 Patent Application Title |
Patent App Num. |
Date |
| Hydrolysis probes | 20130085078 | 20130404 |
 Methods and compositions for the detection and quantification of nucleic acids are provided. In one embodiment, a sample is contacted with a primer complementary to a first region of a target nucleic acid and a probe complementary to a second region of the target nucleic acid downstream of the first region under conditions suitable for hybridization of the target nucleic acid with the primer and the probe. The probe in this embodiment comprises a fluorophore and is attached to a solid support. The hybridized probe is cleaved with a nucleic acid polymerase having exonuclease activity to release the reporter from the solid support. The presence of the target nucleic acid is then detected and optionally quantified by detecting a decrease in signal from the reporter on... |
| Novel fluorescence-based assay for the rapid detection and quantification of deoxyribonucleoside triphosphates | 20130029331 | 20130131 |
 The inventors have developed a rapid and sensitive fluorescence-based assay to quantify dNTPs. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and polymerase-mediated 5-3′ exonuclease hydrolysis of a quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTPs is directly proportional to the fluorescence generated. This assay has important applications in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation.
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| Methods of using fet labeled oligonucleotides that include a 3'-5' exonuclease resistant quencher domain and compositions for practicing the same | 20120245334 | 20120927 |
 Methods and compositions are provided for detecting a primer extension product in a reaction mixture. In the subject methods, a primer extension reaction is conducted in the presence of a polymerase having 3′→5′ exonuclease activity and at least one FET labeled oligonucleotide probe that includes a 3′→5′ exonuclease resistant quencher domain. Also provided are systems and kits for practicing the subject methods. The subject invention finds use in a variety of different applications, and are particularly suited for use in high fidelity PCR based reactions, including SNP detection applications, allelic variation detection applications, and the like.
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| Mutant dna polymerases and uses therof | 20120184017 | 20120719 |
 The present invention relates to mutant DNA polymerases which incorporate dideoxynucleotides with about the same efficiency as deoxynucleotides. The present invention also related to mutant DNA polymerases which also have substantially reduced 5′-to-3′ exonuclease activity or 3′-to-5′ exonuclease activity. The invention also relates to DNA molecules coding for the mutant DNA polymerases, and hosts containing the DNA molecules.
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| Homologous recombination-based dna cloning compositions | 20120129239 | 20120524 |
 Methods and compositions for cloning a donor DNA molecule into an acceptor vector at a predetermined location are described. The methods are based on homologous recombination mediated by in vitro treatment of the donor DNA and the acceptor vector with an enzyme cocktail containing an exonuclease and a single-stranded DNA binding protein.
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| Method and kits for repairing nucleic acid sequences | 20120107806 | 20120503 |
| Methods and kits for DNA repair are provided. The methods and kits described herein repair multiple types of DNA damage. The kit may include a plurality of enzymes to repair a greater variety of lesions than any single enzyme is capable of repairing. Repair of damaged DNA may include releasing damaged bases from the DNA strand, nicking the DNA at the damaged sites, translating the nicks via 5′-3′ exonuclease activity, and sealing the nicks. The enzymes employed in the repair process may then be heat-inactivated, thereby obviating a purification process. The repaired DNA may then be analyzed using a variety of DNA analysis methods.
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| Reagents and methods for pcr | 20120088275 | 20120412 |
| Modified double-stranded oligonucleotides that have terminal regions on each of their strands, that have a hybrid length of 6-50 nucleotides long, that have a melting temperature Tm of at least 32° C., and that include 2-4 modifying groups, each covalently attached to a different terminal region, preferably to a terminal nucleotide, said modifying groups being polycyclic substituents that do not have bulky portions that are non-planar, said modified olgonucleotide being capable of binding to the 5′ ex-nuclease domains of DNA polymerases and, when included in a PCR or other primer-dependent DNA amplification reaction at a concentration, generally not more than 2000 nM, that is effective for at least one of the functions of suppressing mispriming, increasing polymerase selectivity against 3′ terminal mismatches, increasing polymerase selectivity against... |
| Enzyme mixture | 20120082981 | 20120405 |
| Polymorphisms are present throughout an organism's genome, and understanding which alleles are present in a particular organism's genome can be advantageous. When probing the identity of these alleles, one must minimize incorrect readings due to inefficiencies in the system. In hydrolysis probe applications, these inefficiencies may be due to over-activity of an exonuclease functionality that excises nucleotides from probes that are only partially, complementary to a region of a target. The present invention provides a mixture that contains a plurality of polymerases including one that has a 5′→3′ exonuclease functionality and one that lacks or substantially lacks it, each in a sufficient relative amount and concentration to increase efficiencies of the system.
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| Thermostable dna polymerases and methods of use | 20120083018 | 20120405 |
| Thermostable viral and microbial polymerases exhibiting a combination of activities selected from proofreading (3′-5′) exonuclease activity, nick translating (5′-3′) nuclease activity, synthetic primer-initiated polymerase activity, nick-initiated polymerase activity, reverse transcriptase activity, strand displacement activity, terminal transferase activity, primase activity, and/or efficient incorporation of chain terminating analogs. Some of the polymerases provided herein include a first motif and a second motif. The first motif preferably has the sequence X1X2X3DX4PX5IELRX6X7X8, wherein X1 is I or V; X4 is F or Y; X8 is G or A; and X2, X3, X5, X6, and X7 are any amino acid. The second motif preferably has the sequence RX9X10X11KSANX12GX13X14YG, wherein X11 is G or A; X12 is F, L, or Y; X13 is L or V; X14 is I or L; and... |
| Identification of nucleic acid sequences | 20120058471 | 20120308 |
| The invention provides a method for use in the detection of a target nucleic acid comprising the steps of: (i) contacting a single-stranded probe nucleic acid with a sample of interest under conditions effective to generate a probe/target nucleic acid duplex by specific hybridisation of said probe nucleic acid to a target nucleic acid, if said target nucleic acid is present; (ii) contacting any probe/target nucleic acid duplex with an exonuclease to effect digestion of the duplex and release of a label molecule from the duplex; and (iii) detecting the label by Raman spectroscopy.
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| Method for synthesis of double-stranded dna corresponding to rna, and method for amplification of the dna | 20120058520 | 20120308 |
| An object of the present invention is to provide an inexpensive and simple method for synthesis of a double-stranded DNA corresponding to a particular RNA, and a method for amplification of the aforementioned double-stranded DNA. The present invention relates to a method for synthesis of a double-stranded DNA having a nucleotide sequence corresponding to template RNA having polyA, comprising step 1 in which reverse transcription reaction of template RNA is carried out employing oligo(dT)primer to which DNA fragment having a known sequence has been added at the 5′-terminal, to obtain a single-stranded DNA, and step 2 in which double strand formation reaction of single-stranded DNA obtained in step 1 is carried out employing a random primer to which DNA fragment having a known sequence has been... |
| Recombinant polymerases for improved single molecule sequencing | 20120034602 | 20120209 |
| Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.
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| Methods for genotyping | 20120010087 | 20120112 |
| Novel methods and kits are disclosed for reducing the complexity of a nucleic acid sample to interrogate a collection of target sequences, for example, to discriminating between alleles at polymorphic positions in a genome. Complexity reduction can be accomplished by extension of a capture probes followed by amplification of the extended capture probe using common primers. The capture probes may be locus specific and allele-specific. The amplified sample may be hybridized to an array designed to interrogate the desired fragments for the presence or absence of a polymorphism. In some aspects the methods employ allele-specific extension of oligonucleotides that are complementary to one of the alleles at the 3′ end of the oligonucleotide. The allele-specific oligonucleotides are resistant to proof reading activity from a polymerase and... |
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| Enzymatic methods for genotyping on arrays | 20100323914 | 20101223 |
| Disclosed are methods for enzymatic genotyping of polymorphisms on solid supports. In one aspect the method includes hydrolysis of a nucleotide comprising a label on an array-bound probe by a 5′ to 3′ exonuclease activity specific for single-stranded DNA. If there is target-probe sequence mismatch at the polymorphic position (the labeled nucleotide in the probe), the labeled nucleotide is hydrolyzed from the probe by the exonuclease. The presence of a detectable signal on the array is indicative of the identity of the nucleotide at the polymorphic position in the target. In another aspect, the queried position on the probe may be a labeled ribonucleotide, and if there is a sequence mismatch at the polymorphic position on the probe, the labeled ribonucleotide will be hydrolyzed from the... |
| Methods, systems and kits for detecting protein-nucleic acid interactions | 20100323361 | 20101223 |
| Methods, systems and kits for detecting protein-nucleic acid interactions, in particular, detecting the genomic location to near-base pair resolution at which a particular protein (e.g., transcription factor) binds includes combining steps of a convention chromatin immunoprecipitation (ChIP) assay with use of an exonuclease that digests DNA strands in the 5′-3′ or 3′-5′ direction until it reaches a bound protein including a protein crosslinked to DNA. A significant improvement of the resolution and dynamic range of the ChIP assay will increase one's ability to determine with confidence where a particular protein is binding in the genome. Importantly, proteins that inefficiently crosslink to DNA (either intrinsically or due to indirect crosslinking via another protein) and thus are very difficult to detect, are expected to be significantly detected by... |
| Method for in vitro recombination | 20100311126 | 20101209 |
| The present invention relates, e.g., to an in vitro method, using isolated protein reagents, for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising (a) chewing back the DNA molecules with an enzyme having an exonuclease activity, to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence identity to hybridize specifically to each other; (b) specifically annealing the single-stranded overhangs; and (c) repairing single-stranded gaps in the annealed DNA molecules and sealing the nicks thus formed (ligating the nicked DNA molecules). The region of sequence identity generally comprises at least 20 non-palindromic nucleotides... |
| Mutant dna polymerases and their genes from thermococcus | 20100297706 | 20101125 |
| The present invention relates to mutant DNA polymerases and their genes isolated from Thermococcus sp. More specifically, the present invention relates to mutant DNA polymerases which are originally isolated from Thermococcus sp NA1. strain and produced by site-specific mutagenesis, their amino acid sequences, genes encoding said mutant DNA polymerases, their nucleic acids sequences, recombinant vectors containing said nucleic acids sequences, host cells transformed with thereof and methods for producing mutant DNA polymerase protein by using thereof. As mutant DNA polymerases according to the present invention have increased processivity by site-specific mutagenesis on exonuclease active site compared to wild type DNA polymerase, the present invention is broadly applicable for PCR in various molecular genetic technologies.
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| Thermostable dna polymerases and methods of use | 20100291638 | 20101118 |
| Thermostable viral and microbial polymerases exhibiting a combination of activities selected from proofreading (3′-5′) exonuclease activity, nick translating (5′-3′) nuclease activity, synthetic primer-initiated polymerase activity, nick-initiated polymerase activity, reverse transcriptase activity, strand displacement activity, terminal transferase activity, primase activity, and/or efficient incorporation of chain terminating analogs. Some of the polymerases provided herein include a first motif and a second motif. The first motif preferably has the sequence X1X2X3DX4PX5IELRX6X7X8, wherein X1 is I or V; X4 is F or Y; X8 is G or A; and X2, X3, X5, X6, and X7 are any amino acid. The second motif preferably has the sequence RX9X10X11KSANX12GX13X14YG, wherein X11 is G or A; X12 is F, L, or Y; X13 is L or V; X14 is I or L; and... |
| Binary probe and clamp composition and methods for target hybridization detection | 20100291557 | 20101118 |
| Binary probe and clamp compositions conduct methods for target hybridization detection. Where the probe is a substrate for exonuclease cleavage, the composition provides quantitation and detection of PCR products, by real-time and end-point measurements. Where the probe is an amplification primer, the composition provides an improved method for labelling and detection of PCR products. Probes and clamps may be labelled with fluorescent dyes, quenchers, hybridization-stabilizing moieties, chemiluminescent dyes, and affinity ligands. Clamps may be nucleic acid analogs, such as 2-aminoethylglycine PNA.
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| Methods to increase nucleotide signals by raman scattering | 20100267013 | 20101021 |
| The methods and apparatus disclosed herein concern nucleic acid sequencing by enhanced Raman spectroscopy. In certain embodiments of the invention, nucleotides are covalently attached to Raman labels before incorporation into a nucleic acid. In other embodiments, unlabeled nucleic acids are used. Exonuclease treatment of the nucleic acid results in the release of labeled or unlabeled nucleotides that are detected by Raman spectroscopy. In alternative embodiments of the invention, nucleotides released from a nucleic acid by exonuclease treatment are covalently cross-linked to nanoparticles and detected by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS). Other embodiments of the invention concern apparatus for nucleic acid sequencing.
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| Oligonucleotides labeled with a plurality of fluorophores | 20100240103 | 20100923 |
| An embodiment of the invention discloses new methods for designing labeled nucleic acid probes and primers by labeling oligonucleotides with a plurality of spectrally identical or similar dyes and optionally with one or more quencher dyes. Oligonucleotides labeled in accordance with some embodiments of the invention exhibit a detectable increase in signal, for example, fluorescent signal when the labeling dyes are separated from one another. Methods for separating the dye include cleaving the labeled oligonucleotides include using enzymes that have 5′-exonuclease activity. In one embodiment nucleic acid primers of the present invention may fluoresce upon hybridization to a target sequence and incorporation into the amplification product. Nucleic acid probes and primers of the present invention have wide applications ranging from general detection of a target nucleic... |
| In vitro recombination method | 20100184187 | 20100722 |
| The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonuclease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA... |
| Homologous recombination-based dna cloning methods and compositions | 20100167356 | 20100701 |
| Methods and compositions for cloning a donor DNA molecule into an acceptor vector at a predetermined location are described. The methods are based on homologous recombination mediated by in vitro treatment of the donor DNA and the acceptor vector with an enzyme cocktail containing an exonuclease and a single-stranded DNA binding protein.
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| Quantitative amplification with a labeled probe and 3' to 5' exonuclease activity | 20100159447 | 20100624 |
| This invention provides methods and kits for performing a quantitative amplification reaction. The method employs a polymerase enzyme and an enzyme having a 3′ to 5′ exonuclease activity that cleaves the 3′ oligonucleotide of the probe.
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| Systems and methods for nuclease-assisted selection and acquisition of single stranded dna oligomer/polymer aptamers/ligands | 20100152056 | 20100617 |
| A method for identifying aptamers that bind to target molecules may include contacting an oligonucleotide library with target molecule and digesting unbound oligonucleotides with one or more endonucleases, one or more exonucleases, or one or more endonucleases in combination with one or more exonucleases. A method for identifying aptamers may further include optionally subjecting selected aptamers to one or more rounds of selection under conditions of increased stringency. A method for identifying aptamers may include yet further amplifying selected aptamers. The described methods may be performed in a screen for identifying aptamers either alone or in combination with other methods typically employed in the art for selecting aptamers (such as, e.g., SELEX). Also contemplated herein are systems and kits for accomplishing the above.
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| Immobilized nucleic acid complexes for sequence analysis | 20100075328 | 20100325 |
| Provided are methods for sequencing a nucleic acid that include fixing a template to a surface through a template localizing moiety and sequencing the nucleic acid with a sequencing enzyme, e.g. a polymerase or exonuclease. The sequencing enzyme can optionally be exchanged with a second sequencing enzyme, which continues the sequencing of the nucleic acid. The template localizing moiety can optionally anneal with the nucleic acid and/or associate with the sequencing enzyme. Also provided are compositions comprising a nucleic acid fixed to a surface via a template localizing moiety, and a first sequencing enzyme, which can sequence the nucleic acid and optionally exchange with a second sequencing enzyme present in the composition. Compositions in which a template localizing moiety is immobilized on a surface are provided.... |
| Homologous recombination-based dna cloning methods and compositions | 20100062495 | 20100311 |
| Methods and compositions for cloning a donor DNA molecule into an acceptor vector at a predetermined location are described. The methods are based on homologous recombination mediated by in vitro treatment of the donor DNA and the acceptor vector with an enzyme cocktail containing an exonuclease and a single-stranded DNA binding protein.
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| Methods for identification of alleles | 20100041563 | 20100218 |
| The invention provides a method for identification of alleles. In this method, genomic DNA is used as target. Multiple allele-specific PCR amplification are carried out with a group of primers comprising one or more allele-specific primers for a target gene, a universal primer, and a common primer; and a DNA polymerase without 5′ to 3′ exonuclease activity. The PCR products are hybridized with tag probes immobilized on a DNA chip. Results are determined based on the signal intensity and the position of the probe immobilized on the array. Each allele-specific primer comprises a unique tag sequence at the 5′ end. Each tag probe immobilized on the DNA chip comprises a sequence identical to its corresponding tag sequence; and each tag probe hybridizes only with the complementary... |
| Proofreading primer extension | 20100041053 | 20100218 |
| The present invention provides for primer extension reactions, including polymerase chain reactions, in which a polymerase having 3′-5′ exonuclease activity edits a primer that is not fully complementary thereby allowing for amplification and detection of target nucleic acids that may have variability in their sequences.
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| Prevention and alleviation of steric hindrance during single molecule synthesis | 20100035269 | 20100211 |
| The present invention provides compositions and methods for reducing steric hindrance in the product of nucleic acid polymerase reaction. Methods and compositions of the invention encompass application of exonucleases, endonucleases, and uracil-DNA glycosylases to a nucleic acid polymerase reaction such that newly formed nucleic acid strands are modified (e.g., cleaved) while the polymerase reaction continues to proceed.
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| Method and kits for repairing nucleic acid sequences | 20100009411 | 20100114 |
| Methods and kits for DNA repair are provided. The methods and kits described herein repair multiple types of DNA damage. The kit may include a plurality of enzymes to repair a greater variety of lesions than any single enzyme is capable of repairing. Repair of damaged DNA may include releasing damaged bases from the DNA strand, nicking the DNA at the damaged sites, translating the nicks via 5′-3′ exonuclease activity, and sealing the nicks. The enzymes employed in the repair process may then be heat-inactivated, thereby obviating a purification process. The repaired DNA may then be analyzed using a variety of DNA analysis methods.
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| Single copy genomic hybridization probes and methods of generating same | 20100003684 | 20100107 |
| Nucleic acid (e.g., DNA) hybridization probes are described which comprise a labeled, single copy nucleic acid which hybridizes to a deduced single copy sequence interval in target nucleic acid of known sequence. The probes, which are essentially free of repetitive sequences, can be used in hybridization analyses without adding repetitive sequence-blocking nucleic acids. This allows rapid and accurate detection of chromosomal abnormalities. The probes are preferably designed by first determining the sequence of at least one single copy interval in a target nucleic acid sequence, and developing corresponding hybridization probes which hybridize to at least a part of the deduced single copy sequence. In practice, the sequences of the target and of known genomic repetitive sequence representatives are compared in order to deduce locations of the... |
| Single copy genomic hybridization probes and method of generating same | 20090312533 | 20091217 |
| Nucleic acid (e.g., DNA) hybridization probes are described which comprise a labeled, single copy nucleic acid which hybridizes to a deduced single copy sequence interval in target nucleic acid of known sequence. The probes, which are essentially free of repetitive sequences, can be used in hybridization analyses without adding repetitive sequence-blocking nucleic acids. This allows rapid and accurate detection of chromosomal abnormalities. The probes are preferably designed by first determining the sequence of at least one single copy interval in a target nucleic acid sequence, and developing corresponding hybridization probes which hybridize to at least a part of the deduced single copy sequence. In practice, the sequences of the target and of known genomic repetitive sequence representatives are compared in order to deduce locations of the... |
| Methods and compositions for pcr | 20090263869 | 20091022 |
| A modified thermostable Pol B DNA polymerase, produced by a reaction, under essentially aqueous conditions, of a thermostable Pol B DNA polymerase and a modifier reagent of Formula I wherein the reaction results in a thermally reversible inactivation of the thermostable Pol B DNA polymerase activity and the 3′-5′ exonuclease activity, which polymerase is suitable for hot-start PCR. Also disclosed are the method for the modification, a polynucleic acid amplification method and PCR reaction mixture and kit comprising the modified thermostable Pol B DNA polymerase.
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| Methods and reagents for combined pcr amplification | 20090258351 | 20091015 |
| An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5′→3′ exonuclease activity of a polymerase and the 5′→3′ extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification. Additional similar combined PCR hybridization methods are disclosed, such... |
| Anti-bacterial drug targeting of genome maintenance interfaces | 20090203754 | 20090813 |
| Methods for the design and identification of novel antimicrobial compounds are provided, as well as antimicrobial compounds identified using these methods. Pharmaceutical compositions that include these antimicrobial compounds are provided as well. The antimicrobial compounds inhibit the binding of a prokaryotic single-stranded DNA binding protein to a polypeptide. In some examples, the prokaryotic single-stranded DNA binding protein is prokaryotic exonuclease I.
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| Variant scorpion primers for nucleic acid amplification and detection | 20090197254 | 20090806 |
| Disclosed herein are methods of detecting target nucleic acids. In particular, methods for avoiding loss of the fluorescent label form an amplicon that is generated using a Scorpion primer and a polymerase with 5′ exonuclease activity. The methods use a Scorpion primer which comprises a fluorophore, a quencher, and in 5′ to 3′ order, a probe region, a linker region and a primer region, wherein the quencher is located at or near the 5′ end, and, wherein the primer is complementary to the target nucleic acid and the probe region hybridizes to a complementary sequence in an extension product of the primer. The methods provide for detection of target nucleic acids in simplex or multiplex formats.
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| Mutant dna polymerases and uses therof | 20090191560 | 20090730 |
| The present invention relates to mutant DNA polymerases which incorporate dideoxynucleotides with about the same efficiency as deoxynucleotides. The present invention also related to mutant DNA polymerases which also have substantially reduced 5′-to-3′ exonuclease activity or 3′-to-5′ exonuclease activity. The invention also relates to DNA molecules coding for the mutant DNA polymerases, and hosts containing the DNA molecules.
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| Detection format for hot start real time polymerase chain reaction | 20090181401 | 20090716 |
| The present invention is directed to a method and a composition for amplifying and detecting a target nucleic comprising subjecting said target nucleic acid to a real time PCR amplification reaction in the presence of a thermostable DNA polymerase, a thermostable double strand dependent 3′-5′ exonuclease having a temperature optimum above 37° C., a pair of amplification primers, deoxynucleoside triphosphates, a detecting oligonucleotide carrying a first label and a second label, said first label being capable of acting as a fluorescent reporter entity when excited with light of an appropriate wavelength, said second label being capable of acting as a fluorescence quenching entity of said fluorescent reporter entity, characterized in that one label is bound to the 3′ end of said detecting oligonucleotide, and further characterized... |
| Contamination-free reagents for nucleic acid amplification | 20090155859 | 20090618 |
| Methods and kits for generating contamination-free reagents and reagent solutions for use in nucleic acid amplification are provided. Methods include processing of polymerase solutions, nucleotide solutions and primer solutions to render contaminating nucleic acid inert. The methods employ the proofreading activity of the polymerase and/or exonucleases to de-contaminate the reagents and reagent solutions. Methods and kits for contamination-free nucleic acid amplification are provided.
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| Cloned dna polymerases from thermotoga and mutants thereof | 20090155775 | 20090618 |
| The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga (Tne and Tma) and mutants thereof. The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The mutant DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3′→5′ exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5′→3′ exonuclease activity of said DNA polymerase; (3) a third mutation in the O helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides. The present invention also relates to the cloning and expression of the wild type or mutant DNA polymerases in E.... |
| Dna polymerases with enhanced length of primer extension | 20090148911 | 20090611 |
| A formulation and kit of thermostable or other DNA polymerases comprising at least one thermostable or other DNA polymerase which lacks 3′-exonuclease activity, and at least one thermostable DNA polymerase exhibiting 3′-exonuclease activity. Also provided is an improved method for enzymatic extension of DNA strands, especially while, but not limited to, amplifying nucleic acid sequences by polymerase chain reaction wherein the above formulation is made and used to catalyze primer extension.
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| Microarray system with improved sequence specificity | 20090143243 | 20090604 |
| The invention provides a novel array method for nucleic acid sequence detection with improved specificity which allows for detection of genetic variation, from simple SNPs (where the variation occurs at a fixed position and is of limited allelic number) to more complex sequence variation patterns (such as with multigene families or multiple genetic strains of an organism where the sequence variation between the individual members is neither fixed nor consistent). The array is comprised of short, synthetic oligonucleotide probes attached to a solid surface which are hybridized to single-stranded targets. Single stranded targets can be produced using a method that employs primers modified on the 5′ end to prohibit degradation by a 5′-exonuclease that is introduced to degrade the unprotected strand. The invention further provides for... |
| Method for detecting integrated hpv dna | 20090111090 | 20090430 |
| A method for detecting integrated HPV DNA is described herein. This method comprises obtaining first and second samples, obtaining first and second information, and detecting, based on the first and second information, the HPV DNA integrated into the genome of a cell derived from a subject. The second sample comprises DNA derived from the cell, which is treated with an enzyme having exonuclease activity. The first information is related to the amount of HPV DNA in the first sample, and the second information is related to the amount of HPV DNA in the second sample.
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| Method for synthesizing dna | 20090098613 | 20090416 |
| A DNA synthesis reaction-enhancer comprising at least one kind selected from the group consisting of acidic substances and cationic complexes; a DNA synthesis method in which during a DNA synthesis reaction a reaction is carried out in the presence of the above enhancer by using DNA polymerase; a DNA synthesis reaction composition comprising the above enhancer; a DNA synthesis reaction composition comprising two or more kinds of DNA polymerases each having 3′→5′ exonuclease activity; a DNA synthesis method in which during a DNA synthesis reaction two or more kinds of DNA polymerases each having 3′→5′ exonuclease activity are used; a kit for use in in vitro DNA synthesis, comprising two or more kinds of DNA polymerases each having 3′→5′ exonuclease activity; and a kit for use... |
| Thermostable nucleic acid polymerase from thermococcus gorgonarius | 20090093043 | 20090409 |
| A purified thermostable enzyme is derived form the thermophilic archaebacterium Thermococcus gorgonarius. The enzyme can be native or recombinant, retains approximately 90% of its activity after incubation for two hours at 95° C. in the presence of stabilizing agents and possesses 3′-5′ proofreading exonuclease activity. Thermostable DNA polymerases are useful in many recombinant DNA techniques, especially nucleic acid amplification by the polymerase chain reaction (PCR).
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| Nucleic acid sequencing methods and systems | 20090087850 | 20090402 |
| Sequencing methods that use an exonuclease that comprises template dependent nucleobase binding activity are provided. Related compositions and sequencing systems are also provided.
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| Methods of using fet labeled oligonucleotides that include a 3'-5' exonuclease resistant quencher domain and compositions for practicing the same | 20090081676 | 20090326 |
| Methods and compositions are provided for detecting a primer extension product in a reaction mixture. In the subject methods, a primer extension reaction is conducted in the presence of a polymerase having 3′→5′ exonuclease activity and at least one FET labeled oligonucleotide probe that includes a 3′→5′ exonuclease resistant quencher domain. Also provided are systems and kits for practicing the subject methods. The subject invention finds use in a variety of different applications, and are particularly suited for use in high fidelity PCR based reactions, including SNP detection applications, allelic variation detection applications, and the like.
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| Inhibitors of endo-exonuclease activity for treating cancer | 20090068094 | 20090312 |
| The present invention relates to the treatment of cancer with compounds that inhibit the activity of endo-exonuclease. Endo-exonuclease has been shown to be necessary for the repair of damaged DNA. Compounds that inhibit the activity of endo-exonuclease have been shown to be particularly effective for treating cancer when used in combination with drugs that induce DNA breaks such as cisplatin and mitomycin C. These compounds have a synergistic effect when used in combination for inhibiting tumour growth. The invention includes pharmaceutical compositions for inhibiting tumour growth comprising a compound that inhibits endo-exonuclease activity. These pharmaceutical compositions preferably include compounds that induce DNA breaks. The invention includes methods of treating cancer with these pharmaceutical compositions and uses of these compositions to treat cancer. The preferred compounds that... |
| Novel artemis/dna-dependent protein kinase complex and methods of use thereof | 20090017010 | 20090115 |
| In the present invention, it is disclosed that Artemis forms a complex with the 469 kDa DNA-dependent protein kinase (DNA-PKcs) in vitro and in vivo in the absence of DNA. The purified Artemis protein alone possesses single-strand specific 5′ to 3′ exonuclease activity. Upon complex formation, DNA-PKcs phosphorylates Artemis, and Artemis acquires endonucleolytic activity with respect to single-stranded nucleotides, including 5′ and 3′ overhangs, as well as hairpins. Further, the Artemis:DNA-PKcs complex can open hairpins generated by the RAG complex from a 12/23-substrate pair. Thus, DNA-PKcs regulates Artemis by both phosphorylation and complex formation to permit enzymatic activities that are critical for the hairpin opening step of V(D)J recombination and for all of the 5′ and 3′ overhang processing in nonhomologous DNA end joining.
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| Isothermal amplification of nucleic acids | 20080286835 | 20081120 |
| A process of amplifying a nucleic acid template dependent on partial destruction of primer molecules which have extended onto the template molecule followed by strand invasion of the partially destroyed primer template by a replacement primer. The destruction of the primer molecule may be performed by either endonuclease or exonuclease digestion. A signal generation from the amplified products may be obtained by the use of adaptors capable of binding probe molecules as well as the amplified product.
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| Compositions and methods utilizing dna polymerases | 20080280291 | 20081113 |
| The invention features a novel isolated Family B DNA polymerase, a Thermococcus polymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3′ to 5′ exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.
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| Thermostable viral polymerases and methods of use | 20080268498 | 20081030 |
| Thermostable viral polymerases exhibiting a combination of activities selected from, proofreading (3′-5′) exonuclease activity, nick translating (5′-3′) nuclease activity, synthetic primer-initiated polymerase activity, nick-initiated polymerase activity, reverse transcriptase activity, strand displacement activity, and/or decreased discrimination against incorporation of nucleotide analogs. Also provided are compositions including the polymerases, polynucleotides encoding the polymerases and methods of using the polymerases.
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| Dna polymerase blends and mutant dna polymerases | 20080254525 | 20081016 |
| A thermostable DNA polymerase composition comprising at least two DNA polymerases, one of which is substantially reduced in 5′-exonuclease activity and one of which has 5′-exonuclease activity. This polymerase may be used in methods including, but not limited to, nucleic acid synthesis, DNA sequencing, nucleic acid amplification and cDNA synthesis,
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| Method of gene sequence examination | 20080213762 | 20080904 |
| The present invention provides a method for detecting quantitatively a specific nucleic acid sequence and for detecting gene polymorphism or mutation by homogenous system simply, promptly, accurately, and inexpensively, and an oligonucleotide probe used therefor. The nucleic acid having a specific sequence is quantitatively detected and gene polymorphism or mutation is detected in such a way that a probe DNA containing one or two labeling materials is hybridized to the target nucleic acid, and decomposed from the 5′- or the 3′-terminal by exonuclease action to release one of the labeling materials, the signal emitted by which is then detected.
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| Multiplex targeted amplification using flap nuclease | 20080199916 | 20080821 |
| Methods for multiplex amplification of a plurality of targets of distinct sequence from a complex mixture are disclosed. In one aspect targets are circularized using a single circularization probe that is complementary to two regions in the target that flank a region to be amplified. The targets may hybridize to the circularization probe so that 5′ or 3′ flaps are generated and methods for removing flaps and circularizing the resulting product are disclosed. In another aspect targets are hybridized to dU probes so that 5′ and 3′ flaps are generated. The flaps are cleaved using 5′ or 3′ flap endonucleases or 3′ to 5′ exonucleases. The target sequences are then ligated to common primers, the dU probes digested and the ligated targets amplified.
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| Compositions and methods utilizing dna polymerases | 20080199935 | 20080821 |
| The invention features a novel isolated Family B DNA polymerase, a Thermococcus polymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3′ to 5′ exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.
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| Specialized oligonucleotides and their use in nucleic acid amplification and detection | 20080193934 | 20080814 |
| Described herein are labeled probes and unlabeled oligonucleotides that are useful for use in nucleic acid amplification reactions. These probes and oligonucleotides are modified to alter their sensitivity to primer-independent 5′ exonuclease activity of a thermostable DNA polymerase relative to its corresponding unmodified counterpart. Non-symmetric polymerase chain reaction (PCR) amplification and detection methods employing these labeled probes and unlabeled oligonucleotides are also described. Kits for nucleic acid amplification reactions including labeled probes and unlabeled oligonucleotides are also described.
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| Systems and methods for detecting nucleic acids | 20080193940 | 20080814 |
| A method and kit for detecting a target nucleic acid in a sample is described. The sample to be analyzed may include a primer which hybridizes to at least a portion of the target nucleic acid, a probe having a first region which hybridizes to at least a portion of the target nucleic acid and a second region having a detectable label, a polymerase which extends the hybridized primer and an enzyme comprising exonuclease activity that can cleave the hybridized hybridization probe to thereby generate a labeled probe fragment. At least one portion of the hybridization probe hybridizes to another portion of the hybridization probe to thereby form a folded structure. The method can involve melting the sample, reducing the temperature of the sample to allow... |
| Methods and systems for high homologous recombination ("hr") targeting efficiency | 20080187999 | 20080807 |
| Disclosed are vectors, kits and methods useful in the construction of recombinant cells and DNAs via enhanced efficiency homologous recombination. The vectors are targeting vectors that contain a gene-of-interest spliced between two ends that are homologous to a genome target site. The ends of the vector may be protected from exonuclease attack by deploying a cap, such as a hair pin structure. The vector is linked to a nuclear localization signal sequence, and preferably, a bait peptide that binds to RAD51, to facilitate homologous recombination. The vector may be deployed in myriad genetic transformation applications, such as site-directed mutagenesis, gene therapy, and the like.
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| Nucleic acid molecules encoding mismatch endonucleases and methods of use thereof | 20080145913 | 20080619 |
| We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3′ to 5′ exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. Also described are mismatch endonucleases suitable for use in the process.
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| Isothermal snp detection method | 20080118917 | 20080522 |
| In some embodiments, the present teachings provide a method for detecting a nucleotide of interest comprising; forming an amplification reaction mixture comprising a mismatched primer, a target polynucleotide, a strand-displacing polymerase lacking 3′ to 5′ exonuclease activity, a recombinase, and a single-stranded DNA binding protein; hybridizing a mismatched primer to the target polynucleotide to form a primer-target complex; and, detecting the nucleotide of interest by the absence of a primer extension product. In some embodiments, control reactions are performed in which a control polynucleotide is exponentially amplified. Additional methods, as well as reaction mixtures and kits, are also provided.
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| Method of generating nested sets of double stranded dna circles | 20080102466 | 20080501 |
| The invention provides a method of generating nested sets of double stranded DNA (dsDNA) circles that may be used as size ladders in nucleic acid separations and as templates in DNA sequencing operations. In one aspect, the invention provides methods for generating nested sets of double stranded DNA circles in a self-sustaining enzymatic reaction comprising the activities of at least one endonuclease, at least one single stranded exonuclease, and at least one ligase. In another embodiment, such nested sets are generated from linear dsDNA fragments having ligatable terminators that are self-ligated to form corresponding dsDNA circles.
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| Methods and compositions for correcting misincorporation in a nucleic acid synthesis reaction | 20080096765 | 20080424 |
| The invention provides methods for correcting misincorporation of a nucleotide in a primer during a sequencing-by-synthesis reaction by using both a polymerase substantially lacking in exonuclease activity and an enzyme, preferably a polymerase, having exonuclease activity.
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| Single copy genomic hybridization probes and method of generating same | 20080085509 | 20080410 |
| Nucleic acid (e.g., DNA) hybridization probes are described which comprise a labeled, single copy nucleic acid which hybridizes to a deduced single copy sequence interval in target nucleic acid of known sequence. The probes, which are essentially free of repetitive sequences, can be used in hybridization analyses without adding repetitive sequence-blocking nucleic acids. This allows rapid and accurate detection of chromosomal abnormalities. The probes are preferably designed by first determining the sequence of at least one single copy interval in a target nucleic acid sequence, and developing corresponding hybridization probes which hybridize to at least a part of the deduced single copy sequence. In practice, the sequences of the target and of known genomic repetitive sequence representatives are compared in order to deduce locations of the... |
| Methods of detecting an amplified nucleic acid | 20080058216 | 20080306 |
| The present invention is directed to methods of generating a signal indicative of the presence of said target nucleic acid sequence in a sample, comprising, incubating a sample comprising a an amplified target nucleic acid and a nucleic acid polymerase which substantially lacks 5′ to 3′ exonuclease activity, adding a thermostable fen nuclease consisting of 5′ to 3′ exonuclease and/or endonuclease activity so as to cleave a cleavage structure and generate a signal.
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| Methods and reagents for combined pcr amplification | 20080050742 | 20080228 |
| An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5′→3′ exonuclease activity of a polymerase and the 5′→3′ extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification. Additional similar combined PCR hybridization methods are disclosed, such... |
| Methods to increase nucleotide signals by raman scattering | 20080032297 | 20080207 |
| The methods and apparatus disclosed herein concern nucleic acid sequencing by enhanced Raman spectroscopy. In certain embodiments of the invention, nucleotides are covalently attached to Raman labels before incorporation into a nucleic acid. In other embodiments, unlabeled nucleic acids are used. Exonuclease treatment of the nucleic acid results in the release of labeled or unlabeled nucleotides that are detected by Raman spectroscopy. In alternative embodiments of the invention, nucleotides released from a nucleic acid by exonuclease treatment are covalently cross-linked to nanoparticles and detected by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS). Other embodiments of the invention concern apparatus for nucleic acid sequencing.
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| Method of increasing complementarity in a heteroduplex | 20080032346 | 20080207 |
| We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3′ to 5′ exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to... |
| Method to produce single stranded dna of defined length and sequence and dna probes produced thereby | 20080026393 | 20080131 |
| A method for producing a single stranded DNA (ssDNA) molecule of a defined length and sequence is disclosed. This method enables the preparation of, inter alia, probes of greater length than can be chemically synthesized. The method starts with a double stranded molecule, such as genomic, double stranded DNA (dsDNA) from any organism. A fragment of the starting molecule (dsDNA) is amplified by specific primers engineered to introduce cleavage sites on either side of the desired sequence. Cleavage steps on the amplified, engineered fragment are combined with a phosphate removal step, thereby creating a construct that can be digested with an exonuclease without damage to the desired ssDNA. Probes, which hybridize with large gaps between the ends of the probes, are also disclosed.
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| Methods of detection of a target nucleic acid sequence | 20070292863 | 20071220 |
| The present invention is directed to methods of generating a signal indicative of the presence of a target nucleic acid in a sample by forming a cleavage structure comprising duplex and single-stranded nucleic acid by incubating a sample comprising a target nucleic acid with a thermostable nucleic acid polymerase substantially lacking 5′ to 3′ exonuclease activity and cleaving said cleavage structure with a thermostable Fen nuclease lacking 5′ to 3′ synthetic activity to generate a signal.
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| Compositions and methods for detection of a target nucleic acid sequence | 20070292864 | 20071220 |
| The present invention is directed compositions having a Fen nuclease consisting of a 5′ to 3′ exonuclease and/or endonuclease activity and one or more dNTPs.
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| Compositions and methods for detection of a target nucleic acid sequence | 20070292934 | 20071220 |
| The present invention is directed compositions having a Fen nuclease consisting of a 5′ to 3′ exonuclease and/or endonuclease activity and pyrophosphate.
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| Methods for genotyping | 20070269817 | 20071122 |
| The present invention provides for methods for discriminating between alleles at polymorphic positions in a genome. In general the methods employ allele specific extension of oligonucleotides that are complementary to one of the alleles at the 3′ end of the oligonucleotide. The allele specific oligonucleotides are resistant to proof reading activity from a polymerase and may be extended in an allele specific manner by a DNA polymerase with a functional 3′ to 5′ exonuclease activity. The allele specific oligonucleotides may be attached to a solid support such as a chip or a bead.
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| Methods of using fet labeled oligonucleotides that include a 3'-5' exonuclease resistant quencher domain and compositions for practicing the same | 20070231809 | 20071004 |
| Methods and compositions are provided for detecting a primer extension product in a reaction mixture. In the subject methods, a primer extension reaction is conducted in the presence of a polymerase having 3′→5′ exonuclease activity and at least one FET labeled oligonucleotide probe that includes a 3′→5′ exonuclease resistant quencher domain. Also provided are systems and kits for practicing the subject methods. The subject invention finds use in a variety of different applications, and are particularly suited for use in high fidelity PCR based reactions, including SNP detection applications, allelic variation detection applications, and the like.
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| Compositions and kits for detection of a target nucleic acid | 20070231815 | 20071004 |
| The present invention is directed to kits and compositions for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising a nucleic acid polymerase lacking 5′ to 3′ exonuclease activity and a thermostable FEN nuclease consisting of a 5′ to 3′ exonuclease and/or endonuclease activity.
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| Method for long range allele-specific pcr | 20070184457 | 20070809 |
| The present invention is directed to a method and kit for determining the molecular haplotype of a gene in a diploid DNA sample. The method discriminates between two haplotypes on the basis of a difference of one or more nucleotides using allele-specific PCR amplification in combination with long range PCR, using a DNA polymerase enzyme having 3′→5′ exonuclease activity, using annealing temperature conditions sufficiently greater than the predicted annealing temperature (Tm) to effect selective hybridization and extension of an allele-specific extension primer to the target allele relative to the variant allele. The present invention is particularly useful, for example, to determine the haplotype of a gene having multi-allelic genetic loci separated by a distance in which accurate PCR amplification of a single fragment containing the multiple... |
| Methods for genome amplification | 20070178457 | 20070802 |
| A method for whole genome amplification comprising (a) treating genomic DNA with a modifying agent which modifies cytosine bases but does not modify 5′-methyl-cytosine bases under conditions to form single stranded modified DNA; (b) providing a population of random X-mers of exonuclease-resistant primers capable of binding to at least one strand of the modified DNA, wherein X is an integer 3 or greater; (c) providing polymerase capable of amplifying double stranded DNA, together with nucleotides and optionally any suitable buffers or diluents to the modified DNA; and (d) allowing the polymerase to amplify the modified DNA.
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| Compositions and methods for enhanced sensitivity and specificity of nucleic acid synthesis | 20070178489 | 20070802 |
| The present invention relates to polypeptides, compositions and methods for enhancing synthesis of nucleic acid molecules. In a preferred aspect, the invention relates to inhibition or control of nucleic acid synthesis, sequencing or amplification. Specifically, the present invention discloses polypeptides having affinity for double-stranded and/or single-stranded nucleic acid molecules and/or single-stranded/double-stranded nucleic acid complexes (e.g., primer/template complexes, double-stranded templates, single-stranded templates or single-stranded primers) for use in such enhanced synthesis and more particularly to polymerases having reduced polymerase and optionally reduced exonuclease activities (3′ to 5′ and/or 5′ to 3′ exonuclease activity), and to nucleases having reduced nuclease activity. The polypeptides of the invention are capable of inhibiting nonspecific nucleic acid synthesis at ambient temperature. Thus, in a preferred aspect, the invention relates to “hot start”... |
| Compositions for detection of a target nucleic acid sequence | 20070161011 | 20070712 |
| The invention relates to compositions for generating a signal indicative of the presence of a target nucleic acid in a sample, where the compositions include a probe having a 5′ region and a 3′ flap and a P. furiosus polymerase having 3′ exonuclease activity.
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| Method for polymerase chain reactions with use of a dna polymerase with proofreading properties | 20070082355 | 20070412 |
| The present invention concerns a method for a polymerase chain reaction, in which a template nucleic acid, at least one primer, deoxyribonucleoside triphosphates as well as a DNA polymerase with proofreading activity are used. In addition, according to this invention, at least one target substrate is added to the polymerase chain reaction, whereby the efficiency of the DNA polymerase with proofreading activity is significantly increased. Any molecule that reduces or, in the optimal case, blocks the 3′,5′-exonuclease activity of the DNA polymerase used is suitable as target substrate. Technical solutions for the added substrate (target substrate) are in particular single stranded, linear oligonucleotides, hairpin oligonucleotides and RNA and DNA molecules. Furthermore, a kit is disclosed which comprise the required reagents for the implementation of the method... |
| Method for in vitro recombination | 20070037196 | 20070215 |
| The present invention relates, e.g., to an in vitro method, using isolated protein reagents, for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising (a) chewing back the DNA molecules with an enzyme having an exonuclease activity, to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence identity to hybridize specifically to each other; (b) specifically annealing the single-stranded overhangs; and (c) repairing single-stranded gaps in the annealed DNA molecules and sealing the nicks thus formed (ligating the nicked DNA molecules). The region of sequence identity generally comprises at least 20 non-palindromic nucleotides... |
| Binary probe and clamp composition and methods for target hybridization detection | 20070026429 | 20070201 |
| Binary probe and clamp compositions conduct methods for target hybridization detection. Where the probe is a substrate for exonuclease cleavage, the composition provides quantitation and detection of PCR products, by real-time and end-point measurements. Where the probe is an amplification primer, the composition provides an improved method for labelling and detection of PCR products. Probes and clamps may be labelled with fluorescent dyes, quenchers, hybridization-stabilizing moieties, chemiluminescent dyes, and affinity ligands. Clamps may be nucleic acid analogs, such as 2-aminoethylglycine PNA.
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| Mouse model for aging | 20070022488 | 20070125 |
| A mouse model for mammalian aging is disclosed. In one embodiment, the invention comprises a mouse having a genomic mutation in the exonuclease domain II (ExoII) of a mitochondrial DNA polymerase gamma (POlG) gene, wherein the mutation leads to high levels of mutations in polymerase mtDNA.
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| Hot start polymerase reaction using a thermolabile blocker | 20070009922 | 20070111 |
| The invention relates to compositions, methods, and kits for hot start polynucleotide synthesis, including extension of primed polynucleotide templates and polymerase chain reaction (PCR). Hot start is provided by a thermally inactivated blocking polymerase protein that binds primed polynucleotide templates and prevents their access to a thermostable nucleic acid polymerase. High temperatures employed in the synthesis reaction cause the blocking polymerase to denature, thereby permitting the action of a thermostable processive polymerase. Compositions of the invention include a specific blocking polymerase protein which is a mutant of the Klenow fragment of E. coli DNA polymerase. The mutant is essentially devoid of polymerase activity, processivity, and 3′ to 5′ exonuclease activity. Use of the thermally inactivated blocking polymerase together with a thermostable polymerase reduces non-specific priming and... |
| Dna polymerase blends and mutant dna polymerases | 20060292578 | 20061228 |
| A thermostable DNA polymerase composition comprising at least two DNA polymerases, one of which is substantially reduced in 5′-exonuclease activity and one of which has 5′-exonuclease activity. This polymerase may be used in methods including, but not limited to, nucleic acid synthesis, DNA sequencing, nucleic acid amplification and cDNA synthesis,
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| Thiotriphosphate nucleotide dye terminators | 20060281100 | 20061214 |
| Fluorescent reporter compounds having a chain terminating (thio)triphosphate nucleotide derivative, a fluorescent dye, and a linker of sufficient length to connect the nucleotide derivative to the fluorescent dye are provided. The fluorescent reporter compounds are used in DNA sequencing reactions and are substantially inactive toward exonuclease digestion.
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| Inhibitors of endo-exonuclease activity for treating cancer | 20060276548 | 20061207 |
| The present invention relates to the treatment of cancer with compounds that inhibit the activity of endo-exonuclease. Endo-exonuclease has been shown to be necessary for the repair of damaged DNA. Compounds that inhibit the activity of endo-exonuclease have been shown to be particularly effective for treating cancer when used in combination with drugs that induce DNA breaks such as cisplatin and mitomycin C. These compounds have a synergistic effect when used in combination for inhibiting tumour growth. The invention includes pharmaceutical compositions for inhibiting tumour growth comprising a compound that inhibits endo-exonuclease activity. These pharmaceutical compositions preferably include compounds that induce DNA breaks. The invention includes methods of treating cancer with these pharmaceutical compositions and uses of these compositions to treat cancer. The preferred compounds that... |
| Methods of producing interachain fluorophore-quencher fret-aptamers and assays | 20060257914 | 20061116 |
| The present invention describes methods for the production and use of single chain (single-stranded) fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers containing fluorophores (F) and quenchers (Q) at various loci within their structures, such that when its specific matching analyte is bound and the FRET-aptamers are excited by specific wavelengths of light, the fluorescence intensity of the system is modulated (increased or decreased) in proportion to the amount of analyte added. F and Q are covalently linked to nucleotide triphosphates (NTPs), which are incorporated by various nucleic acid polymerases such as Taq polymerase during the polymerase chain reaction (PCR) and then selected by affinity chromatographic, size-exclusion or molecular sieving, and fluorescence techniques. Further separation of related FRET-aptamers can be achieved by ion-pair reverse phase... |
| Compositions and methods employing 5' phosphate-dependent nucleic acid exonucleases | 20060240451 | 20061026 |
| The present invention relates to compositions and methods employing 5′-phosphate-dependent nucleic acid exonucleases. In particular, the present invention provides kits and methods employing 5′-phosphate-dependent nucleic acid exonucleases for selective enrichment, isolation and amplification of a particular set of desired nucleic acid molecules from samples that also contain undesired nucleic acid molecules for a variety of uses. In preferred embodiments, the desired nucleic acid molecules comprise prokaryotic and/or eukaryotic mRNA.
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| Mouse model for aging | 20060212954 | 20060921 |
| A mouse model for mammalian aging is disclosed. In one embodiment, the invention comprises a mouse having a genomic mutation in the exonuclease domain II (ExoII) of a mitochondrial DNA polymerase gamma (PolG) gene, wherein the mutation leads to high levels of mutations in polymerase mtDNA.
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| Nucleic acid molecules encoding endonucleases and methods of use thereof | 20060194288 | 20060831 |
| We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3′ to 5′ exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to... |
| Cleavage of nucleic acids | 20060183109 | 20060817 |
| The present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner. Enzymes, including 5′ nucleases and 3′ exonucleases, are used to detect and identify nucleic acids derived from microorganisms. Methods are provided which allow for the detection and identification of bacterial and viral pathogens in a sample.
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| Method of nucleotide identification using an off-switch through proofreading 3' exonuclease-resistant modified primers by polymerases with 3' exonuclease activity | 20060172307 | 20060803 |
| Methodology for a high fidelity polymerase-mediated primer extension for use in genotyping is provided. The primer extensions are carried out with polymerase having 3′ to 5′ exonuclease activity and primers with their 3′ modification of being exonuclease-resistant. The primers are designed to have their 3′ termini: 1. complementary to nucleotide to be analyzed; and 2. subject to be proofread by the 3′ to 5′ exonuclease of the high fidelity polymerases.
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| Methods and compositions for correcting misincorporation in a nucleic acid synthesis reaction | 20060172328 | 20060803 |
| The invention provides methods for correcting misincorporation of a nucleotide in a primer during a sequencing-by-synthesis reaction by using both a polymerase substantially lacking in exonuclease activity and an enzyme, preferably a polymerase, having exonuclease activity.
... |
| Method for in vitro molecular evolution of protein function | 20060166198 | 20060727 |
| A method for in vitro molecular evolution of protein function The invention provides a method for generating a polynucleotide sequence or population of sequences from parent single-stranded polynucleotide sequences encoding one or more protein motifs, comprising the steps of (a) providing a first population of single-stranded polynucleotide molecules and a second population of single-stranded polynucleotide molecules, the first and second populations together constituting plus and minus strands of parent polynucleotide sequences, (b) carrying out a reaction for digesting the first and second populations of single-stranded polynucleotide molecules with an exonuclease to generate corresponding populations of single-stranded polynucleotide fragments, (c) contacting said fragments generated from the plus strands with fragments generated from the minus strands and optionally, adding primer sequences that anneal to the 3′ and 5′ends... |
| Methods to increase nucleotide signals by raman scattering | 20060166243 | 20060727 |
| The methods and apparatus disclosed herein concern nucleic acid sequencing by enhanced Raman spectroscopy. In certain embodiments of the invention, nucleotides are covalently attached to Raman labels before incorporation into a nucleic acid. In other embodiments, unlabeled nucleic acids are used. Exonuclease treatment of the nucleic acid results in the release of labeled or unlabeled nucleotides that are detected by Raman spectroscopy. In alternative embodiments of the invention, nucleotides released from a nucleic acid by exonuclease treatment are covalently cross-linked to nanoparticles and detected by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS). Other embodiments of the invention concern apparatus for nucleic acid sequencing.
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| Methods for improving rna transcription reactions | 20060105331 | 20060518 |
| Methods are described for eliminating single-stranded oligonucleotides from a sample prior to RNA transcription, thereby reducing non-template derived production of RNA. In one embodiment, a sample containing the template for RNA transcription is treated with one or more exonucleases to remove single-stranded oligonucleotides from the reaction mixture prior to RNA transcription. In another embodiment, the sample containing the template for RNA transcription is contacted with an oligonucleotide complementary to the single-stranded oligonucleotide present in the sample, and allowed to hybridize to form double-stranded oligonucleotides.
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| Novel polymerase compositions and uses thereof | 20060088822 | 20060427 |
| The subject invention provides novel compositions containing a mixture of (a) an enzyme that possesses substantial 3′-5′ exonuclease activity (b) a DNA polymerase with less 3′-5′ exonuclease activity than the enzyme with substantial 3′-5′ exonuclease activity. Preferably, the DNA polymerase for inclusion in the compositions are DNA polymerases that substantially lack 3′-5′ exonuclease activity. A preferred embodiment of the invention is a composition comprising the Taq DNA polymerase (isolated from Thermus aquaticus) and the Pfu DNA polymerase (isolated from Pyrococcus furiosus). Another aspect of the invention is to provide methods for synthesizing polynucleotides, typically DNA, using compositions comprising an enzyme that possesses substantial 3′-5′ exonuclease activity and a DNA polymerase with less 3′-5′ exonuclease activity than the enzymes possessing substantial 3′-5′ exonuclease activity, preferably a DNA... |
| Methods of using fet labeled oligonucleotides that include a 3'-5' exonuclease resistant quencher domain and compositions for practicing the same | 20060088855 | 20060427 |
| Methods and compositions are provided for detecting a primer extension product in a reaction mixture. In the subject methods, a primer extension reaction is conducted in the presence of a polymerase having 3′→5′ exonuclease activity and at least one FET labeled oligonucleotide probe that includes a 3′→5′ exonuclease resistant quencher domain. Also provided are systems and kits for practicing the subject methods. The subject invention finds use in a variety of different applications, and are particularly suited for use in high fidelity PCR based reactions, including SNP detection applications, allelic variation detection applications, and the like.
... |
| Method of controlling gene expression and gene silencing | 20060090218 | 20060427 |
| The present invention relates to methods to regulate gene expression in plants. In particular, manipulation of the expression in a plant cell of a nucleotide sequence encoding a polypeptide comprising a 3′-5′ exonuclease domain is disclosed. More stable and predictable expression is thus obtained. The present invention also relates to method of increasing or decreasing post-transcriptional silencing. The invention further relates to novel nucleic acid molecules comprising nucleotide sequences encoding polypeptides comprising a 3′-5′ exonuclease domain.
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| Novel microarray techniques for nucleic acid expression analyses | 20060078925 | 20060413 |
| Provided are DNA microarray techniques that allow hybridization without RNA amplification, without using cDNA, and without labeling the nucleic acid prior to hybridization. Referred to as the Double-stranded Exonuclease Protection (DEP) assay, the technique permits the sample RNA to be used directly for hybridization, without manipulation in any way. Further provided is a microarray technique for high-throughput miRNA gene expression analyses, termed the RNA-primed, Array-based, Klenow Enzyme (RAKE) assay. The RAKE assay is a sensitive and specific technique for assessing single-stranded DNA and RNA targets, and offers specific advantages over Northern blots.
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| Thermostable enzyme promoting the fidelity of thermostable dna polymerases-for improvement of nucleic acid synthesis and amplification in vitro | 20060078928 | 20060413 |
| A purified thermostable enzyme is derived form the thermophilic archaebacterium Archaeoglobus fulgidus. The enzyme can be native or recombinant, is stable under PCR conditions and exhibits double strand specific exonuclease activity. It is a 3′-5′ exonuclease and cleaves to produce 5′-mononucleotides. Thermostable exonucleases are useful in many recombinant DNA techniques, in combination with a thermostable DNA polymerase like Taq especially for nucleic acid amplification by the polymerase chain reaction (PCR).
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