Methods and compositions for the specific inhibition of gene expression by double-stranded RNA

The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. The dsRNA has a first oligonucleotide sequence that is between 25 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of at least 19 nucleotides is sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger the destruction of the target RNA by the RNAi machinery.

RNase H-Based Assays Utilizing Modified RNA Monomers

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3′ end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein. 1. A hot start enzyme composition comprising an enzyme and a chemical modification , wherein the chemical modification inactivates the enzyme.2Pyrococcus abysii.. The hot start enzyme composition of wherein the enzyme is3Pyrococcus abysii. The hot start enzyme composition of wherein the enzyme comprises the polypeptide of SEQ ID NO. 4.4. The hot start enzyme composition of wherein the chemical modification is a maleic acid anhydride analog.5. The hot start enzyme composition of wherein the maleic acid anhydride analog is citroconic anhydride.6. A hot start enzyme composition comprising an enzyme and an antibody bound to the enzyme claim 4 , wherein the antibody inactivates the enzyme at low temperatures.7Pyrococcus abysii.. The hot start enzyme composition of wherein the enzyme is8Pyrococcus abysii. The hot start enzyme composition of wherein the enzyme comprises the polypeptide of SEQ ID NO. 4.9Pyrococcus abysii. The hot start enzyme composition of wherein the antibody is an antibody that specifically binds Priority is claimed to U.S. provisional application No. 61/049,204 filed on Apr. 30, 2008, which is incorporated by reference in its entirety.This invention pertains to methods of cleaving a nucleic acid strand to initiate, assist, monitor or perform biological assays.The specificity of primer-based amplification reactions, such as the polymerase chain reaction (PCR), largely depends on the specificity of primer hybridization ...

Methods for Predicting Stability and Melting Temperatures of Nucleic Acid Duplexes

The present invention provides methods that more accurately predict melting temperatures for duplex oligomers. The invented methods predict the Tof chimeric duplexes containing various amounts of locked nucleic acid modifications in oligonucleotide strands. 2. The method of claim 1 , wherein ΔH°=ΔH°+ΔΔH°3. The method of claim 1 , wherein ΔS°=ΔS++ΔΔS°6. The method of or claim 1 , wherein the values are determined using nearest neighbor parameters.8. The method of claim 7 , wherein the user interacts with the networked server via a web-browsing application running on the communication device.9. The method of claim 7 , wherein the communication device comprises at least one of a computer claim 7 , a desktop computer claim 7 , or a laptop computer.10. The method of claim 7 , wherein ΔH°=ΔH°+ΔΔH°11. The method of claim 7 , wherein ΔS°=×S°+ΔΔS°14. The method of or claim 7 , wherein the values are determined using nearest neighbor parameters.15. A method of predicting the stability of an oligonucleotide comprising:(a) a computer system receiving a data input from a user, the computer comprising a processor, and instructions executable by the processor; {'br': None, 'i': G°=ΔG°', '+ΔΔG°, 'sub': DNA', 'LNA, 'Δ; and'}, '(b) responsive to the data input from a user, the computer system calculating a free energy value using the equation(c) providing an output to a display,wherein the oligonucleotide comprises at least two Locked Nucleic Acid (LNA) modifications.17. The method of claim 16 , wherein the values are determined using nearest neighbor parameters.18. A method of predicting the stability of an oligonucleotide comprising:(a) a networked server receiving data input from a communication device associated with a user, the server comprising the communication interface, a processor, and instructions executable by the processor; {'br': None, 'i': G°=ΔG°', '+ΔΔG°, 'sub': DNA', 'LNA, 'Δ; and'}, '(b) responsive to receiving the data input, the networked server sending via the ...

FLUORESCENCE QUENCHING AZO DYES, THEIR METHODS OF PREPARATION AND USE

Disclosed is a group of azo quencher compositions useful as fluorescence quenchers having the general structure of formula 1, methods of making or using the compositions, and kits comprising the composition. 2. The method of further comprising altering the spatial relationship between the fluorophore and quencher.3. The method of wherein the altering of the spatial relationship between the fluorophore and quencher is a result of the hybridization of the first and second nucleic acid polymers.4. The method of wherein the fluorophore and quencher composition are linked to a single nucleic acid polymer.5. The method of further comprising claim 1 , when the first and second nucleic acid polymers are hybridized to each other claim 1 , releasing the fluorophore or quencher composition from the hybridized structure.6. A method for synthesizing an oligonucleotide having a fluorescent quencher of Formula 1 claim 1 , comprising:obtaining a first compound having a nitro group and a primary amino group;treating the primary amino group of the first compound in the presence of NaNO2, followed by LiBF4, to form an diazonium salt;reacting the diazonium salt with a second compound having a substituted aryl ring to form a product having an azo group;covalently incorporating the product into an oligonucleotide. This application is a divisional of U.S. application Ser. No. 12/252,721 filed Oct. 16, 2008, which is a divisional of U.S. application Ser. No. 10/987,608 filed Nov. 12, 2004, now U.S. Pat. No. 7,439,341, which claims priority benefits to U.S. Provisional Application No. 60/520,077 filed Nov. 14, 2003. These applications are incorporated herein by reference in their entirety.This invention pertains to compositions that are capable of quenching the fluorescence of fluorophores and to methods for making and using them. The invention also provides kits that contain at least one of the disclosed quencher compositions.Light quenching processes that rely on the interaction of two ...

Modifications for Antisense Compounds

The invention pertains to modifications for antisense oligonucleotides, wherein the modifications are used to improve stability and provide protection from nuclease degradation. The modifications could also be incorporated into double-stranded nucleic acids, such as synthetic siRNAs and miRNAs. 1: An antisense oligonucleotide comprising at least one modification that is incorporated between two bases of the antisense oligonucleotide , wherein the modification increases binding affinity and nuclease resistance of the antisense oligonucleotide.2: The antisense oligonucleotide of claim 1 , wherein the modification is located within three bases of a terminal nucleotide.3: The antisense oligonucleotide of claim 1 , wherein the modification is located between a terminal base and a penultimate base of either the 3′ or the 5′ end of the antisense oligonucleotide.4: The antisense oligonucleotide of claim 1 , wherein a modification is located between the terminal base and the penultimate base at both the 3′ and the 5′ ends of the antisense oligonucleotide.5: The antisense oligonucleotide of claim 1 , wherein the modification is a napthylene-azo compound.8: The antisense oligonucleotide of claim 7 , wherein Ris NO.10: The antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide further comprises at least one 2′-O-methyl RNA.11: The antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide further comprises one or more phosphorothioate linkages.12: The antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide comprises a region of bases linked through phosphodiester bonds claim 1 , wherein the region is flanked at one or both ends by regions containing phosphorothioate linkages. This application claims the benefit of priority from U.S. Provisional Application No. 61/380,586, filed Sep. 7, 2010, the disclosure of which is incorporated by reference herein in its entirety.This invention was supported in part by Small ...

Method for Estimating a Melting Temperature of a Nucleic Acid in Buffers Containing Magnesium Ions

The invention relates to methods and systems for predicting or estimating the melting temperature of duplex nucleic acids, in the presence of divalent cations, particularly duplexes of oligonucleotides which may be used as, for example, but not limited to primers or probes in PCR and/or hybridization assays. The methods and algorithms use novel formulas, having terms and coefficients that are functions of the particular nucleotide sequence, to estimate the effect of divalent cation salt conditions on the melting temperature. 1. A method for estimating a melting temperature , T(X) , for a polynucleotide at a desired divalent cation concentration , [X] , and an optionally present monovalent cation concentration [Mon] , said polynucleotide having a known G-C content value , f , comprising:{'sub': 'm', 'sup': '+0', '(a) obtaining a reference melting temperature, T°, for the polynucleotide, said reference melting temperature being a melting temperature obtained or provided for the polynucleotide at a reference monovalent ion concentration, [Mon]; and'}(b) modifying said reference melting temperature, or reciprocal of said melting temperature, by one or more terms which are a function of fGC to determine the melting temperature of the polynucleotide at the desired monovalent and divalent cation concentrations.2. The method of claim 1 , wherein the said reference melting temperature or the reciprocal of said reference melting temperature is modified by a term which is a function of the fGC multiplied by a term comprising a logarithm of the divalent cation concentration.3. The method of claim 2 , wherein the reciprocal of the reference melting temperature is modified by adding a term comprising a+b ln [X]+f·(c+d ln [X]) claim 2 , wherein each of the coefficients a claim 2 , b claim 2 , c claim 2 , and d is optimized for predicting polynucleotide melting temperatures.8. The method of claim 2 , wherein the reference melting temperature is modified by adding a term comprising ...

RNase H-Based Assays Utilizing Modified RNA Monomers

The present invention provides methods of cleaving a nucleic acid strand to initiate, assist, monitor or perform biological assays. 1. A method of amplifying a target DNA sequence , said method comprising the steps of:a) providing a reaction mixture comprising (i) an upstream oligonucleotide primer and a downstream oligonucleotide primer each having a cleavage domain positioned 5′ of a blocking group, said blocking group linked at or near the end of the 3′-end of each oligonucleotide primer wherein said blocking group prevents primer extension, (ii) a sample nucleic acid that may or may not have the target sequence, (iii) a cleaving enzyme and (iv) a polymerase wherein said cleaving enzyme is a hot start cleaving enzyme which is thermostable and has reduced activity at lower temperatures;b) hybridizing either the upstream or downstream primer to the target DNA sequence to form a double-stranded substrate;c) cleaving the hybridized primer with said cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the primer; andd) extending the primer with the polymerase.2. The method of wherein the hot start cleaving enzyme is an RNase H enzyme.3. The method of wherein said RNase H enzyme is an RNase H2 enzyme.4. The method of wherein said RNase H2 enzyme inherently has lower activity at reduced temperature claim 3 , is reversibly inactivated by chemical modification or by a blocking antibody.5. The method of wherein said cleaving enzyme is a sequence-specific double stranded endonuclease.6. The method of wherein said sequence-specific double stranded endonuclease is a restriction enzyme.7. The method of wherein the blocking group is attached 5′ of the 3′-terminal residue.8. The method of wherein the blocking group includes one or more abasic residues or modified nucleosides.9. The method of wherein the abasic residue is a C3 spacer.10. The method of wherein the modified nucleoside is a 2′-O-methyl ribose residue.11. The method ...

POPPET VALVE ASSEMBLY FOR CONTROLLING A PNEUMATIC ACTUATOR

A poppet valve assembly to control a pneumatic actuator may include a housing having a central bore. A first module may be disposed within the central bore, and the first module may have a first and second poppet valve. A second module may also be disposed within the central bore, and the second module may have a third and fourth poppet valve. A supply of pressurized fluid may be in fluid communication with a plurality of control valves such that pressurized fluid from the control valves opens and closes the poppet valves. The supply of pressurized fluid may also be in fluid communication with the first module and the second module such that the opening and closing of the poppet valves controls the position of the pneumatic actuator. 1. A poppet valve assembly comprising:a valve housing having a central bore;a first module disposed within the central bore, the first module comprising a first normally closed poppet valve and a second normally closed poppet valve, wherein each of the first and second normally closed poppet valves has an open position and a closed position;a central port formed in the first module, wherein the central port of the first module is adapted to be coupled to a first volume of a pneumatic cylinder;an exhaust port formed in the first module such that the central port is in fluid communication with the exhaust port when the first poppet valve is in the open position; anda supply port formed in the first module such that the central port is in fluid communication with the supply port when second poppet valve is in the open position,wherein the supply port is configured to be in fluid communication with a supply of pressurized fluid such that when the second poppet valve is in the open position, pressurized fluid is provided to the first volume of the pneumatic cylinder, andwherein the exhaust port is configured to vent pressurized fluid from the first volume of the pneumatic cylinder when the first poppet valve is in the open position.2. The ...

VALVE SIGNATURE DIAGNOSIS AND LEAK TEST DEVICE

A valve signature diagnosis and leak testing device includes a spool valve operatively connected to a pilot valve, the pilot valve being configured to position the spool valve to one of an open position and a closed position. A blocker valve is fluidly connected to a control fluid outlet of the spool valve. An electrical module is operatively connected to the pilot valve, a supply of control fluid, and the blocker valve, the electrical module being capable of sending pulsed electrical signals to the pilot valve and the blocker valve to selectively position the spool valve and the blocker valve to an open or closed position. 1. A valve signature diagnosis and leak testing device for a control valve , the valve signature diagnosis and leak testing device comprising:a spool valve operatively connected to a pilot valve, the pilot valve being configured to position the spool valve to one of an open position and a closed position, the spool valve including a first control fluid inlet, a first control fluid outlet, and a second control fluid outlet, the first control fluid inlet being fluidly connected to a supply of control fluid and the first control fluid outlet being configured to be connected to a valve actuator;a blocker valve fluidly connected to the second control fluid outlet; andan electrical module operatively connected to the pilot valve, the supply of control fluid, and the blocker valve, the electrical module being capable of sending pulsed electrical signals to the pilot valve and the blocker valve to selectively position the spool valve and the blocker valve to an open or a closed position,wherein the open position of the spool valve fluidly connects the first control fluid inlet to the first control fluid outlet and the closed position of the spool valve fluidly connects the first control fluid outlet to the second control fluid outlet.2. The valve signature diagnosis and leak testing device of claim 1 , wherein the electrical module includes a main ...

WIRELESS MONITORING AND CONTROL OF SAFETY STATIONS IN A PROCESS PLANT

A safety station for use in a process plant includes one or more leverless limit switches to detect activation of one or more parts of the safety station. The safety station further includes a wireless transmitter coupled to the leverless limit switches to transmit signals associated with the safety station to a base station device, which is communicatively coupled to one or more control and/or monitoring devices. 1. A safety station for use in a process plant , the safety station comprising:one or more leverless limit switches to detect activation of one or more parts of the safety station; anda wireless transmitter coupled to the leverless limit switches to transmit signals associated with the safety station to a base station device, wherein the base station device is communicatively coupled to one or more control and/or monitoring modules.2. The safety station of claim 1 , wherein the leverless limit switch is a GO® switch manufactured by the TopWorx corporation.3. The safety station of claim 1 , wherein the leverless limit switch remains latched until physically reset.4702. The safety station of claim 1 , wherein the wireless transmitter is the Rosemount dual input transmitter manufactured by the Emerson corporation.5. The safety station of claim 1 , wherein the wireless transmitter is an intrinsically safe wireless transmitter.6. The safety station of claim 1 , wherein the safety station is a safety shower and/or an eye wash station.7. The safety station of claim 1 , wherein at least one of the one or more control and/or monitoring stations is a remote touch screen panel.8. The safety station of claim 1 , wherein at least one of the one or more control and/or monitoring stations is a paperless recorder.9. The safety station of claim 1 , wherein at least one of the one or more control and/or monitoring stations is a workstation.10. A safety station monitoring and control system in a process plant claim 1 , the system comprising:one or more safety stations ...

SWITCH MODULE

A switch module used for position sensing may be operated in a number of modes for compatibility with a number of legacy position sensing products. A dry switch circuit can be configured to provide a direct output, emulating dry reed, high-side or low-side switched configurations. Alternatively, the dry switch circuit can be connected to an input of a NAMUR circuit to provide the known current output for that standard. In another configuration the dry switch can be selectively coupled to one of two NAMUR circuits allowing the switch module to provide a low-to-high current NAMUR output or a high-to-low current NAMUR output. 1. A switch module arranged and adapted to report a position of a moveable element external to the switch module , the switch module comprising:a first external connection, a second external connection, and a third external connection;a dry contact relay having a relay contact position responsive to a position of a component external to the switch module, the dry contact relay having a common connection, a normally open connection and a normally closed connection, where the normally open connection corresponds to a first position of the moveable element and the normally closed connection corresponding to a second position of the moveable;a NAMUR circuit having a first input connection pair and a first output connection pair;an electrical connection from the normally open connection of the dry relay to a first input of the first input connection pair of the NAMUR circuit;a selector operable to connect the common connection of the dry contact relay to i) a null contact of the selector or ii) a second input of the first input connection pair of the NAMUR circuit;a second electrical connection from the first output of the NAMUR circuit to one of the first external connection or the second external connection; anda third electrical connection between the second output of the NAMUR circuit to the third external connection;a first jumper that removeably ...

FLUORESCENCE QUENCHING AZO DYES, THEIR METHODS OF PREPARATION AND USE

Disclosed is a group of azo quencher compositions useful as fluorescence quenchers having the general structure of formula 1, methods of making or using the compositions, and kits comprising the composition. 2. The method of claim 1 , wherein either Ror Ris a cyanoethylphosphoramidite group.3. The method of claim 1 , wherein either Ror Ris a phosphoramidite group.4. The method of claim 3 , wherein the oligonucleotide is labeled on its 3′-terminus.5. The method of claim 4 , wherein the oligonucleotide is labeled on its 5′-terminus.7. The method of claim 1 , wherein the synthesis step comprises linking the compound of Formula 1 to a solid support.8. The method of claim 7 , wherein the solid support is controlled pore glass.9. The method of claim 1 , further comprising:obtaining a first compound having a nitro group and a primary amino group;treating the primary amino group of the first compound in the presence of NaNO2, followed by LiBF4, to form an diazonium salt;reacting the diazonium salt with a second compound having a substituted aryl ring to form the compound of Formula 1. This application is a continuation of U.S. application Ser. No. 13/298,479 filed Nov. 17, 2011, which is a divisional of U.S. application Ser. No. 12/252,721 filed Oct. 16, 2008, now U.S. Pat. No. 8,084,588, which is a divisional of U.S. application Ser. No. 10/987,608 filed Nov. 12, 2004, now U.S. Pat. No. 7,439,341, which claims priority benefits to U.S. Provisional Application No. 60/520,077 filed Nov. 14, 2003. These applications are incorporated herein by reference in their entirety.This invention pertains to compositions that are capable of quenching the fluorescence of fluorophores and to methods for making and using them. The invention also provides kits that contain at least one of the disclosed quencher compositions.Light quenching processes that rely on the interaction of two dyes as their spatial relationship changes can be used in convenient processes for detecting and/or ...

Modifications for Antisense Compounds

The invention pertains to modifications for antisense oligonucleotides, wherein the modifications are used to improve stability and provide protection from nuclease degradation. The modifications could also be incorporated into double-stranded nucleic acids, such as synthetic siRNAs and miRNAs. 1. An antisense oligonucleotide comprising at least one modification that is incorporated at the terminal end or between two nucleotides of the antisense oligonucleotide , wherein the modification increases binding affinity and nuclease resistance of the antisense oligonucleotide.2. The antisense oligonucleotide of claim 1 , wherein the modification is located at the 5′-end of the antisense oligonucleotide.3. The antisense oligonucleotide of claim 1 , wherein the modification is located at the 3′-end of the antisense oligonucleotide.4. The antisense oligonucleotide of claim 1 , wherein modifications are located at both the 5′- and 3′-ends of the antisense oligonucleotide.5. The antisense oligonucleotide of claim 1 , wherein the modification is inserted between nucleotides and is located near the 3′-end of the antisense oligonucleotide.6. The antisense oligonucleotide of claim 1 , wherein the modification is inserted between nucleotides and is located near the 5′-end of the antisense oligonucleotide.7. The antisense oligonucleotide of claim 1 , wherein modifications are inserted between nucleotides and are located near the 3′-end and the 5′-end of the antisense oligonucleotide.8. The antisense oligonucleotide of claim 1 , wherein a modification is located at the 5′-end and a modification is inserted between nucleotides near the 3′-end of the antisense oligonucleotide.9. The antisense oligonucleotide of claim 1 , wherein a modification is located at the 3′-end and a modification is inserted between nucleotides near the 5′-end of the antisense oligonucleotide.10. The antisense oligonucleotide of claim 1 , wherein the modification is a napthyl-azo compound.13. The antisense ...

Modified rnase h enzymes and their uses

The invention provides a provides improvements to assays that employ RNase H cleavage for biological applications related to nucleic acid amplification and detection, where the RNase H has been reversibly inactivated.

TAILED PRIMER FOR CLONED PRODUCTS USED IN LIBRARY CONSTRUCTION

This invention pertains to improved methods for the synthesis of long, double stranded nucleic acid sequences containing difficult to clone or variable regions. 1a) truncating a gene fragment by cleaving off sequences from both terminal ends;b) inserting the truncated gene into a cloning vector; i. tailed primers that serve to add back the 5′ cleaved regions and', 'ii. bridging primers that serve to introduce new sequences within the gene fragment; and, 'c) amplifying the cloned gene out of the vector using'}d) amplifying the resulting amplicon using primers that target the terminal regions.. A method of constructing a DNA library from a clonally purified synthetic gene with decreased contamination with the wild type background, said method comprising selectively amplifying recombinant sequences, the method comprising: This invention pertains to improved methods for the preparation of long, double stranded nucleic acid sequences containing regions of low complexity, repeating elements, difficult to assemble and clone elements, or variable regions containing mixed bases.Synthetic DNA sequences are a vital tool in molecular biology. They are used in gene therapy, vaccines, DNA libraries, environmental engineering, diagnostics, tissue engineering and research into genetic variants. Long artificially-made nucleic acid sequences are commonly referred to as synthetic genes; however the artificial elements produced do not have to encode for genes, but, for example, can be regulatory or structural elements. Regardless of functional usage, long artificially-assembled nucleic acids can be referred to herein as synthetic genes and the process of manufacturing these species can be referred to as gene synthesis. Gene synthesis provides an advantageous alternative from obtaining genetic elements through traditional means, such as isolation from a genomic DNA library, isolation from a cDNA library, or PCR cloning. Traditional cloning requires availability of a suitable library ...

Methods and compositions for inhibition of crispr re-cleavage events

The disclosure relates to methods and compositions that serve to reduce the potential for targeted nuclease activity for example, but not limited to, CRISPR enzymes such as Cas9 and/or Cas12a. The disclosure also relates to methods and compositions be used as an HDR substrate to prevent Cas9 re-cleavage, and as a result increase the frequency of perfect HDR.

CRISPR-BASED COMPOSITIONS AND METHODS OF USE

This invention pertains to modified compositions for use in CRISPR systems, and their methods of use. In particular, length-modified and chemically-modified forms of crRNA and tracrRNA are described for use as a reconstituted guide RNA for interaction with Cas9 of CRISPR systems. The resultant length-modified and chemically-modified forms of crRNA and tracrRNA are economical to produce and can be tailored to have unique properties relevant to their biochemical and biological activity in the context of the CRISPR Cas9 endonuclease system. 1. An isolated tracrRNA comprising a length-modified form of SEQ ID NO.:18 , wherein the isolated tracrRNA displays activity in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system.2. The isolated tracrRNA of claim 1 , wherein the length-modified form of SEQ ID NO.:18 consists of a shortened form of SEQ ID NO.:18.3. The isolated tracrRNA of claim 2 , wherein the shortened form of SEQ ID NO.:18 consists of a member selected from a group consisting of the following:SEQ ID NO.:18 lacking from 1 to 20 nucleotides at the 5′-end;SEQ ID NO.:18 lacking from 1-10 nucleotides at the 3′-end; andSEQ ID NO.:18 lacking from 1 to 20 nucleotides at the 5′-end and from 1-10 nucleotides at the 3′-end.4. The isolated tracrRNA of claim 2 , wherein the shortened form of SEQ ID NO.:18 consists of a member selected from a group consisting of SEQ ID NOs.: 2 claim 2 , 30-33 and 36-39.5. The isolated tracrRNA of claim 2 , wherein the shortened form of SEQ ID NO.:18 consists of SEQ ID NO.: 2 or 38.6. The isolated tracrRNA of claim 1 , further comprising at least one chemically-modified nucleotide.723-. (canceled) This application is a divisional of U.S. patent application Ser. No. 14/975,709, filed Dec. 18, 2015, which claims benefit of priority under 35 U.S.C. 119 to U.S. provisional patent applications bearing Ser. Nos. 62/093,588 and 62/239,546, filed Dec. 18, 2014 and Oct. 9, 2015, ...

CRISPR-BASED COMPOSITIONS AND METHODS OF USE

This invention pertains to modified compositions for use in CRISPR systems, and their methods of use. In particular, length-modified and chemically-modified forms of crRNA and tracrRNA are described for use as a reconstituted guide RNA for interaction with Cas9 of CRISPR systems. The resultant length-modified and chemically-modified forms of crRNA and tracrRNA are economical to produce and can be tailored to have unique properties relevant to their biochemical and biological activity in the context of the CRISPR Cas9 endonuclease system. 16-. (canceled)7. An isolated crRNA comprising a length-modified form of formula (I):{'br': None, '5′-X-Z-3′\u2003\u2003(I),'}wherein X represents sequences comprising a target-specific protospacer domain comprising about 20 universal nucleotides, and Z represents sequences comprising a tracrRNA-binding domain comprising about 20 nucleotides,wherein the isolated crRNA displays activity in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system.8. The isolated crRNA of claim 7 , wherein the length-modified form of formula (I) consists of a shortened form of formula (I).9. The isolated crRNA of claim 8 , wherein the shortened form of formula (I) consists of a member selected from a group consisting of the following:formula (I) lacking from 1 to 8 nucleotides at the 3′-end of the Z domain; andformula (I) lacking nucleotides at the 5′-end of the X domain to accommodate a target-specific protospacer domain having 17, 18, 19 or 20 nucleotides.10. The isolated crRNA of claim 7 , further comprising at least one chemically-modified nucleotide.11. The isolated crRNA of claim 10 , wherein the length-modified form of formula (I) consists of SEQ ID NOs.:429-439.1223-. (canceled) This application is a divisional of U.S. patent application Ser. No. 14/975,709, filed Dec. 18, 2015, which claims benefit of priority under 35 U.S.C. 119 to U.S. provisional patent applications ...

CRISPR-BASED COMPOSITIONS AND METHODS OF USE

This invention pertains to modified compositions for use in CRISPR systems, and their methods of use. In particular, length-modified and chemically-modified forms of crRNA and tracrRNA are described for use as a reconstituted guide RNA for interaction with Cas9 of CRISPR systems. The resultant length-modified and chemically-modified forms of crRNA and tracrRNA are economical to produce and can be tailored to have unique properties relevant to their biochemical and biological activity in the context of the CRISPR Cas9 endonuclease system. 117-. (canceled)18. An isolated crRNA comprising a chemically-modified form of formula (I):{'br': None, '5′-X-Z-3′\u2003\u2003(I),'}wherein X represents sequences comprising a target-specific protospacer domain comprising from about 17 nucleotides to about 20 nucleotides and Z represents sequences comprising a tracrRNA-binding domain comprising from about 12 nucleotides about 19 nucleotides,wherein the isolated crRNA displays activity in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system.19. The isolated crRNA of claim 18 , wherein the chemically-modified form of formula (I) comprises a chemically-modified nucleotide having a modification selected from a group consisting of a ribose modification claim 18 , an end-modifying group claim 18 , and an internucleotide modifying linkage.20. The isolated crRNA of claim 19 , wherein the chemically-modified nucleotide having a modification consists of a ribose modification selected from a group consisting of 2′OMe claim 19 , 2′F claim 19 , a bicyclic nucleic acid and locked nucleic acid (LNA).21. The isolated crRNA of claim 19 , wherein the chemically-modified nucleotide having a modification consists of an end-modifying group selected from a group consisting of a propanediol (C3) spacer claim 19 , N claim 19 ,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (“ZEN”) claim 19 , and an inverted-dT residue.22. The ...

Rnase h mutants in an emulsion

The invention is directed to methods and kits for performing an RNase H2-mediated cleavage reaction on a sample in an emulsion.

CLEAVABLE HAIRPIN PRIMERS

The invention describes composition and methods of use for novel hairpin blocked-cleavable primers. In one embodiment unblocking occurs through action of RNase H2. The method improves the specificity of PCR and reduces primer dimer events, enabling higher level multiplex reactions. Additionally, the invention protects RNA-containing primers from attack by single-strand RNases. 1. A blocked cleavable hairpin primer for rhPCR , the primer comprising from 5′ to 3′: (i) a complementary target domain that is complementary to the target sequence; (ii) optionally a discrimination domain; (iii) a cleavable domain that , when hybridized to the target , is cleavable by a cleaving enzyme; (iv) a loop domain; (v) a stem domain capable of reducing non-specific cleavage of the cleaving domain , when hybridized to the cleaving domain , by the cleaving enzyme; and (vi) a blocking domain that prevents extension of the primer.2. The blocked cleavable primer of claim 1 , wherein the cleavable domain is comprised of at least 1 RNA base.3. The blocked cleavable primer of claim 1 , wherein the cleaving enzyme is RNase H.4. The blocked cleavable primer of claim 1 , wherein the stem domain comprises DNA or 2′-O-methyl bases.5. The blocked cleavable primer of claim 4 , wherein the RNA containing stem domain comprises 2-15 2′-O-methyl bases.6. The blocked cleavable primer of claim 1 , wherein the stem domain further comprises a non-nucleotide modification capable of reducing RNase H cleavage of the blocked cleavable hairpin primer when the primer is in a hairpin configuration.7. The blocked cleavable primer of wherein the stem domain is complementary to the discrimination domain.8. The blocked cleavable primer of wherein the cleaving enzyme is RNase H2.9. The blocked cleavable primer of wherein the stem domain further comprises a napthyl-azo compound.10. The blocked cleavable primer of wherein the blocking domain further comprises a non-nucleotide modification.11. The blocked cleavable ...

CONFIGURABLE PROCESS CONTROL DEVICE WITH ELECTRONIC DISPLAY ASSEMBLY

A configurable process control device, which includes a field device, such as a valve position controller, that can be configured by a user to emulate any one of a plurality of different types of process control devices, is provided with an electronic display assembly. The electronic display assembly is operatively connected with a control circuit that is arranged to respond to the specific configuration of the field device to cause the electronic display assembly to display information relevant to the specific type of control device the field device has been configured to emulate. The information may include safety certification information specific to each of the different types of process control devices that the field device can be configured to emulate. 1. A configurable process control device for use in a plurality of use environments , wherein at least one of a pre-defined plurality of certifications must be displayed in conjunction with the configurable process control device depending on which of the use environments the configurable process control device is to be used in , the configurable process control device comprising:a field device;a first control circuit operatively coupled to the field device, the first control circuit arranged to control the field device and arranged to be reconfigured by a user to allow the field device to selectively emulate any of a plurality of different process control device types;an electronic display assembly, the electronic display assembly arranged to selectively display different information; anda second control circuit, the second control circuit operatively coupled to the electronic display assembly and the first control circuit, the second control circuit having access to a plurality of different certification information sets, each certification information set corresponding to a different one of the plurality of different process control device types;wherein the second control circuit causes the electronic display ...

RNase H-Based Assays Utilizing Modified RNA Monomers

The present invention provides methods of cleaving a nucleic acid strand to initiate, assist, monitor or perform biological assays.

PIEZOELECTRIC LOUDSPEAKER

The present invention discloses a piezoelectric loudspeaker. The piezoelectric loudspeaker comprises a sound producing plate, a resonant sound-box, a surround and a reflective sound-box. The sound producing plate comprises a piezoelectric ceramic element. The resonant sound-box includes a first opening comprising a first carrying part. The sound producing plate is disposed on the first carrying part. A cavity resonator is formed between the sound producing plate and the resonant sound-box. The surround is disposed between the first carrying part and the sound producing plate. The reflective sound-box includes a second opening and a reflective output opening. The second opening comprises a second carrying part. The resonant sound-box is disposed on the second carrying part. A reflective cavity body is formed between the resonant sound-box and the reflective sound-box, and the reflective cavity body is connected the reflective output opening. 1. A piezoelectric loudspeaker , comprising:a sound producing plate including at least one piezoelectric ceramic element being able to vibrate the sound producing plate;a resonant sound-box including a first opening comprising a first carrying part, the sound producing plate being disposed on the first carrying part, and a cavity resonator being formed between the sound producing plate and the resonant sound-box;a surround disposed between the first carrying part and the sound producing plate; anda reflective sound-box including a second opening and a reflective output opening, a second carrying part formed on a partial of an inner edge of the seconding opening, the resonant sound-box being disposed on the second carrying part, a reflective cavity body being formed between the resonant sound-box and the reflective sound-box, the reflective output opening being disposed on a side of the reflective sound-box, and the reflective cavity body being communicating with the reflective output opening.2. The piezoelectric loudspeaker of ...

Tm-enhanced blocking oligonucleotides and baits for improved target enrichment and reduced off-target selection

The invention is directed to modified oligonucleotide compositions and methods for selectively reducing unwanted nucleic acid contaminants and enriching for desired nucleic acid targets from complex genomic nucleic acid mixtures for sequencing applications. The modified oligonucleotide compositions include one or more modified groups that increase the T m of the resultant oligonucleotide composition.

PRIMERS AND PROBES FOR DETECTION AND DISCRIMINATION OF EBOLA VIRUS

This invention relates to primers and probes for detecting Ebola virus and one or more subtypes of Ebola virus as well as kits including the probes and primers and methods of using the probes and primers. 1. A method of determining the presence of a viral hemorrhagic fever virus nucleic acid in a sample comprising:a) contacting the sample with at least one probe selected from a group consisting of SEQ ID:21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID:30 capable of hybridizing under stringent conditions to the viral hemorrhagic fever nucleic acid; and detecting hybridization between the viral hemorrhagic fever nucleic acid and the probe, wherein the detection of hybridization indicates the presence of the viral hemorrhagic fever virus nucleic acid in the sample.2. The method of claim 1 , wherein the viral hemorrhagic fever nucleic acid is an Ebola virus nucleic acid.3. The method of claim 2 , wherein the Ebola virus nucleic acid is SEQ ID NO:37.4. The method of claim 1 , wherein the probe is labeled.5. The method of claim 4 , wherein the probe is radiolabeled claim 4 , or fluorescently labeled.6. The method of claim 4 , wherein the probe is labeled with FAM and fluorescent quencher.7. The method of claim 1 , further comprising amplifying the viral hemorrhagic fever nucleic acid by polymerase chain reaction (PCR) claim 1 , reverse transcriptase polymerase PCR (RT-PCR) claim 1 , or real time reverse transcriptase PCR. (rt RT-PCR).8. The method of claim 7 , wherein the viral hemorrhagic fever nucleic acid is amplified by rt RT-PCR.9. The method of claim 8 , wherein the amplifying employs primers that consist of the nucleotide sequences set forth as SEQ ID NO:01 claim 8 , SEQ ID NO:02 claim 8 , SEQ ID NO:03 claim 8 , SEQ ID NO:04 claim 8 , SEQ ID NO:05 claim 8 , SEQ ID NO:06 claim 8 , SEQ ID NO:07 claim 8 , SEQ ID NO:08 claim 8 , SEQ ID NO:09 claim 8 , SEQ ID NO:10 claim 8 , SEQ ID NO:11 ...

LONG NUCLEIC ACID SEQUENCES CONTAINING VARIABLE REGIONS

This invention pertains to improved methods for the synthesis of long, double stranded nucleic acid sequences containing difficult to clone or variable regions. 1. A method of constructing a double stranded DNA fragment or library , said method comprising incorporating sequences between clonal or non-clonal double stranded DNA fragments (gene blocks) , the method comprising:a) forming a mixture comprised of a first gene block, a second gene block, and a bridging oligonucleotide set, said bridging oligonucleotide set comprising one or more bridging oligonucleotides, wherein each bridging oligonucleotide contains a first region that is hybridizable to a portion of the first gene block and a second region that is hybridizable to a portion of the second gene block;b) subjecting the mixture to reagents and conditions for PCR to assemble the gene blocks and bridge(s) thereby generating and optionally amplifying a double stranded DNA fragment or library, wherein the sequence generated is comprised of the first gene block, a bridge sequence of the bridging oligonucleotide(s), if any, that did not hybridize to a gene block, and the second gene block.2. The method of wherein the first gene block is greater than 50 base pairs and the second gene block is greater than 50 base pairs.3. The method of wherein the mixture further comprises one or more additional gene blocks wherein the one or more bridging oligonucleotides contain one or more regions that are hybridizable to a portion of the one or more additional gene blocks.4. The method of wherein the mixture further comprises one or more additional gene blocks and one or more additional bridging oligonucleotides wherein the one or more additional bridging oligonucleotides contains (i) a region hybridizable to an additional gene block claim 1 , and (ii) a region hybridizable to another additional gene block claim 1 , the first gene block or the second gene block.5. The method of wherein the mixture is assembled and amplified ...

MODIFIED RNASE H ENZYMES AND THEIR USES

The invention provides a provides improvements to assays that employ RNase H cleavage for biological applications related to nucleic acid amplification and detection, where the RNase H has been reversibly inactivated.

Item Eligibility and/or Promotion Determination Application

In general, the present invention is directed to systems and methods of utilizing an application operating on a mobile device to determine any applicable offers or eligibility and rank such offers and eligibility for user selection. More specifically, a process may be conducted by a processor in communication with a mobile device and data stores including user data, retailer data, and/or product and service data, the process including comparing identification information associated with a product or service received from the mobile device with data in the data stores; determining the product or service and identifying any associated promotional offers or plan eligibility associated with the product or service, the user, and/or the retailer; ranking any promotional offers or plan eligibility based at least in part on size of promotion; transmitting to the mobile device an identifier of applicable promotional offers or plan eligibility. 1. A system , comprising:a processor; user data, including memberships, groups, plans, and/or entitlements associated with a user;', products and services offered by a retailer;', 'any promotional offers offered by the retailer;, 'retailer data, including, 'product and service data, including identification information of each product and service, and any promotional offers or plan eligibility associated with the product or service;, 'one or more data stores, the one or more data stores storing data comprisingwherein the processor is in selective communication with one or more retailers, manufacturers, membership plans, insurance providers, and/or entitlement providers to populate and update the one or more data stores;wherein the processor is in selective communication with a mobile device of the user; comparing first media data comprising identification information associated with a product or service received from the mobile device with product and service data stored in the one or more data stores;', 'based at least in part on the ...

CRISPR-BASED COMPOSITIONS AND METHODS OF USE

This invention pertains to modified compositions for use in CRISPR systems, and their methods of use. In particular, length-modified and chemically-modified forms of crRNA and tracrRNA are described for use as a reconstituted guide RNA for interaction with Cas9 of CRIPSR systems. The resultant length-modified and chemically-modified forms of crRNA and tracrRNA are economical to produce and can be tailored to have unique properties relevant to their biochemical and biological activity in the context of the CRIPSR Cas9 endonuclease system. 1. An isolated tracrRNA comprising a length-modified form of SEQ ID NO.:18 , wherein the isolated tracrRNA displays activity in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system.2. The isolated tracrRNA of claim 1 , wherein the length-modified form of SEQ ID NO.:18 consists of a shortened form of SEQ ID NO.:18.3. The isolated tracrRNA of claim 2 , wherein the shortened form of SEQ ID NO.:18 consists of a member selected from a group consisting of the following:SEQ ID NO.:18 lacking from 1 to 20 nucleotides at the 5′-end;SEQ ID NO.:18 lacking from 1-10 nucleotides at the 3′-end; andSEQ ID NO.:18 lacking from 1 to 20 nucleotides at the 5′-end and from 1-10 nucleotides at the 3′-end.4. The isolated tracrRNA of claim 2 , wherein the shortened form of SEQ ID NO.:18 consists of a member selected from a group consisting of SEQ ID NOs.: 2 claim 2 , 30-33 and 36-39.5. The isolated tracrRNA of claim 2 , wherein the shortened form of SEQ ID NO.:18 consists of SEQ ID NO.: 2 or 38.6. The isolated tracrRNA of claim 1 , further comprising at least one chemically-modified nucleotide.7. An isolated crRNA comprising a length-modified form of formula (I):{'br': None, '5′-X—Z-3′\u2003\u2003(I),'}wherein X represents sequences comprising a target-specific protospacer domain comprising about 20 universal nucleotides, and Z represents sequences comprising a tracrRNA-binding domain ...

QUICK DISCONNECT CONNECTOR ASSEMBLY

A quick-disconnect connector assembly includes a housing having a bore that extends up to but not through a first end of the housing. The connector assembly also includes a proximity switch disposed within the bore, and the proximity switch includes a switch body, a first contact member, and a second contact member. A portion of each of the first and second contact members extends from the switch body towards a second end of the housing. In a first switch position, a contact of a displaceable switching assembly is in contact with the first contact member, and in a second switch position, the contact is in contact with the second contact member. The connector assembly also includes an external connection assembly including a first pin that is electrically coupled to the first contact member and a second pin that is electrically coupled to the second contact member. 1. A quick-disconnect connector assembly comprising:a housing that extends along a longitudinal axis from a first end to a longitudinally-opposite second end, the housing including one or more interior surfaces that cooperate to define a bore that extends from the second end to a point adjacent to the first end such that the bore does not extend through the first end of the housing, wherein the bore includes a first bore portion; a switch body extending along a body longitudinal axis, the switch body having a first end disposed adjacent to the first end of the housing and a longitudinally-opposite second end; and', 'a first contact member and a second contact member, each of the first and second contact members having a first end and a longitudinally-opposite second end, the second end being disposed within the switch body and the first end being disposed external to the switch body, wherein a portion of each of the first and second contact members extends from the second end of the switch body towards the second end of the housing,', 'wherein in a first switch position, a contact of a displaceable ...

Dna polymerase mutants having enhanced template discrimination activity

This invention relates to mutant DNA polymerases having an enhanced template discrimination activity compared with the corresponding unmodified DNA polymerase counterparts, wherein the amino acid sequence of the mutant DNA polymerase includes at least one substitution at residue positions structurally and functionally homologous or orthologous positions 783 or 784 of an unmodified Taq DNA polymerase.

Cleavable primers for isothermal amplification

The invention provides a more efficient and less error-prone method of performing LAMP. The invention also provides a method for utilizing an RNase H2-cleavable probe as a technique for generating signal from the reaction, potentially increasing the specificity of the signal generation.

Construction of next generation sequencing (ngs) libraries using competitive strand displacement

The invention pertains to construction of next-generation DNA sequencing (NGS) libraries for whole genome sequencing, targeted resequencing, sequencing-based screening assays, metagenomics, or any other application requiring sample preparation for NGS.

Adjustable orthotic foot brace and method for adjusting a foot brace

The orthotic foot brace for a person wearing a footwear generally has: a lower leg holder securable to a lower leg of the person for use, a lower leg strut connected to the lower leg holder and extending downwardly towards the footwear during use; a foot strut structurally connected to the lower leg strut and having at least one side portion, each side portion extending forwardly along a respective side of the footwear and extending outwardly along a respective side of the footwear; an instep strut provided in the form of an extension of the foot strut along a long axis of the footwear, the instep strut having a distal portion securable to the footwear, and at least a proximal portion being slidably connected to the foot strut in a manner to allow adjusting the extension distance of the distal portion to a selected position.

DUPLEX ADAPTERS AND DUPLEX SEQUENCING

This invention pertains to the creation of a complex pool of adapters that contain complementary barcodes to be utilized in next generation sequencing library prep methods and methods of using barcoded adapters for next generation sequencing. 1. A method of sequencing DNA comprising:f) Ligating a partially double stranded unique barcoded adapter to a target double stranded DNA, to form an adapter-target-adapter complex;g) Amplifying each strand of the adapter-target-adapter complex to produce a plurality of amplified first strand adapter-target-adapter complexes and a plurality of amplified second strand adapter-target-adapter complexes;h) independently sequencing the amplified adapter-target adapter complexes to form a plurality of first strand reads and a plurality of second strand reads;i) combining at least one first strand read to at least one second strand read and generating a plurality of consensus sequences; andj) analyzing at least one sequence form the consensus sequence and generating an error corrected sequence read of the first and second sequences to generate a consensus sequence of the target double stranded DNA.2. The method of claim 1 , wherein the partially double stranded unique barcoded adapter is Y-shaped or looped.3. The method of claim 1 , wherein the partially double stranded unique barcoded adapter comprises a unique sequence claim 1 , wherein the unique sequence comprises 2 to 6 nucleotide bases.4. The method of claim 3 , wherein the partially double stranded unique barcoded adapter contains a unique sequence claim 3 , wherein the unique sequence is 2 nucleotide bases.5. The method of claim 1 , wherein the partially double stranded unique barcoded adapters consist of 64 unique adapter molecules.6. The method of claim 1 , wherein the partially double stranded unique barcoded adapters consist of 16 unique barcoded adapter molecules.7. A plurality of duplexed barcoded adapters comprising: SEQ ID NO:1 claim 1 , SEQ ID NO:2 claim 1 , SEQ ID NO: ...

TRACEABLE METALLIC PRODUCTS AND METALLIC SUPPORT FOR NANOSTORAGE

The invention relates to traceable metallic products, methods of uses and methods of making same. The metallic products may be made traceable for integrity purposes, identification purposes, counterfeit avoidance and the like. The invention also relates to metallic supports for nanostorage of various compounds and samples. 1. A traceable metallic product , wherein said metallic product comprises a porous surface layer formed by anodization , and wherein said porous surface layer comprises at least one traceable biological compound.2. (canceled)3. The traceable metallic product of claim 1 , wherein said at least one traceable biological compound is selected from the group consisting of nucleic acids claim 1 , peptidic molecules claim 1 , lipids claim 1 , mono and polysaccharides claim 1 , hormones claim 1 , vitamins claim 1 , and derivatives thereof.4. (canceled)5. The traceable metallic product of claim 1 , wherein said at least one traceable biological compound is carried in nanopores of the porous surface layer claim 1 , and wherein said at least one traceable compound is recoverable from said porous surface layer for detection claim 1 , identification and/or utilization purposes.6. (canceled)7. The traceable metallic product of claim 1 , wherein said traceable metallic product consists of a metallic support for nanostorage of compound and samples.8. The traceable metallic product of claim 1 , wherein said porous surface layer is sealed.9. The traceable metallic product of claim 1 , wherein said porous surface layer further comprises an electrodeposit of at least one metal selected from the group consisting of silver claim 1 , gold claim 1 , copper claim 1 , nickel claim 1 , zinc claim 1 , tin claim 1 , cadmium claim 1 , palladium and platinum.10. The traceable metallic product of claim 1 , wherein said traceable metallic product consists of a traceable piece of aluminum comprising a porous surface layer formed by anodization claim 1 , and wherein said porous ...

SYNTHESIS OF LONG NUCLEIC ACID SEQUENCES

The invention provides methods for the synthesis of long oligonucleotides, genes and gene fragments. The methods include the manufacture of genes or gene fragments that can be then inserted into a variety of vectors. 1. A gene fragment library , said library comprising:{'sub': 'a', 'a plurality of gene fragments wherein two or more of the gene fragments are comprised of a constant gene blockof at least 100 bases and a variable gene block of at least 50 bases, wherein the constant gene block sequence is identical for each of the gene fragments and the variable gene block sequence varies.'}2. The gene fragment library of wherein the gene fragments further comprise constant gene block.3. The gene fragment library of wherein the variable gene block is flanked by constant gene blockand constant gene block.4. The gene fragment library of wherein the gene fragments further comprise constant gene blockwherein n represents a plurality of sets of constant gene blocks.5. The gene fragment library of wherein the gene fragments further comprise variable gene blockwherein n represents a plurality of sets of variable gene blocks6. The gene fragment library of wherein the gene fragments are at least 400 bases.7. The gene fragment library of wherein the gene fragments are at least 1000 bases.8. The gene fragment library of wherein the constant gene blocks or variable gene blocks located on terminal ends contain binding sites for forward and reverse amplification primers.9. The gene fragment library of wherein the constant gene blocks and the variable gene blocks contain overlap regions for assembly into a gene fragment.10. The gene fragment library of wherein the overlap regions contain a same sequence of complementarity that allow for assembly with universal primers. This patent application is a continuation of U.S. Nonprovisional patent application Ser. No. 14/865,127 filed Sep. 25, 2015, which claims priority to U.S. Nonprovisional patent application Ser. No. 13/742,959 filed Jan ...

S. pyogenes cas9 mutant genes and polypeptides encoded by same

This invention pertains to mutant Cas9 nucleic acids and proteins for use in CRISPR/Cas endonuclease systems, and their methods of use. In particular, the invention pertains to an isolated mutant Cas9 protein, wherein the isolated mutant Cas9 protein is active in a CRISPR/Cas endonuclease system, wherein the CRISPR/Cas endonuclease system displays reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system. The invention also includes isolated nucleic acids encoding mutant Cas9 proteins, ribonucleoprotein complexes and CRISPR/Cas endonuclease systems having mutant Cas9 proteins that display reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system.

REVERSE COMPLEMENT ADAPTERS FOR THE MITIGATION OF UMI HOPPING

The invention pertains to construction of next-generation DNA sequencing (NGS) libraries for whole genome sequencing, targeted resequencing, sequencing-based screening assays, metagenomics, or any other application requiring sample preparation for NGS. 1. A method of preparing a target nucleic acid fragment for sequencing , the method comprising:a. ligation of a first adaptor sequence to the 5′ end of the target nucleic acid andb. ligation of a second adaptor sequence to the 3′ end of the target nucleic i. the reverse complement of a P7 adapter sequence,', 'ii. an optional degenerate UMI sequence and', 'iii. an optional first sample index sequence, 'c. whereby the first adaptor sequence comprises'} i. a P5 adapter sequence and', 'ii. an optional second sample index sequence., 'd. whereby the second adapter sequence comprises'}2. The method of wherein the sequences of the first and second sample index are different.3. The method of wherein the sequences of the first and second sample index are the same. This application claims benefit of priority under 35 U.S.C. 119 to U.S. provisional patent application bearing Ser. No. 62/511,133, filed May 25, 2017, and entitled “REVERSE COMPLEMENT ADAPTERS FOR THE MITIGATION OF UMI HOPPING,” the contents of which are herein incorporated by reference in their entirety.The instant application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created May 25, 2018, is named Sequence Listing.txt, and is 2,816 bytes in size.The invention pertains to construction of next-generation DNA sequencing (NGS) libraries for whole genome sequencing, whole exome sequencing, targeted resequencing, sequencing-based screening assays, metagenomics, or any other application requiring sample preparation for NGS.Next Generation Sequencing (NGS) has evolved into a very powerful tool in molecular biology, allowing for the rapid progress in fields such as ...

VALVE SIGNATURE DIAGNOSIS AND LEAK TEST DEVICE

A valve signature diagnosis and leak testing device includes a spool valve operatively connected to a pilot valve, the pilot valve being configured to position the spool valve to one of an open position and a closed position. A blocker valve is fluidly connected to a control fluid outlet of the spool valve. An electrical module is operatively connected to the pilot valve, a supply of control fluid, and the blocker valve, the electrical module being capable of sending pulsed electrical signals to the pilot valve and the blocker valve to selectively position the spool valve and the blocker valve to an open or closed position.

Compositions and Methods for siRNA Inhibition of Angiogenesis

System for cleaning and sanitizing

The present invention provides a system ( 10 ) for cleaning and sanitizing surfaces that incorporates a high pressure fluid supply ( 52 ) and a low pressure fluid supply ( 42 ), often carrying an ozonated fluid, and alternates directed streams of the two fluids for application to contaminated surfaces through a single applicator ( 70, 100 ).

COMPOSITION

Compounds and methods for labeling oligonucleotides

A compound having the general formula shown below: where R 1-6 are independently selected from the group consisting of an electron withdrawing group, an alkyl group, an aryl group, hydrogen, a heteroaryl group, and a five or six member ring structure formed from the R 1 and R 2 pair, the R 3 and R 4 pair, the R 4 and R 5 pair, or the R 5 and R 6 pair; R 7 is a substituted or unsubstituted aryl group; and Y is a nucleophile.

Fluorescence quenching azo dyes, their methods of preparation and use

Disclosed is a group of azo quencher compositions useful as fluorescence quenchers having the general structure of formula 1, methods of making or using the compositions, and kits comprising the composition.

Methods for enhancing nucleic acid hybridization

A composition comprising an oligonucleotide having the structure 5′-Y 1 -L 1 -X-L 2 -Y 2 -3′. Y 1 comprises a sequence of one or more DNA or RNA nucleotides, including a first nucleotide N 1 having a 3′ phosphate covalently linked to L 1 . Y 2 comprises a sequence of one or more DNA or RNA nucleotides, including a second nucleotide N 2 having a 5′ phosphate covalently linked to L 2 . L 1 and L 2 each independently are a direct bond or a C 1 -C 7 alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxyl group. X is R 1 is a hydrogen or a C 1 -C 8 alkyl. M is a label or ligand comprising a fused polycyclic aromatic moiety.

Modifications for antisense compounds

The invention pertains to modifications for antisense oligonucleotides, wherein the modifications are used to improve stability and provide protection from nuclease degradation. The modifications could also be incorporated into double-stranded nucleic acids, such as synthetic siRNAs and miRNAs.

Modifications for antisense compounds

The invention pertains to modifications for antisense oligonucleotides, wherein the modifications are used to improve stability and provide protection from nuclease degradation. The modifications could also be incorporated into double-stranded nucleic acids, such as synthetic siRNAs and miRNAs.

Anthraquinone quencher dyes, their methods of preparation and use

The invention provides novel anthraquinone compositions that are useful as broad-spectrum quenchers of fluorescence and provides methods for making and using them. The anthraquinone quenchers can be conjugated to a variety of biologically relevant compounds, including lipids, nucleic acids, polypeptides, and more specifically antigens, steroids, vitamins, drugs, haptens, metabolites, toxins, environmental pollutants, amino acids, peptides, proteins, nucleotides, oligonucleotides, polynucleotides, carbohydrates, and their analogs. The invention also provides kits comprising, in one or more containers, at least one anthraquinone quencher dye composition of the present invention, and instructions for using that composition.

Fluorescence quenching azo dyes, their methods of preparation and use

Disclosed is a group of azo quencher compositions useful as fluorescence quenchers having the general structure of formula 1, methods of making or using the compositions, and kits comprising the composition.

Methods for enhancing nucleic acid hybridization

A composition comprising an oligonucleotide having the structure 5′-Y 1 -L 1 -X-L 2 -Y 2 -3′. Y 1 comprises a sequence of one or more DNA or RNA nucleotides, including a first nucleotide N 1 having a 3′ phosphate covalently linked to L 1 . Y 2 comprises a sequence of one or more DNA or RNA nucleotides, including a second nucleotide N 2 having a 5′ phosphate covalently linked to L 2 . L 1 and L 2 each independently are a direct bond or a C 1 -C 7 alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxyl group. X is R 1 is a hydrogen or a C 1 -C 8 alkyl. M is a label or ligand comprising a fused polycyclic aromatic moiety.

Compounds and methods for labeling oligonucleotides

A compound having the general formula shown below: where R 1-6 are independently selected from the group consisting of an electron withdrawing group, an alkyl group, an aryl group, hydrogen, a heteroaryl group, and a five or six member ring structure formed from the R 1 and R 2 pair, the R 3 and R 4 pair, the R 4 and R 5 pair, or the R 5 and R 6 pair; R 7 is a substituted or unsubstituted aryl group; and Y is a nucleophile.

Di-alpha amino anthraquinone compositions

The invention provides novel anthraquinone compositions that are useful as broad-spectrum quenchers of fluorescence and provides methods for making and using them. The anthraquinone quenchers can be conjugated to a variety of biologically relevant compounds, including lipids, nucleic acids, polypeptides, and more specifically antigens, steroids, vitamins, drugs, haptens, metabolites, toxins, environmental pollutants, amino acids, peptides, proteins, nucleotides, oligonucleotides, polynucleotides, carbohydrates, and their analogs. The invention also provides kits comprising, in one or more containers, at least one anthraquinone quencher dye composition of the present invention, and instructions for using that composition.

Method for variant detection

本发明可用来提供检测DNA中的变体诸如SNP和插入/缺失的更有效和不太易错的方法。本发明还提供了用于进行便宜的多重测定的方法。

Cysteine engineered antibodies and conjugates withCysteine engineered antibodies and conjugates with a free cysteine amino acid in the heavy chain a free cysteine amino acid in the heavy chain

Disclosed is a cysteine engineered antibody compriDisclosed is a cysteine engineered antibody comprising a free cysteine amino acid having a thiol reasing a free cysteine amino acid having a thiol reactivity value in the range of 0.6 to 1.0; and a sectivity value in the range of 0.6 to 1.0; and a sequence in the heavy chain selected from SEQ ID NOSquence in the heavy chain selected from SEQ ID NOS: 11, 12, 13, and 15: LVTVCSASTKGPS SEQ ID NO:11 : 11, 12, 13, and 15: LVTVCSASTKGPS SEQ ID NO:11 LVTVSCASTKGPS SEQ ID NO:12 LVTVSSCSTKGPS SEQ ID LVTVSCASTKGPS SEQ ID NO:12 LVTVSSCSTKGPS SEQ ID NO:13 HTFPCVLQSSGLYS SEQIDNO:15 where the cysteiNO:13 HTFPCVLQSSGLYS SEQIDNO:15 where the cysteine in SEQ ID NOS: 11, 12, 13, and 15 is the free cne in SEQ ID NOS: 11, 12, 13, and 15 is the free cysteine amino acid. Also disclosed are antibody-drysteine amino acid. Also disclosed are antibody-drug conjugate compounds with the cysteine engineereug conjugate compounds with the cysteine engineered antibody described above and a drug moiety selecd antibody described above and a drug moiety selected from a maytansinoid, an auristatin, a dolastatted from a maytansinoid, an auristatin, a dolastatin, and a calicheamicin. in, and a calicheamicin.

RNase-H-based assays utilizing modified RNA monomers

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3' end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

RNase H-based assays utilizing modified RNA monomers

Methods and compositions are provided for improving specificity during amplification of a target DNA sequence. The methods and compositions rely upon the use of an RNase H enzyme, a polymerase, and RNase H enzyme-sensitive, blocked-cleavable oligonucleotide primers in the amplification reactions, wherein the reaction mixtures include either an optimized final concentration of a divalent metal salt comprising 2.0 mM or less of free Mg++ cation and/or an optimized final concentration of a non-ionic detergent comprising at least about 0.001% polyethylene glycol hexadecyl ether.

DNA polymerase mutants having enhanced template discrimination activity

This invention relates to mutant DNA polymerases having an enhanced template discrimination activity compared with the corresponding unmodified DNA polymerase counterparts, wherein the amino acid sequence of the mutant DNA polymerase includes at least one substitution at residue positions structurally and functionally homologous or orthologous positions 783 or 784 of an unmodified Taq DNA polymerase.

Rnase h2 mutants that reduce primer dimers and off-target amplification in rhpcr-based amplicon sequencing with high-fidelity dna polymerases

The present invention pertains to hybrid RNase H2 proteins that include fragments of amino acid sequences from Pyrococcus abyssi ( P.a.), Thermococcus kodakarensis (T.kod), and Pyrococcus furiosus organisms, as well as methods of using the same to improve mismatch discrimination and activity in a high-fidelity DNA polymerase buffer.

RNase-H-based assays utilizing modified RNA monomers

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3' end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

RNASE-H-based assays utilizing modified RNA monomers

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3' end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

Rnase-h-based assays utilizing modified rna monomers

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3' end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

RNase-H-based assays utilizing modified RNA monomers

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3' end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

RNase H-based assays utilizing modified RNA monomers

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3′ end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

Solenoid actuated butterfly valve

A butterfly valve (100) and a valve system using the butterfly valve are disclosed. The butterfly valve includes a valve body (102) and a valve disk (112) disposed in the body such that the valve disk may be rotated between at least a first position and a second position using a linear actuator. The actuator may be a linear solenoid coupled to the valve disk spaced a distance from the axis of rotation of the valve disk.

Chemical modification of small hairpin rnas for inhibition of gene expression

Aspects of the present invention include the production and use of chemically modified RNAi agents (e.g., shRNAs) in gene silencing applications. The chemically modified RNAi agents disclosed herein have reduced immunostimulatory activity, increased serum stability, or both, as compared to a corresponding RNAi agent not having the chemical modification. Compositions containing chemically modified RNAi agents according to aspects of the present invention (including pharmaceutical compositions) and kits containing the same are also provided.

Multivalent antibodies and uses therefor. Multivalent antibodies and uses thereof.

Cleavable hairpin primers

The invention describes composition and methods of use for novel hairpin blocked-cleavable primers. In one embodiment unblocking occurs through action of RNase H2. The method improves the specificity of PCR and reduces primer dimer events, enabling higher level multiplex reactions. Additionally, the invention protects RNA-containing primers from attack by single-strand RNases.

Energy activated printing process

给出了反应性油墨和在基材上使用反应性且热激活的油墨产生图像的方法。在基材上印刷图像，而不使该油墨中的反应试剂反应。随后，使该反应试剂反应而将图像显著持久且牢固地固定到基材上。还将印刷的升华或类似热激活的染料印刷在该基材上。该升华或类似热激活的染料被激活，并且对施加到基材上的聚合物具有亲合性。

Method and apparatus for making insulating translucent panel assemblies

A method of making an insulating translucent panel assembly (10) such as a window or door lite is provided. In the method, a spacer (16) between two translucent panels (12) of glass or plastic includes a thermo- responsive sealing material (22). When heat and compression are applied to the spacer (16), the thermo-responsive sealing material (22) softens and fully seals the spacer (16) to the two translucent panels (12). A spacer (16) designed to be used in this method is also provided.

Methods and compositions for the specific inhibition of gene expression by double-stranded rna

Methods and compositions for the specific inhibition of gene expression by double-stranded RNA

The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. The dsRNA has a first oligonucleotide sequence that is between 25 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of at least 19 nucleotides is sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger the destruction of the target RNA by the RNAi machinery.

Patent DE4028660C2

Liquid flourescent dye concentrate for flow cytometry evaluation of virus-size particles and related products and methods

A kit, and method for flow cytometry include a liquid dye concentrate for fluorescent staining of virus-size particles with a plurality of fluorogenic dyes in a liquid medium. The liquid dye concentrate includes a plurality of fluorogenic dyes and one or both of (i) the liquid medium comprising a liquid mixture including water and liquid phase organic material and (ii) disaccharide dissolved in the liquid medium.

Compounds and methods for labeling oligonucleotides

The invention provides a novel method of labeling oligonucleotides, with reporter moieties, including but not limited to, quenchers, fluorophores, biotin, digoxigenin, peptides and proteins. In addition, this invention provides a method of detecting hybridization of oligonucleotides. This invention also provides novel azo quenchers having the general formula shown below. -Ry R4" R6 The invention further provides compositions comprising labeled oligonucleotides and solid supports. The invention also provides kits comprising at least one composition of the present invention.

Microarray system with improved sequence specificity

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 printing buffers/solutions for the immobilization of oligonucleotide probes to an array surface. The invention also provides hybridization and wash buffers and conditions to maximize hybridization specificity and signal intensity, and reduce hybridization times.

Oligonucleotides containing high concentrations of guanine monomers

This invention pertains to methods for oligonucleotide synthesis, specifically the synthesis of oligonucleotides that contain a high content of guanine monomers. In more detail, the invention relates to a method for coupling a nucleoside phosphoramidite during the synthesis of an oligonucleotide to a universal support, to a first nucleoside, or to an extending oligonucleotide. The invention further relates to oligonucleotides obtainable by the methods of the invention.

S. pyogenes cas9 mutant genes and polypeptides encoded by same

Compounds and methods for labeling oligonucleotides

Long nuceic acid sequences containing variable regions

This invention pertains to improved methods for the synthesis of long, double stranded nucleic acid sequences containing difficult to clone or variable regions.

Rnase h-based assays utilizing modified rna monomers

Methods and compositions are provided for improving specificity during amplification of a target DNA sequence. The methods and compositions rely upon the use of an RNase H enzyme, a polymerase, and RNase H enzyme-sensitive, blocked-cleavable oligonucleotide primers in the amplification reactions, wherein the reaction mixtures include either an optimized final concentration of a divalent metal salt comprising 2.0 mM or less of free Mg++ cation and/or an optimized final concentration of a non-ionic detergent comprising at least about 0.001% polyethylene glycol hexadecyl ether.

Method of constructing and executing a process

Disclosed is a method of constructing and executing a process. A conventional process is minutely divided into minimum unit subprocesses, and the minutely divided subprocesses are classified into a decision subprocesses and a routine subprocess by whether they require decision-making. Any subprocess which is executable using the setup condition in a specific decision subprocess is classified into the routine subprocess in such a manner that the classified routine subprocess follows on the specific decision subprocess. One or a series of decision subprocesses are combined with one or a series of routine subprocesses which are executable on the condition of the completion of the decision subprocesses to form one unit process, and a job-support computer program is created to allow the plurality of subprocesses included in the one unit process to be successively executed. A plurality of subprocesses which are executable in accordance with common input data are detected from the minutely divided minimum unit subprocesses, and a job flow is constructed to allow the respective jobs in the plurality of subprocesses to be simultaneously initiated and executed in parallel. The present invention can drastically reduce the lead-time of a process while facilitating execution of the entire process with high efficiency.

Enclosed proximity switch assembly

An enclosed proximity switch assembly includes a top enclosure and a bottom enclosure that are coupled to form an interior volume. A shaft protrusion upwardly extends from a top surface of the top enclosure, and an interior bore portion having an enclosed volume is defined within the shaft protrusion to form a portion of the interior volume. A first end of a vertical shaft is rotatably disposed within the interior bore portion such that the shaft rotates relative to the top and bottom enclosures. A samarium cobalt target magnet is coupled to the shaft, and the target magnet interacts with a samarium cobalt driver magnet within a proximity switch when the target magnet is rotated within a predetermined distance of a top portion of the proximity switch. The interaction causes a switch to move from a first state to a second state, or vice versa.

RNase-H-Based Assays Utilizing Modified RNA Monomers

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid 5 domain and a blocking group at or near the 3' end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

Component of printed circuit boards

A component (10) for use in manufacturing articles, such as printed circuit boards, comprising a laminate constructed of a sheet of copper foil (20) that, in a finished circuit board, constitutes a functional element, and a sheet of carbon steel (14) having a layer of an inert metal (16) thereon, the sheet of carbon steel (14) constituting a discardable element. One surface (22) of the copper sheet (20) and the surface (18) of the inert metal layer (16) on the carbon steel sheet (14) are essentially uncontaminated and engageable with each other at interfaces. The copper sheet (20) is attached to the inner metal layer (16) of the carbon steel (14) at their borders and defines substantially uncontaminated central zones inwardly of the edges of the sheets that are unjoined at the interfaces.

Data generating device, data generating method and data generating program

Methods and compositions for the specific inhibition of gene expression by double-stranded rna

The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. The dsRNA has a first oligonucleotide sequence that is between 25 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of at least 19 nucleotides is sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger the destruction of the target RNA by the RNAi machinery.

Siloxy oligonucleotide analogs

CRISPR-based compositions and methods of use

This invention pertains to modified compositions for use in CRISPR systems, and their methods of use. In particular, length-modified and chemically-modified forms of crRNA and tracrRNA are described for use as a reconstituted guide RNA for interaction with Cas9 of CRIPSR systems. The resultant length-modified and chemically-modified forms of crRNA and tracrRNA are economical to produce and can be tailored to have unique properties relevant to their biochemical and biological activity in the context of the CRIPSR Cas9 endonuclease system.

Tm-enhanced blocking oligonucleotides and baits for improved target enrichment and reduced off-target selection

The invention is directed to modified oligonucleotide compositions and methods for selectively reducing unwanted nucleic acid contaminants and enriching for desired nucleic acid targets from complex genomic nucleic acid mixtures for sequencing applications. The modified oligonucleotide compositions include one or more modified groups that increase the Tm of the resultant oligonucleotide composition.

Methods for ligation-coupled-PCR

The present disclosure provides methods and kits for ligation-coupled PCR. Methods for performing ligation and PCR and, optionally, enzymatic digestion in a single closed tube are provided. Methods and kits for splint-mediated primer assembly and ligation-coupled PCR are also provided.

Systems and methods for providing, reloading, and redeeming stored value cards used in transit applications

The invention is directed to systems and methods of conducting transactions associated with a transit card. A method of conducting transactions may be conducted between a processor and a transit processor. Steps may include receiving a redemption request and determining if it is a pre-authorization request or a redemption; if the redemption request is a pre-authorization request, determining if the account is authorized for a particular transit type. If the account is authorized, communicating approval to the transit processor. If the account is not authorized communicating denial. If the redemption request is a redemption: determining if account value is sufficient to pay the redemption amount; if not, denying and if so approving the transaction and deducting the amount, and determining if the account value is below a pre-determined threshold for a particular transit types, and if so, updating the status of the account at a data store.

Efficient and rapid method for assembling and cloning double-stranded DNA fragments

The invention is directed to an in vitro method for joining a first set of double-stranded (ds) DNA molecules. Small molecules acting as chaperone agents are identified that promote efficient and rapid assembly (that is, joining) of overlapping double-stranded DNA fragments.

Methods for amplifying polymeric nucleic acids

The invention provides compositions and methods for amplifying nucleic acid polymer sequences in a high complexity nucleic acid sample. The unique compositions of the invention include a primer set composed of a mixture of two types of primers for DNA synthesis. For extension in one direction, the primers all contain modifications that destroy their ability to serve as templates that can be copied by DNA polymerases. For extension in the opposite direction the set includes at least one primer that can serve as a template and be replicated by DNA polymerases throughout its length. The method can be carried out by mixing the nucleic acid polymer sequence of interest with the set of DNA synthesis primers in an amplification reaction mixture. The reaction mixture is then subjected to temperature cycling analogous to the temperature cycling in PCR reactions. At least one primer in the primer set hybridizes to the nucleic acid polymer. It is preferred that the non-replicable primer hybridizes to the nucleic acid polymer and is extended to produce an extension product that contains sequence from the nucleic acid polymer to which the replicable primer then hybridizes. Of course, if the nucleic acid polymer is double stranded, both the replicable and nonreplicable primers will hybridize and be extended by DNA polymerase.

Crispr-based compositions and methods of use

This invention pertains to modified compositions for use in CRISPR systems, and their methods of use. In particular, length-modified and chemically-modified forms of crRNA are described for use as a reconstituted guide RNA for interaction with Cas9 of CRISPR systems. The resultant length-modified and chemically-modified forms of crRNA are economical to produce and can be tailored to have unique properties relevant to their biochemical and biological activity in the context of the CRISPR Cas9 endonuclease system.

RNase-H-Based Assays Utilizing Modified RNA Monomers

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid 5 domain and a blocking group at or near the 3' end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

Fingerprint analysis for a plurality of oligonucleotides

Methods and compositions for the specific inhibition of gene expression by double-stranded rna

The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. The dsRNA has a first oligonucleotide sequence that is between 25 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of at least 19 nucleotides is sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger the destruction of the target RNA by the RNAi machinery.

Dual/variable gain oil pump control valve

A solenoid fluid control valve is disclosed for controlling a variable displacement pump. The solenoid fluid control valve comprises a fixed solenoid component (702), a movable armature component (710), a fixed nozzle body (722), a movable spool (720) within the fixed nozzle body, and a valve member (714). The valve member regulates fluid pressure in a first (746, 748) and second feedback chamber. Fluid in the second feedback chamber establishes a second feedback pressure that acts on the movable spool with a motive feedback force in a first axial direction. The movable spool moves in the first axial direction in response to the motive feedback force.

Primer extension methods for production of high specific activity nucleic acid probes