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Patent Issued for Separation of Pyrophosphate Release and Pyrophosphate Detection

July 14, 2014



By a News Reporter-Staff News Editor at Biotech Business Week -- A patent by the inventors Hirschbein, Bernard (San Francisco, CA); Gorpe-Yasar, Filiz (Redwood City, CA), filed on April 29, 2013, was published online on July 1, 2014, according to news reporting originating from Alexandria, Virginia, by NewsRx correspondents (see also Biotechnology Companies).

Patent number 8765419 is assigned to Illumina, Inc. (San Diego, CA).

The following quote was obtained by the news editors from the background information supplied by the inventors: "The detection of specific nucleic acid sequences present in a biological sample has a wide variety of applications, such as identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to disease, and measuring response to various types of treatment. A valuable technique for detecting specific nucleic acid sequences in a biological sample is nucleic acid sequencing.

"Nucleic acid sequencing methodology has evolved significantly from the chemical degradation methods used by Maxam and Gilbert and the strand elongation methods used by Sanger. Today one of the sequencing methodologies in use is pyrosequencing, which is based on the concept of sequencing-by-synthesis. The technique can be applied to massively parallel sequencing projects. For example, using an automated platform, it is possible to carry out hundreds of thousands of sequencing reactions simultaneously. Sequencing-by-synthesis differs from the classic dideoxy sequencing approach in that, instead of generating a large number of sequences and then characterizing them at a later step, real time monitoring of the incorporation of each base into a growing chain is employed. Although this approach is slow in the context of an individual sequencing reaction, it can be used for generating large amounts of sequence information in each cycle when hundreds of thousands to millions of reactions are performed in parallel. Despite these advantages, there are still limitations in the pyrosequencing approach."

In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventors' summary information for this patent: "The present technology relates to methods and systems for detection of pyrophosphate, which can either be used alone or in connection with other technologies, such as pyrosequencing. In some embodiments of the present invention, such methods and systems permit the detection of pyrophosphate with reduced background. In some embodiments, methods and systems are described for pyrosequencing of nucleic acids with reduced background.

"In some embodiments of the technology described herein, methods of delaying pyrophosphate detection are provided. The methods can include the steps of providing a pyrophosphate sequestering agent, generating pyrophosphate in the presence of the sequestering agent, whereby the pyrophosphate is reversibly sequestered, releasing the pyrophosphate from the sequestering agent and detecting the pyrophosphate.

"In certain aspects, the pyrophosphate is reversibly sequestered by adsorption with the sequestering agent. In certain aspects, the sequestering agent comprises a cationic agent capable of sequestering pyrophosphate through chelation, complexation, or adsorption. In certain aspects, the cationic agent comprises an agent selected from the group consisting of a metal, metal salt, a metal oxide or other agent as exemplified herein. In certain aspects, the metal or metal oxide comprises Ti or TiO.sub.2. In other aspects, the pyrophosphate sequestering agent comprises hydroxyapatite. In other aspects the sequestering agent comprises an ammonium or substituted ammonium salt, or a resin or bead that contains such groups. In certain aspects of the above embodiments, the pyrophosphate sequestering agent comprises particles or beads.

"In certain aspects of the methods described herein, the step of releasing the pyrophosphate from the sequestering agent comprises providing a release reagent to the sequestering agent. In some aspects, the release reagent comprises an anion capable of displacing the pyrophosphate from the sequestering agent, for example, by preferentially complexing or chelating the cation of the sequestering agent. In certain aspects, the release reagent comprises an agent selected from the group consisting of an acid or salt of an acid such as oxalic acid, an oxalate salt, sulfamic acid, a sulfamate salt, ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis-.beta.-amino-ethyl ether N,N, N',N'-tetra-acetic acid (EGTA) citric acid, tartaric acid, acetic or other carboxylic acids or their salts. In other aspects, the release reagent comprises phosphate. In other aspects the release reagent comprises a bisphosphonate. In certain aspects the release reagent is the enzyme ATP sulfurylase. In this particular aspect the ATP sulfurylase is in solution rather than being bound to a bead or other surface. The ATP sulfurylase can release the pyrophosphate from the sequestering agent by transforming the pyrophosphate into ATP in the presence of adenysine phosphosulfate (APS). Typically, the ATP will have a lower binding affinity for the sequestering agent than does pyrophosphate.

"In some methods described herein, the step of detecting the pyrophosphate comprises providing a pyrophosphate detecting agent. In certain aspects, the pyrophosphate detecting agent comprises luciferase. In certain aspects, the pyrophosphate detecting agent further comprises ATP sulfurylase. In certain aspects, the luciferase is attached to a bead. In certain aspects, the ATP sulfurylase is attached to a bead. In certain aspects, the ATP sulfurylase and the luciferase are both attached to the same bead.

"In certain aspects of the above-described methods, the step of generating pyrophosphate in the presence of the sequestering agent comprises incorporating a nucleotide or nucleotide analog into a polynucleotide. In certain aspects, the polynucleotide is attached to a surface. In some aspects, the surface comprises a bead or a well. In certain aspects, the sequestering agent is disposed between the pyrophosphate detecting agent and the polynucleotide. Polynucleotides can comprise any type of nucleic acid but most often comprise DNA. In certain aspects, the DNA comprises genomic DNA and/or cDNA. Such genomic and/or cDNAs can include copies of genomic DNA sequences and/or cDNA sequences. Alternatively, the genomic DNA and/or cDNA can include fragments or copies of fragments of genomic DNA and/or cDNA sequences.

"In certain aspects, the methods further comprise the step of washing the sequestering agent. In certain aspects, washing comprises adding a washing enzyme to the sequestering agent. In certain aspects, the washing enzyme is pyrophosphatase, apyrase, alkaline phosphatase and/or ATP sulfurylase. In certain aspects, the methods further comprise adding a washing enzyme inhibitor to the sequestering agent. In certain aspects, the washing enzyme inhibitor is sodium azide. In certain aspects, the washing step comprises electric-field assisted removal of pyrophosphate from the sequestering agent.

"Also provided herein are methods for sequencing a nucleic acid. The method can include the steps of providing nucleotides or nucleotide analogs in the presence of a pyrophosphate sequestering agent and a pyrophosphate detecting agent, incorporating one or more of the nucleotides or nucleotide analogs into a polynucleotide so as to extend the polynucleotide in the presence of the pyrophosphate sequestering agent, thereby generating sequestered pyrophosphate, removing the unincorporated nucleotides or nucleotide analogs from the presence of the pyrophosphate detecting agent, releasing the pyrophosphate from the sequestering agent in the presence of the pyrophosphate detecting agent and detecting released pyrophosphate, wherein released pyrophosphate indicates that one or more nucleotides or nucleotide analogs have been incorporated into the polynucleotide.

"In certain aspects, the pyrophosphate is reversibly sequestered by adsorption with the sequestering agent. In certain aspects, the sequestering agent comprises a cationic agent capable of sequestering pyrophosphate through chelation, complexation, or adsorption. In certain aspects, the cationic agent comprises an agent selected from the group consisting of a metal, metal salt, a metal oxide or other agent set forth below. In certain aspects, the metal or metal oxide comprises Ti or TiO.sub.2. In other aspects, the pyrophosphate sequestering agent comprises hydroxyapatite. In other aspects the sequestering agent comprises an ammonium or substituted ammonium salt, or a resin or bead that contains such groups. In certain aspects of the above embodiments, the pyrophosphate sequestering agent comprises particles or beads.

"In certain aspects, the step of releasing the pyrophosphate from the sequestering agent comprises providing a release reagent to the sequestering agent. In certain aspects, the release reagent comprises an anion capable of displacing the pyrophosphate from the sequestering agent, for example, by preferentially complexing or chelating the cation of the sequestering agent. In certain aspects, the release reagent comprises an agent selected from the group consisting of an acid or salt of an acid such as oxalic acid, an oxalate salt, sulfamic acid, a sulfamate salt, ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis-.beta.-amino-ethyl ether N,N,N',N'-tetra-acetic acid (EGTA) citric acid, tartaric acid, acetic or other carboxylic acids or their salts. In other aspects the release reagent comprises phosphate. In other aspects the release reagent comprises a bisphosphonate. In certain aspects the release reagent is the enzyme ATP sulfurylase. In this particular aspect, the ATP sulfurylase is in solution rather than being bound to a bead or other surface. The ATP sulfurylase can release the pyrophosphate from the sequestering agent by transforming the pyrophosphate into ATP in the presence of adenysine phosphosulfate (APS). Typically, the ATP will have a lower binding affinity for the sequestering agent than does pyrophosphate.

"In some of the sequencing methods described herein, the pyrophosphate detecting agent comprises luciferase. In certain aspects, the pyrophosphate detecting agent further comprises ATP sulfurylase. In certain aspects, the luciferase is attached to a bead. In certain aspects, the ATP sulfurylase is attached to a bead. In certain aspects, the ATP sulfurylase and the luciferase are both attached to the same bead.

"In other aspects of the above-described methods, the polynucleotide is attached to a surface. In some aspects, the surface comprises a bead or a well. In certain aspects, the sequestering agent is disposed between the pyrophosphate detecting agent and the polynucleotide. Polynucleotides can comprise any type of nucleic acid but most often comprise DNA. In certain aspects, the DNA comprises genomic DNA and/or cDNA. Such genomic and/or cDNAs can include copies of genomic DNA sequences and/or cDNA sequences. Alternatively, the genomic DNA and/or cDNA can include fragments or copies of fragments of genomic DNA and/or cDNA sequences.

"In some aspects, the sequencing methods further comprise the step of washing the sequestering agent. In some aspects, washing comprises adding a washing enzyme to the sequestering agent. In certain aspects, the washing enzyme is pyrophosphatase apyrase, alkaline phosphatase and/or ATP sulfurylase. In certain aspects, the methods further comprise adding a washing enzyme inhibitor to the sequestering agent. In some aspects, the washing enzyme inhibitor is sodium azide. In some aspects, the washing step comprises electric-field assisted removal of pyrophosphate from the sequestering agent.

"In further aspects of the methods described herein, the removing step comprises washing the pyrophosphate detecting agent. In certain aspects, washing comprises adding a washing enzyme to the pyrophosphate detecting agent. In some aspects, the washing enzyme is a nucleotide degrading enzyme such as alkaline phosphatase or apyrase. In certain aspects, washing comprises electric-field assisted removal of pyrophosphate from the detecting agent.

"Also provided herein are methods of modulating the availability of free pyrophosphate during sequencing of a nucleic acid molecule. These methods can include the steps of combining nucleotides or nucleotide analogs with a nucleic acid template; incubating the nucleic acid template and the nucleotides or nucleotide analogs together with a polymerase and a pyrophosphate sequestering agent under conditions sufficient to form a polynucleotide complementary to all or a portion of the nucleic acid template, wherein pyrophosphate generated during the incubating is reversibly sequestered by the sequestering agent, removing from the nucleic acid template the nucleotides or nucleotide analogs that have not been incorporated into the polynucleotide and releasing the pyrophosphate from the pyrophosphate sequestering agent by providing a release reagent.

"In certain aspects, the pyrophosphate is reversibly sequestered by adsorption with the sequestering agent. In certain aspects, the sequestering agent comprises a cationic agent capable of sequestering pyrophosphate through chelation, complexation, or adsorption. In certain aspects, the cationic agent comprises an agent selected from the group consisting of a metal, metal salt, a metal oxide or other agent set forth below. In certain aspects, the metal or metal oxide comprises Ti or TiO.sub.2. In other aspects, the pyrophosphate sequestering agent comprises hydroxyapatite. In other aspects the sequestering agent comprises an ammonium or substituted ammonium salt, or a resin or bead that contains such groups. In certain aspects of the above embodiments, the pyrophosphate sequestering agent comprises particles or beads.

"In certain aspects, the step of releasing the pyrophosphate from the sequestering agent comprises providing a release reagent to the sequestering agent. In certain aspects, the release reagent comprises an anion capable of displacing the pyrophosphate from the sequestering agent, for example, by preferentially complexing or chelating the cation of the sequestering agent. In certain aspects, the release reagent comprises an agent selected from the group consisting of an acid or salt of an acid such as oxalic acid, an oxalate salt, sulfamic acid, a sulfamate salt, ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis-.beta.-amino-ethyl ether N,N,N',N'-tetra-acetic acid (EGTA) citric acid, tartaric acid, acetic or other carboxylic acids or their salts. In other aspects, the release reagent comprises phosphate. In other aspects, the release reagent comprises a bisphosphonate. In certain aspects, the release reagent is the enzyme ATP sulfurylase. In this particular aspect, the ATP sulfurylase is in solution rather than being bound to a bead or other surface. The ATP sulfurylase can release the pyrophosphate from the sequestering agent by transforming the pyrophosphate into ATP in the presence of adenysine phosphosulfate (APS). Typically, the ATP will have a lower binding affinity for the sequestering agent than does pyrophosphate.

"In some of the above-described methods, the pyrophosphate detecting agent comprises luciferase. In certain aspects, the pyrophosphate detecting agent further comprises ATP sulfurylase. In certain aspects, the luciferase is attached to a bead. In certain aspects, the ATP sulfurylase is attached to a bead. In certain aspects, the ATP sulfurylase and the luciferase are both attached to the same bead.

"In other aspects of the above-described methods, the polynucleotide is attached to a surface. In some aspects, the surface comprises a bead or a well. In certain aspects, the sequestering agent is disposed between the pyrophosphate detecting agent and the polynucleotide. Polynucleotides can comprise any type of nucleic acid but most often comprise DNA. In certain aspects, the DNA comprises genomic DNA and/or cDNA. Such genomic and/or cDNAs can include copies of genomic DNA sequences and/or cDNA sequences. Alternatively, the genomic DNA and/or cDNA can include fragments or copies of fragments of genomic DNA and/or cDNA sequences.

"In some aspects, the above-described methods further comprise the step of washing the sequestering agent. In some aspects, washing comprises adding a washing enzyme to the sequestering agent. In certain aspects, the washing enzyme is pyrophosphatase apyrase, alkaline phosphatase and/or ATP sulfurylase. In certain aspects, the methods further comprise adding a washing enzyme inhibitor to the sequestering agent. In some aspects, the washing enzyme inhibitor is sodium azide. In some aspects, the washing step comprises electric-field assisted removal of pyrophosphate from the sequestering agent.

"In further aspects of the methods described herein, the removing step comprises washing the pyrophosphate detecting agent. In certain aspects, washing comprises adding a washing enzyme to the pyrophosphate detecting agent. In some aspects, the washing enzyme is a nucleotide degrading enzyme such as alkaline phosphatase or apyrase. In certain aspects, washing comprises electric-field assisted removal of pyrophosphate from the detecting agent.

"Certain embodiments of the methods described above and elsewhere herein relate to sequestering pyrophosphate with a sequestering agent that involves a relatively weak interaction. In such embodiments, the unincorporated nucleotide triphosphates can be washed from the detection volume while the removal of pyrophosphate from the detection volume is retarded by the interaction of the pyrophosphate with the sequestering agent. In this regard, the method can function to permit separation of the nucleotide triphosphates from the pyrophosphate by permitting the nucleotide triphosphates to be eluted out of the detection volume before the pyrophosphate. In some aspects, the sequestering agent can function in ion-exchange, metal chelation or both. In such aspects, the release reagent can include, but is not limited to, substances that effect solvent composition, pH, concentration of ionic species and the like.

"Also provided herein are arrays comprising a solid support having a plurality of sites distributed thereon, wherein at least a portion of the sites comprise a template nucleic acid and a pyrophosphate sequestering agent capable of reversibly sequestering pyrophosphate.

"In certain aspects, the sites comprise wells. In certain aspects, the template nucleic acid is attached to a particle or bead within the wells.

"In certain aspects, the wells further comprise beads having a pyrophosphate detecting agent attached thereto. In certain aspects, the pyrophosphate sequestering agent is disposed between the template nucleic acid and the pyrophosphate detecting agent. In certain aspects, the pyrophosphate detecting agent comprises ATP sulfurylase and luciferase. In certain aspects, the wells further comprise packing beads.

"With respect to some embodiments of the arrays described herein, pyrophosphate is reversibly sequestered by adsorption with the sequestering agent. In certain aspects, the sequestering agent comprises a cationic agent capable of sequestering pyrophosphate through chelation, complexation, or adsorption. In certain aspects, the cationic agent comprises an agent selected from the group consisting of a metal, metal salt, a metal oxide or other agent set forth below. In certain aspects, the metal or metal oxide comprises Ti or TiO.sub.2. In other aspects, the pyrophosphate sequestering agent comprises hydroxyapatite. In other aspects the sequestering agent comprises an ammonium or substituted ammonium salt, or a resin or bead that contains such groups. In certain aspects of the above embodiments, the pyrophosphate sequestering agent comprises particles or beads.

"In addition to the foregoing, in some embodiments of the arrays described herein, pyrophosphate can be released from the sequestering agent by providing a release reagent to the sequestering agent. In certain aspects, the release reagent comprises an anion capable of displacing the pyrophosphate from the sequestering agent, for example, by preferentially complexing or chelating the cation of the sequestering agent. In certain aspects, the release reagent comprises an agent selected from the group consisting of an acid or salt of an acid such as oxalic acid, an oxalate salt, sulfamic acid, a sulfamate salt, ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis-.beta.-amino-ethyl ether N,N,N',N'-tetra-acetic acid (EGTA) citric acid, tartaric acid, acetic or other carboxylic acids or their salts. In other aspects the release reagent comprises phosphate. In other aspects the release reagent comprises a bisphosphonate. In certain aspects the release reagent is the enzyme ATP sulfurylase. In this particular aspect the ATP sulfurylase is in solution rather than being bound to a bead or other surface. The ATP sulfurylase can release the pyrophosphate from the sequestering agent by transforming the pyrophosphate into ATP in the presence of adenysine phosphosulfate (APS). Typically, the ATP will have a lower binding affinity for the sequestering agent than does pyrophosphate.

"In some aspects, arrays described herein can include sites that further comprise a polymerase and nucleotides or nucleotide analogs.

"In certain aspects, arrays described herein can further comprises at least one electrode capable of producing an electric field in the presence of the sites.

"Also provided herein are methods of making an array. The methods can include the steps of providing a solid support having a plurality of sites distributed thereon and providing a template nucleic acid and a pyrophosphate sequestering agent capable of reversibly sequestering pyrophosphate to at least a portion of the sites.

"In certain aspects, the step of providing the template nucleic acid to the plurality of sites occurs prior to providing the pyrophosphate sequestering agent. In certain aspects, the step of providing the template nucleic acid to the plurality of sites occurs subsequent to providing the pyrophosphate sequestering agent. In certain aspects, the step of providing the template nucleic acid to the plurality of sites occurs at the same time as providing the pyrophosphate sequestering agent.

"In certain aspects, the sites comprise wells. In certain aspects, a reagent useful in the methods is attached to a bead. For example, a template nucleic acid, pyrophosphate sequestering agent or pyrophosphate detecting agent can be attached to a particle or bead. In certain aspects the beads are provided to wells by packing, for example, via centrifugation.

"In certain aspects, the methods further comprise the step of providing to the wells beads having a pyrophosphate detecting agent attached thereto. In certain aspects, the pyrophosphate detecting agent comprises ATP sulfurylase and luciferase. In certain aspects, the step of providing said beads having a pyrophosphate detecting agent to the wells occurs prior to providing the pyrophosphate sequestering agent. In certain aspects, the step of providing said beads having a pyrophosphate detecting agent to the wells occurs subsequent to providing the pyrophosphate sequestering agent. In certain aspects, the step of providing said beads having a pyrophosphate detecting agent to the wells occurs at the same time as providing the pyrophosphate sequestering agent.

"In certain aspects, the methods further comprise the step of providing packing beads to the wells. In certain aspects, the beads having a pyrophosphate detecting agent attached thereto and the packing beads are provided to the wells at the same time.

"In certain aspects, the methods further comprise the step of providing a polymerase and nucleotides or nucleotide analogs to the sites.

"In certain aspects of the above-described methods of making an array, the step of providing comprises applying a voltage to the plurality of sites.

"Arrays manufactured according to the methods described herein can be employed in the sequencing and/or pyrophosphate sequestering and release processes described above and elsewhere herein. For example, in some embodiments, the pyrophosphate is reversibly sequestered by adsorption with the sequestering agent. In certain aspects, the sequestering agent comprises a cationic agent capable of sequestering pyrophosphate through chelation, complexation, or adsorption. In certain aspects, the cationic agent comprises an agent selected from the group consisting of a metal, metal salt, a metal oxide or other agent set forth below. In certain aspects, the metal or metal oxide comprises Ti or TiO.sub.2. In other aspects, the pyrophosphate sequestering agent comprises hydroxyapatite. In other aspects the sequestering agent comprises an ammonium or substituted ammonium salt, or a resin or bead that contains such groups. In certain aspects of the above embodiments, the pyrophosphate sequestering agent comprises particles or beads.

"In addition to the foregoing, in some embodiments, arrays manufactured according to the methods described herein can be utilized in processes in which pyrophosphate can be released from the sequestering agent by providing a release reagent to the sequestering agent. In certain aspects, the release reagent comprises an anion capable of displacing the pyrophosphate from the sequestering agent, for example, by preferentially complexing or chelating the cation of the sequestering agent. In certain aspects, the release reagent comprises an agent selected from the group consisting of an acid or salt of an acid such as oxalic acid, an oxalate salt, sulfamic acid, a sulfamate salt, ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis-.beta.-amino-ethyl ether N,N, N',N'-tetra-acetic acid (EGTA) citric acid, tartaric acid, acetic or other carboxylic acids or their salts. In other aspects the release reagent comprises phosphate. In other aspects the release reagent comprises a bisphosphonate. In certain aspects the release reagent is the enzyme ATP sulfurylase. In this particular aspect the ATP sulfurylase is in solution rather than being bound to a bead or other surface. The ATP sulfurylase can release the pyrophosphate from the sequestering agent by transforming the pyrophosphate into ATP in the presence of adenysine phosphosulfate (APS). Typically, the ATP will have a lower binding affinity for the sequestering agent than does pyrophosphate."

URL and more information on this patent, see: Hirschbein, Bernard; Gorpe-Yasar, Filiz. Separation of Pyrophosphate Release and Pyrophosphate Detection. U.S. Patent Number 8765419, filed April 29, 2013, and published online on July 1, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8765419.PN.&OS=PN/8765419RS=PN/8765419

Keywords for this news article include: Anions, Alkenes, Genetics, Chemistry, Phosphates, Polymerase, Luciferases, Acetic Acids, DNA Research, Oxalic Acids, Acyclic Acids, Illumina Inc., Sulfamic Acid, Hydroxyapatites, Ethylene Glycols, Phosphoric Acids, Organic Chemicals, Dicarboxylic Acids, Alkaline Phosphatase, Luminescent Proteins, Enzymes and Coenzymes.

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