amino acid sequence that is specifically recognized as a modification substrate by a cognate enzyme. The fluorogen-activating peptide and the blocking peptide at least partially disassociate when the linker is modified by an enzyme, thereby allowing the fluorogen-activating peptide to bind a cognate fluorogen and modulate a fluorescence signal.

Abstract: The invention generally relates to methods for obtaining a sequence, such as a consensus sequence or a haplotype sequence. In certain embodiments, methods of the invention involve determining an amount of amplifiable nucleic acid present in a sample, partitioning the nucleic acid based upon results of the determining step such that each partitioned portion includes, on average, a subset of unique sequences, sequencing the nucleic acid to obtain sequence reads, and assembling a consensus sequence from the reads.

Abstract: A method is provided for generating a preparation in which more than 70% of the oligonucleotides are adenylated. The method includes reacting an oligonucleotide with an ATP-sensitive ligase where the ligase is characterized by its ability to efficiently generate adenylated oligonucleotides at ATP concentrations at which ligation and circularization of the oligonucleotide is minimal.

Abstract: Compositions, methods and kits are disclosed for synthesizing and amplifying pools of probes using precursor oligonucleotides. In some aspects the precursor is amplified and nicking enzymes are used to separate the full length probes from the amplification products. The methods enable the preparation of single stranded DNA probes of defined sequence and length that are suitable for use in target detection assays.

Abstract: Multiple properties of plasmonic assemblies are determined by their geometrical organization. This patent focuses on the formation of Janus structure of the asymmetric assembly structure of the gold nanorods and gold nanoparticles. Chiral structure of gold nanorods and gold nanoparticles can be obtained through the characterization of optical spectra of the Janus structure. And it opens the door for the explanation of the mechanism of the chirality, plays a strong guiding role in the negative refractive material above and has good application prospects.

Abstract: Aspects of the invention relate to methods, compositions and algorithms for designing and producing a target nucleic acid. The method can include: (1) providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends thereof; (2) producing via enzymatic digestion a plurality of cohesive-end double-stranded nucleic acid fragments each having two different and non-complementary overhangs; (3) ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase; and (4) forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid.

Abstract: The present invention provides methods and compositions for asymmetrically tagging a nucleic acid fragment using asymmetric adapters.