US20030064366A1 - Real-time sequence determination - Google Patents

Real-time sequence determination Download PDF

Info

Publication number
US20030064366A1
US20030064366A1 US09/901,782 US90178201A US2003064366A1 US 20030064366 A1 US20030064366 A1 US 20030064366A1 US 90178201 A US90178201 A US 90178201A US 2003064366 A1 US2003064366 A1 US 2003064366A1
Authority
US
United States
Prior art keywords
polymerase
tag
dna
monomer
tagged
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US09/901,782
Other languages
English (en)
Inventor
Susan Hardin
Xiaolian Gao
James Briggs
Richard Willson
Shiao-Chun Tu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Life Technologies Corp
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=22807697&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20030064366(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Individual filed Critical Individual
Priority to US09/901,782 priority Critical patent/US20030064366A1/en
Publication of US20030064366A1 publication Critical patent/US20030064366A1/en
Assigned to VISIGEN, INC. reassignment VISIGEN, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GAO, XIAOLIAN, BRIGGS, JAMES M., HARDIN, SUSAN H., TU, SHIAO-CHUN, WILLSON, RICHARD C.
Priority to US11/007,797 priority patent/US7329492B2/en
Priority to US11/007,642 priority patent/US20050266424A1/en
Priority to US11/648,713 priority patent/US20070184475A1/en
Priority to US11/648,184 priority patent/US20100255463A1/en
Priority to US11/648,856 priority patent/US20070275395A1/en
Priority to US11/648,115 priority patent/US20070172861A1/en
Priority to US11/648,137 priority patent/US20070292867A1/en
Priority to US11/648,191 priority patent/US20070172867A1/en
Priority to US11/648,174 priority patent/US20070172865A1/en
Priority to US11/648,106 priority patent/US20070172858A1/en
Priority to US11/648,164 priority patent/US20070172864A1/en
Priority to US11/648,182 priority patent/US20070172866A1/en
Priority to US11/648,722 priority patent/US20070172868A1/en
Priority to US11/648,138 priority patent/US20070172863A1/en
Priority to US11/648,136 priority patent/US20070172862A1/en
Assigned to Life Technologies Corporation reassignment Life Technologies Corporation ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: VISIGEN BIOTECHNOLOGIES, INC
Priority to US12/410,370 priority patent/US20110014604A1/en
Priority to US12/412,208 priority patent/US20100304367A1/en
Priority to US12/411,997 priority patent/US20110021383A1/en
Priority to US12/414,417 priority patent/US20090275036A1/en
Priority to US12/419,214 priority patent/US20090305278A1/en
Priority to US12/419,660 priority patent/US20110059436A1/en
Priority to US12/724,392 priority patent/US20100317005A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to a single-molecule sequencing apparatus and methods.
  • the present invention relates to a single-molecule sequencing apparatus and methods using tagged polymerizing agents and/or tagged monomers where the tagged polymerizing agent and/or the tagged monomers undergo a change in a detectable property before, during and/or after monomer insertion into a growing polymer chain.
  • the apparatus and methods are ideally-suited for sequencing DNA, RNA, polypeptide, carbohydrate or similar bio-molecular sequences under near real-time or real-time conditions.
  • the present invention also relates to a single-molecule sequencing apparatus and methods using tagged depolymerizing agents and/or tagged depolymerizable polymer where the tagged depolymerizing agent and/or the tagged depolymerizable polymer undergo a change in a detectable property before, during and/or after monomer removal from the depolymerizable polymer chain.
  • the apparatus and methods are ideally-suited for sequencing DNA, RNA, polypeptide, carbohydrate or similar bio-molecular sequences.
  • the present invention also relates to detecting a signal evidencing interactions between the tagged polymerizing agent or depolymerizing agent and a tagged or untagged polymer subunit such as a monomer or collection of monomers, where the detected signal provides information about monomer order.
  • the methods are carried out in real-time or near real-time.
  • DNA sequence information provides scientists with information critical to a wide range of biological processes.
  • the order of bases in DNA specifies the order of bases in RNA, the molecule within the cell that directly encodes the informational content of proteins.
  • DNA sequence information is routinely used to deduce protein sequence information. Base order dictates DNA structure and its function, and provides a molecular program that can specify normal development, manifestation of a genetic disease, or cancer.
  • DNA sequence information may provide a way to uniquely identify individuals.
  • a DNA molecule is comprised of four bases, adenine (A), guanine (G), cytosine (C), and thymine (T). These bases interact with each other in very specific ways through hydrogen bonds, such that A interacts with T, and G interacts with C. These specific interactions between the bases are referred to as base-pairings. In fact, it is these base-pairings (and base stacking interactions) that stabilize double-stranded DNA.
  • the two strands of a DNA molecule occur in an antiparallel orientation, where one strand is positioned in the 5′ to 3′ direction, and the other strand is positioned in the 3′ to 5′ direction.
  • 5′ and 3′ refer to the directionality of the DNA backbone, and are critical to describing the order of the bases.
  • the convention for describing base order in a DNA sequence uses the 5′ to 3′ direction, and is written from left to right. Thus, if one knows the sequence of one DNA strand, the complementary sequence can be deduced.
  • Sanger sequencing is currently the most commonly used method to sequence DNA (Sanger et al., 1977). This method exploits several features of a DNA polymerase: its ability to make an exact copy of a DNA molecule, its directionality of synthesis (5′ to 3′), its requirement of a DNA strand (a ‘primer’) from which to begin synthesis, and its requirement for a 3′ OH at the end of the primer. If a 3′ OH is not available, then the DNA strand cannot be extended by the polymerase.
  • ddNTP dideoxynucleotide
  • ddNTP dideoxynucleotide
  • ddATP dideoxynucleotide
  • ddTTP dideoxynucleotide
  • ddGTP dideoxynucleotide
  • ddCTP a base analogue lacking a 3′ OH
  • the polymerase is unable to add any additional bases to the end of the strand.
  • ddNTPs are incorporated by the polymerase into the DNA strand using the same base incorporation rules that dictate incorporation of natural nucleotides, where A specifies incorporation of T, and G specifies incorporation of C (and vice versa).
  • the automated sequencer is used to separate sequencing reaction products, detect and collect (via computer) the data from the reactions, and analyze the order of the bases to automatically deduce the base sequence of a DNA fragment.
  • Automated sequencers detect extension products containing a fluorescent tag. Sequence read lengths obtained using an automated sequencer are dependent upon a variety of parameters, but typically range between 500 to 1,000 bases (3-18 hours of data collection). At maximum capacity an automated sequencer can collect data from 96 samples in parallel.
  • dye-labeled terminator chemistry When dye-labeled terminator chemistry is used to detect the sequencing products, base identity is determined by the color of the fluorescent tag attached to the ddNTP. After the reaction is assembled and processed through the appropriate number of cycles (3-12 hours), the extension products are prepared for loading into a single lane on an automated sequencer (unincorporated, dye-labeled ddNTPs are removed and the reaction is concentrated; 1-2 hours).
  • An advantage of dye-terminator chemistry is that extension products are visualized only if they terminate with a dye-labeled dNTP; prematurely terminated products are not detected. Thus, reduced background noise typically results with this chemistry.
  • dRhodamine dichlororhodamine
  • the d-Rhodamine acceptor dyes associated with the terminators are dichloro[R110], dichloro[R6G], dichloro[TAMRA] or dichloro[ROX], for the G-, A-, T- or C-terminators, respectively.
  • the donor dye (6-FAM) efficiently absorbs energy from the argon ion laser in the automated sequencing machine and transfers that energy to the linked acceptor dye.
  • the linker connecting the donor and acceptor portions of the terminator is optimally spaced to achieve essentially 100% efficient energy transfer.
  • the fluorescence signals emitted from these acceptor dyes exhibit minimal spectral overlap and are collected by an ABI PRISM 377 DNA sequencer using 10 nm virtual filters centered at 540, 570, 595 and 625 nm, for G-, A-, T- or C-terminators, respectively.
  • energy transfer dye-labeled terminators produce brighter signals and improve spectral resolution.
  • the predominant enzyme used in automated DNA sequencing reactions is a genetically engineered form of DNA polymerase I from Thermus aquaticus .
  • This enzyme, AmpliTaq DNA Polymerase, FS was optimized to more efficiently incorporate ddNTPs and to eliminate the 3′ to 5′ and 5′ to 3′ exonuclease activities.
  • Replacing a naturally occurring phenylalanine at position 667 in T. aquaticus DNA polymerase with a tyrosine reduced the preferential incorporation of a dNTP, relative to a ddNTP (Tabor and Richardson, 1995; Reeve and Fuller, 1995).
  • a single hydroxyl group within the polymerase is responsible for discrimination between dNTPs and ddNTPs.
  • the 3′ to 5′ exonuclease activity which enables the polymerase to remove a mis-incorporated base from the newly replicated DNA strand (proofreading activity), was eliminated because it also allows the polymerase to remove an incorporated ddNTP.
  • the 5′ to 3′ exonuclease activity was eliminated because it removes bases from the 5′ end of the reaction products. Since the reaction products are size separated during gel electrophoresis, interpretable sequence data is only obtained if the reaction products share a common endpoint.
  • the primer defines the 5′ end of the extension product and the incorporated, color-coded ddNTP defines base identity at the 3′ end of the molecule.
  • conventional DNA sequencing involves analysis of a population of DNA molecules sharing the same 5′ endpoint, but differing in the location of the ddNTP at the 3′ end of the DNA chain.
  • a directed or primer-walking sequencing strategy can be used to fill-in gaps remaining after the random phase of large-fragment sequencing, and as an efficient approach for sequencing smaller DNA fragments.
  • This strategy uses DNA primers that anneal to the template at a single site and act as a start site for chain elongation. This approach requires knowledge of some sequence information to design the primer. The sequence obtained from the first reaction is used to design the primer for the next reaction and these steps are repeated until the complete sequence is determined.
  • a primer-based strategy involves repeated sequencing steps from known into unknown DNA regions, the process minimizes redundancy, and it does not require additional cloning steps. However, this strategy requires the synthesis of a new primer for each round of sequencing.
  • This method requires that the DNA strand is synthesized to contain the flourescently-labeled base(s). This requirement limits the length of sequence that can be determined, and increases the number of manipulations that must be performed before any sequence data is obtained.
  • a related approach proposes to sequentially separate single (unlabeled) nucleotides from a strand of DNA, confine them in their original order in a solid matrix, and detect the spectroscopic emission of the separated nucleotides to reconstruct DNA sequence information (Ulmer, 1997; Mitsis and Kwagh, 1999; Dapprich, 1999). This is the approach that is being developed by Praelux, Inc., a company with a goal to develop single-molecule DNA sequencing. Theoretically, this latter method should not be as susceptible to length limitations as the former enzymatic degradation method, but it does require numerous manipulations before any sequence information can be obtained.
  • Li-cor, Inc. is developing an enzyme synthesis based strategy for single-molecule sequencing as set forth in PCT application WO 00/36151.
  • the Li-cor method involves multiply modifying each dNTP by attaching a fluorescent tag to the ⁇ -phosphate and a quenching moiety to the another site on the dNTP, preferably on the base.
  • the quenching moiety is added to prevent emission from the fluorescent tag attached to an unincorporated dNTP.
  • the fluorescent tag and quenching moiety are separated, resulting in emission from the tag.
  • the tag (contained on the pyrophosphate) flows away from the polymerase active site, but the modified (quenched) base becomes part of the DNA polymer.
  • the A-form DNA may be important for maximizing minor groove contacts between the enzyme and the DNA. If the DNA structure is affected due to base modification, enzyme fidelity and/or function maybe altered. Thus, there is still a need in the art for a fast and efficient enzymatic DNA sequencing system for single molecular DNA sequences.
  • the present invention provides a polymerizing agent modified with at least one molecular or atomic tag located at or near, associated with or covalently bonded to a site on the polymerizing agent, where a detectable property of the tag undergoes a change before, during and/or after monomer incorporation.
  • the monomers can be organic, inorganic or bio-organic monomers such as nucleotides for DNA, RNA, mixed DNA/RNA sequences, amino acids, monosaccharides, synthetic analogs of naturally occurring nucleotides, synthetic analogs of naturally occurring amino acids or synthetic analogs of naturally occurring monosaccharides, synthetic organic or inorganic monomers, or the like.
  • the present invention provides a depolymerizing agent modified with at least one molecular or atomic tag located at or near, associated with or covalently bonded to a site on the depolymerizing agent, where a detectable property of the tag undergoes a change before, during and/or after monomer removal.
  • the polymers can be DNA, RNA, mixed DNA/RNA sequences containing only naturally occurring nucleotides or a mixture of naturally occurring nucleotides and synthetic analogs thereof, polypeptide sequences containing only naturally occurring amino acids or a mixture of naturally occurring amino acids and synthetic analogs thereof, polysaccharide or carbohydrate sequences containing only naturally occurring monosaccharides or a mixture of naturally occurring monosaccharides and synthetic analogs thereof, or polymers containing synthetic organic or inorganic monomers, or the like.
  • the present invention also provides a system that enables detecting a signal corresponding to a detectable property evidencing changes in interactions between a synthesizing/polymerizing agent or a depolymerizing agent (molecule) and its substrates (monomers or depolymerizable polymers) and decoding the signal into monomer order specific information or monomer sequence information, preferably in real-time or near real-time.
  • the present invention provides a polymerase modified with at least one molecular or atomic tag located at or near, associated with, or covalently bonded to a site on the polymerase, where a detectable property of the tag undergoes a change before, during and/or after monomer incorporation.
  • the monomers can be nucleotides for DNA, RNA or mixed DNA/RNA monomers or synthetic analogs polymerizable by the polymerase.
  • the present invention provides an exonuclease modified with at least one molecular or atomic tag located at or near, associated with, or covalently bonded to a site on the exonuclease, where a detectable property of the tag undergoes a change before, during and/or after monomer release.
  • the polymers can be DNA, RNA or mixed DNA/RNA sequences comprised of naturally occurring monomers or synthetic analogs depolymerizable by the exonuclease.
  • the present invention provides a polymerase modified with at least one molecular or atomic tag located at or near, associated with, or covalently bonded to a site that undergoes a conformational change before, during and/or after monomer incorporation, where the tag has a first detection propensity when the polymerase is in a first conformational state and a second detection propensity when the polymerase is in a second conformational state.
  • the present invention provides a polymerase modified with at least one chromophore located at or near, associated with, or covalently bonded to a site that undergoes a conformational change before, during and/or after monomer incorporation, where an intensity and/or frequency of emitted light of the chromophore has a first value when the polymerase is in a first conformational state and a second value when the polymerase is in a second conformational state.
  • the present invention provides a polymerase modified with at least one fluorescently active molecular tag located at or near, associated with, or covalently bonded to a site that undergoes a conformational change before, during and/or after monomer incorporation, where the tag has a first fluorescence propensity when the polymerase is in a first conformational state and a second fluorescence propensity when the polymerase is in a second conformational state.
  • the present invention provides a polymerase modified with a molecular tag located at or near, associated with, or covalently bonded to a site that undergoes a conformational change before, during and/or after monomer incorporation, where the tag is substantially detectable when the polymerase is in a first conformational state and substantially non-detectable when the polymerase is in a second conformational state or substantially non-detectable when the polymerase is in the first conformational state and substantially detectable when the polymerase is in the second conformational state.
  • the present invention provides a polymerase modified with at least one molecular or atomic tag located at or near, associated with, or covalently bonded to a site that interacts with a tag on the released pyrophosphate group, where the polymerase tag has a first detection propensity before interacting with the tag on the released pyrophosphate group and a second detection propensity when interacting with the tag on the released pyrophosphate group.
  • this change in detection propensity is cyclical occurring as each pyrophosphate group is released.
  • the present invention provides a polymerase modified with at least one chromophore located at or near, associated with, or covalently bonded to a site that interacts with a tag on the released pyrophosphate group, where an intensity and/or frequency of light emitted by the chromophore has a first value before the chromophore interacts with the tag on the released pyrophosphate and a second value when interacting with the tag on the released pyrophosphate group.
  • this change in detection propensity is cyclical occurring as each pyrophosphate group is released.
  • the present invention provides a polymerase modified with at least one fluorescently active molecular tag located at or near, associated with, or covalently bonded to a site that interacts with a tag on the released pyrophosphate group, where the polymerase tag changes from a first state prior to release of the pyrophosphate group and a second state as the pyrophosphate group diffuses away from the site of release.
  • this change in detection propensity is cyclical occurring as each pyrophosphate group is released.
  • the present invention provides a polymerase modified with a molecular tag located at or near, associated with, or covalently bonded to a site that interacts with a tag on the released pyrophosphate group, where the polymerase tag changes from a substantially detectable state prior to pyrophosphate release to a substantially non-detectable state when the polymerase tag interacts with the tag on the pyrophosphate group after group release, or changes from a substantially non-detectable state prior to pyrophosphate release to a substantially detectable state when the polymerase tag interacts with the tag on the pyrophosphate group after group release.
  • the present invention provides a monomer polymerizing agent modified with at least one pair of molecular and/or atomic tags located at or near, associated with, or covalently bonded to sites on the polymerizing agent, where a detectable property of at least one tag of the pair undergoes a change before, during and/or after monomer incorporation or where a detectable property of at least one tag of the pair undergoes a change before, during and/or after monomer incorporation due to a change in inter-tag interaction.
  • the present invention provides a depolymerizing agent modified with at least one pair of molecular and/or atomic tags located at or near, associated with, or covalently bonded to sites on the depolymerizing agent, where a detectable property of at least one tag of the pair undergoes a change before, during and/or after monomer release or where a detectable property of at least one tag of the pair undergoes a change before, during and/or after monomer release due to a change in inter-tag interaction.
  • the present invention provides a monomer polymerizing agent modified with at least one pair of molecular and/or atomic tags located at or near, associated with, or covalently bonded to sites on the polymerizing agent, where a detectable property of at least one tag of the pair has a first value when the polymerizing agent is in a first state and a second value when the polymerizing agent is in a second state, where the polymerizing agent changes from the first state to the second state and back to the first state during a monomer incorporation cycle.
  • the present invention provides a depolymerizing agent modified with at least one pair of molecular and/or atomic tags located at or near, associated with or covalently bonded to sites on the polymerizing agent, where a detectable property of at least one tag of the pair has a first value when the depolymerizing agent is in a first state and a second value when the depolymerizing agent is in a second state, where the depolymerizing agent changes from the first state to the second state and back to the first state during a monomer release cycle.
  • the first and second states are different so that a change in the detected signal occurs.
  • a no-change result may evidence other properties of the polymerizing media or depolymerizing media.
  • the present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with, or covalently bonded to sites at least one of the tags undergoes a change during monomer incorporation, where a detectable property of the pair has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state, where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.
  • the present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites at least one of the tags undergoes conformational change during monomer incorporation, where the detectably property of the pair has a first value when the polymerase is in a first conformational state and a second value when the polymerase is in a second conformational state, where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.
  • the present invention provides a polymerase modified with at least one pair of molecules or atoms located at or near, associated with or covalently bonded to sites at least one of the tags undergoes conformational change during monomer incorporation, where the pair interact to form a chromophore when the polymerase is in a first conformational state or a second conformational state, where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.
  • the present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites at least one of the tags undergoes conformational change during monomer incorporation, where the tags have a first fluorescence propensity when the polymerase is in a first conformational state and a second fluorescence propensity when the polymerase is in a second conformational state, where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.
  • the present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites at least one of the tags undergoes conformational change during monomer incorporation, where the pair is substantially active when the polymerase is in a first conformational state and substantially inactive when the polymerase is in a second conformational state or substantially inactive when the polymerase is in the first conformational state and substantially active when the polymerase is in the second conformational state, where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.
  • the present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with, or covalently bonded to sites at least one of the tags undergoes a change during and/or after pyrophosphate release during the monomer incorporation process, where a detectable property of the pair has a first value when the tag is in a first state prior to pyrophosphate release and a second value when the tag is in a second state during and/or after pyrophosphate release, where the tag changes from its first state to its second state and back to its first state during a monomer incorporation cycle.
  • the present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites at least one of the tags undergoes a change in position due to a conformational change in the polymerase during the pyrophosphate release process, where the detectably property of the pair has a first value when the tag is in its first position and a second value when the tag is in its second position, where the tag changes from its first position to its second position and back to its first position during a release cycle.
  • the present invention provides a polymerase modified with at least one pair of molecules or atoms located at or near, associated with or covalently bonded to sites, where the tags change relative separation due to a conformational change in the polymerase during pyrophosphate release, where the tags interact to form a chromophore having a first emission profile when the tags are a first distance apart and a second profile when the tags are a second distance apart, where the separation distance changes from its first state to its second state and back to its first state during a pyrophosphate release cycle.
  • the present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites, where the tags change relative separation due to a conformational change in the polymerase during pyrophosphate release, where the tags have a first fluorescence propensity when the polymerase is in a first conformational state and a second fluorescence propensity when the polymerase is in a second conformational state, where the propensity changes from its the first value to its second value and back again during a pyrophosphate release cycle.
  • the present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites, where the tags change relative separation due to a conformational change in the polymerase during pyrophosphate release, where the pair is substantially fluorescently active when the tags have a first separation and substantially fluorescently inactive when the tags have a second separation or substantially fluorescently inactive when the tags have the first separation and substantially fluorescently active when the tags have the second separation, where the fluorescence activity undergoes one cycle during a pyrophosphate release cycle.
  • the present invention provides a method for determining when a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of an atomic or molecular tag, where the tag is located at or near, associated with, or covalently bonded to a site on a polymerizing agent, where the detectable property of the tag undergoes a change before, during and/or after monomer incorporation.
  • the present invention provides a method for determining when a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of an atomic or molecular tag, where the tag is located at or near, associated with, or covalently bonded to a site on a polymerizing agent, where the detectable property has a first value when the agent is in a first state and a second value when the agent is in a second state, where the agent changes from the first state to the second state and back to the first state during a monomer incorporation cycle.
  • the first and second states are different so that a change in the detected signal occurs.
  • a no-change result may evidence other properties of the polymerizing medium.
  • the present invention provides a method for determining when or whether a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of a tag, where the tag is located at or near, associated with, or covalently bonded to a site on a polymerase, where the site undergoes a change during monomer incorporation and where the detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state, where the values signify that the site has undergone the change and where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.
  • the present invention provides a method for determining when or whether a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of a tag, where the tag is located at or near, associated with, or covalently bonded to a site on a polymerase, where the site undergoes a conformational change during monomer incorporation and where the detectable property has a first value when the polymerase is in a first conformational state and a second value when the polymerase is in a second conformational state, where the values signify that the site has undergone the change and where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.
  • the present invention provides a method for determining when or whether a monomer is incorporated into a growing molecular chain comprising the steps of exposing a tagged polymerase to light, monitoring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase, where the tagged polymerase comprises a polymerase including a tag located at or near, associated with, or covalently bonded to a site that undergoes conformational change during monomer incorporation and where the tag emits fluorescent light at a first intensity and/or frequency when the polymerase is in a first conformational state and a second intensity and/or frequency when the polymerase is in a second conformational state, where the change in intensities and/or frequencies signifies that the site has undergone the change and where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.
  • the present invention also provides the above methods using a plurality of tagged polymerases permitting parallel and/or massively parallel sequencing simultaneously. Such parallelism can be used to ensure confidence. Such parallelism can also be used to quickly detect the degree of homology in DNA sequences for a given gene across species or to quickly screen patient DNA for specific genetic traits or to quickly screen DNA sequences for polymorphisms.
  • the present invention also provides a method for determining if or when a monomer is incorporated into a growing DNA chain associated with a polymerase, where a tag is located on the polymerase so that as the pyrophosphate group is released after base incorporation and prior to its diffusion away from the polymerase, the polymerase tag interacts with the tag on the pyrophosphate causing a change in a detectable property of one of the tags or a detectable property associated with both tags in the case of a fluorescent pair.
  • the first and second states are different so that a change in the detected signal occurs.
  • a no-change result may evidence other properties of the polymerizing media.
  • the present invention provides a single-molecule sequencing apparatus comprising a substrate having deposited thereon at least one tagged polymerizing agent.
  • the tagged polymerizing agent can be placed on the surface of the substrate in an appropriate polymerizing medium or the polymerizing agent can be confined in a region, area, well, groove, channel or other similar structure on the substrate.
  • the substrate can also include a monomer region, area, well, groove, channel, reservoir or other similar structure on the substrate connected to the polymerizing agent confinement structure by at least one connecting structure capable of supporting molecular transport of monomer to the polymerizing agent such as a channel, groove, or the like.
  • the substrate can include structures containing each monomer, where each structure is connected to the polymerizing agent confinement structure by a connecting structure capable of supporting molecular transport of monomer to the polymerizing agent.
  • the substrate can also be subdivided into a plurality of polymerizing agent confinement structures, where each structure is connected to a monomer reservoir.
  • each polymerizing agent confinement structure can have its own monomer reservoir or sufficient monomer reservoirs so that each reservoir contains a specific monomer.
  • the present invention also provides a single-molecule sequencing apparatus comprising a substrate having at least one tagged polymerizing agent attached to the surface of the substrate by a molecular tether or linking group, where one end of the tether or linking group is bonded to a site on the surface of the substrate and the other end is bonded to a site on the polymerizing agent or bonded to a site on a molecule strongly associated with the polymerizing agent.
  • a single-molecule sequencing apparatus comprising a substrate having at least one tagged polymerizing agent attached to the surface of the substrate by a molecular tether or linking group, where one end of the tether or linking group is bonded to a site on the surface of the substrate and the other end is bonded to a site on the polymerizing agent or bonded to a site on a molecule strongly associated with the polymerizing agent.
  • bonded to means that chemical and/or physical interactions sufficient to maintain the polymerizing agent within a given region of the substrate under normal polymerizing conditions.
  • the chemical and/or physical interactions include, without limitation, covalent bonding, ionic bonding, hydrogen bonding, a polar bonding, attractive electrostatic interactions, dipole interactions, or any other electrical or quantum mechanical interaction sufficient in to to maintain the polymerizing agent in a desired region of the substrate.
  • the substrate having tethered tagged polymerizing agent attached thereon can be placed in container containing an appropriate polymerizing medium.
  • the tagged polymerizing agent can be tethered or anchored on or within a region, area, well, groove, channel or other similar structure on the substrate capable of being filled with an appropriate polymerizing medium.
  • the substrate can also include a monomer region, area, well, groove, channel or other similar structure on the substrate connected to the polymerizing agent structure by at least one a connecting structure capable of supporting molecular transports of monomer to the polymerizing agent.
  • the substrate can include structures containing each monomer, where each structure is connected to the polymerizing agent structure by a connecting structure capable of supporting molecular transports of monomer to the polymerizing agent.
  • the substrate can also be subdivided into a plurality of polymerizing agent structures each having at least one tethered polymerizing agent, where each structure is connected to a monomer reservoir.
  • each polymerizing agent structure can have its own monomer reservoir or sufficient monomer reservoirs, one reservoir of each specific monomer.
  • the monomers for use in these apparatus including, without limitation, dNTPs, tagged dNTPs, ddNTPs, tagged ddNTPs, amino acids, tagged amino acids, mono saccharides, tagged monosaccharides or appropriate mixtures or combinations thereof depending on the type of polymer being sequenced.
  • the present invention provides a single-molecule sequencing apparatus comprising a substrate having deposited thereon at least one tagged polymerase.
  • the tagged polymerase can be placed on the surface of the substrate in an appropriate polymerizing medium or the polymerase can be confined in a region, area, well, groove, channel or other similar structure on the substrate capable of being filled with an appropriate polymerizing medium.
  • the substrate can also include a monomer region, area, well, groove, channel or other similar structure on the substrate connected to the polymerase confinement structure by at least one connecting structure capable of supporting molecular transports of monomer to the polymerase.
  • the substrate can include structures containing each monomer, where each structure is connected to the polymerase confinement structure by a connecting structure capable of supporting molecular transports of the monomer to the polymerase in the polymerase confinement structures.
  • the substrate can also be subdivided into a plurality of polymerase confinement structures, where each structure is connected to a monomer reservoir.
  • each polymerase confinement structure can have its own monomer reservoir or four reservoirs, each reservoir containing a specific monomer.
  • the present invention also provides a single-molecule sequencing apparatus comprising a substrate having at least one tagged polymerase attached to the surface of the substrate by a molecular tether or linking group, where one end of the tether or linking group is bonded to a site on the surface of the substrate and the other end is bonded (either directly or indirectly) to a site on the polymerase or bonded to a site on a molecule strongly associated with the polymerase.
  • bonded to means that chemical and/or physical interactions sufficient to maintain the polymerase within a given region of the substrate under normal polymerizing conditions.
  • the chemical and/or physical interactions include, without limitation, covalent bonding, ionic bonding, hydrogen bonding, a polar bonding, attractive electrostatic interactions, dipole interactions, or any other electrical or quantum mechanical interaction sufficient in toto to maintain the polymerase in its desired region.
  • the substrate having tethered tagged polymerizing agent attached thereon can be placed in container containing an appropriate polymerizing medium.
  • the tagged polymerizing agent can be tethered or anchored on or within a region, area, well, groove, channel or other similar structure on the substrate capable of being filled with an appropriate polymerizing medium.
  • the substrate can also include a monomer region, area, well, groove, channel or other similar structure on the substrate connected to the polymerase structure by at least one channel.
  • the substrate can include structures containing each monomer, where each structure is connected to the polymerase structure by a connecting structure that supports molecular transports of the monomer to the polymerase in the polymerase confinement structures.
  • the substrate can also be subdivided into a plurality of polymerase structures each having at least one tethered polymerase, where each structure is connected to a monomer reservoir.
  • each polymerase structure can have its own monomer reservoir or four reservoirs, each reservoir containing a specific monomer.
  • the monomers for use in these apparatus including, without limitation, dNTPs, tagged dNTPs, ddNTPs, tagged ddNTPs, or mixtures or combinations thereof.
  • the present invention provides a method for single-molecule sequencing comprising the step of supplying a plurality of monomers to a tagged polymerizing agent confined on or tethered to a substrate and monitoring a detectable property of the tag over time.
  • the method can also include a step of relating changes in the detectable property to the occurrence (timing) of monomer addition and/or to the identity of each incorporated monomer and/or to the near simultaneous determination of the sequence of incorporated monomers.
  • the present invention provides a method for single-molecule sequencing comprising the step of supplying a plurality of monomers to a tagged polymerizing agent confined on or tethered to a substrate, exposing the tagged polymerizing agent to light either continuously or periodically and measuring an intensity and/or frequency of fluorescent light emitted by the tag over time.
  • the method can further comprise relating the changes in the measured intensity and/or frequency of emitted fluorescent light from the tag over time to the occurrence (timing) of monomer addition and/or to the identity of each incorporated monomer and/or to the near simultaneous determination of the sequence of the incorporated monomers.
  • the present invention provides a method for single-molecule sequencing comprising the step of supplying a plurality of monomers to a tagged polymerase confined on or tethered to a substrate and monitoring a detectable property of the tag over time.
  • the method can also include a step of relating changes in the detectable property over time to the occurrence (timing) of monomer addition and/or to the identity of each incorporated monomer and/or to the near simultaneous determination of the sequence of the incorporated monomers.
  • the present invention provides a method for single-molecule sequencing comprising the step of supplying a plurality of monomers to a tagged polymerase confined on a substrate, exposing the tagged polymerase to light continuously or periodically and measuring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase over time.
  • the method can further comprise relating changes in the measured intensity and/or frequency of emitted fluorescent light from the tag over time to the occurrence (timing) of monomer addition and/or to the identity of each incorporated monomer and/or to the near simultaneous determination of the sequence of the incorporated monomers.
  • the present invention provides cooperatively tagged polymerizing agents and tagged monomers, where a detectable property of at least one of the tags changes when the tags interact before, during and/or after monomer insertion.
  • the tag on the polymerase is positioned such that the tags interact before, during and/or after each monomer insertion.
  • tags that are released from the monomers after monomer insert such as of ⁇ and/or ⁇ phosphate tagged dNTPs, i.e., the tags reside on the ⁇ and/or ⁇ phosphate groups
  • the tag on the polymerizing agent can be designed to interact with the tag on the monomer only after the tag is released from the polymerizing agent after monomer insertion.
  • Tag placement within a polymerizing agent can be optimized to enhance interaction between the polymerase and dNTP tags by attaching the polymerase tag to sites on the polymerase that move during an incorporation event changing the relative separation of the two tags or optimized to enhance interaction between the polymerase tag and the tag on the pyrophosphate as it is release during base incorporation and prior to its diffusion away from the polymerizing agent.
  • the present invention provides cooperatively tagged polymerizing agents and tagged monomers, where a detectable property of at least one of the tags changes when the tags are within a distance sufficient to cause a measurable change in the detectable property. If the detectable property is fluorescence induced in one tag by energy transfer to the other tag or due to one tag quenching the fluorescence of the other tag or causing a measurable change in the fluorescence intensity and/or frequency, the measurable change is caused by bringing the tags into close proximity to each other, i.e., decrease the distance separating the tags.
  • the distance needed to cause a measurable change in the detectable property is within (less than or equal to) about 100 ⁇ , preferably within about 50 ⁇ , particularly within about 25 ⁇ , especially within about 15 ⁇ and most preferably within about 10 ⁇ .
  • a distance sufficient to cause a measurable change in a detectable property of a tag will depend on many parameters including the location of the tag, the nature of the tag, the solvent system, external fields, excitation source intensity and frequency band width, temperature, pressure, etc.
  • the present invention provides a tagged polymerizing agent and tagged monomer precursor(s), where an intensity and/or frequency of fluorescence light emitted by at least one tag changes when the tags interact before, during and/or after monomer insertion.
  • the present invention provides cooperatively tagged depolymerizing agents and tagged depolymerizable polymer, where a detectable property of at least one of the tags changes when the tags interact before, during and/or after monomer release.
  • the tag on the depolymerizing agent can be designed so that the tags interact before, during and/or after each monomer release.
  • the present invention provides cooperatively tagged depolymerizing agents and tagged polymers, where a detectable property of at least one of the tags changes when the tags are within a distance sufficient to cause a change in measurable change in the detectable property. If the detectable property is fluorescence induced in one tag by energy transfer to the other tag or due to one tag quenching the fluorescence of the other tag or causing a measurable change in the fluorescence intensity and/or frequency, the measurable change is caused by bringing to tags into close proximity to each other, i.e., decrease the distance separating the tags.
  • the distance needed to cause a measurable change in the detectable property is within (less than or equal to) about 100 ⁇ , preferably within about 50 ⁇ , particularly within about 25 ⁇ , especially within about 15 ⁇ and most preferably within about 10 ⁇ .
  • a distance sufficient to cause a measurable change in a detectable property of a tag will depend on many parameters including the location of the tag, the nature of the tag, the solvent system, external fields, excitation source intensity and frequency band width, temperature, pressure, etc.
  • the present invention provides a tagged depolymerizing agents and a tagged polymer, where an intensity and/or frequency of fluorescence light emitted by at least one tag changes when the tags interact before, during and/or after monomer release.
  • the present invention provides cooperatively tagged polymerase and tagged monomers, where a detectable property of at least one of the tags changes when the tags interact before, during and/or after monomer insertion.
  • the tag on the polymerase can be designed so that the tags interact before, during and/or after each monomer insertion.
  • tags that are released from the monomers after monomer insert such as of ⁇ and/or ⁇ phosphate tagged dNTPs, i.e., the tags reside on the ⁇ and/or ⁇ phosphate groups
  • the tag on the polymerizing agent can be designed to interact with the tag on the monomer only after the tag is released from the polymerizing agent after monomer insertion.
  • the polymerase tag In the first case, the polymerase tag must be located on a site of the polymerase which allows the polymerase tag to interact with the monomer tag during the monomer insertion process—initial binding and bonding into the growing polymer. While in the second case, the polymerase tag must be located on a site of the polymerase which allows the polymerase tag to interact with the monomer tag now on the released pyrophosphate prior to its diffusion away from the polymerase and into the polymerizing medium.
  • the present invention provides cooperatively tagged polymerase and tagged monomers, where a detectable property of at least one of the tags changes when the tags are within a distance sufficient or in close proximity to cause a measurable change in the detectable property.
  • the detectable property is fluorescence induced in one tag by energy transfer to the other tag or due to one tag quenching the fluorescence of the other tag or causing a measurable change in the fluorescence intensity and/or frequency
  • the measurable change is caused by bringing to tags into close proximity to each other, i.e., decrease the distance separating the tags.
  • the distance or close proximity is a distance between about 100 ⁇ and about 10 ⁇ .
  • the distance is less than or equal to about 100 ⁇ , preferably less than or equal to about 50 ⁇ , particularly less than or equal to about 25 ⁇ , especially less than or equal to about 15 ⁇ and most preferably less than or equal to about 10 ⁇ .
  • a distance sufficient to cause a measurable change in a detectable property of a tag will depend on many parameters including the location of the tags, the nature of the tags, the solvent system (polymerizing medium), external fields, excitation source intensity and frequency band width, temperature, pressure, etc.
  • the present invention provides a tagged polymerase and tagged monomer precursors, where the tags form a fluorescently active pair such as a donor-acceptor pair and an intensity and/or frequency of fluorescence light emitted by at least one tag (generally the acceptor tag in donor-acceptor pairs) changes when the tags interact.
  • the present invention provides a tagged polymerase and a tagged monomer precursors, where the tags form a fluorescently active pair such as a donor-acceptor pair and an intensity and/or frequency of fluorescence light emitted by at least one tag (generally the acceptor tag in donor-acceptor pairs) changes when the tags are a distance sufficient or in close proximity to change either the intensity and/or frequency of the fluorescent light.
  • the distance or close proximity is a distance between about 100 ⁇ and about 10 ⁇ .
  • the distance is less than or equal to about 100 ⁇ , preferably less than or equal to about 50 ⁇ , particularly less than or equal to about 25 ⁇ , especially less than or equal to about 15 ⁇ and most preferably less than or equal to about 10 ⁇ .
  • a distance sufficient to cause a measurable change in a detectable property of a tag will depend on many parameters including the location of the tag, the nature of the tag, the solvent system, external fields, excitation source intensity and frequency band width, temperature, pressure, etc.
  • the present invention provides a single-molecule sequencing apparatus comprising a container having at least one tagged polymerase confined on or tethered to an interior surface thereof and having a solution containing a plurality of tagged monomers in contact with the interior surface.
  • the present invention provides a method for single-molecule sequencing comprising the step of supplying a plurality of tagged monomers to a tagged polymerase confined on an interior surface of a container, exposing the tagged polymerase to light and measuring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase during each successive monomer addition or insertion into a growing polymer chain.
  • the method can further comprise relating the measured intensity and/or frequency of emitted fluorescent light to incorporation events and/or to the identification of each inserted or added monomer resulting in a near real-time or real-time readout of the sequence of the a growing nucleic acid sequence—DNA sequence, RNA sequence or mixed DNA/RNA sequences.
  • the present invention also provides a system for retrieving stored information comprising a molecule having a sequence of known elements representing a data stream, a single-molecule sequencer comprising a polymerase having at least one tag associated therewith, an excitation source adapted to excite at least one tag on the polymerase, and a detector adapted to detect a response from the excited tag on the polymerase, where the response from the at least one tag changes during polymerization of a complementary sequence of elements and the change in response represents a content of the data stream.
  • the present invention also provides a system for determining sequence information from a single-molecule comprising a molecule having a sequence of known elements, a single-molecule sequencer comprising a polymerase having at least one tag associated therewith, a excitation source adapted to excite at least one tag on the polymerase, and a detector adapted to detect a response from the excited tag on the polymerase, where the response from at least one tag changes during polymerization of a complementary sequence of elements representing the element sequence of the molecule.
  • the present invention also provides a system for determining sequence information from a single-molecule comprising a molecule having a sequence of known elements, a single-molecule sequencer comprising a polymerase having at least one fluorescent tag associated therewith, an excitation light source adapted to excite at least one fluorescent tag on the polymerase and/or monomer and a fluorescent light detector adapted to detect at least an intensity of emitted fluorescent light from at least one fluorescent tag on the polymerase and/or monomer, where the signal intensity changes each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.
  • the present invention also provides a system for storing and retrieving data comprising a sequence of nucleotides or nucleotide analogs representing a given data stream; a single-molecule sequencer comprising a polymerase having at least one fluorescent tag covalently attached thereto; an excitation light source adapted to excite the at least one fluorescent tag on the polymerase and/or monomer; and a fluorescent light detector adapted to detect emitted fluorescent light from at least one fluorescent tag on the polymerase and/or monomer, where at least one fluorescent tag emits or fails to emit fluorescent light each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.
  • monomer as used herein means any compound that can be incorporated into a growing molecular chain by a given polymerase.
  • monomers include, without limitations, naturally occurring nucleotides (e.g., ATP, GTP, TTP, UTP, CTP, DATP, dGTP, dTTP, dUTP, dCTP, synthetic analogs), precursors for each nucleotide, non-naturally occurring nucleotides and their precursors or any other molecule that can be incorporated into a growing polymer chain by a given polymerase.
  • amino acids naturally occurring for protein or protein analog synthesis, mono saccharides for carbohydrate synthesis or other monomeric syntheses.
  • polymerase as used herein means any molecule or molecular assembly that can polymerize a set of monomers into a polymer having a predetermined sequence of the monomers, including, without limitation, naturally occurring polymerases or reverse transcriptases, mutated naturally occurring polymerases or reverse transcriptases, where the mutation involves the replacement of one or more or many amino acids with other amino acids, the insertion or deletion of one or more or many amino acids from the polymerases or reverse transcriptases, or the conjugation of parts of one or more polymerases or reverse transcriptases, non-naturally occurring polymerases or reverse transcriptases.
  • polymerase also embraces synthetic molecules or molecular assembly that can polymerize a polymer having a pre-determined sequence of monomers, or any other molecule or molecular assembly that may have additional sequences that facilitate purification and/or immobilization and/or molecular interaction of the tags, and that can polymerize a polymer having a pre-determined or specified or templated sequence of monomers.
  • the present invention provides a composition
  • a composition comprising a polymerizing agent including at least one molecular and/or atomic tag located at or near, associated with or covalently bonded to a site on the agent, where a detectable property of the tag undergoes a change before, during and/or after monomer incorporation.
  • the present invention provides a composition
  • a composition comprising a polymerizing agent including at least one molecular and/or atomic tag located at or near, associated with or covalently bonded to a site on the agent, where a detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state during monomer incorporation.
  • the present invention provides a composition
  • a depolymerizing agent including at least one molecular and/or atomic tag located at or near, associated with or covalently bonded to a site on the agent, where a detectable property of the tag undergoes a change before, during and/or after monomer removal.
  • the present invention provides a composition
  • a composition comprising a polymerizing agent including at least one molecular and/or atomic tag located at or near, associated with or covalently bonded to a site on the agent, where a detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state during monomer removal.
  • the present invention provides a composition
  • a composition comprising a polymerase including at least one molecular and/or atomic tag located at or near, associated with or covalently bonded to a site on the polymerase, where a detectable property of the tag undergoes a change before, during and/or after monomer incorporation.
  • the present invention provides a composition
  • a composition comprising a polymerase including at least one molecular and/or atomic tag located at or near, associated with or covalently bonded to a site on the polymerase, where a detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state during monomer incorporation.
  • the present invention provides a composition
  • a composition comprising an exonuclease including at least one molecular and/or atomic tag located at or near, associated with or covalently bonded to a site on the agent, where a detectable property of the tag undergoes a change before, during and/or after monomer removal.
  • the present invention provides a A composition
  • a composition comprising an exonuclease including at least one molecular and/or atomic tag located at or near, associated with or covalently bonded to a site on the agent, where a detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state during monomer removal.
  • the present invention provides a composition comprising an enzyme modified to produce a detectable response prior to, during and/or after interaction with an appropriately modified monomer, where the monomers are nucleotides, nucleotide analogs, amino acids, amino acid analogs, monosaccarides, monosaccaride analogs or mixtures or combinations thereof.
  • the present invention provides a composition
  • a composition comprising a polymerase including at least one molecular tag located at or near, associated with or covalently bonded to a site that undergoes conformational change during monomer incorporation, where the tag has a first detection propensity when the polymerase is in a first conformational state and a second detection propensity when the polymerase is in a second conformational state.
  • the present invention provides a composition
  • a composition comprising a polymerase including at least one chromophore located at or near, associated with or covalently bonded to a site that undergoes conformational change during monomer incorporation, where an intensity and/or frequency of emitted light of the tag has a first value when the polymerase is in a first conformational state and a second value when the polymerase is in a second conformational state.
  • the present invention provides a composition
  • a composition comprising a polymerase including at least one molecular tag located at or near, associated with or covalently bonded to a site that undergoes conformational change during monomer incorporation, where the tag has a first fluorescence propensity when the polymerase is in a first conformational state and a second fluorescence propensity when the polymerase is in a second conformational state.
  • the present invention provides a composition
  • a composition comprising a polymerase including a molecular tag located at or near, associated with or covalently bonded to a site that undergoes conformational change during monomer incorporation, where the tag is substantially active when the polymerase is in a first conformational state and substantially inactive when the polymerase is in a second conformational state or substantially inactive when the polymerase is in the first conformational state and substantially active when the polymerase is in the second conformational state.
  • the present invention provides a composition
  • a composition comprising a polymerizing agent including at least one pair of molecular tags located at or near, associated with or covalently bonded to a site of the agent, where a detectable property of at least one of the tags undergoes a change before, during and/or after monomer incorporation.
  • the present invention provides a composition
  • a composition comprising a polymerizing agent including at least one pair of molecular tags located at or near, associated with or covalently bonded to a site of the agent, where a detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state during monomer incorporation.
  • the present invention provides a composition
  • a depolymerizing agent including at least one pair of molecular tags located at or near, associated with or covalently bonded to a site of the agent, where a detectable property of at least one of the tags undergoes a change before, during and/or after monomer removal.
  • the present invention provides a composition
  • a depolymerizing agent including at least one pair of molecular tags located at or near, associated with or covalently bonded to a site of the agent, where a detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state during monomer removal.
  • the present invention provides a composition
  • a composition comprising a polymerase including at least one pair of molecular tags located at or near, associated with or covalently bonded to a site of the polymerase, where a detectable property of at least one of the tags undergoes a change before, during and/or after monomer incorporation.
  • the present invention provides a composition
  • a composition comprising a polymerase including at least one pair of molecular tags located at or near, associated with or covalently bonded to a site of the polymerase, where a detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state during monomer incorporation.
  • the present invention provides a composition
  • a composition comprising an exonuclease including at least one pair of molecular tags located at or near, associated with or covalently bonded to a site of the polymerase, where a detectable property of at least one of the tags undergoes a change before, during and/or after monomer removal.
  • the present invention provides a composition
  • a composition comprising an exonuclease including at least one pair of molecular tags located at or near, associated with or covalently bonded to a site of the polymerase, where a detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state during monomer removal.
  • the present invention provides a composition
  • a composition comprising a polymerase including at least one pair of molecular tags located at or near, associated with or covalently bonded to a site that undergoes conformational change during monomer incorporation, where the detectable property of the pair has a first value when the polymerase is in a first conformational state and a second value when the polymerase is in a second conformational state.
  • the present invention provides a composition
  • a composition comprising a polymerase including at least one pair of molecules or atoms located at or near, associated with or covalently bonded to a site that undergoes conformational change during monomer incorporation, where the pair interact to form a chromophore when the polymerase is in a first conformational state or a second conformational state.
  • the present invention provides a composition
  • a composition comprising a polymerase including at least one pair of molecular tags located at or near, associated with or covalently bonded to a site that undergoes conformational change during monomer incorporation, where the tags have a first fluorescence propensity when the polymerase is in a first conformational state and a second fluorescence propensity when the polymerase is in a second conformational state.
  • the present invention provides a composition
  • a composition comprising a polymerase including at least one pair of molecular tags located at or near, associated with or covalently bonded to a site that undergoes conformational change during monomer incorporation, where the pair is substantially active when the polymerase is in a first conformational state and substantially inactive when the polymerase is in a second conformational state or substantially inactive when the polymerase is in the first conformational state and substantially active when the polymerase is in the second conformational state.
  • the present invention provides a method for determining when a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of a tag, where the tag is located at or near, associated with or covalently bonded to a site on a polymerase or associated with or covalently bonded to a site on the monomer, where the site undergoes a change during monomer incorporation and where the detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state and cycles from the first value to the second value during each monomer addition.
  • the present invention provides a method for determining when a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of a tag, where the tag is located at or near, associated with or covalently bonded to a site on a polymerase or associated with or covalently bonded to a site on the monomer, where the site undergoes a conformational change during monomer incorporation and where the detectable property has a first value when the polymerase is in a first conformational state and a second value when the polymerase is in a second conformational state and cycles from the first value to the second value during each monomer addition.
  • the present invention provides a method for determining when a monomer is incorporated into a growing molecular chain comprising the steps of exposing a tagged polymerase to light, monitoring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase and/or monomer, where the tagged polymerase comprises a polymerase including a tag located at or near, associated with or covalently bonded to a site that undergoes conformational change during monomer incorporation or associated with or covalently bonded to a site on the monomer and where the tag emits fluorescent light at a first intensity and/or frequency when the polymerase is in a first conformational state and a second intensity and/or frequency when the polymerase is in a second conformational state and cycles from the first value to the second value during each monomer addition.
  • the present invention provides a composition comprising a single-molecule sequencing apparatus comprising a substrate having a chamber or chip surface in which at least one tagged polymerase is confined therein and a plurality of chambers, each of which includes a specific monomer and a plurality of channels interconnecting the chambers, where each replication complex is sufficiently distant to enable data collection from each complex individually.
  • the present invention provides a method for single-molecule sequencing comprising the steps of supplying a plurality of monomers to a tagged polymerase confined on a substrate, exposing the tagged polymerase to light and measuring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase.
  • the method can further comprise the step of relating the measured intensity and/or frequency of emitted fluorescent light to incorporation of a specific monomer into a growing DNA chain.
  • the present invention provides a composition comprising a cooperatively tagged polymerizing agent and tagged monomers, where a detectable property of at least one of the tags changes when the tags interact.
  • the present invention provides a composition comprising a cooperatively tagged depolymerizing agent and tagged depolymerizable monomers, where a detectable property of at least one of the tags changes when the tags interact.
  • the present invention provides a composition comprising a cooperatively tagged polymerase and tagged monomers, where a detectable property of at least one of the tags changes when the tags interact.
  • the present invention provides a composition comprising a cooperatively tagged polymerase and tagged monomers, where a detectable property of at least one of the tags changes when the tag are within within a distance sufficient to cause a change in the intensity and/or frequency of emitted fluorescent light.
  • the present invention provides a composition comprising a tagged polymerase and tagged monomer precursors, where an intensity and/or frequency of fluorescence light emitted by at least one tag changes when the tags interact.
  • the present invention provides a composition comprising a tagged polymerase and a tagged monomer precursors, where an intensity and/or frequency of fluorescence light emitted by at least one tag changes when the tags are within a distance sufficient to cause a change in the intensity and/or frequency of emitted fluorescent light.
  • the present invention provides a single-molecule sequencing apparatus comprising a container having at least one tagged polymerase confined on an interior surface thereof and having a solution containing a plurality of tagged monomers in contact with the interior surface or a subset of tagged monomers and a subset of untagged monomers which together provide all monomers precursor for polymerization.
  • the present invention provides a method for single-molecule sequencing comprising the steps of supplying a plurality of tagged monomers to a tagged polymerase confined on an interior surface of a container, exposing the tagged polymerase to light and measuring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase.
  • the method can further comprise relating the measured intensity and/or frequency of emitted fluorescent light to incorporation of a specific monomer into a growing DNA chain.
  • the present invention provides a system for retrieving stored information comprising: (a) a molecule having a sequence of elements representing a data stream; (b) a single-molecule sequencer comprising a polymerase having at least one tag associated therewith; (c) an excitation source adapted to excite the at least one tag on the polymerase; and (d) a detector adapted to detect a response from the tag on the polymerase or on the monomers; where the response from at least one tag changes during polymerization of a complementary sequence of elements and the change in response represents a data stream content.
  • the present invention provides a system for determining sequence information from a single-molecule comprising: (a) a molecule having a sequence of elements; (b) a single-molecule sequencer comprising a polymerase having at least one tag associated therewith; (c) an excitation source adapted to excite at least one tag on the polymerase or on the monomers; and (d) a detector adapted to detect a response from the tag on the polymerase; where the response from at least one tag changes during polymerization of a complementary sequence of elements representing the element sequence of the molecule.
  • the present invention provides a system for determining sequence information from an individual molecule comprising: (a) a molecule having a sequence of elements; (b) a single-molecule sequencer comprising a polymerase having at least one fluorescent tag associated therewith; (c) an excitation light source adapted to excite the at least one fluorescent tag on the polymerase or on the monomers; and (d) a fluorescent light detector adapted to detect at least an intensity of emitted fluorescent light from the at least one fluorescent tag on the polymerase; where the intensity change of at least one fluorescent tag emits or fails to emit fluorescent light each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.
  • the present invention provides a system for storing and retrieving data comprising: (a) a sequence of nucleotides or nucleotide analogs representing a given data stream; (b) a single-molecule sequencer comprising a polymerase having at least one fluorescent tag covalently attached thereto; (c) an excitation light source adapted to excite at least one fluorescent tag on the polymerase; and (d) a fluorescent light detector adapted to detect emitted fluorescent light from at least one fluorescent tag on the polymerase; where at least one fluorescent tag emits or fails to emit fluorescent light each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.
  • the present invention provides a system for storing and retrieving data comprising: (a) a sequence of nucleotides or nucleotide analogs representing a given data stream; (b) a single-molecule sequencer comprising a polymerase having at least one fluorescent tag covalently attached thereto; (c) an excitation light source adapted to excite the at least one fluorescent tag on the polymerase or the monomers; and (d) a fluorescent light detector adapted to detect emitted fluorescent light from at least one fluorescent tag on the polymerase or the monomers; where at least one fluorescent tag emits or fails to emit fluorescent light each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.
  • the present invention provides a method for sequencing a molecular sequence comprising the steps of: (a) a sequenced of nucleotides or nucleotide analogs representing a given data stream; (b) a single-molecule sequencer comprising a polymerase having at least one fluorescent tag covalently attached thereto; (c) an excitation light source adapted to excite at least one fluorescent tag on the polymerase or the monomers; and (d) a fluorescent light detector adapted to detect emitted fluorescent light from at least one fluorescent tag on the polymerase; where at least one fluorescent tag emits or fails to emit fluorescent light each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.
  • the present invention provides a method for synthesizing a ⁇ -phosphate modified nucleotide comprising the steps of attaching a molecular tag to a pyrophosphate group and contacting the modified pyrophosphate with a dNMP to produce a ⁇ -phosphate tagged dNTP.
  • the present invention provides a method for 5′ end-labeling a biomolecule comprising the step of contacting the biomolecule with a kinase able to transfer a ⁇ -phosphate of a ⁇ -phosphate labeled ATP to the 5′ end of the biomolecule resulting in a covalently modified biomolecule.
  • the present invention provides a method for end-labeling a polypeptide or carbohydrate comprising the step of contacting the polypeptide or carbohydrate with an agent able to transfer an atomic or molecular tag to either a carboxy or amino end of a protein or polypeptide or to either the ⁇ -phosphate of a ⁇ -phosphate labeled ATP to the 5′ end of the biomolecule resulting in a covalently modified biomolecule.
  • FIG. 1 depicts FRET activity as a function of distance separating the fluorescent donor and acceptor
  • FIG. 2 depicts the open and closed ternary complex forms of the large fragment of Taq DNA pol I (Klentaq 1);
  • FIGS. 3 A-C depicts an overlay between 3 ktq (closed ‘black’) and 1 tau (open ‘light blue’), the large fragment of faq DNA polymerase I;
  • FIG. 4 depicts an image of a 20% denaturing polyacryamide gel containing size separated radiolabeled products from DNA extension experiments involving ⁇ -ANS-phosphate-dATP;
  • FIG. 5 depicts an image of (A) the actual gel, (B) a lightened phosphorimage and (C) an enhanced phosphorimage of products generated in DNA extension reactions using ⁇ -ANS-phosphate-dNTPs;
  • FIG. 6 depicts an image of (A) 6% denaturing polyacrylamide gel, (B) a lightened phosphorimage of the actual gel, and (C) an enhanced phosphorimage of the actual gel containing products generated in DNA extension reactions using ⁇ -ANS-phosphate-dNTPs;
  • the inventors have devised a methodology using tagged monomers such as dNTPs and/or tagged polymerizing agents such as polymerase and/or tagged agents associated with the polymerizing agent such as polymerase associated proteins or probes to directly readout the exact monomer sequence such as a base sequence of an RNA or DNA sequence during polymerase activity.
  • the methodology of this invention is adaptable to protein synthesis or to carbohydrate synthesis or to the synthesis of any molecular sequence where the sequence of monomers provides usable information such as the sequence of a RNA or DNA molecule, a protein, a carbohydrate, a mixed biomolecule or an inorganic or organic sequence of monomers which stores a data stream.
  • the methods and apparatuses using these methods are designed to create new ways to address basic research questions such as monitoring conformation changes occurring during replication and assaying polymerase incorporation fidelity in a variety of sequence contexts.
  • the single-molecule detection systems of this invention are designed to improve fluorescent molecule chemistry, computer modeling, base-calling algorithms, and genetic engineering of biomolecules, especially for real-time or near real-time sequencing.
  • the inventors have also found that the methodology can be adapted to depolymerizing agents such as exonucleases where the polymer sequence is determined by depolymerization instead of polymerization.
  • the single-molecule systems of this invention are amendable to parallel and/or massively parallel assays, where tagged polymerases are patterned in arrays on a substrate. The data collected from such arrays can be used to improve sequence confidence and/or to simultaneously sequence DNA regions from many different sources to identify similarities or differences.
  • the pattern of emission signals is collected, either directly, such as by an Intensitifed Charge Coupled Devise (ICCD) or through an intermediate or series of intermediates to amplify signal prior to electronic detection, where the signals are decoded and confidence values are assigned to each base to reveal the sequence complementary to that of the template.
  • ICCD Intensitifed Charge Coupled Devise
  • the present invention also provides techniques for amplifying the fluorescent light emitted from a fluorescent tag using physical light amplification techniques or molecular cascading agent to amplify the light produced by single-molecular fluorescent events.
  • the single-molecule DNA sequencing systems of this invention have the potential to replace current DNA sequencing technologies, because the methodology can decrease time, labor, and costs associated with the sequencing process, and can lead to highly scalable sequencing systems, improving the DNA sequence discovery process by at least one to two orders of magnitude per reaction.
  • the single-molecule DNA sequencing technology of this invention can: (1) make it easier to classify an organism or identify variations within an organism by simply sequencing the genome or a portion thereof; (2) make rapid identification of a pathogen or a genetically-modified pathogen easier, especially in extreme circumstances such as in pathogens used in warfare; and (3) make rapid identification of persons for either law enforcement and military applications easier.
  • One embodiment of the single-molecule sequencing technology of this invention involves strategically positioning a pair of tags on a DNA polymerase so that as a dNTP is incorporated during the polymerization reaction, the tags change relative separation.
  • This relative change causes a change in a detectable property, such as the intensity and/or frequency of fluorescence from one or both of the tags.
  • a time profile of these changes in the detectable property evidences each monomer incorporation event and provides evidence about which particular dNTP is being incorporated at each incorporation event.
  • the pair of tags do not have to be covalently attached to the polymerase, but can be attached to molecules that associate with the polymerase in such a way that the relative separation of the tags change during base incorporation.
  • Another embodiment of the single-molecule sequencing technology of this invention involves a single tag strategically positioned on a DNA polymerase that interacts with a tag on a dNTP or separate tags on each dNTP.
  • the tags could be different for each dNTP such as color-coded tags which emit a different color of fluorescent light.
  • the identity of the base is indicated by a signature fluorescent signal (color) or a change in a fluorescent signal intensity and/or frequency.
  • the rate of polymerase incorporation can be varied and/or controlled to create an essentially “real-time” or near “real-time” or real-time readout of polymerase activity and base sequence. Sequence data can be collected at a rate of >100,000 bases per hour from each polymerase.
  • the tagged polymerases each include a donor tag and an acceptor tag situated or located on or within the polymerase, where the distance between the tags changes during dNTP binding, dNTP incorporation and/or chain extension. This change in inter-tag distance results in a change in the intensity and/or wavelength of emitted fluorescent light from the fluorescing tag. Monitoring the changes in intensity and/or frequency of the emitted light provides information or data about polymerization events and the identity of incorporated bases.
  • the tags on the polymerases are designed to interact with the tags on the dNTPs, where the interaction changes a detectable property of one or both of the tags.
  • Each fluorescently tagged polymerase is monitored for polymerization using tagged dNTPs to determine the efficacy of base incorporation data derived therefrom.
  • Specific assays and protocols have been developed along with specific analytical equipment to measure and quantify the fluorescent data allowing the determination and identification of each incorporated dNTP.
  • the inventors have identified tagged dNTPs that are polymerized by suitable polymerases and have developed software that analyze the fluorescence emitted from the reaction and interpret base identity.
  • appropriate fluorescently active pairs are well-known in the art and commercially available from such vendors as Molecular Probes located in Oregon or Biosearch Technologies, Inc. in Novato, Calif.
  • the tagged DNA polymerase for use in this invention are genetically engineered to provide one or more tag binding sites that allow the different embodiments of this invention to operate.
  • a suitable polymerase candidate is identified, specific amino acids within the polymerase are mutated and/or modified such reactions well-known in the art; provided, however, that the mutation and/or modification do not significantly adversely affect polymerization efficiency.
  • the mutated and/or modified amino acids are adapted to facilitate tag attachment such as a dye or fluorescent donor or acceptor molecule in the case of light activated tags.
  • the engineered polymerase can be contacted with one or more appropriate tags and used in the apparatuses and methods of this invention.
  • the single-molecule DNA sequencing system of this invention can significantly reduce time, labor, and costs associated with the sequencing process and is highly scalable.
  • the single-molecule DNA sequencing system of this invention (1) can improve the sequence discovery process by at least two orders of magnitude per reaction; (2) is not constrained by the length limitations associated with the degradation-based, single-molecule methods; and (3) allows direct sequencing of desired (target) DNA sequences, especially genomes without the need for cloning or PCR amplification, both of which introduce errors in the sequence.
  • the systems of this invention can make easier the task of classifying an organism or identifying variations within an organism by simply sequencing the genome in question or any desired portion of the genome.
  • the system of this invention is adapted to rapidly identify pathogens or engineered pathogens, which has importance for assessing health-related effects, and for general DNA diagnostics, including cancer detection and/or characterization, genome analysis, or a more comprehensive form of genetic variation detection.
  • the single-molecule DNA sequencing system of this invention can become an enabling platform technology for single-molecule genetic analysis.
  • the single-molecule sequencing systems of this invention have the following advantages: (1) the systems eliminates sequencing reaction processing, gel or capillary loading, electrophoresis, and data assembly; (2) the systems results in significant savings in labor, time, and costs; (3) the systems allows near real-time or real-time data acquisition, processing and determination of incorporation events (timing, duration, etc.), base sequence, etc.; (4) the systems allows parallel or massively parallel sample processing in microarray format; (5) the systems allows rapid genome sequencing, in time frames of a day or less; (6) the systems requires very small amount of material for analysis; (7) the systems allows rapid genetic identification, screening and characterization of animals including humans or pathogen; (8) the systems allows large increases in sequence throughput; (9) the system can avoid error introduced in PCR, RT-PCR, and transcription processes; (10) the systems can allow accurate sequence information for allele-specific mutation detection; (11) the systems allows rapid medical diagnostics, e.g., Single Nucleotide Polymorphism (SNP) detection; (12) the systems allows improvement
  • a single tag is attached to an appropriate site on a polymerase and a unique tag is attached to each of the four nucleotides: dATP, dTTP, dCTP and dGTP.
  • the tags on each dNTPs are designed to have a unique emission signature (i.e., different emission frequency spectrum or color), which is directly detected upon incorporation.
  • a characteristic fluorescent signal or base emission signature is emitted due to the interaction of polymerase tag and the dNTP tag.
  • the fluorescent signals i.e., the emission intensity and/or frequency, are then detected and analyzed to determine DNA base sequence.
  • One criteria for selection of the tagged polymerase and/or dNTPs for use in this invention is that the tags on either the polymerase and/or the dNTPs do not interfere with Watson-Crick base-pairing or significantly adversely impact polymerase activity.
  • the inventors have found that dNTPs containing tags attached to the terminal (gamma) phosphate are incorporated by a native Taq polymerase either in combination with untagged dNTPs or using only tagged dNTPs. Tagging the dNTPs on the ⁇ and/or ⁇ phosphate group is preferred because the resulting DNA strands do not include any of the dNTP tags in their molecular make up, minimizing enzyme distortion and background fluorescence.
  • One embodiment of the sequencing system of this invention involves placing a fluorescent donor such as fluorescein or a fluorescein-type molecule on the polymerase and unique fluorescent acceptors such as a d-rhodamine or a similar molecule on each dNTP, where each unique acceptor, when interacting with the donor on the polymerase, generates a fluorescent spectrum including at least one distinguishable frequency or spectral feature.
  • a fluorescent donor such as fluorescein or a fluorescein-type molecule
  • unique fluorescent acceptors such as a d-rhodamine or a similar molecule on each dNTP
  • Another embodiment of the sequencing system of this invention involves a fluorescent tag on the polymerase and unique quenchers on the dNTPs, where the quenchers preferably have distinguishable quenching efficiencies for the polymerase tag. Consequently, the identity of each incoming quencher tagged dNTP is determined by its unique quenching efficiency of the emission of the polymerase fluorescent tag. Again, the signals produced during incorporation are detected and analyzed to determine each base incorporated, the sequence of which generates the DNA base sequence.
  • Suitable polymerizing agents for use in this invention include, without limitation, any polymerizing agent that polymerizes monomers relative to a specific template such as a DNA or RNA polymerase, reverse transcriptase, or the like or that polymerizes monomers in a step-wise fashion.
  • Suitable polymerases for use in this invention include, without limitation, any polymerase that can be isolated from its host in sufficient amounts for purification and use and/or genetically engineered into other organisms for expression, isolation and purification in amounts sufficient for use in this invention such as DNA or RNA polymerases that polymerize DNA, RNA or mixed sequences, into extended nucleic acid polymers.
  • Preferred polymerases for use in this invention include mutants or mutated variants of native polymerases where the mutants have one or more amino acids replaced by amino acids amenable to attaching an atomic or molecular tag, which have a detectable property.
  • Exemplary DNA polymerases include, without limitation, HIV1-Reverse Transcriptase using either RNA or DNA templates, DNA pol I from T. aquaticus or E. coli , Bateriophage T4 DNA pol, T7 DNA pol or the like.
  • Exemplary RNA polymerases include, without limitation, T7 RNA polymerase or the like.
  • Suitable depolymerizing agents for use in this invention include, without limitation, any depolymerizing agent that depolymerizes monomers in a step-wise fashion such as exonucleases in the case of DNA, RNA or mixed DNA/RNA polymers, proteases in the case of polypeptides and enzymes or enzyme systems that sequentially depolymerize polysaccharides.
  • Suitable monomers for use in this invention include, without limitation, any monomer that can be step-wise polymerized into a polymer using a polymerizing agent.
  • Suitable nucleotides for use in this invention include, without limitation, naturally occurring nucleotides, synthetic analogs thereof, analog having atomic and/or molecular tags attached thereto, or mixtures or combinations thereof.
  • Suitable atomic tag for use in this invention include, without limitation, any atomic element amenable to attachment to a specific site in a polymerizing agent or DNTP, especially Europium shift agents, nmr active atoms or the like.
  • Suitable atomic tag for use in this invention include, without limitation, any atomic element amenable to attachment to a specific site in a polymerizing agent or dNTP, especially fluorescent dyes such as d-Rhodamine acceptor dyes including dichloro[R110], dichloro[R6G], dichloro[TAMRA], dichloro [ROX] or the like, fluorescein donor dye including fluorescein, 6-FAM, or the like; Acridine including Acridine orange, Acridine yellow, Proflavin, pH 7, or the like; Aromatic Hydrocarbon including 2-Methylbenzoxazole, Ethyl p-dimethylaminobenzoate, Phenol, Pyrrole, benzene, toluene, or the like; Arylmethine Dyes including Auramine O, Crystal violet, H2O, Crystal violet, glycerol, Malachite Green or the like; Coumarin dyes including 7-Methoxycoumarin-4-acetic
  • Taq DNA polymerase I Thermus aquaticus —is ideally suited for use in the single-molecule apparatuses, systems and methods of this invention.
  • Taq DNA Polymerase sometimes simply referred to herein as Taq, has many attributes that the inventors can utilize in constructing tagged polymerases for use in the inventions disclosed in this application.
  • ordinary artisans will recognize that other polymerases can be adapted for use in the single-molecule sequencing systems of this invention.
  • Taq DNA polymerase I tolerates so many mutations within or near its active site (as reviewed in Patel et al, J. Mol Biol., volume 308, pages 823-837, and incorporated herein by reference), the enzyme is more tolerant of enzyme tagging modification(s) and also able to incorporate a wider range of modified nucleotide substrates.
  • the program compares structural data associated with the Taq polymerase in its open and closed form to identify regions in the polymerase structure that are optimally positioned to optimize the difference in conformation extremes between a tag on the polymerase and the dNTP or to optimize a change in separation between two tags on the polymerase, thereby increasing or maximizing changes in a detectable property of one of the tags or tag pair.
  • Taq DNA Polymerase is Efficiently Expressed in E. coli
  • the Taq DNA polymerase is efficiently expressed in E. coli allowing efficient production and purification of the nascent polymerase and variants thereof for rapid identification, characterization and optimization of an engineered Taq DNA polymerase for use in the single-molecule DNA sequencing systems of this invention.
  • the Taq DNA polymerase contains no cysteines, which allows the easy generation of cysteine-containing mutants in which a single cysteine is placed or substituted for an existing amino acid at strategic sites, where the inserted cysteine serves as a tag attachment site.
  • native Taq DNA polymerase may not represent an optimal polymerase for sequencing system of this invention because it is not a very processive polymerase (50-80 nucleotides are incorporated before dissociation), the low processivity may be compensated for by appropriately modifying the base calling software.
  • the processivity of the Taq DNA Polymerase can be enhanced through genetic engineering by inserting into the polymerase gene a processivity enhancing sequence. Highly processive polymerases are expected to minimize complications that may arise from template dissociation effects, which can alter polymerization rate.
  • the processivity of Taq can be genetically altered by introducing the 76 amino acid ‘processivity domain’ from T7 DNA polymerase between the H and H, helices (at the tip of ‘thumb’ region within the polymerase) of Taq.
  • the processivity domain also includes the thioredoxin binding domain (TBD) from T7 DNA polymerase causing the Taq polymerase to be thioredoxin-dependent increasing both the processivity and specific activity of Taq polymerase.
  • TBD thioredoxin binding domain
  • Taq DNA Polymerase Possesses a 5′ to 3′ Exonuclease Activity and is Thermostable
  • Single-stranded M13 DNA and synthetic oligonucleotides are used in the initial studies. After polymerase activity is optimized, the sequencing system can be used to directly determine sequence information from an isolated chromosome —a double-stranded DNA molecule. Generally, heating a sample of double-stranded DNA is sufficient to produce or maintain the double-stranded DNA in stranded DNA form for sequencing.
  • the 5′ to 3′ exonuclease activity of the native Taq DNA polymerase in the enzyme engineered for single-molecule DNA sequencing is retained. This activity of the polymerase is exploited by the ‘TaqMan’ assay.
  • the exonuclease activity removes a duplex strand that may denature downstream from the replication site using a nick-translation reaction mechanism. Synthesis from the engineered polymerase is initiated either by a synthetic oligonucleotide primer (if a specific reaction start is necessary) or by a nick in the DNA molecule (if multiple reactions are processed) to determine the sequence of an entire DNA molecule.
  • the Polymerase is Free from 3′ to 5′ Exonuclease Activity
  • the Taq DNA polymerase is does not contain 3′ to 5′ exonuclease activity, which means that the polymerase cannot replace a base, for which fluorescent signal was detected, with another base which would produce another signature fluorescent signal.
  • the error rate of engineered polymerases of this invention are assayed by determining their error rates in synthesizing known sequences.
  • the error rate determines the optimal number of reactions to be run in parallel so that sequencing information can be assigned with confidence.
  • the optimal number can be 1 or 10 or more.
  • base context influences polymerase accuracy and reaction kinetics, and this information is used to assign confidence values to individual base calls.
  • Taq DNA Polymerase is the Enzyme of Choice for Single-molecule DNA Sequencing
  • FRET-based fluorescence resonance energy transfer-based
  • a FRET-based method exists when the emission from an acceptor is more intense than the emission from a donor, i.e., the acceptor has a higher fluorescence quantum yield than the donor at the excitation frequency.
  • the efficiency of FRET method can be estimated form computational models. See, e.g., Furey et al., 1998; Clegg et al., 1993; Mathies et al., 1990.
  • the efficiency of energy transfer (E) is computed from the equation (1):
  • R 0 is calculated from equation (2):
  • ⁇ 2 is a geometric orientation factor related to the relative angle of the two transition dipoles ( ⁇ 2 is generally assumed to be 2 ⁇ 3)
  • J DA [M ⁇ 1 cm 3 ] is the overlap integral representing the normalized spectral overlap of the donor emission and acceptor absorption
  • Q D is the quantum yield.
  • the overlap integral is computed from equation (3):
  • I D and I RF are the fluorescence intensities of donor and a reference compound (fluorescein in 0.1 N NaOH), and A RF and A D are the absorbances of the reference compound and donor.
  • Q RF is the quantum yield of fluorescein in 0.1N NaOH and is taken to be 0.90.
  • R the distance between the donor and acceptor
  • R is measured by looking at different configurations (e.g., conformations) of the polymerase in order to obtain a conformationally averaged value. If both tags are on the polymerase, then R is the distance between the donor and acceptor in the open and closed conformation, while if the donor is on the polymerase and the acceptor on the dNTP, R is the distance between the donor and acceptor when the dNTP is bound to the polymerase and the polymerase is its closed form.
  • configurations e.g., conformations
  • Dye sets are chosen to maximize energy transfer efficiency between a tagged dNTP and a tag on the polymerase when the polymerase is in its closed configuration and to minimize energy transfer efficiency between the tag on the DNTP (either non-productively bound or in solution) and the tag on the polymerase when the polymerase is in its open configuration.
  • a molarity of each nucleotide in the reaction medium of no more than about 1 ⁇ M
  • an average distance between tagged nucleotides is calculated to be greater than or equal to about 250 ⁇ . Because this distance is several fold larger than the distance separating sites on the polymerase in its open to closed conformational, minimal FRET background between the polymerase and free dNTPs is observed.
  • nucleotide concentrations are reduced below 1 ⁇ M.
  • Reducing dNTP concentrations to levels of at least ⁇ 10% of the K m further minimizes background fluorescence and provides a convenient method for controlling the rate of the polymerase reaction for the real-time monitoring. Under such conditions, the velocity of the polymerization reaction is linearly proportional to the dNTP concentration and, thus, highly sensitive to regulation.
  • the use of a single excitation wavelength allows improved identification of unique tags on each DNTP. A single, lower-wavelength excitation laser is used to achieve high selectivity.
  • a fluorescence donor is attached to a site on the polymerase comprising a replaced amino acid more amenable to donor attachment such as cysteine and four unique fluorescence acceptors are attached to each dNTP.
  • fluorescein is attached to a site on the polymerase and rhodamine, rhodamine derivatives and/or fluorescein derivatives are attached to each dNTP.
  • Each donor-acceptor fluorophore pair is designed to have an absorption spectra sufficiently distinct from the other pairs to allow separate identification after excitation.
  • the donor is selected such that the excitation light activates the donor, which then efficiency transfers the excitation energy to one of the acceptors.
  • the acceptor After energy transfer, the acceptor emits it unique fluorescence signature.
  • the emission of the fluorescence donor must significant overlap with the absorption spectra of the fluorescence acceptors for efficient energy transfer.
  • the methods of this invention can also be performed using two, three or four unique fluorescence donor-acceptor pairs, by running parallel reactions.
  • Fluorophore choice is a function of not only its enzyme compatibility, but also its spectral and photophysical properties. For instance, it is critical that the acceptor fluorophore does not have any significant absorption at the excitation wavelength of the donor fluorophore, and less critical (but also desirable) is that the donor fluorophore does not have emission at the detection wavelength of the acceptor fluorophore. These spectral properties can be attenuated by chemical modifications of the fluorophore ring systems.
  • the dNTPs are amenable to tagging at several sites including the base, the sugar and the phosphate groups, the dNTPs are preferably tagged at either the ⁇ and/or ⁇ phosphate. Tagging the terminal phosphates of dNTP has a unique advantage. When the incoming, tagged dNTP is bound to the active site of the polymerase, significant FRET from the donor on the polymerase to the acceptor on the dNTP occurs. The unique fluorescence of the acceptor identifies which dNTP is incorporated.
  • the fluorescence acceptor which is now attached to the pyrophosphate group, is released to the medium with the cleaved pyrophosphate group.
  • the growing DNA chain includes no fluorescence acceptor molecules at all.
  • FRET occurs only between the donor on the polymerase and incoming acceptor-labeled dNTP, one at a time. This approach is better than the alternative attachment of the acceptor to a site within the dNMP moiety of the dNTP or the use of multiply-modified dNTPs. If the acceptor is attached to a site other than the ⁇ or ⁇ phosphate group, it becomes part of the growing DNA chain and the DNA chain will contain multiple fluorescence acceptors. Interference with the polymerization reaction and FRET measurements would likely occur.
  • collisional quenchers can be added to the polymerizing medium that do not covalently interact with the acceptors on the dNTPs and quench fluorescence from the tagged dNTPs in the medium.
  • the quenchers are also adapted to have insignificant contact with the donor on the polymerase.
  • the polymerase tag is preferably localized internally and shielded from the collisional quenchers or the collisional quencher can be made sterically bulky or associate with a sterically bulky group to decrease interaction between the quencher and the polymerase.
  • Another preferred method for detecting polymerase-nucleotide interactions involves using nucleotide-specific quenching agents to quench the emission of a fluorescent tag on the polymerase.
  • the polymerase is tagged with a fluorophore, while each dNTP is labeled with a quencher for the fluorophore.
  • DABCYL 4-(4′-dimethylaminophenylazo) benzoic acid is a universal quencher, which absorbs energy from a fluorophore, such as 5-(2′-aminoethyl) aminonaphthalene-1-sulfonic acid (AEANS) and dissipates heat.
  • AEANS 5-(2′-aminoethyl) aminonaphthalene-1-sulfonic acid
  • a quencher is selected for each dNTP so that when each quencher is brought into close proximity to the fluorophore, a distinguishable quenching efficiency is obtained. Therefore, the degree of quenching is used to identify each dNTP as it is being incorporated into the growing DNA chain.
  • One advantage of this preferred detection method is that fluorescence emission comes from a single source rendering background noise negligible.
  • two or three suitable quenchers are identified, then two or three of the four dNTPs are labeled and a series of polymerization reaction are made each time with a different pair of the labeled dNTPs. Combining the results from these runs, generates a complete sequence of the DNA molecule.
  • the present invention is directed to attaching any type of atomic and/or molecular tag that has a detectable property
  • the processes for site selection and tag attachment are illustrated using a preferred class of tags, namely fluorescent tags.
  • the fluorescence probes or quenchers attached to the polymerase or dNTPs are designed to minimize adverse effects on the DNA polymerization reaction.
  • the inventors have developed synthetic methods for chemically tagging the polymerase and dNTPs with fluorescence probes or quenchers.
  • the polymerase is tagged by replacing a selected amino acid codon in the DNA sequence encoding the polymerase with a codon for an amino acid that more easily reacts with a molecular tag such as cysteine via mutagenesis.
  • a molecular tag such as cysteine via mutagenesis.
  • the mutant is inserted into E. coli for expression.
  • the mutant polymerase is isolated and purified.
  • the purified mutant polymerase is then tested for polymerase activity.
  • the mutant polymerase is reacted with a slight molar excess of a desired tag to achieve near stoichiometric labeling.
  • the polymerase can be treated with an excess amount of the tag and labeling followed as a function of time. The tagging reaction is than stopped when near stoichiometric labeling is obtained.
  • the mutant polymerase includes several sites including the target residue that can undergo tagging with the desired molecular tag
  • the tagging reaction can also be carried out under special reaction conditions such as using a protecting group or competitive inhibitor and a reversible blocking group, which are later removed.
  • a protecting group or competitive inhibitor is first added to protect the target residue and a reversible blocking group is subsequently added to inactivate non-target residues.
  • the protecting group or competitive inhibitor is then removed from the target residue, and the mutant polymerase is treated with the desired tag to label the target residue.
  • the blocking groups are chemically removed from non-target residues in the mutant polymerase and removed to obtain a tagged mutant polymerase with the tag substantially to completely isolated on the target residue.
  • the polymerase can be treated with a blocking group to inactivate non-target residues. After removal of unreacted blocking group, the mutant polymerase is treated with the desired tag for labeling the target residue. Finally, the blocking groups are chemically removed from the non-target residues in the mutant polymerase and removed to obtain the tagged mutant polymerase.
  • the inventors have identified amino acids in the Taq polymerase that are likely to withstand mutation and subsequent tag attachment such as the attachment of a fluorescent tag. While many sites are capable of cysteine replacement and tag attachment, preferred sites in the polymerase were identified using the following criteria: (1) they are not in contact with other proteins; (2) they do not alter the conformation or folding of the polymerase; and (3) they are not involved in the function of the protein. The selections were accomplished using a combination of mutational studies including sequence analyses data, computational studies including molecular docking data and assaying for polymerase activity and fidelity. After site mutation, computational studies will be used to refine the molecular models and help to identify other potential sites for mutation.
  • Regions of the protein surface that are not important for function were identified, indirectly, by investigating the variation in sequence as a function of evolutionary time and protein function using the evolutionary trace method. See, e.g., Lichtarge et al., 1996. In this approach, amino acid residues that are important for structure or function are found by comparing evolutionary mutations and structural homologies.
  • the polymerases are ideal systems for this type of study, as there are many crystal and co-crystal structures and many available sequences. The inventors have excluded regions of structural/functional importance from sites selection for mutation/labeling.
  • visual inspection and overlays of available crystal structures of the polymerase in different conformational states provided further assistance in identifying amino acid sites near the binding site for dNTPs.
  • Some of the chosen amino acids sites are somewhat internally located and preferably surround active regions in the polymerase that undergo changes during base incorporation, such as the dNTP binding regions, base incorporation regions, pyrophosphate release regions, etc. These internal sites are preferred because a tag on these sites show reduced background signals during detection, i.e., reduce interaction between the polymerase enzyme and non-specifically associated tagged dNTPs, when fluorescently tagged dNTPs are used.
  • Another aspect of this invention involves the construction of molecular mechanics force field parameters for atomic and/or molecular tags such as fluorescent tags used to tag the dNTPs and the polymerase and parameters for the fluorescent tagged amino acid on the polymerase and/or dNTP.
  • Force field parameters are using quantum mechanical studies to obtain partial charge distributions and energies for relevant intramolecular conformations (i.e., for the dihedral angle definitions) derived from known polymerase crystal structures.
  • Ionization states of each ionizable residue are estimated using an electrostatic model in which the protein is treated as a low dielectric region and the solvent as a high dielectric, using the UHBD program. See, e.g., Antosiewicz et al., 1994; Briggs and Antosiewicz, 1999; Madura et al., 1995.
  • the electrostatic free energies of ionization of each ionizable residue are computed by solving the Poisson-Boltzmann equation for each residue. These individual ionization free energies are modified to take into account coupled titration behavior resulting in a set of self-consistent predicted ionization states.
  • an electrostatic potential map is generated from properties of the molecular surface of the Taq polymerase/DNA complex, screened by solvent and, optionally, by dissolved ions (i.e., ionic strength) using mainly the UHBD program.
  • the map provides guidance about binding locations for the dNTPs and the electrostatic environment at proposed mutation/labeling sites.
  • the molecular models generated are designed to be continually refined taking into account new experimental data, allowing the construction of improved molecular models, improved molecular dynamics calculations and improved force field parameters so that the models better predict system behavior for refining tag chemistry and/or tag positioning, predicting new polymerase mutants, base incorporation rates and polymerase fidelity.
  • Co-crystal structures solved for DNA polymerase I (DNA pol I) from E. coli, T. aquaticus, B. stearothermophilus , T7 bacteriophage, and human pol ⁇ demonstrate that (replicative) polymerases share mechanistic and structural features.
  • the structures that capture Taq DNA polymerase in an ‘open’ (non-productive) conformation and in a ‘closed’ (productive) conformation are of particular importance for identifying regions of the polymerase that undergo changes during base incorporation.
  • the addition of the nucleotide to the polymerase/primer/template complex is responsible for the transition from its open to its closed conformation.
  • the open and closed ternary complex forms of the large fragment of Taq DNA pol I (Klentaq 1) are shown in a superimposition of their C ⁇ tracings.
  • the ternary complex contains the enzyme, the ddCTP and the primer/template duplex DNA.
  • the open structure is shown in magenta and the closed structure is shown in yellow.
  • the disorganized appearance in the upper left portion of the protein shows movement of the ‘fingers’ domain in open and closed conformations.
  • amino acids represent preferred amino acid sites for cysteine replacement and subsequent tag attachment, because these sites represent the sites in the Taq polymerase the undergo significant changes in position during base incorporation.
  • amino acid site selection visualization of the polymerase in its open and closed conformational extremes for these identified amino acid sites is used so that the final selected amino acid sites maximize signal and minimize background noise, when modified to carry fluorescent tags for analysis using the FRET methodology.
  • Amino acid changes that are not predicted to significantly affect the protein's secondary structure or activity make up a refined set of amino acid sites in the Taq polymerase for mutagenesis and fluorescent modification so that the tag is shielded from interaction with free dNTPs.
  • the following three panels illustrate the protocol used in this invention to refine amino acid site selection from the about list of amino acids that undergo the largest change in position relative to a bound ddGTP as the polymerase transitions from the open to the closed form.
  • FIGS. 3 A-C an overlay between 3 ktq (closed ‘black’) and 1 tau (open ‘light blue’), the large fragment of Taq DNA polymerase I is shown.
  • the bound DNA from 3 ktq is shown in red while the ddCTP bound to 3 ktq is in green.
  • Three residues were visually identified as moving the most when the polymerase goes from open (1 tau) to closed (3 ktq), namely, Asp655, Pro656, and Leu657. Based on further analyses of the structures, Pro656 appears to have the role of capping the O-helix. Leu657's side chain is very close to another part of the protein in the closed (3 ktq) form.
  • FIG. 3B a close-up view of the active site from the overlay of the 3 ktq (closed) and 1 tau (open) conformations of Taq polymerase is shown. The large displacements between the open and closed conformations are evident.
  • FIG. 3C a close-up view of a molecular surface representation of 3 ktq (in the absence of DNA and ddCTP). The molecular surface is colored in two areas, blue for Asp655 and green for Leu657.
  • Leu657 is in close proximity to another part of the protein, because the green part of the molecular surface, in the thumb domain, is “connected” to a part of the fingers domain.
  • This view shows this region of the polymerase looking into the palm of the hand with fingers to the right and thumb to the left.
  • the gene encoding Taq DNA polymerase was obtained and will be expressed in pTTQ 18 in E. coli strain DH1. See, e.g., Engelke et al., 1990.
  • the inventors have identified candidate amino acids for mutagenesis including the amino acids in Tables I-IV, the refined lists or mixtures or combinations thereof.
  • the inventors using standard molecular methods well-known in the art introduced a cysteine codon, individually, at each of target amino acid sites. See, e.g., Sambrook et al., 1989 and Allen et al., 1998.
  • DNA is purified from isolated colonies expressing the mutant polymerase, sequenced using dye-terminator fluorescent chemistry, detected on an ABI PRISM 377 Automated Sequencer, and analyzed using SequencherTM available from GeneCodes, Inc.
  • the inventors have demonstrated that the Taq polymerase is capable of incorporating ⁇ -tagged dNTPs to synthesize extended DNA sequences.
  • the next step involves the construction of mutants capable of carrying a tag designed to interact with the tags on the dNTPS and optimization of the polymerase for single-molecule sequencing.
  • the mutants are constructed using standard site specific mutagenesis as described above and in the experimental section.
  • the constructs are then inserted into and expressed in E. coli .
  • Mutant Taq polymerase is then obtained after sufficient E. coli is grown for subsequence polymerase isolation and purification.
  • E. coli can be grown to optical densities exceeding 100 by computer-controlled feedback-based supply of non-fermentation substrates, the resulting three kg of E. coli cell paste will be excessive during polymerase optimization.
  • optimized polymerases construct are prepared, then this large scale production will be used.
  • the mutants are derived from E. coli cell masses grown in 10 L well-oxygenated batch cultures using a rich medium available from Amgen.
  • the mutants are prepared by growing E. coli in 2 L baffled shake glasses. Cell paste are then harvested using a 6 L preparative centrifuge, lysed by French press, and cleared of cell debris by centrifugation. To reduce interference from E.
  • nucleic acid sequences it is preferably to also remove other nucleic acids. Removal is achieved using either nucleases (and subsequent heat denaturation of the nuclease) or, preferably using a variation of the compaction agent-based nucleic acid precipitation protocol as described in Murphy et al., Nature Biotechnology 17, 822, 1999.
  • a single anion-exchange step typically on Q Sepharose at pH 8.0, is generally sufficient to produce a product pure enough to these tests.
  • a second purification step will also be performed to insure that contamination does not cloud the results of subsequent testing.
  • the second purification step involves SDS-PAGE and CD-monitored melting experiments.
  • the dNTPs are tagged either by reacting a dNTP with a desired tag or by reacting a precursor such as the pyrophosphate group or the base with a desired tag and then completing the synthesis of the dNTP.
  • the inventors have developed syntheses for modifying fluorophore and fluorescence energy transfer compounds to have distinct optical properties for differential signal detection, for nucleotide/nucleoside synthons for incorporation of modifications on base, sugar or phosphate backbone positions, and for producing complementary sets of four deoxynucleotide triphosphates (dNTPs) containing substituents on nucleobases, sugar or phosphate backbone.
  • dNTPs deoxynucleotide triphosphates
  • the native Taq polymerase is capable of polymerizing phosphate-modified dNTPs or ddNTPs. Again, tagging the dNPTs or ddNTPs at the beta and/or gamma phosphate groups is a preferred because the replicated DNA contains no unnatural bases, polymerase activity is not significantly adversely affected and long DNA strands are produced.
  • the inventors have synthesized ⁇ -ANS-phosphate dNTPs, where the ANS is attached to the phosphate through a phosphamide bond. Although these tagged dNTPs are readily incorporated by the native Taq polymerase and by HIV reverse transcriptase, ANS is only one of a wide range of tags that can be attached through either the ⁇ and/or ⁇ phosphate groups.
  • the present invention uses tagged dNTPs or ddNTPs in combination with polymerase for signal detection.
  • the dNTPs are modified at phosphate positions (alpha, beta and/or gamma) and/or other positions of nucleotides through a covalent bond or affinity association.
  • the tags are designed to be removed from the base before the next monomer is added to the sequence.
  • One method for removing the tag is to place the tag on the gamma and/or beta phosphates.
  • the tag is removed as pyrrophosphate dissociates from the growing DNA sequence.
  • Another method is to attach the tag to a position of on the monomer through a cleavable bond.
  • the tag is then removed after incorporation and before the next monomer incorporation cleaving the cleavable bond using light, a chemical bond cleaving reagent in the polymerization medium, and/or heat.
  • FR is a fluorescent tag
  • L is a linker group
  • X is either H or a counterioin depending on the pH of the reaction medium
  • Z is a group capable of reaction with the hydroxyl group of the pyrophosphate and Z′ is group after reaction with the dNMP.
  • Z is Cl, Br, I, OH, SH, NH 2 , NHR, CO 2 H, CO 2 R, SiOH, SiOR, GeOH, GeOR, or similar reactive functional groups, where R is an alkyl, aryl, aralkyl, alkaryl, halogenated analogs thereof or hetero atom analogs thereof and Z′ is O, NH, NR, CO 2 , SiO, GeO, where R is an alkyl, aryl, aralkyl, alkaryl, halogenated analogs thereof or hetero atom analogs thereof .
  • the synthesis involves reacting Z terminated fluorescent tag, FR-L-Z with a pyrophosphate group, P 2 O 6 X 3 H, in DCC and dichloromethane to produce a fluorescent tagged pyrophosphate.
  • a fluorescent tagged pyrophosphate is prepared, it is reacted with a morpholine terminated dNMP in acidic THF to produce a dNTP having a fluorescent tag on its ⁇ -phosphate. Because the final reaction bears a fluorescent tag and is larger than starting materials, separation from unmodified starting material and tagged pyrophosphate is straight forward.
  • Fluorescein (FR) is first reacted with isobutyryl anhydride in pyridine in the presence of diisopropyl amine to produce a fluorescein having both ring hydroxy groups protected for subsequent linker attachment.
  • the hydroxy protected fluorescein is then reacted with N-hydroxylsuccinimide in DCC and dichloromethane to produce followed by the addition of 1-hydroxy-6-amino hexane to produce an hydroxy terminated FR-L group.
  • This group can then be reacted either with pyrophosphate to tag the dNTPs at their ⁇ -phosphate group or to tag amino acids. See, e.g., Ward et al., 1987; Engelhardt et al., 1993; Little et al., 2000; Hobbs, 1991.
  • tags can be designed so that each tag emits a distinguishable emission spectra.
  • the emission spectra can be distinguished by producing tags with non-overlapping emission frequencies—multicolor—or each tag can have a non-overlapping spectral feature such a unique emission band, a unique absorption band and/or a unique intensity feature.
  • System that use a distinguishable tag on each dNTP improves confidence values associated with the base calling algorithm.
  • fluorescein is adaptable to other dyes as well such as tetrachlorofluorescein (JOE) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA).
  • JE tetrachlorofluorescein
  • TAMRA N,N,N′,N′-tetramethyl-6-carboxyrhodamine
  • the gamma phosphate tagged reactions are carried out in basic aqueous solutions and a carbodiimide, such as DEC.
  • DEC carbodiimide
  • Other fluorophore molecules and dNTPs can be similarly modified.
  • the dNTPs can also be tagged on the base and/or sugar moieties while maintaining their polymerase reaction activity.
  • the sites for modifications are preferably selected to not interfere with Watson-Crick base pairing.
  • a generalized scheme for base modification is shown below:
  • polymerase variants The activities of polymerase variants are monitored throughout polymerase development. Polymerase activity is assayed after a candidate amino acid is mutated to cysteine and after fluorescent tagging of the cysteine.
  • the assay used to monitor the ability of the native Taq polymerase to incorporate fluorescently-tagged dNTPs is also used to screen polymerase variants. Since the mutant Taq polymerases have altered amino acid sequences, the assays provide mutant characterization data such as thermostability, fidelity, polymerization rate, affinity for modified versus natural bases.
  • Mutant Taq polymerase activity assays are carried out under conditions similar to those used to examine the incorporation of fluorescently-tagged dNTPs into DNA polymers by the native Taq polymerase.
  • the purified mutant Taq polymerase is incubated in polymerase reaction buffer with a 5′- 32 P end-labeled primer/single-stranded template duplex, and appropriate tagged dNTP(s).
  • the polymerase's ability to incorporate a fluorescently-tagged dNTP is monitored by assaying the relative amount of fluorescence associated with the extended primer on either an AB1377 DNA Sequencer (for fluorescently tagged bases), a Fuji BAS1000 phosphorimaging system, or other appropriate or similar detectors or detection systems. This assay is used to confirm that the mutant polymerase incorporates tagged DNT ⁇ and to confirm that fluorescent signatures are obtained during base incorporation. These assays use an end-labeled primer, the fluorescently-tagged dNT ⁇ and the appropriate base beyond the fluorescent tag. The products are then size separated and analyzed for extension. Reactions are either performed under constant temperature reaction conditions or thermocycled, as necessary.
  • Taq DNA polymerase to incorporate a ⁇ -phosphate dNTP variant is assayed using conditions similar to those developed to examine single base incorporation by a fluorescently-tagged DNA polymerase. See, e.g. Furey et al., 1998. These experiments demonstrate that polymerases bearing a fluorescent tag do not a priori have reduced polymerization activity. The inventors have demonstrated that the native Taq polymerase incorporates ⁇ -tagged dNTP, singly or collectively to produce long DNA chains.
  • polymerase reaction buffer such as Taq DNA polymerase buffer available from Promega Corporation of Madison, Wisconsin with either a 5′- 32 P or a fluorescently end-labeled primer (TOP)/single-stranded template (BOT-‘X’) duplex, and appropriate dNTP(s) as shown in Table V. Reactions are carried out either at constant temperature or thermocycled, as desired or as is necessary. Reaction products are then size-separated and quantified using a phosphorimaging or fluorescent detection system. The relative efficiency of incorporation for each tagged dNTP is determined by comparison with its natural counterpart.
  • polymerase reaction buffer such as Taq DNA polymerase buffer available from Promega Corporation of Madison, Wisconsin with either a 5′- 32 P or a fluorescently end-labeled primer (TOP)/single-stranded template (BOT-‘X’) duplex, and appropriate dNTP(s) as shown in Table V. Reactions are carried out either at constant temperature or thermocycled,
  • ‘TOP’ represents the primer strand of an assay DNA duplex. Variants of the template strand are represented by ‘BOT’. The relevant feature of the DNA template is indicated after the hyphen.
  • BOT-T, BOT-C, BOT-G, BOT-A are used to monitor polymerase incorporation efficiency and fidelity for either nucleotides or nucleotide variants of dA, dG, dC, and dT, respectively.
  • Preliminary assays are performed prior to exhaustive purification of the tagged dNTP to ensure that the polymerase is not inhibited by a chemical that co-purifies with the tagged dNTP, using the ‘BOT-Sau’ template.
  • the ‘BOT-Sau’ template was designed to monitor incorporation of natural dGTP prior to tagged dATP (i.e., a positive control for polymerase activity). More extensive purification is then performed for promising tagged nucleotides.
  • experiments are carried out to determine whether the polymerase continues extension following incorporation of the tagged dNTPs, individually or collectively, using the same end-labeled ‘TOP’ primer, the appropriate ‘BOT’ primer, the fluorescently-tagged dNTP, and the appropriate base 3′ of the tagged nucleotide.
  • the products are then size-separated and analyzed to determine the relative extension efficiency.
  • the Taq DNA polymerase lacks 3′ to 5′ exonuclease activity (proofreading activity). If the polymerase used in single-molecule DNA sequencing possessed a 3′ to 5′ exonuclease activity, the polymerase would be capable of adding another base to replace one that would be removed by the proofreading activity. This newly added base would produce a signature fluorescent signal evidencing the incorporation of an additional base in the template, resulting in a misidentified DNA sequence, a situation that would render the single-molecule sequencing systems of this invention problematic.
  • the sequencing reactions are preferably run in parallel, with the optimal number required to produce sequence information with a high degree of confidence for each base call determined by the error rate. Larger error rates require more parallel run, while smaller error rates require fewer parallel runs. In fact, if the error rate is low enough, generally less than 1 error in 1,000, preferably 1 error in 5,000 and particularly 1 error in 10,000 incorporated base, then no parallel runs are required. Insertions or deletions are, potentially, more serious types of errors and warrant a minimal redundancy of 3 repeats per sample. If 2 reactions were run, one could not be certain which was correct. Thus, 3 reactions are needed for the high quality data produced by this system.
  • the BOT-variant templates are used to characterize the accuracy at which each ⁇ -tagged dNTP is incorporated by an engineered polymerase as set forth in Table V. Oligonucleotides serve as DNA templates, and each differing only in the identity of the first base incorporated. Experiments using these templates are used to examine the relative incorporation efficiency of each base and the ability of the polymerase to discriminate between the tagged dNTPs. Initially, experiments with polymerase variants are carried out using relatively simple-sequence, single-stranded DNA templates. A wide array of sequence-characterized templates is available from the University of Houston in Dr. Hardin's laboratory, including a resource of over 300 purified templates. For example, one series of templates contains variable length polyA or polyT sequences. Additional defined-sequence templates are constructed as necessary, facilitating the development of the base-calling algorithms.
  • SPEX 212 instrument Direct detection of polymerase action on the tagged dNTP is obtained by solution fluorescence measurements, using SPEX 212 instrument or similar instrument. This instrument was used to successfully detect fluorescent signals from ANS tagged ⁇ -phosphate dNTPs, being incorporated by Taq polymerase at nanomolar concentration levels.
  • the SPEX 212 instrument includes a 450 watt xenon arc source, dual emission and dual excitation monochromators, cooled PMT (recently upgraded to simultaneous T-format anisotropy data collection), and a Hi-Tech stopped-flow accessory.
  • This instrument is capable of detecting an increase in fluorescence intensity and/or change in absorption spectra upon liberation of the tagged pyrophosphate from ANS tagged ⁇ -phosphate dNTPs, as was verified for ANS-pyrophosphate released by Taq and RNA polymerase and venom phosphodiesterase.
  • the detection of fluorescence from single molecules is preferably carried out using microscopy. Confocal-scanning microscopy can be used in this application, but a non-scanning approach is preferred.
  • An microscope useful for detecting fluorescent signals due to polymerase activity include any type of microscope, with oil-immersion type microscopes being preferred.
  • the microscopes are preferably located in an environment in which vibration and temperature variations are controlled, and fitted with a highly-sensitive digital camera. While many different cameras can be to record the fluorescent signals, the preferred cameras are intensified CCD type cameras such as the iPentaMax from Princeton Instruments.
  • the method of detection involves illuminating the samples at wavelengths sufficient to induce fluorescence of the tags, preferably in an internal-reflection format. If the fluorescent tags are a donor-acceptor pair, then the excitation frequency must be sufficient to excite the donor.
  • the preferred light source is a laser. It will often be advantageous to image the same sample in multiple fluorescence emission wavelengths, either in rapid succession or simultaneously. For simultaneous multi-color imaging, an image splitter is preferred to allow the same CCD to collect all of the color images simultaneously. Alternatively, multiple cameras can be used, each viewing the sample through emission optical filters of different wavelength specificity.
  • Tag detection in practice depends upon many variables including the specific tag used as well electrical, fluorescent, chemical, physical, electrochemical, mass isotope, or other properties.
  • Single-molecule fluorescence imaging is obtainable employing a research-grade Nikon Diaphot TMD inverted epifluorescence microscope, upgraded with laser illumination and a more-sensitive camera.
  • single-molecule technology is a well-developed and commercially available technology.
  • the epifluorescence microscope can be retrofitted for evanescent-wave excitation using an argon ion laser at 488 nm.
  • the inventors have previously used this illumination geometry in assays for nucleic acid hybridization studies.
  • the existing setup has also been upgraded by replacement of the current CCD camera with a 12-bit 512 ⁇ 512 pixel Princeton Instruments I-PentaMAX generation IV intensified CCD camera, which has been used successfully in a variety of similar single-molecule applications. This camera achieves a quantum efficiency of over 45% in the entire range of emission wavelengths of the dyes to be used, and considerably beyond this range.
  • the vertical alignment of their existing microscope tends to minimize vibration problems, and the instrument is currently mounted on an anti-vibration table.
  • a preferred high-sensitivity imaging system is based on an Olympus IX70-S8F inverted epifluorescence microscope.
  • the system incorporates low-background components and enables capture of single molecule fluorescence images at rates of greater than 80 frames per second with quantum efficiency between 60-70% in the range of emission wavelengths of the fluorescently active tags.
  • a data collection channel such as a single pixel or pixel-bin of the viewing field of the CCD or other digital imaging system.
  • a finite number of data collection channels such as pixels are available in any given digital imaging apparatus. Randomly-spaced, densely-positioned fluorescent emitters generally produce an increased fraction of pixels or pixel bins that are multiply-occupied and problematic in data analysis. As the density of emitters in the viewing field increases so does the number of problematic data channels. While multiple occupancy of distinguishable data collection regions within the viewing field can be reduced by reducing the concentration of emitters in the viewing field, this decrease in concentration of emitters increases the fraction of data collection channels or pixels that see no emitter at all, therefore, leading to inefficient data collection.
  • a preferred method for increasing and/or maximizing the data collection efficiency involves controlling the spacing between emitters (tagged polymerase molecules). This spacing is achieved in a number of ways.
  • the polymerases can be immobilized on a substrate so that only a single polymerase is localized within each data collection channel or pixel region within the viewing field of the imaging system.
  • the immobilization is accomplished by anchoring a capture agent or linking group chemically attached to the substrate.
  • Capture or linking agents can be spaced to useful distances by choosing inherently large capture agents, by conjugating them with or bonding them to molecules which enhance their steric bulk or electrostatic repulsion bulk, or by immobilizing under conditions chosen to maximize repulsion between polymerizing molecular assembly (e.g., low ionic strength to maximize electrostatic repulsion).
  • the polymerase can be associated with associated proteins that increase the steric bulk of the polymerase or the electrostatic repulsion bulk of the polymerizing system so that each polymerizing molecular assembly cannot approach any closer than a distance greater than the data channel resolution size of the imaging system.
  • assays are performed essentially as described in for polymerase activity assays described herein.
  • the primary difference between assaying polymerase activity for screening purposes involves the immobilization of some part of the polymerizing assembly such as the polymerase, target DNA or a primer associated protein to a solid support to enable viewing of individual replication events.
  • immobilization options are available, including, without limitation, covalent and/or non-covalent attachment of one of the molecular assemblies on a surface such as an organic surface, an inorganic surface, in or on a nanotubes or other similar nano-structures and/or in or on porous matrices.
  • a preferred data collection method for single-molecule sequencing is to ensure that the fluorescently tagged polymerases are spaced apart within the viewing field of the imagining apparatus so that each data collection channel sees the activity of only a single polymerase.
  • the raw data generated by the detector represents between one to four time-dependent fluorescence data streams comprising wavelengths and intensities: one data stream for each fluorescently labeled base being monitored. Assignment of base identities and reliabilities are calculated using the PHRED computer program. If needed, the inventors will write computer programs to interpret the data streams having partial and overlapping data. In such cases, multiple experiments are run so that confidence limits are assigned to each base identity according to the variation in the reliability indices and the difficulties associated with assembling stretches of sequence from fragments. The reliability indices represent the goodness of the fit between the observed wavelengths and intensities of fluorescence compared with ideal values. The result of the signal analyses is a linear DNA sequence with associated probabilities of certainty.
  • the data is stored in a database for dynamic querying for identification and comparison purposes.
  • a search term sequence of 6-10, 11-16, 17-20, 21-30 bases can be compared against reference sequences to quickly identify perfectly matched sequences or those sharing a user defined level of identity.
  • Multiple experiments are run so that confidence limits can be assigned to each base identity according to the variation in the reliability indices and the difficulties associated with assembling stretches of sequence from fragments.
  • the reliability indices represent the goodness of the fit between the observed wavelengths and intensities of fluorescence compared with the ideal values.
  • the result of the signal analyses is a linear DNA sequence with associated probabilities of certainty.
  • Data collection allows data to be assembled from partial information to obtain sequence information from multiple polymerase molecules in order to determine the overall sequence of the template or target molecule.
  • An important driving force for convolving together results obtained with multiple single-molecules is the impossibility of obtaining data from a single molecule over an indefinite period of time.
  • a typical dye photobleaching efficiency of 2*10 ⁇ 5 a typical dye molecule is expected to undergo 50,000 excitation/emission cycles before permanent photobleaching.
  • Data collection from a given molecule may also be interrupted by intersystem crossing to an optically inactive (on the time scales of interest) triplet state. Even with precautions against photobleaching, therefore, data obtained from any given molecule is necessarily fragmentary for template sequences of substantial length, and these subsequences are co-processed in order to derive the overall sequence of a target DNA molecule.
  • Taq pol I Bacteriophage lambda host strain Charon 35 harboring the full-length of the Thermus aquaticus gene encoding DNA polymerase I (Taq pol I) was obtained from the American Type Culture Collection (ATCC; Manassas, Va.). Taq pol I was amplified directly from the lysate of the infected E. coli host using the following DNA oligonucleotide primers:
  • each synthetic DNA oligonucleotide represents engineered EcoRI restriction sites immediately preceding and following the Taq pol I gene.
  • PCR amplification using the reverse primer described above and the following forward primer created an additional construct with an N-terminal deletion of the gene:
  • the underlined segment corresponds to an engineered NcoI restriction site with the first codon encoding for an alanine (ATG start representing an expression vector following the ribosome binding site).
  • ATG start representing an expression vector following the ribosome binding site.
  • the full-length and truncated constructs of the Taq pol I gene is ligated to a single EcoRI site (full-length) and in an Ncoo/EcoRI digested pRSET-b expression vector.
  • E. coli strain JM109 is used as host for all in vivo manipulation of the engineered vectors.
  • Overlapping primers are used to introduce point mutations into the native gene by PCR based mutagenesis (using Pfu DNA polymerase).
  • Complementary forward and reverse primers each contain a codon that encodes the desired mutated amino acid residue.
  • PCR using these primers results in a knicked, non-methylated, double-stranded plasmid containing the desired mutation.
  • DpnI restriction enzyme cuts at methylated guanosines in the sequence GATC.
  • the resulting mutant Taq polymerases are then reacted with a desired atomic or molecular tag to tag the cysteine in the mutant structure through the SH group of the cysteine residue and screened for native and/or tagged dNTP incorporation and incorporation efficiency.
  • the mutant polymerases are also screened for fluorescent activity during base incorporation.
  • the present invention also relates to mutant Taq polymerase having a cysteine residue added one or more of the sites selected from the group consisting of 513-518, 643, 647, 649 and 653-661. After cysteine replacement and verification of polymerase activity using the modified dNTPs, the mutant Taq polymerases are reacted with a tag through the SH group of the inserted cysteine residue.
  • This example illustrates the preparation of gamma ANS tagged dATP, shown graphically in FIG. 4.
  • reaction was diluted to 50 mL and adjusted to a solution of 0.05 M triethylammonium bicarbonate buffer (TEAB, pH ⁇ 7.5).
  • TEAB triethylammonium bicarbonate buffer
  • the reaction product was chromatographed on a 50 mL DEAE-SEPHADEX ion exchanger (A-25-120) column at low temperature that was equilibrated with ⁇ pH 7.5 1.0 M aqueous TEAB (100 mL), 1.0 M aqueous sodiumbicarbonate (1100 mL), and ⁇ pH 7.5, 0.05 Maqueous TEAB (100 mL).
  • the column was eluted with a linear gradient of ⁇ pH 7.5 aqueous TEAB from 0.05 to 0.9 M.
  • Lane 1 contains 5′ radiolabeled ‘TOP’ probe in extension buffer.
  • Lane 2 contains Taq DNA polymerase, 50 ⁇ M dGTP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-Sau’).
  • Lane 3 contains Taq DNA polymerase, 50 ⁇ M dATP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-Sau’).
  • Lane 4 contains Taq DNA polymerase, 50 ⁇ M ANS- ⁇ -dATP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-Sau’).
  • Lane 5 contains Taq DNA polymerase, 50 ⁇ M dGTP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-T’).
  • Lane 6 contains spill-over from lane 5.
  • Lane 7 contains Taq DNA polymerase, 50 ⁇ M dATP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-T’).
  • Lane 8 contains Taq DNA polymerase, 50 ⁇ M ANS- ⁇ -dATP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-T’).
  • Lane 9 contains Taq DNA polymerase, 50 ⁇ M dGTP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-3T’).
  • Lane 10 contains Taq DNA polymerase, 50 ⁇ M dATP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-3T’).
  • Lane 11 contains Taq DNA polymerase, ANS- ⁇ -dATP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-3T’).
  • Lane 12 contains 5′ radiolabeled ‘TOP’ probe in extension buffer.
  • Lane 13 contains 5′ radiolabeled ‘TOP’ probe and Taq DNA polymerase in extension buffer. Oligonucleotide sequences are shown in Table V.
  • This next example illustrates the synthesis of extended DNA polymers using all four ANS tagged ⁇ -phosphate dNTPs. Products generated in these reactions were separated on a 20% denaturing polyacrylamide gel, the gel was dried and imaged following overnight exposure to a Fuji BAS1000 imaging plate. Referring now to FIG. 6, an image of (A) the actual gel, (B) a lightened phosphorimage and (C) an enhanced phosphorimage. Lane descriptions for A, B, and C follow: Lane 1 is the control containing purified 10-base primer extended to 11 and 12 bases by template-mediated addition of alpha- 32 P dCTP. Lane 2 includes the same primer that was incubated with double-stranded plasmid DNA at 96° C.
  • Lane 3 includes the same labeled primer that was incubated with double-stranded DNA plasmid at 96° C. for 3 minutes, the reaction was DNA polymerase and all four gamma-modified DNTPs (100 uM, each) were added and the reaction was incubated at 37° C. for 60 minutes.
  • Lane 4 includes the control, purified 10-base primer that was extended to 11 and 12 bases by the addition of alpha- 32 P-dCTP was cycled in parallel with lanes 5-8 reactions.
  • Lane 5 includes the same 32 P-labeled primer that was incubated with double-stranded plasmid DNA at 96° C. for 3 minutes, the reaction was brought to 37° C. for 10 minutes, during which time Taq DNA polymerase and all four natural dNTPs (100 uM, each) were added. The reaction was cycled 25 times at 96° C. for 10 seconds, 37° C. for 1 minute, and 70° C. for 5 minutes.
  • Lane 6 includes the same 32 P-labeled primer that was incubated with double-stranded plasmid DNA at 96° C.
  • the reaction was brought to 37° C. for 10 minutes, during which time Taq DNA polymerase and all four gamma-modified dNTPs (100 uM, each) were added.
  • the reaction was cycled 25 times at 96° C. for 10 seconds, 37° C. for 1 minute, and 70° C. for 5 minutes.
  • Lane 7 includes nonpurified, 10-base, 32 P-labeled primer that was incubated with double-stranded DNA plasmid at 96° C. for 3 minutes, the reaction was brought to 37° C. for 10 minutes, during which time Taq DNA polymerase and all four natural dNTPs (100 uM, each) were added.
  • the reaction was cycled 25 times at 96° C. for 10 seconds, 37° C.
  • Lane 8 includes nonpurified, 10-base, 32 P-labeled primer that was incubated with double-stranded DNA plasmid at 96° C. for 3 minutes, the reaction was brought to 37° C. for 10 minutes, during which time Taq DNA polymerase and all four gamma-modified dNTPs were added. The reaction was cycled 25 times at 96° C. for 10 seconds, 37° C. for 1 minute, and 70° C. for 5 minutes.
  • Evident in the reactions involving tagged dNTPs is a substantial decrease in pyrophosphorolysis as compared to reactions involving natural nucleotides.
  • This next example illustrates the synthesis of long DNA polymers using all four ANS tagged ⁇ -phosphate dNTPs.
  • Each primer extension reaction was split into two fractions, and one fraction was electrophoresed through a 20% denaturing gel (as described above), while the other was electrophoresed through a 6% denaturing gel to better estimate product lengths.
  • the gel was dried and imaged (overnight) to a Fuji BAS1000 imaging plate. Referring now to FIG. 7, an image of (A) the actual gel, (B) a lightened phosphorimage of the actual gel, and (C) an enhanced phosphorimage of the actual gel.
  • Lane descriptions for A, B, and C follow: Lane 1 includes 123 Marker with size standards indicated at the left of each panel.
  • Lane 2 contains the control, purified 10-base primer extended to 11 and 12 bases by template-mediated addition of alpha- 32 P dCTP.
  • Lane 3 contains the same 32 P -labeled primer that was incubated with double-stranded plasmid DNA at 96° C. for 3 minutes (to denature template), the reaction was brought to 37° C. (to anneal primer-template), Taq DNA polymerase and all four natural dNTPs (100 uM, each) were added and the reaction was incubated at 37° C. for 60 minutes.
  • Lane 4 includes the same 32 P -labeled primer that was incubated with double-stranded DNA plasmid at 96° C.
  • Lane 5 includes the control, purified 10-base primer that was extended to 11 and 12 bases by the addition of alpha- 32 P -dCTP was cycled in parallel with lanes 5-8 reactions.
  • Lane 6 includes the same 32 P -labeled primer that was incubated with double-stranded plasmid DNA at 96° C. for 3 minutes, the reaction was brought to 37° C. for 10 minutes, during which time Taq DNA polymerase and all four natural dNTPs (100 uM, each) were added.
  • the reaction was cycled 25 times at 96° C. for 10 seconds, 37° C. for 1 minute, and 70° C. for 5 minutes.
  • Lane 7 includes the same 32 P -labeled primer that was incubated with double-stranded plasmid DNA at 96° C. for 3 minutes, the reaction was brought to 37° C. for 10 minutes, during which time Taq DNA polymerase and all four gamma-modified dNTPs (100 uM, each) were added.
  • the reaction was cycled 25 times at 96° C. for 10 seconds, 37° C. for 1 minute, and 70° C. for 5 minutes.
  • Lane 8 includes nonpurified, 10-base, 32 P -labeled primer that was incubated with double-stranded DNA plasmid at 96° C. for 3 minutes, the reaction was brought to 37° C. for 10 minutes, during which time Taq DNA polymerase and all four natural dNTPs (100 uM, each) were added. The reaction was cycled 25 times at 96° C. for 10 seconds, 37° C. for 1 minute, and 70° C. for 5 minutes. Lane 9 includes nonpurified, 10-base, 32 P -labeled primer that was incubated with double-stranded DNA plasmid at 96° C. for 3 minutes, the reaction was brought to 37° C.
  • the indicated enzyme (Taq DNA Polymerase, Sequenase, HIV-1 Reverse Transcriptase, T7 DNA Polymerase, Klenow Fragment, Pfu DNA Polymerase) were incubated in the manufacturers suggested reaction buffer, 50 ⁇ M of the indicated nucleotide at 37° C. for 30-60 minutes, and the reaction products were analyzed by size separation through a 20% denaturing gel.
  • Taq DNA polymerase efficiently uses the gamma-modified nucleotides to synthesize extended DNA polymers at increased accuracy as shown in FIGS. 4 - 6 .
  • Pfu DNA polymerase does not efficiently use gamma-modified nucleotides and is, thus, not a preferred enzyme for the single-molecule sequencing system as shown in FIG. 9.
  • HIV-1 reverse transcriptase efficiently uses the gamma-tagged nucleotides, and significant fidelity improvement results as shown in FIG. 10.
  • the fidelity of nucleic acid synthesis is a limiting factor in achieving amplification of long target molecules using PCR.
  • the misincorporation of nucleotides during the synthesis of primer extension products limits the length of target that can be efficiently amplified.
  • the effect on primer extension of a 3′-terminal base that is mismatched with the template is described in Huang et al., 1992, Nucl. Acids Res. 20:4567-4573, incorporated herein by reference.
  • the presence of misincorporated nucleotides may result in prematurely terminated strand synthesis, reducing the number of template strands for future rounds of amplification, and thus reducing the efficiency of long target amplification.
  • the present invention provides an improved PCR system for generating increased extension length PCR amplified DNA products comprising contacting a native Taq polymerase with gamma tagged dNTPs of this invention under PCR reaction conditions.
  • the extended length PCR products are due to improved accuracy of base incorporation, resulting from the use of the gamma-modified dNTPs of this invention.
US09/901,782 2000-07-07 2001-07-09 Real-time sequence determination Abandoned US20030064366A1 (en)

Priority Applications (23)

Application Number Priority Date Filing Date Title
US09/901,782 US20030064366A1 (en) 2000-07-07 2001-07-09 Real-time sequence determination
US11/007,797 US7329492B2 (en) 2000-07-07 2004-12-08 Methods for real-time single molecule sequence determination
US11/007,642 US20050266424A1 (en) 2000-07-07 2004-12-08 Real-time sequence determination
US11/648,136 US20070172862A1 (en) 2000-07-07 2006-12-29 Data stream determination
US11/648,722 US20070172868A1 (en) 2000-07-07 2006-12-29 Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US11/648,106 US20070172858A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11/648,184 US20100255463A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US11/648,138 US20070172863A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US11/648,856 US20070275395A1 (en) 2000-07-07 2006-12-29 Tagged monomers for use in sequence determination
US11/648,115 US20070172861A1 (en) 2000-07-07 2006-12-29 Mutant polymerases
US11/648,137 US20070292867A1 (en) 2000-07-07 2006-12-29 Sequence determination using multiply tagged polymerizing agents
US11/648,191 US20070172867A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11/648,174 US20070172865A1 (en) 2000-07-07 2006-12-29 Sequence determination in confined regions
US11/648,713 US20070184475A1 (en) 2000-07-07 2006-12-29 Sequence determination by direct detection
US11/648,164 US20070172864A1 (en) 2000-07-07 2006-12-29 Composition for sequence determination
US11/648,182 US20070172866A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination using depolymerizing agent
US12/410,370 US20110014604A1 (en) 2000-07-07 2009-03-24 Methods for sequence determination
US12/412,208 US20100304367A1 (en) 2000-07-07 2009-03-26 Compositions for real-time, single molecule sequence determination
US12/411,997 US20110021383A1 (en) 2000-07-07 2009-03-26 Apparatuses for real-time, single molecule sequence determination
US12/414,417 US20090275036A1 (en) 2000-07-07 2009-03-30 Systems and methods for real time single molecule sequence determination
US12/419,214 US20090305278A1 (en) 2000-07-07 2009-04-06 Sequence determination in confined regions
US12/419,660 US20110059436A1 (en) 2000-07-07 2009-04-07 Methods for sequence determination
US12/724,392 US20100317005A1 (en) 2000-07-07 2010-03-15 Modified Nucleotides and Methods for Making and Use Same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US21659400P 2000-07-07 2000-07-07
US09/901,782 US20030064366A1 (en) 2000-07-07 2001-07-09 Real-time sequence determination

Related Child Applications (17)

Application Number Title Priority Date Filing Date
US10/007,621 Continuation-In-Part US7211414B2 (en) 2000-12-01 2001-12-03 Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
US11/007,797 Division US7329492B2 (en) 2000-07-07 2004-12-08 Methods for real-time single molecule sequence determination
US779404A Continuation-In-Part 2000-07-07 2004-12-08
US11/007,642 Division US20050266424A1 (en) 2000-07-07 2004-12-08 Real-time sequence determination
US11/648,722 Continuation US20070172868A1 (en) 2000-07-07 2006-12-29 Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US11/648,164 Continuation US20070172864A1 (en) 2000-07-07 2006-12-29 Composition for sequence determination
US11/648,191 Continuation US20070172867A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11/648,182 Continuation-In-Part US20070172866A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination using depolymerizing agent
US11/648,856 Division US20070275395A1 (en) 2000-07-07 2006-12-29 Tagged monomers for use in sequence determination
US11/648,138 Continuation US20070172863A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US11/648,713 Continuation US20070184475A1 (en) 2000-07-07 2006-12-29 Sequence determination by direct detection
US11/648,106 Continuation US20070172858A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11/648,136 Continuation US20070172862A1 (en) 2000-07-07 2006-12-29 Data stream determination
US11/648,115 Division US20070172861A1 (en) 2000-07-07 2006-12-29 Mutant polymerases
US11/648,137 Division US20070292867A1 (en) 2000-07-07 2006-12-29 Sequence determination using multiply tagged polymerizing agents
US11/648,184 Continuation US20100255463A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US11/648,174 Division US20070172865A1 (en) 2000-07-07 2006-12-29 Sequence determination in confined regions

Publications (1)

Publication Number Publication Date
US20030064366A1 true US20030064366A1 (en) 2003-04-03

Family

ID=22807697

Family Applications (22)

Application Number Title Priority Date Filing Date
US09/901,782 Abandoned US20030064366A1 (en) 2000-07-07 2001-07-09 Real-time sequence determination
US11/007,642 Abandoned US20050266424A1 (en) 2000-07-07 2004-12-08 Real-time sequence determination
US11/007,797 Expired - Lifetime US7329492B2 (en) 2000-07-07 2004-12-08 Methods for real-time single molecule sequence determination
US11/648,191 Abandoned US20070172867A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11/648,106 Abandoned US20070172858A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11/648,856 Abandoned US20070275395A1 (en) 2000-07-07 2006-12-29 Tagged monomers for use in sequence determination
US11/648,174 Abandoned US20070172865A1 (en) 2000-07-07 2006-12-29 Sequence determination in confined regions
US11/648,722 Abandoned US20070172868A1 (en) 2000-07-07 2006-12-29 Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US11/648,713 Abandoned US20070184475A1 (en) 2000-07-07 2006-12-29 Sequence determination by direct detection
US11/648,137 Abandoned US20070292867A1 (en) 2000-07-07 2006-12-29 Sequence determination using multiply tagged polymerizing agents
US11/648,164 Abandoned US20070172864A1 (en) 2000-07-07 2006-12-29 Composition for sequence determination
US11/648,136 Abandoned US20070172862A1 (en) 2000-07-07 2006-12-29 Data stream determination
US11/648,184 Abandoned US20100255463A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US11/648,115 Abandoned US20070172861A1 (en) 2000-07-07 2006-12-29 Mutant polymerases
US11/648,138 Abandoned US20070172863A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US12/410,370 Abandoned US20110014604A1 (en) 2000-07-07 2009-03-24 Methods for sequence determination
US12/412,208 Abandoned US20100304367A1 (en) 2000-07-07 2009-03-26 Compositions for real-time, single molecule sequence determination
US12/411,997 Abandoned US20110021383A1 (en) 2000-07-07 2009-03-26 Apparatuses for real-time, single molecule sequence determination
US12/414,417 Abandoned US20090275036A1 (en) 2000-07-07 2009-03-30 Systems and methods for real time single molecule sequence determination
US12/419,214 Abandoned US20090305278A1 (en) 2000-07-07 2009-04-06 Sequence determination in confined regions
US12/419,660 Abandoned US20110059436A1 (en) 2000-07-07 2009-04-07 Methods for sequence determination
US12/724,392 Abandoned US20100317005A1 (en) 2000-07-07 2010-03-15 Modified Nucleotides and Methods for Making and Use Same

Family Applications After (21)

Application Number Title Priority Date Filing Date
US11/007,642 Abandoned US20050266424A1 (en) 2000-07-07 2004-12-08 Real-time sequence determination
US11/007,797 Expired - Lifetime US7329492B2 (en) 2000-07-07 2004-12-08 Methods for real-time single molecule sequence determination
US11/648,191 Abandoned US20070172867A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11/648,106 Abandoned US20070172858A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11/648,856 Abandoned US20070275395A1 (en) 2000-07-07 2006-12-29 Tagged monomers for use in sequence determination
US11/648,174 Abandoned US20070172865A1 (en) 2000-07-07 2006-12-29 Sequence determination in confined regions
US11/648,722 Abandoned US20070172868A1 (en) 2000-07-07 2006-12-29 Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US11/648,713 Abandoned US20070184475A1 (en) 2000-07-07 2006-12-29 Sequence determination by direct detection
US11/648,137 Abandoned US20070292867A1 (en) 2000-07-07 2006-12-29 Sequence determination using multiply tagged polymerizing agents
US11/648,164 Abandoned US20070172864A1 (en) 2000-07-07 2006-12-29 Composition for sequence determination
US11/648,136 Abandoned US20070172862A1 (en) 2000-07-07 2006-12-29 Data stream determination
US11/648,184 Abandoned US20100255463A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US11/648,115 Abandoned US20070172861A1 (en) 2000-07-07 2006-12-29 Mutant polymerases
US11/648,138 Abandoned US20070172863A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US12/410,370 Abandoned US20110014604A1 (en) 2000-07-07 2009-03-24 Methods for sequence determination
US12/412,208 Abandoned US20100304367A1 (en) 2000-07-07 2009-03-26 Compositions for real-time, single molecule sequence determination
US12/411,997 Abandoned US20110021383A1 (en) 2000-07-07 2009-03-26 Apparatuses for real-time, single molecule sequence determination
US12/414,417 Abandoned US20090275036A1 (en) 2000-07-07 2009-03-30 Systems and methods for real time single molecule sequence determination
US12/419,214 Abandoned US20090305278A1 (en) 2000-07-07 2009-04-06 Sequence determination in confined regions
US12/419,660 Abandoned US20110059436A1 (en) 2000-07-07 2009-04-07 Methods for sequence determination
US12/724,392 Abandoned US20100317005A1 (en) 2000-07-07 2010-03-15 Modified Nucleotides and Methods for Making and Use Same

Country Status (9)

Country Link
US (22) US20030064366A1 (zh)
EP (3) EP1368460B1 (zh)
JP (3) JP2004513619A (zh)
CN (2) CN100462433C (zh)
AT (1) ATE377093T1 (zh)
AU (2) AU2001282881B2 (zh)
CA (1) CA2415897A1 (zh)
DE (1) DE60131194T2 (zh)
WO (1) WO2002004680A2 (zh)

Cited By (110)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020025529A1 (en) * 1999-06-28 2002-02-28 Stephen Quake Methods and apparatus for analyzing polynucleotide sequences
US20020085891A1 (en) * 1996-02-16 2002-07-04 Moore Richard A. Twist drill bit
US20020164629A1 (en) * 2001-03-12 2002-11-07 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension
US20030077610A1 (en) * 2001-08-29 2003-04-24 John Nelson Terminal-phosphate-labeled nucleotides and methods of use
US20030096253A1 (en) * 2001-08-29 2003-05-22 John Nelson Single nucleotide amplification and detection by polymerase
US20030124576A1 (en) * 2001-08-29 2003-07-03 Shiv Kumar Labeled nucleoside polyphosphates
US20030162213A1 (en) * 2001-08-29 2003-08-28 Carl Fuller Terminal-phosphate-labeled nucleotides and methods of use
US20040014096A1 (en) * 2002-04-12 2004-01-22 Stratagene Dual-labeled nucleotides
US20040152104A1 (en) * 2003-02-05 2004-08-05 Anup Sood Nucleic acid amplification
US20040241716A1 (en) * 2003-02-05 2004-12-02 Shiv Kumar Terminal-phosphate-labeled nucleotides with new linkers
US20040248186A1 (en) * 2001-09-24 2004-12-09 Intel Corporation Nucleic acid sequencing by Raman monitoring of uptake of precursors during molecular replication
US20050158761A1 (en) * 1999-05-19 2005-07-21 Jonas Korlach Method for sequencing nucleic acid molecules
US6982146B1 (en) 1999-08-30 2006-01-03 The United States Of America As Represented By The Department Of Health And Human Services High speed parallel molecular nucleic acid sequencing
US20060063264A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for performing nucleic acid analysis
US20060060766A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and methods for optical analysis of molecules
US20070105123A1 (en) * 2005-11-04 2007-05-10 Mannkind Corporation IRE-1alpha substrates
US20070172868A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US20070172866A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination using depolymerizing agent
US20070172819A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: compositions including pyrophosphorolysis inhibitors
US20070211467A1 (en) * 2006-03-08 2007-09-13 Helicos Biosciences Corporation Systems and methods for reducing detected intensity non-uniformity in a laser beam
US20070250274A1 (en) * 2006-02-06 2007-10-25 Visigen Biotechnologies, Inc. Method for analyzing dynamic detectable events at the single molecule level
US20080091005A1 (en) * 2006-07-20 2008-04-17 Visigen Biotechnologies, Inc. Modified nucleotides, methods for making and using same
US20080241951A1 (en) * 2006-07-20 2008-10-02 Visigen Biotechnologies, Inc. Method and apparatus for moving stage detection of single molecular events
US20080241938A1 (en) * 2006-07-20 2008-10-02 Visigen Biotechnologies, Inc. Automated synthesis or sequencing apparatus and method for making and using same
US20080299565A1 (en) * 2005-12-12 2008-12-04 Schneider Thomas D Probe for Nucleic Acid Sequencing and Methods of Use
WO2008154317A1 (en) * 2007-06-06 2008-12-18 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
US20080309926A1 (en) * 2006-03-08 2008-12-18 Aaron Weber Systems and methods for reducing detected intensity non uniformity in a laser beam
US20090026082A1 (en) * 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090065471A1 (en) * 2003-02-10 2009-03-12 Faris Sadeg M Micro-nozzle, nano-nozzle, manufacturing methods therefor, applications therefor
US20090081644A1 (en) * 2001-08-29 2009-03-26 General Electric Company Ligation amplification
US20090092970A1 (en) * 2003-04-08 2009-04-09 Pacific Biosciences Composition and method for nucleic acid sequencing
US20090186343A1 (en) * 2003-01-28 2009-07-23 Visigen Biotechnologies, Inc. Methods for preparing modified biomolecules, modified biomolecules and methods for using same
US7645596B2 (en) 1998-05-01 2010-01-12 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
US20100188073A1 (en) * 2006-12-14 2010-07-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale fet arrays
US20100203504A1 (en) * 2005-07-07 2010-08-12 Yoichi Katsumoto Substance-Information Acquisition Method Using Evanescent Light Beam, Substance-Information Measurement Apparatus, Base-Sequence Determination Method and Base-Sequence Determination Apparatus
US20100311597A1 (en) * 2005-07-20 2010-12-09 Harold Philip Swerdlow Methods for sequence a polynucleotide template
US20100311061A1 (en) * 2009-04-27 2010-12-09 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
US20110003343A1 (en) * 2009-03-27 2011-01-06 Life Technologies Corporation Conjugates of biomolecules to nanoparticles
US7897345B2 (en) 2003-11-12 2011-03-01 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
US7981604B2 (en) 2004-02-19 2011-07-19 California Institute Of Technology Methods and kits for analyzing polynucleotide sequences
US20110281737A1 (en) * 2008-10-22 2011-11-17 Life Technologies Corporation Method and Apparatus for Rapid Nucleic Acid Sequencing
US8217433B1 (en) 2010-06-30 2012-07-10 Life Technologies Corporation One-transistor pixel array
WO2012104851A1 (en) 2011-01-31 2012-08-09 Yeda Research And Development Co. Ltd. Methods of diagnosing disease using overlap extension pcr
US8263336B2 (en) 2009-05-29 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes
US20130004950A1 (en) * 2010-08-06 2013-01-03 Tandem Diagnostics, Inc. Assay systems for genetic analysis
US8349167B2 (en) 2006-12-14 2013-01-08 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US20130078622A1 (en) * 2011-09-26 2013-03-28 The Regents Of The University Of California Electronic device for monitoring single molecule dynamics
JP2013094149A (ja) * 2011-11-04 2013-05-20 Hitachi Ltd Ｄｎａ配列解読システム、ｄｎａ配列解読方法及びプログラム
US8470164B2 (en) 2008-06-25 2013-06-25 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US20130345391A1 (en) * 2009-12-28 2013-12-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv Composite Probes and Use Thereof in Super Resolution Methods
US8653567B2 (en) 2010-07-03 2014-02-18 Life Technologies Corporation Chemically sensitive sensor with lightly doped drains
US8673627B2 (en) 2009-05-29 2014-03-18 Life Technologies Corporation Apparatus and methods for performing electrochemical reactions
US8685324B2 (en) 2010-09-24 2014-04-01 Life Technologies Corporation Matched pair transistor circuits
US8700338B2 (en) 2011-01-25 2014-04-15 Ariosa Diagnosis, Inc. Risk calculation for evaluation of fetal aneuploidy
US8703734B2 (en) 2005-12-12 2014-04-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Nanoprobes for detection or modification of molecules
US8703422B2 (en) 2007-06-06 2014-04-22 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
US8712697B2 (en) 2011-09-07 2014-04-29 Ariosa Diagnostics, Inc. Determination of copy number variations using binomial probability calculations
US8747748B2 (en) 2012-01-19 2014-06-10 Life Technologies Corporation Chemical sensor with conductive cup-shaped sensor surface
US8756020B2 (en) 2011-01-25 2014-06-17 Ariosa Diagnostics, Inc. Enhanced risk probabilities using biomolecule estimations
US8753812B2 (en) 2004-11-12 2014-06-17 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection method for DNA and other molecules
US8771491B2 (en) 2009-09-30 2014-07-08 Quantapore, Inc. Ultrafast sequencing of biological polymers using a labeled nanopore
US8776573B2 (en) 2009-05-29 2014-07-15 Life Technologies Corporation Methods and apparatus for measuring analytes
US8821798B2 (en) 2012-01-19 2014-09-02 Life Technologies Corporation Titanium nitride as sensing layer for microwell structure
US8858782B2 (en) 2010-06-30 2014-10-14 Life Technologies Corporation Ion-sensing charge-accumulation circuits and methods
US9080968B2 (en) 2013-01-04 2015-07-14 Life Technologies Corporation Methods and systems for point of use removal of sacrificial material
US9096898B2 (en) 1998-05-01 2015-08-04 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9109251B2 (en) 2004-06-25 2015-08-18 University Of Hawaii Ultrasensitive biosensors
US9206417B2 (en) 2012-07-19 2015-12-08 Ariosa Diagnostics, Inc. Multiplexed sequential ligation-based detection of genetic variants
US9270264B2 (en) 2012-05-29 2016-02-23 Life Technologies Corporation System for reducing noise in a chemical sensor array
US9482615B2 (en) 2010-03-15 2016-11-01 Industrial Technology Research Institute Single-molecule detection system and methods
US9567639B2 (en) 2010-08-06 2017-02-14 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US9618475B2 (en) 2010-09-15 2017-04-11 Life Technologies Corporation Methods and apparatus for measuring analytes
US9624537B2 (en) 2014-10-24 2017-04-18 Quantapore, Inc. Efficient optical analysis of polymers using arrays of nanostructures
US9651539B2 (en) 2012-10-28 2017-05-16 Quantapore, Inc. Reducing background fluorescence in MEMS materials by low energy ion beam treatment
US9671363B2 (en) 2013-03-15 2017-06-06 Life Technologies Corporation Chemical sensor with consistent sensor surface areas
US9670243B2 (en) 2010-06-02 2017-06-06 Industrial Technology Research Institute Compositions and methods for sequencing nucleic acids
US9778188B2 (en) 2009-03-11 2017-10-03 Industrial Technology Research Institute Apparatus and method for detection and discrimination molecular object
US9823217B2 (en) 2013-03-15 2017-11-21 Life Technologies Corporation Chemical device with thin conductive element
US9835585B2 (en) 2013-03-15 2017-12-05 Life Technologies Corporation Chemical sensor with protruded sensor surface
US9841398B2 (en) 2013-01-08 2017-12-12 Life Technologies Corporation Methods for manufacturing well structures for low-noise chemical sensors
US9862997B2 (en) 2013-05-24 2018-01-09 Quantapore, Inc. Nanopore-based nucleic acid analysis with mixed FRET detection
US9885079B2 (en) 2014-10-10 2018-02-06 Quantapore, Inc. Nanopore-based polymer analysis with mutually-quenching fluorescent labels
US9903820B2 (en) 2007-05-08 2018-02-27 The Trustees Of Boston University Chemical functionalization of solid-state nanopores and nanopore arrays and applications thereof
US9970984B2 (en) 2011-12-01 2018-05-15 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array
US9995683B2 (en) 2010-06-11 2018-06-12 Industrial Technology Research Institute Apparatus for single-molecule detection
US9995708B2 (en) 2013-03-13 2018-06-12 Life Technologies Corporation Chemical sensor with sidewall spacer sensor surface
US9994897B2 (en) 2013-03-08 2018-06-12 Ariosa Diagnostics, Inc. Non-invasive fetal sex determination
US10077472B2 (en) 2014-12-18 2018-09-18 Life Technologies Corporation High data rate integrated circuit with power management
US10100357B2 (en) 2013-05-09 2018-10-16 Life Technologies Corporation Windowed sequencing
US10131947B2 (en) 2011-01-25 2018-11-20 Ariosa Diagnostics, Inc. Noninvasive detection of fetal aneuploidy in egg donor pregnancies
US10131951B2 (en) 2010-08-06 2018-11-20 Ariosa Diagnostics, Inc. Assay systems for genetic analysis
EP3438285A1 (en) 2012-05-02 2019-02-06 Ibis Biosciences, Inc. Dna sequencing
EP3438286A1 (en) 2012-05-02 2019-02-06 Ibis Biosciences, Inc. Dna sequencing
US10233496B2 (en) 2010-08-06 2019-03-19 Ariosa Diagnostics, Inc. Ligation-based detection of genetic variants
US10289800B2 (en) 2012-05-21 2019-05-14 Ariosa Diagnostics, Inc. Processes for calculating phased fetal genomic sequences
US10364469B2 (en) * 2014-01-16 2019-07-30 Illumina, Inc Gene expression panel for prognosis of prostate cancer recurrence
US10379079B2 (en) 2014-12-18 2019-08-13 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US10451585B2 (en) 2009-05-29 2019-10-22 Life Technologies Corporation Methods and apparatus for measuring analytes
US10458942B2 (en) 2013-06-10 2019-10-29 Life Technologies Corporation Chemical sensor array having multiple sensors per well
US10533223B2 (en) 2010-08-06 2020-01-14 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US10605767B2 (en) 2014-12-18 2020-03-31 Life Technologies Corporation High data rate integrated circuit with transmitter configuration
US10823721B2 (en) 2016-07-05 2020-11-03 Quantapore, Inc. Optically based nanopore sequencing
US11031095B2 (en) 2010-08-06 2021-06-08 Ariosa Diagnostics, Inc. Assay systems for determination of fetal copy number variation
US11203786B2 (en) 2010-08-06 2021-12-21 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US11231451B2 (en) 2010-06-30 2022-01-25 Life Technologies Corporation Methods and apparatus for testing ISFET arrays
US11270781B2 (en) 2011-01-25 2022-03-08 Ariosa Diagnostics, Inc. Statistical analysis for non-invasive sex chromosome aneuploidy determination
US11307166B2 (en) 2010-07-01 2022-04-19 Life Technologies Corporation Column ADC
US11339430B2 (en) 2007-07-10 2022-05-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US11705217B2 (en) 2008-03-28 2023-07-18 Pacific Biosciences Of California, Inc. Sequencing using concatemers of copies of sense and antisense strands

Families Citing this family (572)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9213691D0 (en) * 1992-06-27 1992-08-12 Hurlstone Gary Personal alarm torch
AU2180200A (en) * 1998-12-14 2000-07-03 Li-Cor Inc. A heterogeneous assay for pyrophosphate detection
US20050009124A1 (en) * 2001-11-26 2005-01-13 Echelon Biosciences Incorporated Assays for detection of phosphoinositide kinase and phosphatase activity
CA2496182C (en) * 2002-08-29 2012-06-05 Amersham Biosciences Corp Terminal phosphate blocked nucleoside polyphosphates
US7563600B2 (en) 2002-09-12 2009-07-21 Combimatrix Corporation Microarray synthesis and assembly of gene-length polynucleotides
EP1572105B1 (en) * 2002-11-14 2011-08-31 John Wayne Cancer Institute Detection of micro metastasis of melanoma and breast cancer in paraffin-embedded tumor draining lymph nodes by multimaker quantitative rt-pcr
GB0324456D0 (en) * 2003-10-20 2003-11-19 Isis Innovation Parallel DNA sequencing methods
US7311794B2 (en) 2004-05-28 2007-12-25 Wafergen, Inc. Methods of sealing micro wells
US7405434B2 (en) * 2004-11-16 2008-07-29 Cornell Research Foundation, Inc. Quantum dot conjugates in a sub-micrometer fluidic channel
JP2006197832A (ja) * 2005-01-19 2006-08-03 Tohoku Univ 環境感受性蛍光プローブを用いたシステイン残基が変異導入されたａｂｃトランスポーターの汎用的な基質親和性検出方法
EP1846758A2 (en) * 2005-02-09 2007-10-24 Pacific Biosciences of California, Inc. Nucleotide compositions and uses thereof
US20070190542A1 (en) * 2005-10-03 2007-08-16 Ling Xinsheng S Hybridization assisted nanopore sequencing
US9156004B2 (en) 2005-10-17 2015-10-13 Stc.Unm Fabrication of enclosed nanochannels using silica nanoparticles
US10060904B1 (en) 2005-10-17 2018-08-28 Stc.Unm Fabrication of enclosed nanochannels using silica nanoparticles
US7825037B2 (en) * 2005-10-17 2010-11-02 Stc.Unm Fabrication of enclosed nanochannels using silica nanoparticles
US7897737B2 (en) 2006-12-05 2011-03-01 Lasergen, Inc. 3′-OH unblocked, nucleotides and nucleosides, base modified with photocleavable, terminating groups and methods for their use in DNA sequencing
WO2008103848A1 (en) * 2007-02-21 2008-08-28 Invitrogen Corporation Materials and methods for single molecule nucleic acid sequencing
WO2008147879A1 (en) * 2007-05-22 2008-12-04 Ryan Golhar Automated method and device for dna isolation, sequence determination, and identification
WO2008151023A2 (en) 2007-06-01 2008-12-11 Ibis Biosciences, Inc. Methods and compositions for multiple displacement amplification of nucleic acids
US8278047B2 (en) 2007-10-01 2012-10-02 Nabsys, Inc. Biopolymer sequencing by hybridization of probes to form ternary complexes and variable range alignment
WO2009055508A1 (en) * 2007-10-22 2009-04-30 Life Technologies Corporation A method and system for obtaining ordered, segmented sequence fragments along a nucleic acid molecule
GB0721340D0 (en) * 2007-10-30 2007-12-12 Isis Innovation Polymerase-based single-molecule DNA sequencing
US8101353B2 (en) * 2007-12-18 2012-01-24 Advanced Analytical Technologies, Inc. System and method for nucleotide sequence profiling for sample identification
CN101519698B (zh) * 2008-03-10 2013-05-22 周国华 一种利用序列标签定量测定核酸的方法
JP4586081B2 (ja) * 2008-03-31 2010-11-24 株式会社日立ハイテクノロジーズ 蛍光分析装置
US20090269746A1 (en) * 2008-04-25 2009-10-29 Gil Atzmon Microsequencer-whole genome sequencer
EP2307565B1 (en) 2008-06-11 2017-11-29 Lasergen, Inc. Reversible nucleosides and nucleotides terminators and their use in dna sequencing
JP5268444B2 (ja) * 2008-06-23 2013-08-21 株式会社日立ハイテクノロジーズ 単分子リアルタイムシーケンス装置，核酸分析装置及び単分子リアルタイムシーケンス方法
US20110281740A1 (en) * 2008-06-30 2011-11-17 Joseph Beechem Methods for Real Time Single Molecule Sequencing
US20100036110A1 (en) * 2008-08-08 2010-02-11 Xiaoliang Sunney Xie Methods and compositions for continuous single-molecule nucleic acid sequencing by synthesis with fluorogenic nucleotides
US20100035252A1 (en) 2008-08-08 2010-02-11 Ion Torrent Systems Incorporated Methods for sequencing individual nucleic acids under tension
US20100227327A1 (en) * 2008-08-08 2010-09-09 Xiaoliang Sunney Xie Methods and compositions for continuous single-molecule nucleic acid sequencing by synthesis with fluorogenic nucleotides
US9650668B2 (en) 2008-09-03 2017-05-16 Nabsys 2.0 Llc Use of longitudinally displaced nanoscale electrodes for voltage sensing of biomolecules and other analytes in fluidic channels
EP2342362B1 (en) * 2008-09-03 2017-03-01 Nabsys 2.0 LLC Use of longitudinally displaced nanoscale electrodes for voltage sensing of biomolecules and other analytes in fluidic channels
US8262879B2 (en) 2008-09-03 2012-09-11 Nabsys, Inc. Devices and methods for determining the length of biopolymers and distances between probes bound thereto
US8383345B2 (en) 2008-09-12 2013-02-26 University Of Washington Sequence tag directed subassembly of short sequencing reads into long sequencing reads
WO2010039189A2 (en) * 2008-09-23 2010-04-08 Helicos Biosciences Corporation Methods for sequencing degraded or modified nucleic acids
EP2358913B1 (en) 2008-11-03 2017-03-22 The Regents of The University of California Methods for detecting modification resistant nucleic acids
US8236532B2 (en) 2008-12-23 2012-08-07 Illumina, Inc. Multibase delivery for long reads in sequencing by synthesis protocols
US8455260B2 (en) * 2009-03-27 2013-06-04 Massachusetts Institute Of Technology Tagged-fragment map assembly
WO2010111605A2 (en) * 2009-03-27 2010-09-30 Nabsys, Inc. Devices and methods for analyzing biomolecules and probes bound thereto
WO2010127304A2 (en) * 2009-05-01 2010-11-04 Illumina, Inc. Sequencing methods
US8246799B2 (en) * 2009-05-28 2012-08-21 Nabsys, Inc. Devices and methods for analyzing biomolecules and probes bound thereto
US20100311144A1 (en) 2009-06-05 2010-12-09 Life Technologies Corporation Mutant dna polymerases
WO2010144151A2 (en) * 2009-06-12 2010-12-16 Pacific Biosciences Of California, Inc. Single-molecule real-time analysis of protein synthesis
WO2011014811A1 (en) 2009-07-31 2011-02-03 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests
EP2464729B9 (en) 2009-08-14 2014-12-31 Epicentre Technologies Corporation METHODS, COMPOSITIONS, AND KITS FOR GENERATING rRNA-DEPLETED SAMPLES OR ISOLATING rRNA FROM SAMPLES
WO2011028296A2 (en) * 2009-09-07 2011-03-10 Caerus Molecular Diagnostics Incorporated Sequence determination by use of opposing forces
WO2011078897A1 (en) * 2009-09-15 2011-06-30 Life Technologies Corporation Improved sequencing methods, compositions, systems, kits and apparatuses
US9890408B2 (en) 2009-10-15 2018-02-13 Ibis Biosciences, Inc. Multiple displacement amplification
AU2010326349B2 (en) 2009-12-01 2015-10-29 Oxford Nanopore Technologies Limited Biochemical analysis instrument
US8835358B2 (en) 2009-12-15 2014-09-16 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
US9315857B2 (en) 2009-12-15 2016-04-19 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse label-tags
US8911972B2 (en) 2009-12-16 2014-12-16 Pacific Biosciences Of California, Inc. Sequencing methods using enzyme conformation
EP3151052A1 (en) 2010-02-01 2017-04-05 Illumina, Inc. Focusing methods and optical systems and assemblies using the same
US8603741B2 (en) 2010-02-18 2013-12-10 Pacific Biosciences Of California, Inc. Single molecule sequencing with two distinct chemistry steps
DE202011003570U1 (de) 2010-03-06 2012-01-30 Illumina, Inc. Systeme und Vorrichtungen zum Detektieren optischer Signale aus einer Probe
ES2555106T3 (es) 2010-04-05 2015-12-29 Prognosys Biosciences, Inc. Ensayos biológicos codificados espacialmente
US20190300945A1 (en) 2010-04-05 2019-10-03 Prognosys Biosciences, Inc. Spatially Encoded Biological Assays
US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
SG10201503540QA (en) 2010-05-06 2015-06-29 Ibis Biosciences Inc Integrated sample preparation systems and stabilized enzyme mixtures
US8865077B2 (en) 2010-06-11 2014-10-21 Industrial Technology Research Institute Apparatus for single-molecule detection
WO2011159942A1 (en) 2010-06-18 2011-12-22 Illumina, Inc. Conformational probes and methods for sequencing nucleic acids
WO2012027618A2 (en) 2010-08-25 2012-03-01 Pacific Biosciences Of California, Inc. Functionalized cyanine dyes
CA2811333C (en) 2010-09-16 2020-05-12 Gen-Probe Incorporated Capture probes immobilizable via l-nucleotide tail
US20120070830A1 (en) 2010-09-16 2012-03-22 lbis Biosciences, Inc. Stabilization of ozone-labile fluorescent dyes by thiourea
US8715933B2 (en) 2010-09-27 2014-05-06 Nabsys, Inc. Assay methods using nicking endonucleases
EP2633069B1 (en) 2010-10-26 2015-07-01 Illumina, Inc. Sequencing methods
US9255293B2 (en) 2010-11-01 2016-02-09 Gen-Probe Incorporated Integrated capture and amplification of target nucleic acid for sequencing
EP2635679B1 (en) 2010-11-05 2017-04-19 Illumina, Inc. Linking sequence reads using paired code tags
US9074251B2 (en) 2011-02-10 2015-07-07 Illumina, Inc. Linking sequence reads using paired code tags
WO2012067911A1 (en) 2010-11-16 2012-05-24 Nabsys, Inc. Methods for sequencing a biomolecule by detecting relative positions of hybridized probes
WO2012074855A2 (en) 2010-11-22 2012-06-07 The Regents Of The University Of California Methods of identifying a cellular nascent rna transcript
SG191409A1 (en) 2010-12-27 2013-08-30 Ibis Biosciences Inc Nucleic acid sample preparation methods and compositions
US8951781B2 (en) 2011-01-10 2015-02-10 Illumina, Inc. Systems, methods, and apparatuses to image a sample for biological or chemical analysis
WO2012106081A2 (en) 2011-01-31 2012-08-09 Illumina, Inc. Methods for reducing nucleic acid damage
US10457936B2 (en) 2011-02-02 2019-10-29 University Of Washington Through Its Center For Commercialization Massively parallel contiguity mapping
WO2012109315A1 (en) * 2011-02-08 2012-08-16 Life Technologies Corporation Linking methods, compositions, systems, kits and apparatuses
US11274341B2 (en) 2011-02-11 2022-03-15 NABsys, 2.0 LLC Assay methods using DNA binding proteins
ES2538694T3 (es) * 2011-04-01 2015-06-23 F. Hoffmann-La Roche Ag Variantes de la T7 ARN polimerasa con sustituciones de Cisteína-Serina
US20120258871A1 (en) 2011-04-08 2012-10-11 Prognosys Biosciences, Inc. Peptide constructs and assay systems
GB201106254D0 (en) 2011-04-13 2011-05-25 Frisen Jonas Method and product
WO2012170936A2 (en) 2011-06-09 2012-12-13 Illumina, Inc. Patterned flow-cells useful for nucleic acid analysis
GB201113430D0 (en) 2011-08-03 2011-09-21 Fermentas Uab DNA polymerases
US9670538B2 (en) 2011-08-05 2017-06-06 Ibis Biosciences, Inc. Nucleic acid sequencing by electrochemical detection
US11208636B2 (en) 2011-08-10 2021-12-28 Life Technologies Corporation Polymerase compositions, methods of making and using same
LT2742151T (lt) 2011-08-10 2018-02-12 Life Technologies Corporation Polimerazės kompozicija
CA2848049C (en) 2011-09-13 2021-01-26 Lasergen, Inc. 5-methoxy, 3'-oh unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing
WO2013049135A1 (en) 2011-09-26 2013-04-04 Gen-Probe Incorporated Algorithms for sequence determinations
WO2013063308A1 (en) 2011-10-25 2013-05-02 University Of Massachusetts An enzymatic method to enrich for capped rna, kits for performing same, and compositions derived therefrom
EP3305400A3 (en) 2011-10-28 2018-06-06 Illumina, Inc. Microarray fabrication system and method
EP2776165A2 (en) 2011-11-07 2014-09-17 Illumina, Inc. Integrated sequencing apparatuses and methods of use
DK2788499T3 (en) 2011-12-09 2016-03-21 Illumina Inc Enhanced root for polymer tags
WO2013096692A1 (en) * 2011-12-21 2013-06-27 Illumina, Inc. Apparatus and methods for kinetic analysis and determination of nucleic acid sequences
EP3211100A1 (en) 2011-12-22 2017-08-30 Ibis Biosciences, Inc. Amplification primers and methods
ES2645418T3 (es) 2011-12-22 2017-12-05 Ibis Biosciences, Inc. Amplificación de una secuencia de un ácido ribonucleico
US9334491B2 (en) 2011-12-22 2016-05-10 Ibis Biosciences, Inc. Systems and methods for isolating nucleic acids from cellular samples
US10150993B2 (en) 2011-12-22 2018-12-11 Ibis Biosciences, Inc. Macromolecule positioning by electrical potential
WO2013102091A1 (en) 2011-12-28 2013-07-04 Ibis Biosciences, Inc. Nucleic acid ligation systems and methods
US9803231B2 (en) 2011-12-29 2017-10-31 Ibis Biosciences, Inc. Macromolecule delivery to nanowells
WO2013101741A1 (en) 2011-12-30 2013-07-04 Abbott Molecular, Inc. Channels with cross-sectional thermal gradients
US9822417B2 (en) 2012-01-09 2017-11-21 Oslo Universitetssykehus Hf Methods and biomarkers for analysis of colorectal cancer
CN108611398A (zh) 2012-01-13 2018-10-02 Data生物有限公司 通过新一代测序进行基因分型
US10655165B2 (en) 2012-02-01 2020-05-19 Gen-Probe Incorporated Asymmetric hairpin target capture oligomers
CN114717296A (zh) * 2012-02-03 2022-07-08 加州理工学院 多路生化测定中信号的编码和解码
ES2776673T3 (es) 2012-02-27 2020-07-31 Univ North Carolina Chapel Hill Métodos y usos para etiquetas moleculares
GB2513024B (en) 2012-02-27 2016-08-31 Cellular Res Inc A clonal amplification method
NO2694769T3 (zh) 2012-03-06 2018-03-03
US20130261984A1 (en) 2012-03-30 2013-10-03 Illumina, Inc. Methods and systems for determining fetal chromosomal abnormalities
US9732387B2 (en) 2012-04-03 2017-08-15 The Regents Of The University Of Michigan Biomarker associated with irritable bowel syndrome and Crohn's disease
EP2834622B1 (en) 2012-04-03 2023-04-12 Illumina, Inc. Integrated optoelectronic read head and fluidic cartridge useful for nucleic acid sequencing
US20130274148A1 (en) 2012-04-11 2013-10-17 Illumina, Inc. Portable genetic detection and analysis system and method
ES2683707T3 (es) 2012-05-02 2018-09-27 Ibis Biosciences, Inc. Secuenciación de ADN
US20150133310A1 (en) 2012-05-02 2015-05-14 Ibis Biosciences, Inc. Nucleic acid sequencing systems and methods
US9315864B2 (en) 2012-05-18 2016-04-19 Pacific Biosciences Of California, Inc. Heteroarylcyanine dyes with sulfonic acid substituents
WO2013173844A1 (en) 2012-05-18 2013-11-21 Pacific Biosciences Of California, Inc. Heteroarylcyanine dyes
US9012022B2 (en) 2012-06-08 2015-04-21 Illumina, Inc. Polymer coatings
US8895249B2 (en) 2012-06-15 2014-11-25 Illumina, Inc. Kinetic exclusion amplification of nucleic acid libraries
WO2014005076A2 (en) 2012-06-29 2014-01-03 The Regents Of The University Of Michigan Methods and biomarkers for detection of kidney disorders
WO2014008448A1 (en) 2012-07-03 2014-01-09 Sloan Kettering Institute For Cancer Research Quantitative assessment of human t-cell repertoire recovery after allogeneic hematopoietic stem cell transplantation
NL2017959B1 (en) 2016-12-08 2018-06-19 Illumina Inc Cartridge assembly
WO2014031157A1 (en) 2012-08-20 2014-02-27 Illumina, Inc. Method and system for fluorescence lifetime based sequencing
EP2909343B1 (en) 2012-10-16 2018-10-10 Abbott Molecular Inc. Methods to sequence a nucleic acid
US9181583B2 (en) 2012-10-23 2015-11-10 Illumina, Inc. HLA typing using selective amplification and sequencing
US9605309B2 (en) * 2012-11-09 2017-03-28 Genia Technologies, Inc. Nucleic acid sequencing using tags
US9914966B1 (en) 2012-12-20 2018-03-13 Nabsys 2.0 Llc Apparatus and methods for analysis of biomolecules using high frequency alternating current excitation
US9683230B2 (en) 2013-01-09 2017-06-20 Illumina Cambridge Limited Sample preparation on a solid support
EP2956550B1 (en) 2013-01-18 2020-04-08 Nabsys 2.0 LLC Enhanced probe binding
US9805407B2 (en) 2013-01-25 2017-10-31 Illumina, Inc. Methods and systems for using a cloud computing environment to configure and sell a biological sample preparation cartridge and share related data
US9512422B2 (en) 2013-02-26 2016-12-06 Illumina, Inc. Gel patterned surfaces
EP3919617A1 (en) 2013-03-13 2021-12-08 Illumina, Inc. Methods and compositions for nucleic acid sequencing
CA2898453C (en) 2013-03-13 2021-07-27 Illumina, Inc. Multilayer fluidic devices and methods for their fabrication
EP2971154A4 (en) 2013-03-14 2017-03-01 Ibis Biosciences, Inc. Nucleic acid control panels
EP2971070B2 (en) 2013-03-14 2021-03-03 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues
CN105378107A (zh) 2013-03-14 2016-03-02 雅培分子公司 多重甲基化-特异性扩增系统和方法
WO2014150910A1 (en) 2013-03-15 2014-09-25 Ibis Biosciences, Inc. Dna sequences to assess contamination in dna sequencing
US9890425B2 (en) 2013-03-15 2018-02-13 Abbott Molecular Inc. Systems and methods for detection of genomic copy number changes
US9593373B2 (en) 2013-03-15 2017-03-14 Illumina Cambridge Limited Modified nucleosides or nucleotides
US20140274747A1 (en) 2013-03-15 2014-09-18 Illumina, Inc. Super resolution imaging
US9193998B2 (en) 2013-03-15 2015-11-24 Illumina, Inc. Super resolution imaging
DK3013983T3 (da) 2013-06-25 2023-03-06 Prognosys Biosciences Inc Spatialt kodede biologiske assays ved brug af en mikrofluidisk anordning
IL273519B2 (en) 2013-07-01 2023-04-01 Illumina Inc Surface activation and polymer assembly without a catalyst
ES2719579T3 (es) 2013-07-03 2019-07-11 Illumina Inc Sistema para secuenciación por síntesis ortogonal
CN103333680B (zh) * 2013-07-16 2015-05-27 北京化工大学 一种具有多色荧光特性的二苯基恶唑共晶材料及其制备方法
DK3030645T3 (da) 2013-08-08 2023-01-30 Illumina Inc Fluidsystem til levering af reagenser til en flowcelle
WO2015026853A2 (en) 2013-08-19 2015-02-26 Abbott Molecular Inc. Next-generation sequencing libraries
GB2546833B (en) 2013-08-28 2018-04-18 Cellular Res Inc Microwell for single cell analysis comprising single cell and single bead oligonucleotide capture labels
JP2016539343A (ja) 2013-08-30 2016-12-15 イルミナ インコーポレイテッド 親水性または斑状親水性表面上の液滴の操作
WO2015042708A1 (en) 2013-09-25 2015-04-02 Bio-Id Diagnostic Inc. Methods for detecting nucleic acid fragments
ES2938585T3 (es) 2013-09-30 2023-04-12 Life Technologies Corp Composiciones de polimerasa, métodos de producción y uso de estas
JP2017504307A (ja) 2013-10-07 2017-02-09 セルラー リサーチ， インコーポレイテッド アレイ上のフィーチャーをデジタルカウントするための方法およびシステム
US10540783B2 (en) * 2013-11-01 2020-01-21 Illumina, Inc. Image analysis useful for patterned objects
PT3077943T (pt) 2013-12-03 2020-08-21 Illumina Inc Métodos e sistemas para analisar dados de imagem
NZ720871A (en) 2013-12-10 2020-03-27 Illumina Inc Biosensors for biological or chemical analysis and methods of manufacturing the same
EP3083700B1 (en) 2013-12-17 2023-10-11 The Brigham and Women's Hospital, Inc. Detection of an antibody against a pathogen
CA2932283A1 (en) 2013-12-20 2015-06-25 Illumina, Inc. Preserving genomic connectivity information in fragmented genomic dna samples
KR102333635B1 (ko) 2013-12-23 2021-11-30 일루미나, 인코포레이티드 광 방출의 검출을 개선시키기 위한 구조화 기판 및 이와 관련한 방법
US9677132B2 (en) 2014-01-16 2017-06-13 Illumina, Inc. Polynucleotide modification on solid support
WO2015107430A2 (en) 2014-01-16 2015-07-23 Oslo Universitetssykehus Hf Methods and biomarkers for detection and prognosis of cervical cancer
JP6484636B2 (ja) 2014-01-16 2019-03-13 イラミーナ インコーポレーテッド 固相担体におけるアンプリコン調製および配列決定
RU2752700C2 (ru) 2014-02-18 2021-07-30 Иллумина, Инк. Способы и композиции для днк-профилирования
US10767219B2 (en) 2014-03-11 2020-09-08 Illumina, Inc. Disposable, integrated microfluidic cartridge and methods of making and using same
JP6412954B2 (ja) 2014-04-29 2018-10-24 イルミナ インコーポレイテッド 鋳型切換え及びタグメンテーションを用いる単一細胞の遺伝子発現の多重分析
EP3143161B1 (en) 2014-05-16 2021-04-21 Illumina, Inc. Nucleic acid synthesis techniques
CA3127071A1 (en) 2014-05-27 2015-12-03 Alex Aravanis Systems and methods for biochemical analysis including a base instrument and a removable cartridge
EP3152320B1 (en) 2014-06-03 2020-10-28 Illumina, Inc. Compositions, systems, and methods for detecting events using tethers anchored to or adjacent to nanopores
EP3151733B1 (en) 2014-06-06 2020-04-15 The Regents Of The University Of Michigan Compositions and methods for characterizing and diagnosing periodontal disease
US20150353989A1 (en) 2014-06-09 2015-12-10 Illumina Cambridge Limited Sample preparation for nucleic acid amplification
WO2015189636A1 (en) 2014-06-13 2015-12-17 Illumina Cambridge Limited Methods and compositions for preparing sequencing libraries
KR102598819B1 (ko) 2014-06-23 2023-11-03 더 제너럴 하스피탈 코포레이션 서열결정에 의해 평가된 DSB의 게놈 전체에 걸친 비편향된 확인 (GUIDE-Seq)
US11155809B2 (en) 2014-06-24 2021-10-26 Bio-Rad Laboratories, Inc. Digital PCR barcoding
US10017759B2 (en) 2014-06-26 2018-07-10 Illumina, Inc. Library preparation of tagged nucleic acid
MX2016017136A (es) 2014-06-27 2017-05-10 Abbott Lab Composiciones y metodos para detectar pegivirus 2 de humano (hpgv-2).
DK3161154T3 (da) 2014-06-27 2020-06-15 Illumina Inc Modificerede polymeraser til forbedret inkorporering af nukleotidanaloger
CN112430641A (zh) 2014-06-30 2021-03-02 亿明达股份有限公司 使用单侧转座的方法和组合物
DK3460075T3 (da) 2014-07-15 2021-02-01 Illumina Inc Biokemisk aktiveret elektronisk anordning
CA2955382C (en) 2014-07-21 2023-07-18 Illumina, Inc. Polynucleotide enrichment using crispr-cas systems
BR112017001500A2 (pt) 2014-07-24 2018-02-20 Abbott Molecular Inc composições e métodos para a detecção e análise de tuberculose por micobactéria
GB201414098D0 (en) 2014-08-08 2014-09-24 Illumina Cambridge Ltd Modified nucleotide linkers
WO2016026924A1 (en) 2014-08-21 2016-02-25 Illumina Cambridge Limited Reversible surface functionalization
WO2016040602A1 (en) 2014-09-11 2016-03-17 Epicentre Technologies Corporation Reduced representation bisulfite sequencing using uracil n-glycosylase (ung) and endonuclease iv
ES2803079T3 (es) 2014-09-12 2021-01-22 Illumina Inc Métodos para detectar la presencia de subunidades de polímeros usando quimioluminiscencia
KR102538753B1 (ko) 2014-09-18 2023-05-31 일루미나, 인코포레이티드 핵산 서열결정 데이터를 분석하기 위한 방법 및 시스템
CN107002051A (zh) 2014-09-30 2017-08-01 亿明达股份有限公司 用于核苷酸类似物的改善的掺入的经修饰的聚合酶
WO2016153999A1 (en) 2015-03-25 2016-09-29 Life Technologies Corporation Modified nucleotides and uses thereof
JP6668336B2 (ja) 2014-10-09 2020-03-18 イラミーナ インコーポレーテッド 非混和性液体を分離して少なくとも１つの液体を効果的に単離する方法及び装置
US9897791B2 (en) 2014-10-16 2018-02-20 Illumina, Inc. Optical scanning systems for in situ genetic analysis
AU2015331739B2 (en) 2014-10-17 2021-12-02 Illumina Cambridge Limited Contiguity preserving transposition
EP3970849A1 (en) 2014-10-31 2022-03-23 Illumina Cambridge Limited Polymers and dna copolymer coatings
US9828627B2 (en) 2014-11-05 2017-11-28 Illumina Cambridge Limited Reducing DNA damage during sample preparation and sequencing using siderophore chelators
GB201419731D0 (en) 2014-11-05 2014-12-17 Illumina Cambridge Ltd Sequencing from multiple primers to increase data rate and density
EP3218511B1 (en) 2014-11-11 2020-04-22 Illumina Cambridge Limited Methods and arrays for producing and sequencing monoclonal clusters of nucleic acid
CN114438174A (zh) 2014-11-11 2022-05-06 伊鲁米那股份有限公司 使用crispr-cas系统的多核苷酸扩增
RU2582198C1 (ru) * 2014-11-20 2016-04-20 Федеральное государственное бюджетное учреждение науки Лимнологический институт Сибирского отделения Российской академии наук (ЛИН СО РАН) Аналоги природных дезоксирибонуклеозидтрифосфатов и рибонуклеозидтрифосфатов, содержащие репортёрные флуоресцентные группы, для использования в аналитической биоорганической химии
CN104458686B (zh) * 2014-12-02 2017-01-18 公安部第一研究所 一种基于特征分子量内标定量分析的dna荧光光谱采集方法
EP3234187B1 (en) 2014-12-15 2021-02-17 Illumina, Inc. Method for single molecular placement on a substrate
CN113403293A (zh) 2014-12-16 2021-09-17 生命技术公司 聚合酶组合物和制造与使用其的方法
EP3256604B1 (en) 2015-02-10 2020-03-25 Illumina, Inc. Methods and compositions for analyzing cellular components
EP3766988B1 (en) 2015-02-19 2024-02-14 Becton, Dickinson and Company High-throughput single-cell analysis combining proteomic and genomic information
US10208339B2 (en) 2015-02-19 2019-02-19 Takara Bio Usa, Inc. Systems and methods for whole genome amplification
JP6620160B2 (ja) 2015-02-20 2019-12-11 タカラ バイオ ユーエスエー， インコーポレイテッド 単一細胞の迅速かつ正確な分注、視覚化及び解析のための方法
CN107208158B (zh) 2015-02-27 2022-01-28 贝克顿迪金森公司 空间上可寻址的分子条形编码
EP3271073B1 (en) 2015-03-20 2019-06-12 Illumina, Inc. Fluidics cartridge for use in the vertical or substantially vertical position
CN112326557A (zh) 2015-03-24 2021-02-05 伊鲁米那股份有限公司 对样品成像用于生物或化学分析的方法、载体组件和系统
WO2016160844A2 (en) 2015-03-30 2016-10-06 Cellular Research, Inc. Methods and compositions for combinatorial barcoding
WO2016156845A1 (en) 2015-03-31 2016-10-06 Illumina Cambridge Limited Surface concatamerization of templates
JP6828007B2 (ja) 2015-04-10 2021-02-10 スペーシャル トランスクリプトミクス アクチボラグ 生物学的試料の空間識別されるマルチプレックスな核酸分析
WO2016168386A1 (en) 2015-04-14 2016-10-20 Illumina, Inc. Structured substrates for improving detection of light emissions and methods relating to the same
KR20170136555A (ko) 2015-04-15 2017-12-11 더 제너럴 하스피탈 코포레이션 Lna-기반 돌연변이체 농축 차세대 서열분석 검정
EP3286326A1 (en) 2015-04-23 2018-02-28 Cellular Research, Inc. Methods and compositions for whole transcriptome amplification
US10844428B2 (en) 2015-04-28 2020-11-24 Illumina, Inc. Error suppression in sequenced DNA fragments using redundant reads with unique molecular indices (UMIS)
EP3294911B1 (en) 2015-05-11 2020-08-19 Illumina, Inc. Platform for discovery and analysis of therapeutic agents
KR102054571B1 (ko) 2015-05-29 2019-12-10 일루미나 케임브리지 리미티드 클러스터에서 표면 프라이머의 향상된 이용
US11014089B2 (en) 2015-05-29 2021-05-25 Illumina, Inc. Sample carrier and assay system for conducting designated reactions
EP3303584B1 (en) 2015-05-29 2019-10-09 Epicentre Technologies Corporation Methods of analyzing nucleic acids
US11124823B2 (en) 2015-06-01 2021-09-21 Becton, Dickinson And Company Methods for RNA quantification
CA2986074A1 (en) 2015-06-03 2016-12-08 Illumina, Inc. Compositions, systems, and methods for sequencing polynucleotides using tethers anchored to polymerases adjacent to nanopores
EP3878974A1 (en) 2015-07-06 2021-09-15 Illumina Cambridge Limited Sample preparation for nucleic acid amplification
WO2017007757A1 (en) 2015-07-06 2017-01-12 Illumina, Inc. Balanced ac modulation for driving droplet operations electrodes
WO2017007753A1 (en) 2015-07-07 2017-01-12 Illumina, Inc. Selective surface patterning via nanoimrinting
EP3988658A1 (en) 2015-07-14 2022-04-27 Abbott Molecular Inc. Purification of nucleic acids using copper-titanium oxides
US10526664B2 (en) 2015-07-14 2020-01-07 Abbott Molecular Inc. Compositions and methods for identifying drug resistant tuberculosis
CN107835857A (zh) 2015-07-17 2018-03-23 亿明达股份有限公司 用于测序应用的聚合物片层
EP3325642B1 (en) * 2015-07-21 2020-05-27 Omniome, Inc. Nucleic acid sequencing method
US10077470B2 (en) 2015-07-21 2018-09-18 Omniome, Inc. Nucleic acid sequencing methods and systems
DK3329012T3 (da) 2015-07-27 2021-10-11 Illumina Inc Rumlig kortlægning af nukleinsyresekvensinformation
US11155864B2 (en) 2015-07-30 2021-10-26 Illumina, Inc. Orthogonal deblocking of nucleotides
DK3334839T3 (da) 2015-08-14 2021-04-26 Illumina Inc Systemer og fremgangsmåder under anvendelse af magnetisk reagerende sensorer til bestemmelse af en genetisk egenskab
CN116338219A (zh) 2015-08-24 2023-06-27 亿明达股份有限公司 用于生物和化学测定的线路内蓄压器和流量控制系统
EP4086357A1 (en) 2015-08-28 2022-11-09 Illumina, Inc. Nucleic acid sequence analysis from single cells
US10906044B2 (en) 2015-09-02 2021-02-02 Illumina Cambridge Limited Methods of improving droplet operations in fluidic systems with a filler fluid including a surface regenerative silane
US10450598B2 (en) 2015-09-11 2019-10-22 Illumina, Inc. Systems and methods for obtaining a droplet having a designated concentration of a substance-of-interest
CA3000816A1 (en) 2015-09-11 2017-03-16 The General Hospital Corporation Full interrogation of nuclease dsbs and sequencing (find-seq)
US10619186B2 (en) 2015-09-11 2020-04-14 Cellular Research, Inc. Methods and compositions for library normalization
KR20180053748A (ko) 2015-09-30 2018-05-23 더 제너럴 하스피탈 코포레이션 시퀀싱(circle-seq)에 의한 절단 반응의 포괄적인 시험관내 보고
EP3356557B1 (en) 2015-10-01 2022-07-20 Life Technologies Corporation Polymerase compositions and kits, and methods of using and making the same
US20190217300A1 (en) 2015-10-22 2019-07-18 Illumina, Inc. Filler fluid for fluidic devices
US11203016B2 (en) 2015-12-01 2021-12-21 Illumina, Inc. Digital microfluidic system for single-cell isolation and characterization of analytes
CA3168463A1 (en) 2015-12-17 2017-06-22 Illumina, Inc. Distinguishing methylation levels in complex biological samples
WO2017120531A1 (en) 2016-01-08 2017-07-13 Bio-Rad Laboratories, Inc. Multiple beads per droplet resolution
WO2017123533A1 (en) 2016-01-11 2017-07-20 Illumina, Inc. Detection apparatus having a microfluorometer, a fluidic system, and a flow cell latch clamp module
JP6902052B2 (ja) 2016-02-08 2021-07-14 アールジーン・インコーポレイテッドＲｇｅｎｅ， Ｉｎｃ． 複数のリガーゼ組成物、システム、および方法
WO2017160788A2 (en) 2016-03-14 2017-09-21 RGENE, Inc. HYPER-THERMOSTABLE LYSINE-MUTANT ssDNA/RNA LIGASES
CN108474744B (zh) 2016-03-24 2019-11-05 伊鲁米那股份有限公司 在发光成像中使用的基于光子超晶格的设备和组成物及其使用方法
SG11201805665UA (en) 2016-03-28 2018-10-30 Illumina Inc Multi-plane microarrays
AU2017246899B2 (en) 2016-04-07 2020-04-09 Illumina, Inc. Methods and systems for construction of normalized nucleic acid libraries
US11326206B2 (en) 2016-04-07 2022-05-10 Pacific Biosciences Of California, Inc. Methods of quantifying target nucleic acids and identifying sequence variants
AU2017254689B2 (en) 2016-04-22 2022-07-07 Illumina, Inc. Photonic stucture-based devices and compositions for use in luminescent imaging of multiple sites within a pixel, and methods of using the same
CN109072297B (zh) 2016-04-22 2022-09-13 加利福尼亚太平洋生物科学股份有限公司 使用增强的核苷酸特异性三元复合物形成检测的核酸测序方法和系统
AU2017258619B2 (en) 2016-04-29 2020-05-14 Pacific Biosciences Of California, Inc. Sequencing method employing ternary complex destabilization to identify cognate nucleotides
AU2017261189B2 (en) 2016-05-02 2023-02-09 Becton, Dickinson And Company Accurate molecular barcoding
EP3455372B1 (en) 2016-05-11 2019-10-30 Illumina, Inc. Polynucleotide enrichment and amplification using argonaute systems
JP6824290B2 (ja) 2016-05-18 2021-02-03 イルミナ インコーポレイテッド パターン化された疎水性表面を用いる自己組織化パターニング
KR102425463B1 (ko) * 2016-05-20 2022-07-27 퀀텀-에스아이 인코포레이티드 핵산 서열분석을 위한 표지된 뉴클레오티드 조성물 및 방법
US10301677B2 (en) 2016-05-25 2019-05-28 Cellular Research, Inc. Normalization of nucleic acid libraries
JP7046007B2 (ja) 2016-05-26 2022-04-01 ベクトン・ディキンソン・アンド・カンパニー 分子標識カウントの調節方法
US10202641B2 (en) 2016-05-31 2019-02-12 Cellular Research, Inc. Error correction in amplification of samples
US10640763B2 (en) 2016-05-31 2020-05-05 Cellular Research, Inc. Molecular indexing of internal sequences
US11091795B2 (en) 2016-07-11 2021-08-17 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions and methods for diagnosing and treating arrhythmias
WO2018013558A1 (en) 2016-07-12 2018-01-18 Life Technologies Corporation Compositions and methods for detecting nucleic acid regions
EP3487616B1 (en) 2016-07-21 2023-08-09 Takara Bio USA, Inc. Multi-z imaging of wells of multi-well devices and liquid dispensing into the wells
KR102475710B1 (ko) 2016-07-22 2022-12-08 오레곤 헬스 앤드 사이언스 유니버시티 단일 세포 전체 게놈 라이브러리 및 이의 제조를 위한 조합 인덱싱 방법
WO2018035134A1 (en) 2016-08-15 2018-02-22 Omniome, Inc. Method and system for sequencing nucleic acids
WO2018034780A1 (en) 2016-08-15 2018-02-22 Omniome, Inc. Sequencing method for rapid identification and processing of cognate nucleotide pairs
US11543417B2 (en) 2016-08-29 2023-01-03 Oslo Universitetssykehus Hf ChIP-seq assays
JP7091348B2 (ja) 2016-09-26 2022-06-27 ベクトン・ディキンソン・アンド・カンパニー バーコード付きオリゴヌクレオチド配列を有する試薬を用いたタンパク質発現の測定
WO2018064116A1 (en) 2016-09-28 2018-04-05 Illumina, Inc. Methods and systems for data compression
CN111781139B (zh) 2016-10-14 2023-09-12 亿明达股份有限公司 夹盒组件
CN110100009B (zh) 2016-10-19 2023-11-21 伊鲁米那股份有限公司 核酸的化学连接方法
CN117056774A (zh) 2016-11-08 2023-11-14 贝克顿迪金森公司 用于细胞标记分类的方法
JP7232180B2 (ja) 2016-11-08 2023-03-02 ベクトン・ディキンソン・アンド・カンパニー 発現プロファイル分類の方法
CN110168648A (zh) 2016-11-16 2019-08-23 伊路米纳有限公司 序列变异识别的验证方法和系统
GB201619458D0 (en) 2016-11-17 2017-01-04 Spatial Transcriptomics Ab Method for spatial tagging and analysing nucleic acids in a biological specimen
PT3551753T (pt) 2016-12-09 2022-09-02 Harvard College Diagnósticos baseados num sistema efetor de crispr
WO2018118971A1 (en) 2016-12-19 2018-06-28 Bio-Rad Laboratories, Inc. Droplet tagging contiguity preserved tagmented dna
RU2760391C2 (ru) 2016-12-22 2021-11-24 Иллюмина, Инк. Биочипы, включающие пленку смолы и структурированный слой полимера
EP3559262A4 (en) 2016-12-22 2020-07-22 Illumina, Inc. NETWORKS PRESENTING QUALITY CONTROL TRACERS
ES2936079T3 (es) 2016-12-22 2023-03-14 Illumina Inc Matriz que incluye cebador de secuenciación y entidad no secuenciador
CA3049667A1 (en) * 2016-12-27 2018-07-05 Bgi Shenzhen Single fluorescent dye-based sequencing method
CA3048415C (en) 2016-12-30 2023-02-28 Omniome, Inc. Method and system employing distinguishable polymerases for detecting ternary complexes and identifying cognate nucleotides
GB201704754D0 (en) 2017-01-05 2017-05-10 Illumina Inc Kinetic exclusion amplification of nucleic acid libraries
LT3566158T (lt) 2017-01-06 2022-06-27 Illumina, Inc. Fazinė korekcija
AU2018208462B2 (en) 2017-01-10 2021-07-29 Pacific Biosciences Of California, Inc. Polymerases engineered to reduce nucleotide-independent DNA binding
JP7104048B2 (ja) 2017-01-13 2022-07-20 セルラー リサーチ， インコーポレイテッド 流体チャネルの親水性コーティング
KR102326612B1 (ko) 2017-01-17 2021-11-15 일루미나, 인코포레이티드 종양원성 스플라이스 변이체 결정
CA3050247A1 (en) 2017-01-18 2018-07-26 Illumina, Inc. Methods and systems for generation and error-correction of unique molecular index sets with heterogeneous molecular lengths
EP3565900B1 (en) 2017-01-20 2021-04-07 Omniome, Inc. Genotyping by polymerase binding
WO2018136487A1 (en) 2017-01-20 2018-07-26 Omniome, Inc. Process for cognate nucleotide detection in a nucleic acid sequencing workflow
WO2018136117A1 (en) 2017-01-20 2018-07-26 Omniome, Inc. Allele-specific capture of nucleic acids
EP3354746B1 (en) 2017-01-30 2019-05-29 Gregor Mendel Institute of Molecular Plant Biology GmbH Novel spike-in oligonucleotides for normalization of sequence data
EP3577232A1 (en) 2017-02-01 2019-12-11 Cellular Research, Inc. Selective amplification using blocking oligonucleotides
GB201701686D0 (en) 2017-02-01 2017-03-15 Illunina Inc System & method with fiducials having offset layouts
GB201701688D0 (en) 2017-02-01 2017-03-15 Illumia Inc System and method with fiducials in non-recliner layouts
GB201701689D0 (en) 2017-02-01 2017-03-15 Illumia Inc System and method with fiducials of non-closed shapes
WO2018152162A1 (en) 2017-02-15 2018-08-23 Omniome, Inc. Distinguishing sequences by detecting polymerase dissociation
JP7164276B2 (ja) 2017-02-21 2022-11-01 イルミナ インコーポレイテッド リンカーを用いた固定化トランスポソームを使用するタグメンテーション
US11174515B2 (en) 2017-03-15 2021-11-16 The Broad Institute, Inc. CRISPR effector system based diagnostics
US11021740B2 (en) 2017-03-15 2021-06-01 The Broad Institute, Inc. Devices for CRISPR effector system based diagnostics
JP2020513815A (ja) 2017-03-15 2020-05-21 ザ・ブロード・インスティテュート・インコーポレイテッド クラスター化短鎖反復回文配列エフェクター系に基づくウイルス検出用診断法
US11104937B2 (en) 2017-03-15 2021-08-31 The Broad Institute, Inc. CRISPR effector system based diagnostics
EP3601560A1 (en) 2017-03-20 2020-02-05 Illumina, Inc. Methods and compositions for preparing nucleic acid libraries
CN110446787A (zh) 2017-03-24 2019-11-12 生物辐射实验室股份有限公司 通用发夹引物
US10737267B2 (en) 2017-04-04 2020-08-11 Omniome, Inc. Fluidic apparatus and methods useful for chemical and biological reactions
EP3615671B1 (en) 2017-04-23 2021-07-21 Illumina Cambridge Limited Compositions and methods for improving sample identification in indexed nucleic acid libraries
AU2018260633B2 (en) 2017-04-23 2021-04-15 Illumina Cambridge Limited Compositions and methods for improving sample identification in indexed nucleic acid libraries
WO2018200380A1 (en) 2017-04-23 2018-11-01 Illumina, Inc. Compositions and methods for improving sample identification in indexed nucleic acid libraries
US9951385B1 (en) 2017-04-25 2018-04-24 Omniome, Inc. Methods and apparatus that increase sequencing-by-binding efficiency
US10161003B2 (en) 2017-04-25 2018-12-25 Omniome, Inc. Methods and apparatus that increase sequencing-by-binding efficiency
WO2018204423A1 (en) 2017-05-01 2018-11-08 Illumina, Inc. Optimal index sequences for multiplex massively parallel sequencing
AU2018266377A1 (en) 2017-05-08 2019-11-14 Illumina, Inc. Universal short adapters for indexing of polynucleotide samples
EP3635135A1 (en) 2017-06-05 2020-04-15 Becton, Dickinson and Company Sample indexing for single cells
FI3981884T3 (fi) 2017-06-07 2023-09-21 Univ Oregon Health & Science Yksittäissolujen kokogenomikirjastoja metylaatiosekvensointia varten
WO2018227091A1 (en) 2017-06-08 2018-12-13 The Brigham And Women's Hospital, Inc. Methods and compositions for identifying epitopes
CN111032882A (zh) 2017-06-20 2020-04-17 伊鲁米那股份有限公司 解决扩增反应中低效的方法和组合物
WO2018236918A1 (en) 2017-06-20 2018-12-27 Bio-Rad Laboratories, Inc. MDA USING A BALL OLIGONUCLEOTIDE
WO2019002265A1 (en) 2017-06-26 2019-01-03 Universität Für Bodenkultur Wien NOVEL BIOMARKERS FOR THE DETECTION OF SENESCENT CELLS
US11578180B2 (en) 2017-07-18 2023-02-14 Pacific Biosciences Of California, Inc. Method of chemically modifying plastic surfaces
KR102626317B1 (ko) 2017-07-24 2024-01-18 퀀텀-에스아이 인코포레이티드 고강도 표지된 반응물 조성물 및 서열분석 방법
WO2019027767A1 (en) 2017-07-31 2019-02-07 Illumina Inc. SEQUENCING SYSTEM COMPRISING AGGREGATION OF MULTIPLEXED BIOLOGICAL SAMPLES
JP6998404B2 (ja) 2017-08-01 2022-02-04 深▲セン▼恒特基因有限公司 標的ヌクレオチド配列の富化及び決定方法
US11352668B2 (en) 2017-08-01 2022-06-07 Illumina, Inc. Spatial indexing of genetic material and library preparation using hydrogel beads and flow cells
JP7032452B2 (ja) 2017-08-01 2022-03-08 イルミナ インコーポレイテッド ヌクレオチド配列決定のためのヒドロゲルビーズ
AU2018317826B2 (en) 2017-08-15 2022-11-24 Pacific Biosciences Of California, Inc. Scanning apparatus and methods useful for detection of chemical and biological analytes
US11447818B2 (en) 2017-09-15 2022-09-20 Illumina, Inc. Universal short adapters with variable length non-random unique molecular identifiers
CA3075932A1 (en) 2017-09-20 2019-03-28 Guardant Health, Inc. Methods and systems for differentiating somatic and germline variants
NZ759818A (en) 2017-10-16 2022-04-29 Illumina Inc Semi-supervised learning for training an ensemble of deep convolutional neural networks
MX2019014689A (es) 2017-10-16 2020-10-19 Illumina Inc Clasificacion de sitio de escision y empalme basado en aprendizaje profundo.
CA3079411C (en) 2017-10-19 2023-12-05 Omniome, Inc. Simultaneous background reduction and complex stabilization in binding assay workflows
US10907205B2 (en) 2017-11-02 2021-02-02 Bio-Rad Laboratories, Inc. Transposase-based genomic analysis
CN111492068A (zh) 2017-12-19 2020-08-04 贝克顿迪金森公司 与寡核苷酸相关联的颗粒
KR102239487B1 (ko) 2018-01-08 2021-04-14 일루미나, 인코포레이티드 반도체-기반 검출을 사용한 고-처리율 서열분석
WO2019136388A1 (en) 2018-01-08 2019-07-11 Illumina, Inc. Systems and devices for high-throughput sequencing with semiconductor-based detection
SG11201911805VA (en) 2018-01-15 2020-01-30 Illumina Inc Deep learning-based variant classifier
DK3746568T3 (da) 2018-01-29 2023-12-18 Broad Inst Inc Crispr-effektorsystembaseret diagnostik
CN111699253A (zh) 2018-01-31 2020-09-22 生物辐射实验室股份有限公司 用于解卷积分区条码的方法和组合物
BR112020015905A2 (pt) 2018-02-06 2020-12-15 Omniome, Inc. Composições e técnicas para extensão de iniciador de ácido nucleico
CA3186025A1 (en) 2018-02-13 2019-08-22 Illumina, Inc. Dna sequencing using hydrogel beads
EP3775278A1 (en) 2018-04-02 2021-02-17 Illumina Inc. Compositions and methods for making controls for sequence-based genetic testing
US20190318806A1 (en) 2018-04-12 2019-10-17 Illumina, Inc. Variant Classifier Based on Deep Neural Networks
US11512002B2 (en) 2018-04-18 2022-11-29 University Of Virginia Patent Foundation Silica materials and methods of making thereof
US20190338352A1 (en) 2018-04-19 2019-11-07 Omniome, Inc. Accuracy of base calls in nucleic acid sequencing methods
CN111051525A (zh) 2018-04-20 2020-04-21 伊鲁米纳公司 包封单细胞的方法、包封的单细胞及其用途
CA3098296A1 (en) 2018-04-26 2019-10-31 Omniome, Inc. Methods and compositions for stabilizing nucleic acid-nucleotide-polymerase complexes
WO2019213237A1 (en) 2018-05-03 2019-11-07 Becton, Dickinson And Company Molecular barcoding on opposite transcript ends
ES2945191T3 (es) 2018-05-03 2023-06-29 Becton Dickinson Co Análisis de muestras multiómicas de alto rendimiento
US20210239700A1 (en) 2018-05-04 2021-08-05 Abbott Laboratories Hbv diagnostic, prognostic, and therapeutic methods and products
US20190352327A1 (en) 2018-05-15 2019-11-21 Illumina, Inc. Compositions and methods for chemical cleavage and deprotection of surface-bound oligonucleotides
CA3062226A1 (en) 2018-05-25 2019-11-25 Illumina, Inc. Circulating rna signatures specific to preeclampsia
AU2019276719A1 (en) 2018-05-31 2020-11-26 Pacific Biosciences Of California, Inc. Increased signal to noise in nucleic acid sequencing
CN111247248A (zh) 2018-06-04 2020-06-05 伊鲁米纳公司 高通量单细胞转录组文库及制备和使用方法
EP3802878A1 (en) 2018-06-04 2021-04-14 Guardant Health, Inc. Methods and systems for determining the cellular origin of cell-free nucleic acids
US20200251183A1 (en) 2018-07-11 2020-08-06 Illumina, Inc. Deep Learning-Based Framework for Identifying Sequence Patterns that Cause Sequence-Specific Errors (SSEs)
EP3827100A2 (en) 2018-07-23 2021-06-02 Guardant Health, Inc. Methods and systems for adjusting tumor mutational burden by tumor fraction and coverage
WO2020023362A1 (en) 2018-07-24 2020-01-30 Omniome, Inc. Serial formation of ternary complex species
AU2019318079A1 (en) 2018-08-07 2021-01-28 Massachusetts Institute Of Technology Novel Cas12b enzymes and systems
BR112021002779A2 (pt) 2018-08-15 2021-05-04 Illumina, Inc. composições e métodos para melhorar o enriquecimento de bibliotecas
CN109161536B (zh) * 2018-08-20 2022-04-08 天津科技大学 制备尿苷酸用酶制剂以及酶催化制备尿苷酸的方法
EP3841202B1 (en) 2018-08-20 2023-10-04 Bio-Rad Laboratories, Inc. Nucleotide sequence generation by barcode bead-colocalization in partitions
US11519033B2 (en) 2018-08-28 2022-12-06 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic DNA in a biological sample
JP2021536232A (ja) 2018-08-30 2021-12-27 ガーダント ヘルス， インコーポレイテッド 試料間の汚染を検出するための方法およびシステム
EP3844761A1 (en) 2018-08-31 2021-07-07 Guardant Health, Inc. Microsatellite instability detection in cell-free dna
EP3847276A2 (en) 2018-09-04 2021-07-14 Guardant Health, Inc. Methods and systems for detecting allelic imbalance in cell-free nucleic acid samples
WO2020060811A1 (en) 2018-09-17 2020-03-26 Omniome, Inc. Engineered polymerases for improved sequencing
ES2899203T3 (es) 2018-09-20 2022-03-10 Tamirna Gmbh Firmas de micro-ARN para la predicción de la disfunción hepática
EP3861134A1 (en) 2018-10-01 2021-08-11 Becton, Dickinson and Company Determining 5' transcript sequences
US20210396756A1 (en) 2018-10-03 2021-12-23 The Broad Institute, Inc. Crispr effector system based diagnostics for hemorrhagic fever detection
CN111328419B (zh) 2018-10-15 2021-10-19 因美纳有限公司 基于神经网络实现的方法和系统
CN113272449B (zh) 2018-10-26 2024-03-12 Illumina公司 调整聚合物小珠以进行dna处理
CA3116176A1 (en) 2018-10-31 2020-05-07 Guardant Health, Inc. Methods, compositions and systems for calibrating epigenetic partitioning assays
EP3874036A1 (en) 2018-10-31 2021-09-08 Illumina, Inc. Polymerases, compositions, and methods of use
WO2020097315A1 (en) 2018-11-08 2020-05-14 Cellular Research, Inc. Whole transcriptome analysis of single cells using random priming
NL2022043B1 (en) 2018-11-21 2020-06-03 Akershus Univ Hf Tagmentation-Associated Multiplex PCR Enrichment Sequencing
JP2022513561A (ja) 2018-11-30 2022-02-09 イルミナ インコーポレイテッド 単一アッセイを使用した複数の分析物の分析
EP3891304A1 (en) 2018-12-04 2021-10-13 Omniome, Inc. Mixed-phase fluids for nucleic acid sequencing and other analytical assays
EP3891305A1 (en) 2018-12-05 2021-10-13 Illumina Cambridge Limited Methods and compositions for cluster generation by bridge amplification
JP2022511206A (ja) 2018-12-05 2022-01-31 イラミーナ インコーポレーテッド ポリメラーゼ、組成物、および使用方法
EP3894586A2 (en) 2018-12-10 2021-10-20 10X Genomics, Inc. Methods for determining a location of a biological analyte in a biological sample
GB201820341D0 (en) 2018-12-13 2019-01-30 10X Genomics Inc Method for transposase-mediated spatial tagging and analysing genomic DNA in a biological specimen
GB201820300D0 (en) 2018-12-13 2019-01-30 10X Genomics Inc Method for spatial tagging and analysing genomic DNA in a biological specimen
WO2020123384A1 (en) 2018-12-13 2020-06-18 Cellular Research, Inc. Selective extension in single cell whole transcriptome analysis
WO2020120179A1 (en) 2018-12-14 2020-06-18 Illumina Cambridge Limited Decreasing phasing with unlabeled nucleotides during sequencing
JP2022512265A (ja) 2018-12-17 2022-02-03 イルミナ ケンブリッジ リミテッド シーケンシング用プライマーオリゴヌクレオチド
EP3899040A1 (en) 2018-12-17 2021-10-27 Illumina Cambridge Limited Compositions for use in polyunucleotide sequencing
SG11202012807YA (en) 2018-12-18 2021-01-28 Illumina Cambridge Ltd Methods and compositions for paired end sequencing using a single surface primer
FI3899037T3 (fi) 2018-12-19 2023-11-21 Illumina Inc Menetelmiä polynukleotidiklusterin klonaalisuusprioriteetin parantamiseksi
WO2020132350A2 (en) 2018-12-20 2020-06-25 Omniome, Inc. Temperature control for analysis of nucleic acids and other analytes
CN113454218A (zh) 2018-12-20 2021-09-28 夸登特健康公司 用于改进核酸分子的回收的方法、组合物和系统
US11293061B2 (en) 2018-12-26 2022-04-05 Illumina Cambridge Limited Sequencing methods using nucleotides with 3′ AOM blocking group
US11926867B2 (en) 2019-01-06 2024-03-12 10X Genomics, Inc. Generating capture probes for spatial analysis
US11649485B2 (en) 2019-01-06 2023-05-16 10X Genomics, Inc. Generating capture probes for spatial analysis
EP3908672A1 (en) 2019-01-11 2021-11-17 Illumina Cambridge Limited Complex surface-bound transposome complexes
WO2020150356A1 (en) 2019-01-16 2020-07-23 Becton, Dickinson And Company Polymerase chain reaction normalization through primer titration
ES2945227T3 (es) 2019-01-23 2023-06-29 Becton Dickinson Co Oligonucleótidos asociados con anticuerpos
MX2021008867A (es) 2019-01-23 2021-08-19 Quantum Si Inc Composiciones reactantes marcadas de intensidad alta y metodos de secuenciamiento.
EP3918089A1 (en) 2019-01-31 2021-12-08 Guardant Health, Inc. Compositions and methods for isolating cell-free dna
WO2020167574A1 (en) 2019-02-14 2020-08-20 Omniome, Inc. Mitigating adverse impacts of detection systems on nucleic acids and other biological analytes
US11680950B2 (en) 2019-02-20 2023-06-20 Pacific Biosciences Of California, Inc. Scanning apparatus and methods for detecting chemical and biological analytes
WO2020176659A1 (en) 2019-02-27 2020-09-03 Guardant Health, Inc. Methods and systems for determining the cellular origin of cell-free dna
CA3113841A1 (en) 2019-03-01 2020-09-10 Illumina, Inc. High-throughput single-nuclei and single-cell libraries and methods of making and of using
NL2023310B1 (en) 2019-03-21 2020-09-28 Illumina Inc Training data generation for artificial intelligence-based sequencing
NL2023314B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based quality scoring
NL2023316B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based sequencing
NL2023311B9 (en) 2019-03-21 2021-03-12 Illumina Inc Artificial intelligence-based generation of sequencing metadata
NL2023312B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based base calling
WO2020191387A1 (en) 2019-03-21 2020-09-24 Illumina, Inc. Artificial intelligence-based base calling
US11210554B2 (en) 2019-03-21 2021-12-28 Illumina, Inc. Artificial intelligence-based generation of sequencing metadata
US11783917B2 (en) 2019-03-21 2023-10-10 Illumina, Inc. Artificial intelligence-based base calling
US11965208B2 (en) 2019-04-19 2024-04-23 Becton, Dickinson And Company Methods of associating phenotypical data and single cell sequencing data
US11593649B2 (en) 2019-05-16 2023-02-28 Illumina, Inc. Base calling using convolutions
EP3976820A1 (en) 2019-05-30 2022-04-06 10X Genomics, Inc. Methods of detecting spatial heterogeneity of a biological sample
US11939636B2 (en) 2019-05-31 2024-03-26 Guardant Health, Inc. Methods and systems for improving patient monitoring after surgery
WO2020252186A1 (en) 2019-06-11 2020-12-17 Omniome, Inc. Calibrated focus sensing
BR112021012751A2 (pt) 2019-07-12 2021-12-14 Illumina Cambridge Ltd Preparação de biblioteca de ácidos nucleicos usando eletroforese
JP2022540268A (ja) 2019-07-12 2022-09-15 イルミナ ケンブリッジ リミテッド 固体支持体上で固定化されたｃｒｉｓｐｒ／ｃａｓ９を使用する核酸シークエンシングライブラリを調製するための組成物及び方法
US11377655B2 (en) 2019-07-16 2022-07-05 Pacific Biosciences Of California, Inc. Synthetic nucleic acids having non-natural structures
CN114051534A (zh) 2019-07-22 2022-02-15 贝克顿迪金森公司 单细胞染色质免疫沉淀测序测定
US10656368B1 (en) 2019-07-24 2020-05-19 Omniome, Inc. Method and system for biological imaging using a wide field objective lens
EP4265628A3 (en) 2019-09-10 2024-01-03 Pacific Biosciences of California, Inc. Reversible modification of nucleotides
US20220290245A1 (en) 2019-09-11 2022-09-15 The United States Of America, As Represented By The Secretary, Department Of Health And Human Servic Cancer detection and classification
WO2021076152A1 (en) 2019-10-18 2021-04-22 Omniome, Inc. Methods and compositions for capping nucleic acids
ES2938896T3 (es) 2019-10-21 2023-04-17 Univ Freiburg Albert Ludwigs Un ensayo in vitro verdaderamente imparcial para perfilar la actividad fuera de objetivo de una o más nucleasas programables específicas de objetivo en células (ABNOBA-SEQ)
US20210139867A1 (en) 2019-11-08 2021-05-13 Omniome, Inc. Engineered polymerases for improved sequencing by binding
JP2023500679A (ja) 2019-11-08 2023-01-10 ベクトン・ディキンソン・アンド・カンパニー 免疫レパートリーシーケンシングのための完全長ｖ（ｄ）ｊ情報を得るためのランダムプライミングの使用
WO2021091611A1 (en) 2019-11-08 2021-05-14 10X Genomics, Inc. Spatially-tagged analyte capture agents for analyte multiplexing
WO2021092433A2 (en) 2019-11-08 2021-05-14 10X Genomics, Inc. Enhancing specificity of analyte binding
US11753685B2 (en) 2019-11-22 2023-09-12 Illumina, Inc. Circulating RNA signatures specific to preeclampsia
CN114746560A (zh) 2019-11-26 2022-07-12 夸登特健康公司 改进甲基化多核苷酸结合的方法、组合物和系统
DE202019106694U1 (de) 2019-12-02 2020-03-19 Omniome, Inc. System zur Sequenzierung von Nukleinsäuren in Fluidschaum
DE202019106695U1 (de) 2019-12-02 2020-03-19 Omniome, Inc. System zur Sequenzierung von Nukleinsäuren in Fluidschaum
CN113631721A (zh) 2019-12-04 2021-11-09 因美纳有限公司 用于检测血浆中dna病原体的dna测序文库的制备
US20220356461A1 (en) 2019-12-19 2022-11-10 Illumina, Inc. High-throughput single-cell libraries and methods of making and of using
EP3891300B1 (en) 2019-12-23 2023-03-29 10X Genomics, Inc. Methods for spatial analysis using rna-templated ligation
CN115244184A (zh) 2020-01-13 2022-10-25 贝克顿迪金森公司 用于定量蛋白和rna的方法和组合物
US11732299B2 (en) 2020-01-21 2023-08-22 10X Genomics, Inc. Spatial assays with perturbed cells
US11702693B2 (en) 2020-01-21 2023-07-18 10X Genomics, Inc. Methods for printing cells and generating arrays of barcoded cells
US11821035B1 (en) 2020-01-29 2023-11-21 10X Genomics, Inc. Compositions and methods of making gene expression libraries
WO2021152586A1 (en) 2020-01-30 2021-08-05 Yeda Research And Development Co. Ltd. Methods of analyzing microbiome, immunoglobulin profile and physiological state
US11898205B2 (en) 2020-02-03 2024-02-13 10X Genomics, Inc. Increasing capture efficiency of spatial assays
US20230054204A1 (en) 2020-02-04 2023-02-23 Pacific Biosciences Of California, Inc. Flow cells and methods for their manufacture and use
US11732300B2 (en) 2020-02-05 2023-08-22 10X Genomics, Inc. Increasing efficiency of spatial analysis in a biological sample
US11835462B2 (en) 2020-02-11 2023-12-05 10X Genomics, Inc. Methods and compositions for partitioning a biological sample
CN111311265B (zh) * 2020-02-13 2023-07-25 布比（北京）网络技术有限公司 区块链私密交易证明方法、装置、计算机设备和存储介质
US20210265018A1 (en) 2020-02-20 2021-08-26 Illumina, Inc. Knowledge Distillation and Gradient Pruning-Based Compression of Artificial Intelligence-Based Base Caller
AU2021224871A1 (en) 2020-02-20 2022-09-08 Illumina, Inc. Artificial intelligence-based many-to-many base calling
US20210265016A1 (en) 2020-02-20 2021-08-26 Illumina, Inc. Data Compression for Artificial Intelligence-Based Base Calling
US20210265015A1 (en) 2020-02-20 2021-08-26 Illumina, Inc. Hardware Execution and Acceleration of Artificial Intelligence-Based Base Caller
US11891654B2 (en) 2020-02-24 2024-02-06 10X Genomics, Inc. Methods of making gene expression libraries
US11926863B1 (en) 2020-02-27 2024-03-12 10X Genomics, Inc. Solid state single cell method for analyzing fixed biological cells
CN115516104A (zh) 2020-03-03 2022-12-23 加利福尼亚太平洋生物科学股份有限公司 用于对双链核酸进行测序的方法和组合物
US11768175B1 (en) 2020-03-04 2023-09-26 10X Genomics, Inc. Electrophoretic methods for spatial analysis
CA3176615A1 (en) 2020-03-30 2021-10-07 Illumina, Inc. Methods and compositions for preparing nucleic acid libraries
WO2021214766A1 (en) 2020-04-21 2021-10-28 Yeda Research And Development Co. Ltd. Methods of diagnosing viral infections and vaccines thereto
EP4242325A3 (en) 2020-04-22 2023-10-04 10X Genomics, Inc. Methods for spatial analysis using targeted rna depletion
CA3177127A1 (en) 2020-04-30 2021-11-04 Guardant Health, Inc. Methods for sequence determination using partitioned nucleic acids
WO2021224677A1 (en) 2020-05-05 2021-11-11 Akershus Universitetssykehus Hf Compositions and methods for characterizing bowel cancer
CN115836135A (zh) 2020-05-05 2023-03-21 加利福尼亚太平洋生物科学股份有限公司 用于修饰聚合酶-核酸复合物的组合物和方法
US11188778B1 (en) 2020-05-05 2021-11-30 Illumina, Inc. Equalization-based image processing and spatial crosstalk attenuator
JP2023526062A (ja) 2020-05-12 2023-06-20 イルミナ インコーポレイテッド 組換え末端デオキシヌクレオチジルトランスフェラーゼを使用して修飾塩基を有する核酸の生成
US20220025468A1 (en) 2020-05-14 2022-01-27 Guardant Health, Inc. Homologous recombination repair deficiency detection
WO2021231779A1 (en) 2020-05-14 2021-11-18 Becton, Dickinson And Company Primers for immune repertoire profiling
EP4153775A1 (en) 2020-05-22 2023-03-29 10X Genomics, Inc. Simultaneous spatio-temporal measurement of gene expression and cellular activity
AU2021275906A1 (en) 2020-05-22 2022-12-22 10X Genomics, Inc. Spatial analysis to detect sequence variants
WO2021242834A1 (en) 2020-05-26 2021-12-02 10X Genomics, Inc. Method for resetting an array
WO2021247543A2 (en) 2020-06-02 2021-12-09 10X Genomics, Inc. Nucleic acid library methods
AU2021283184A1 (en) 2020-06-02 2023-01-05 10X Genomics, Inc. Spatial transcriptomics for antigen-receptors
EP4162074B1 (en) 2020-06-08 2024-04-24 10X Genomics, Inc. Methods of determining a surgical margin and methods of use thereof
WO2021252617A1 (en) 2020-06-09 2021-12-16 Illumina, Inc. Methods for increasing yield of sequencing libraries
WO2021252591A1 (en) 2020-06-10 2021-12-16 10X Genomics, Inc. Methods for determining a location of an analyte in a biological sample
US11935311B2 (en) 2020-06-11 2024-03-19 Nautilus Subsidiary, Inc. Methods and systems for computational decoding of biological, chemical, and physical entities
IL297992A (en) 2020-06-22 2023-01-01 Illumina Cambridge Ltd Nucleotides and nucleosides with a 3' mask group
WO2021263111A1 (en) 2020-06-25 2021-12-30 10X Genomics, Inc. Spatial analysis of dna methylation
AU2021300252A1 (en) 2020-07-02 2023-01-05 Illumina, Inc. A method to calibrate nucleic acid library seeding efficiency in flowcells
US11761038B1 (en) 2020-07-06 2023-09-19 10X Genomics, Inc. Methods for identifying a location of an RNA in a biological sample
MX2023000345A (es) 2020-07-08 2023-02-13 Illumina Inc Globulos como portadores de transposomas.
WO2023282916A1 (en) 2021-07-09 2023-01-12 Guardant Health, Inc. Methods of detecting genomic rearrangements using cell free nucleic acids
US11932901B2 (en) 2020-07-13 2024-03-19 Becton, Dickinson And Company Target enrichment using nucleic acid probes for scRNAseq
EP4189111A1 (en) 2020-07-30 2023-06-07 Guardant Health, Inc. Methods for isolating cell-free dna
MX2023000872A (es) 2020-08-06 2023-02-22 Illumina Inc Preparacion de genotecas de secuenciacion de arn y adn usando transposomas enlazados por globulos.
IL299783A (en) 2020-08-18 2023-03-01 Illumina Inc Sequence-Specific Targeted Transposition, Selection and Sorting of Nucleic Acids
WO2022046947A1 (en) 2020-08-25 2022-03-03 Guardant Health, Inc. Methods and systems for predicting an origin of a variant
US20220067489A1 (en) 2020-08-28 2022-03-03 Illumina, Inc. Detecting and Filtering Clusters Based on Artificial Intelligence-Predicted Base Calls
WO2022053610A1 (en) 2020-09-11 2022-03-17 Illumina Cambridge Limited Methods of enriching a target sequence from a sequencing library using hairpin adaptors
US11926822B1 (en) 2020-09-23 2024-03-12 10X Genomics, Inc. Three-dimensional spatial analysis
WO2022073011A1 (en) 2020-09-30 2022-04-07 Guardant Health, Inc. Methods and systems to improve the signal to noise ratio of dna methylation partitioning assays
CA3198842A1 (en) 2020-10-21 2022-04-28 Illumina, Inc. Sequencing templates comprising multiple inserts and compositions and methods for improving sequencing throughput
US11827935B1 (en) 2020-11-19 2023-11-28 10X Genomics, Inc. Methods for spatial analysis using rolling circle amplification and detection probes
EP4247967A1 (en) 2020-11-20 2023-09-27 Becton, Dickinson and Company Profiling of highly expressed and lowly expressed proteins
AU2021409136A1 (en) 2020-12-21 2023-06-29 10X Genomics, Inc. Methods, compositions, and systems for capturing probes and/or barcodes
WO2022140629A1 (en) 2020-12-23 2022-06-30 Guardant Health, Inc. Methods and systems for analyzing methylated polynucleotides
CA3204784A1 (en) 2021-01-13 2022-07-21 Alex Nemiroski Surface structuring with colloidal assembly
EP4284944A1 (en) 2021-01-29 2023-12-06 Illumina, Inc. Methods, compositions and kits to improve seeding efficiency of flow cells with polynucleotides
CA3208854A1 (en) 2021-02-04 2022-08-11 Illumina, Inc. Long indexed-linked read generation on transposome bound beads
US20240043915A1 (en) 2021-02-13 2024-02-08 The General Hospital Corporation Methods and compositions for in situ macromolecule detection and uses thereof
JP2024513668A (ja) 2021-03-05 2024-03-27 ガーダント ヘルス， インコーポレイテッド 分子応答を分析するための方法および関連する態様
JP2024512372A (ja) 2021-03-09 2024-03-19 ガーダント ヘルス， インコーポレイテッド オフターゲットポリヌクレオチド配列決定データに基づく腫瘍の存在の検出
WO2022197752A1 (en) 2021-03-16 2022-09-22 Illumina, Inc. Tile location and/or cycle based weight set selection for base calling
AU2022238446A1 (en) 2021-03-18 2023-09-07 10X Genomics, Inc. Multiplex capture of gene and protein expression from a biological sample
EP4314327A1 (en) 2021-03-22 2024-02-07 Illumina Cambridge Limited Methods for improving nucleic acid cluster clonality
EP4314279A1 (en) 2021-03-29 2024-02-07 Illumina, Inc. Improved methods of library preparation
KR20230163434A (ko) 2021-03-29 2023-11-30 일루미나, 인코포레이티드 라이브러리에서 dna 손상을 평가하고 앰플리콘 크기 바이어스를 정규화하기 위한 조성물 및 방법
AU2022246579A1 (en) 2021-03-30 2023-09-21 Illumina, Inc. Improved methods of isothermal complementary dna and library preparation
JP2024511760A (ja) 2021-03-31 2024-03-15 イルミナ インコーポレイテッド エラー補正のための固有分子識別子を有するトランスポゾンベースの技術を使用した指向性タグメンテーション配列決定ライブラリーの調製方法
CN115803816A (zh) 2021-03-31 2023-03-14 因美纳有限公司 具有情境感知的基于人工智能的碱基检出器
CA3214148A1 (en) 2021-04-02 2022-10-06 Brandon Tyler WESTERBERG Machine-learning model for detecting a bubble within a nucleotide-sample slide for sequencing
US20220336054A1 (en) 2021-04-15 2022-10-20 Illumina, Inc. Deep Convolutional Neural Networks to Predict Variant Pathogenicity using Three-Dimensional (3D) Protein Structures
US20220356519A1 (en) 2021-05-10 2022-11-10 Pacific Biosciences Of California, Inc. Single-molecule seeding and amplification on a surface
US20220356515A1 (en) 2021-05-10 2022-11-10 Pacific Biosciences Of California, Inc. Dna amplification buffer replenishment during rolling circle amplification
CA3216735A1 (en) 2021-05-20 2022-11-24 Patrizia IAVICOLI Compositions and methods for sequencing by synthesis
CN117677435A (zh) 2021-06-15 2024-03-08 伊鲁米纳公司 用于测序的不含水凝胶的表面官能化
US20220411864A1 (en) 2021-06-23 2022-12-29 Illumina, Inc. Compositions, methods, kits, cartridges, and systems for sequencing reagents
WO2023278184A1 (en) 2021-06-29 2023-01-05 Illumina, Inc. Methods and systems to correct crosstalk in illumination emitted from reaction sites
AU2022301321A1 (en) 2021-06-29 2024-01-18 Illumina, Inc. Machine-learning model for generating confidence classifications for genomic coordinates
KR20240027599A (ko) 2021-06-29 2024-03-04 일루미나, 인코포레이티드 올리고 서열을 사용하여 훈련된 자체-학습 염기 호출자
WO2023278927A1 (en) 2021-06-29 2023-01-05 Illumina Software, Inc. Signal-to-noise-ratio metric for determining nucleotide-base calls and base-call quality
US20230005253A1 (en) 2021-07-01 2023-01-05 Illumina, Inc. Efficient artificial intelligence-based base calling of index sequences
WO2023287617A1 (en) 2021-07-13 2023-01-19 Illumina, Inc. Methods and systems for real time extraction of crosstalk in illumination emitted from reaction sites
WO2023003757A1 (en) 2021-07-19 2023-01-26 Illumina Software, Inc. Intensity extraction with interpolation and adaptation for base calling
US11455487B1 (en) 2021-10-26 2022-09-27 Illumina Software, Inc. Intensity extraction and crosstalk attenuation using interpolation and adaptation for base calling
US20230021577A1 (en) 2021-07-23 2023-01-26 Illumina Software, Inc. Machine-learning model for recalibrating nucleotide-base calls
WO2023004357A1 (en) 2021-07-23 2023-01-26 Illumina, Inc. Methods for preparing substrate surface for dna sequencing
AU2022319125A1 (en) 2021-07-28 2024-01-18 Illumina, Inc. Quality score calibration of basecalling systems
WO2023014741A1 (en) 2021-08-03 2023-02-09 Illumina Software, Inc. Base calling using multiple base caller models
US20230047225A1 (en) 2021-08-14 2023-02-16 Illumina, Inc. Polymerases, compositions, and methods of use
WO2023023500A1 (en) 2021-08-17 2023-02-23 Illumina, Inc. Methods and compositions for identifying methylated cytosines
EP4196605A1 (en) 2021-09-01 2023-06-21 10X Genomics, Inc. Methods, compositions, and kits for blocking a capture probe on a spatial array
US20230093253A1 (en) 2021-09-17 2023-03-23 Illumina, Inc. Automatically identifying failure sources in nucleotide sequencing from base-call-error patterns
US20230095961A1 (en) 2021-09-21 2023-03-30 Illumina, Inc. Graph reference genome and base-calling approach using imputed haplotypes
WO2023049212A2 (en) 2021-09-22 2023-03-30 Illumina, Inc. State-based base calling
US20230096386A1 (en) 2021-09-30 2023-03-30 Illumina Cambridge Limited Polynucleotide sequencing
WO2023056328A2 (en) 2021-09-30 2023-04-06 Illumina, Inc. Solid supports and methods for depleting and/or enriching library fragments prepared from biosamples
AU2022367166A1 (en) 2021-10-11 2024-04-04 Nautilus Subsidiary, Inc. Highly multiplexable analysis of proteins and proteomes
CA3234961A1 (en) 2021-10-20 2023-04-27 Illumina, Inc. Methods for capturing library dna for sequencing
WO2023081485A1 (en) 2021-11-08 2023-05-11 Pacific Biosciences Of California, Inc. Stepwise sequencing of a polynucleotide with a homogenous reaction mixture
CN117581303A (zh) 2021-12-02 2024-02-20 因美纳有限公司 产生用于确定核苷酸碱基检出的簇特异性信号校正
WO2023122363A1 (en) 2021-12-23 2023-06-29 Illumina Software, Inc. Dynamic graphical status summaries for nucelotide sequencing
US20230215515A1 (en) 2021-12-23 2023-07-06 Illumina Software, Inc. Facilitating secure execution of external workflows for genomic sequencing diagnostics
US20230207050A1 (en) 2021-12-28 2023-06-29 Illumina Software, Inc. Machine learning model for recalibrating nucleotide base calls corresponding to target variants
WO2023129764A1 (en) 2021-12-29 2023-07-06 Illumina Software, Inc. Automatically switching variant analysis model versions for genomic analysis applications
CA3223362A1 (en) 2022-01-20 2023-07-27 Xiaolin Wu Methods of detecting methylcytosine and hydroxymethylcytosine by sequencing
AU2023225949A1 (en) 2022-02-25 2024-01-18 Illumina, Inc. Machine-learning models for detecting and adjusting values for nucleotide methylation levels
US20230410944A1 (en) 2022-02-25 2023-12-21 Illumina, Inc. Calibration sequences for nucelotide sequencing
WO2023183937A1 (en) 2022-03-25 2023-09-28 Illumina, Inc. Sequence-to-sequence base calling
WO2023192917A1 (en) 2022-03-29 2023-10-05 Nautilus Subsidiary, Inc. Integrated arrays for single-analyte processes
CA3223722A1 (en) 2022-04-07 2023-10-12 Illumina, Inc. Altered cytidine deaminases and methods of use
WO2023212490A1 (en) 2022-04-25 2023-11-02 Nautilus Subsidiary, Inc. Systems and methods for assessing and improving the quality of multiplex molecular assays
US20230340571A1 (en) 2022-04-26 2023-10-26 Illumina, Inc. Machine-learning models for selecting oligonucleotide probes for array technologies
US20230348967A1 (en) 2022-04-29 2023-11-02 Illumina Cambridge Limited Methods and systems for encapsulating lyophilised microspheres
US20230360725A1 (en) 2022-05-09 2023-11-09 Guardant Health, Inc. Detecting degradation based on strand bias
US20230368866A1 (en) 2022-05-10 2023-11-16 Illumina Software, Inc. Adaptive neural network for nucelotide sequencing
US20230392207A1 (en) 2022-06-03 2023-12-07 Illumina, Inc. Circulating rna biomarkers for preeclampsia
US20230407386A1 (en) 2022-06-09 2023-12-21 Illumina, Inc. Dependence of base calling on flow cell tilt
WO2023240494A1 (zh) * 2022-06-15 2023-12-21 深圳华大智造科技股份有限公司 测序缓冲液及提高具有可逆阻断基团修饰的dNTP的稳定性的方法
US20230420080A1 (en) 2022-06-24 2023-12-28 Illumina Software, Inc. Split-read alignment by intelligently identifying and scoring candidate split groups
US20230420082A1 (en) 2022-06-27 2023-12-28 Illumina Software, Inc. Generating and implementing a structural variation graph genome
WO2024006779A1 (en) 2022-06-27 2024-01-04 Illumina, Inc. Accelerators for a genotype imputation model
WO2024006705A1 (en) 2022-06-27 2024-01-04 Illumina Software, Inc. Improved human leukocyte antigen (hla) genotyping
CN116024320A (zh) * 2022-07-13 2023-04-28 上海翔琼生物技术有限公司 一种用于检测核酸的荧光定量pcr方法
WO2024015962A1 (en) 2022-07-15 2024-01-18 Pacific Biosciences Of California, Inc. Blocked asymmetric hairpin adaptors
US20240038327A1 (en) 2022-07-26 2024-02-01 Illumina Software, Inc. Rapid single-cell multiomics processing using an executable file
WO2024039516A1 (en) 2022-08-19 2024-02-22 Illumina, Inc. Third dna base pair site-specific dna detection
US20240094215A1 (en) 2022-09-15 2024-03-21 Nautilus Subsidiary, Inc. Characterizing accessibility of macromolecule structures
WO2024073516A1 (en) 2022-09-29 2024-04-04 Illumina, Inc. A target-variant-reference panel for imputing target variants
WO2024073519A1 (en) 2022-09-30 2024-04-04 Illumina, Inc. Machine-learning model for refining structural variant calls
WO2024073047A1 (en) 2022-09-30 2024-04-04 Illumina, Inc. Cytidine deaminases and methods of use in mapping modified cytosine nucleotides
WO2024069581A1 (en) 2022-09-30 2024-04-04 Illumina Singapore Pte. Ltd. Helicase-cytidine deaminase complexes and methods of use
WO2024073043A1 (en) 2022-09-30 2024-04-04 Illumina, Inc. Methods of using cpg binding proteins in mapping modified cytosine nucleotides
WO2024068971A1 (en) 2022-09-30 2024-04-04 Illumina, Inc. Polymerases, compositions, and methods of use
US20240127905A1 (en) 2022-10-05 2024-04-18 Illumina, Inc. Integrating variant calls from multiple sequencing pipelines utilizing a machine learning architecture
WO2024077202A2 (en) 2022-10-06 2024-04-11 Illumina, Inc. Probes for improving environmental sample surveillance
WO2024077162A2 (en) 2022-10-06 2024-04-11 Illumina, Inc. Probes for improving coronavirus sample surveillance
WO2024077152A1 (en) 2022-10-06 2024-04-11 Illumina, Inc. Probes for depleting abundant small noncoding rna
WO2024081649A1 (en) 2022-10-11 2024-04-18 Illumina, Inc. Detecting and correcting methylation values from methylation sequencing assays

Citations (65)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4994373A (en) * 1983-01-27 1991-02-19 Enzo Biochem, Inc. Method and structures employing chemically-labelled polynucleotide probes
US4997928A (en) * 1988-09-15 1991-03-05 E. I. Du Pont De Nemours And Company Fluorescent reagents for the preparation of 5'-tagged oligonucleotides
US5200313A (en) * 1983-08-05 1993-04-06 Miles Inc. Nucleic acid hybridization assay employing detectable anti-hybrid antibodies
US5230781A (en) * 1984-03-29 1993-07-27 Li-Cor, Inc. Sequencing near infrared and infrared fluorescence labeled DNA for detecting using laser diodes
US5232075A (en) * 1991-06-25 1993-08-03 New Venture Gear, Inc. Viscous coupling apparatus with coined plates
US5241060A (en) * 1982-06-23 1993-08-31 Enzo Diagnostics, Inc. Base moiety-labeled detectable nucleatide
US5302509A (en) * 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
US5403708A (en) * 1992-07-06 1995-04-04 Brennan; Thomas M. Methods and compositions for determining the sequence of nucleic acids
US5405747A (en) * 1991-09-25 1995-04-11 The Regents Of The University Of California Office Of Technology Transfer Method for rapid base sequencing in DNA and RNA with two base labeling
US5534125A (en) * 1984-03-29 1996-07-09 Li-Cor, Inc. DNA sequencing
US5547839A (en) * 1989-06-07 1996-08-20 Affymax Technologies N.V. Sequencing of surface immobilized polymers utilizing microflourescence detection
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
US5601982A (en) * 1995-02-07 1997-02-11 Sargent; Jeannine P. Method and apparatus for determining the sequence of polynucleotides
US5620854A (en) * 1993-08-25 1997-04-15 Regents Of The University Of California Method for identifying biochemical and chemical reactions and micromechanical processes using nanomechanical and electronic signal identification
US5631134A (en) * 1992-11-06 1997-05-20 The Trustees Of Boston University Methods of preparing probe array by hybridation
US5639874A (en) * 1984-03-29 1997-06-17 Li-Cor, Inc. Method for preparing fluorescent-labeled DNA
US5646264A (en) * 1990-03-14 1997-07-08 The Regents Of The University Of California DNA complexes with dyes designed for energy transfer as fluorescent markers
US5661028A (en) * 1995-09-29 1997-08-26 Lockheed Martin Energy Systems, Inc. Large scale DNA microsequencing device
US5707804A (en) * 1994-02-01 1998-01-13 The Regents Of The University Of California Primers labeled with energy transfer coupled dyes for DNA sequencing
US5723298A (en) * 1996-09-16 1998-03-03 Li-Cor, Inc. Cycle labeling and sequencing with thermostable polymerases
US5858671A (en) * 1996-11-01 1999-01-12 The University Of Iowa Research Foundation Iterative and regenerative DNA sequencing method
US5922591A (en) * 1995-06-29 1999-07-13 Affymetrix, Inc. Integrated nucleic acid diagnostic device
US6027709A (en) * 1997-01-10 2000-02-22 Li-Cor Inc. Fluorescent cyanine dyes
US6027890A (en) * 1996-01-23 2000-02-22 Rapigene, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
US6044744A (en) * 1998-10-29 2000-04-04 At&T Corp. Fiber optic cable sheath removal tool
US6048690A (en) * 1991-11-07 2000-04-11 Nanogen, Inc. Methods for electronic fluorescent perturbation for analysis and electronic perturbation catalysis for synthesis
US6086737A (en) * 1984-03-29 2000-07-11 Li-Cor, Inc. Sequencing near infrared and infrared fluorescence labeled DNA for detecting using laser diodes and suitable labels therefor
US6207421B1 (en) * 1984-03-29 2001-03-27 Li-Cor, Inc. DNA sequencing and DNA terminators
US6210896B1 (en) * 1998-08-13 2001-04-03 Us Genomics Molecular motors
US6221592B1 (en) * 1998-10-20 2001-04-24 Wisconsin Alumi Research Foundation Computer-based methods and systems for sequencing of individual nucleic acid molecules
US6255083B1 (en) * 1998-12-14 2001-07-03 Li Cor Inc System and methods for nucleic acid sequencing of single molecules by polymerase synthesis
US6263286B1 (en) * 1998-08-13 2001-07-17 U.S. Genomics, Inc. Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer
US6280939B1 (en) * 1998-09-01 2001-08-28 Veeco Instruments, Inc. Method and apparatus for DNA sequencing using a local sensitive force detector
US20020025529A1 (en) * 1999-06-28 2002-02-28 Stephen Quake Methods and apparatus for analyzing polynucleotide sequences
US6355420B1 (en) * 1997-02-12 2002-03-12 Us Genomics Methods and products for analyzing polymers
US6399335B1 (en) * 1999-11-16 2002-06-04 Advanced Research And Technology Institute, Inc. γ-phosphoester nucleoside triphosphates
US6403311B1 (en) * 1997-02-12 2002-06-11 Us Genomics Methods of analyzing polymers using ordered label strategies
US6524829B1 (en) * 1998-09-30 2003-02-25 Molecular Machines & Industries Gmbh Method for DNA- or RNA-sequencing
US20030044781A1 (en) * 1999-05-19 2003-03-06 Jonas Korlach Method for sequencing nucleic acid molecules
US20030064400A1 (en) * 2001-08-24 2003-04-03 Li-Cor, Inc. Microfluidics system for single molecule DNA sequencing
US6558945B1 (en) * 1999-03-08 2003-05-06 Aclara Biosciences, Inc. Method and device for rapid color detection
US6593148B1 (en) * 1994-03-01 2003-07-15 Li-Cor, Inc. Cyanine dye compounds and labeling methods
US20030134807A1 (en) * 2000-12-01 2003-07-17 Hardin Susan H. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
US20040015964A1 (en) * 2001-04-25 2004-01-22 Mccann Thomas Matthew Methods and systems for load sharing signaling messages among signaling links in networks utilizing international signaling protocols
US20050042633A1 (en) * 2003-04-08 2005-02-24 Li-Cor, Inc. Composition and method for nucleic acid sequencing
US6869764B2 (en) * 2000-06-07 2005-03-22 L--Cor, Inc. Nucleic acid sequencing using charge-switch nucleotides
US6982186B2 (en) * 2003-09-25 2006-01-03 Dongbuanam Semiconductor Inc. CMOS image sensor and method for manufacturing the same
US6982146B1 (en) * 1999-08-30 2006-01-03 The United States Of America As Represented By The Department Of Health And Human Services High speed parallel molecular nucleic acid sequencing
US6995274B2 (en) * 2000-09-19 2006-02-07 Li-Cor, Inc. Cyanine dyes
US7005518B2 (en) * 2002-10-25 2006-02-28 Li-Cor, Inc. Phthalocyanine dyes
US20060061755A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for analysis of molecules
US20060060766A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and methods for optical analysis of molecules
US20060063173A1 (en) * 2000-06-07 2006-03-23 Li-Cor, Inc. Charge switch nucleotides
US7037687B2 (en) * 1998-05-01 2006-05-02 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US20070036502A1 (en) * 2001-09-27 2007-02-15 Levene Michael J Zero-mode waveguides
US20070042398A1 (en) * 2005-06-30 2007-02-22 Li-Cor, Inc. Cyanine dyes and methods of use
US20070048748A1 (en) * 2004-09-24 2007-03-01 Li-Cor, Inc. Mutant polymerases for sequencing and genotyping
US20070044538A1 (en) * 2005-09-01 2007-03-01 Li-Cor, Inc. Gas flux system chamber design and positioning method
US20070134128A1 (en) * 2005-11-28 2007-06-14 Pacific Biosciences Of California, Inc. Uniform surfaces for hybrid material substrate and methods for making and using same
US20070154921A1 (en) * 2005-12-16 2007-07-05 Applera Corporation Method and System for Phase-Locked Sequencing
US20070172868A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US20070172866A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination using depolymerizing agent
US20080076189A1 (en) * 2006-03-30 2008-03-27 Visigen Biotechnologies, Inc. Modified surfaces for the detection of biomolecules at the single molecule level
US20080091005A1 (en) * 2006-07-20 2008-04-17 Visigen Biotechnologies, Inc. Modified nucleotides, methods for making and using same
US7393640B2 (en) * 2003-02-05 2008-07-01 Ge Healthcare Bio-Sciences Corp. Terminal-phosphate-labeled nucleotides with new linkers

Family Cites Families (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5571388A (en) * 1984-03-29 1996-11-05 Li-Cor, Inc. Sequencing near infrared and infrared fluorescense labeled DNA for detecting using laser diodes and suitable labels thereof
JP3227189B2 (ja) * 1991-12-25 2001-11-12 キヤノン株式会社 フレキシブルケーブルを備えた装置及び該装置を具備するインクジェット記録装置
US5677196A (en) * 1993-05-18 1997-10-14 University Of Utah Research Foundation Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays
US5512462A (en) 1994-02-25 1996-04-30 Hoffmann-La Roche Inc. Methods and reagents for the polymerase chain reaction amplification of long DNA sequences
US5961923A (en) * 1995-04-25 1999-10-05 Irori Matrices with memories and uses thereof
US6165765A (en) * 1995-10-18 2000-12-26 Shanghai Institute Of Biochemistry, Chinese Academy Of Sciences DNA polymerase having ability to reduce innate selective discrimination against fluorescent dye-labeled dideoxynucleotides
US5972603A (en) * 1996-02-09 1999-10-26 President And Fellows Of Harvard College DNA polymerase with modified processivity
US5804386A (en) * 1997-01-15 1998-09-08 Incyte Pharmaceuticals, Inc. Sets of labeled energy transfer fluorescent primers and their use in multi component analysis
AU743025B2 (en) * 1997-03-12 2002-01-17 Applera Corporation DNA polymerases having improved labeled nucleotide incorporation properties
ATE225858T1 (de) 1997-07-28 2002-10-15 Medical Biosystems Ltd Sequenzanalyse von nukleinsäuren
JP2002521064A (ja) 1998-07-30 2002-07-16 ソレックサ リミテッド アレイ生体分子およびシークエンシングにおけるその使用
JP3542967B2 (ja) 1998-12-15 2004-07-14 松下電器産業株式会社 クロック位相調整方法、及び集積回路とその設計方法
ATE397092T1 (de) * 1999-03-10 2008-06-15 Asm Scient Inc Methode zur direkten sequenzierung von nukleinsäuren
GB9907812D0 (en) 1999-04-06 1999-06-02 Medical Biosystems Ltd Sequencing
AU7086800A (en) * 1999-08-30 2001-03-26 Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The High speed parallel molecular nucleic acid sequencing
WO2001023610A2 (en) 1999-09-29 2001-04-05 Solexa Ltd. Polynucleotide sequencing
GB9923644D0 (en) 1999-10-06 1999-12-08 Medical Biosystems Ltd DNA sequencing
US20040161741A1 (en) * 2001-06-30 2004-08-19 Elazar Rabani Novel compositions and processes for analyte detection, quantification and amplification
US7052839B2 (en) * 2001-08-29 2006-05-30 Amersham Biosciences Corp Terminal-phosphate-labeled nucleotides and methods of use
US7223541B2 (en) * 2001-08-29 2007-05-29 Ge Healthcare Bio-Sciences Corp. Terminal-phosphate-labeled nucleotides and methods of use
US7033762B2 (en) * 2001-08-29 2006-04-25 Amersham Biosciences Corp Single nucleotide amplification and detection by polymerase
EP1421213B1 (en) * 2001-08-29 2010-02-17 Amersham Biosciences Corp. Labeled nucleoside polyphosphates
EP1590479B1 (en) * 2003-02-05 2010-11-24 GE Healthcare Bio-Sciences Corp. Nucleic acid amplification
US7482120B2 (en) * 2005-01-28 2009-01-27 Helicos Biosciences Corporation Methods and compositions for improving fidelity in a nucleic acid synthesis reaction
EP1846758A2 (en) * 2005-02-09 2007-10-24 Pacific Biosciences of California, Inc. Nucleotide compositions and uses thereof
US7130041B2 (en) * 2005-03-02 2006-10-31 Li-Cor, Inc. On-chip spectral filtering using CCD array for imaging and spectroscopy

Patent Citations (98)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5241060A (en) * 1982-06-23 1993-08-31 Enzo Diagnostics, Inc. Base moiety-labeled detectable nucleatide
US4994373A (en) * 1983-01-27 1991-02-19 Enzo Biochem, Inc. Method and structures employing chemically-labelled polynucleotide probes
US5200313A (en) * 1983-08-05 1993-04-06 Miles Inc. Nucleic acid hybridization assay employing detectable anti-hybrid antibodies
US5639874A (en) * 1984-03-29 1997-06-17 Li-Cor, Inc. Method for preparing fluorescent-labeled DNA
US5230781A (en) * 1984-03-29 1993-07-27 Li-Cor, Inc. Sequencing near infrared and infrared fluorescence labeled DNA for detecting using laser diodes
US6207421B1 (en) * 1984-03-29 2001-03-27 Li-Cor, Inc. DNA sequencing and DNA terminators
US6086737A (en) * 1984-03-29 2000-07-11 Li-Cor, Inc. Sequencing near infrared and infrared fluorescence labeled DNA for detecting using laser diodes and suitable labels therefor
US5755943A (en) * 1984-03-29 1998-05-26 Li-Cor, Inc. DNA sequencing
US5534125A (en) * 1984-03-29 1996-07-09 Li-Cor, Inc. DNA sequencing
US4997928A (en) * 1988-09-15 1991-03-05 E. I. Du Pont De Nemours And Company Fluorescent reagents for the preparation of 5'-tagged oligonucleotides
US5547839A (en) * 1989-06-07 1996-08-20 Affymax Technologies N.V. Sequencing of surface immobilized polymers utilizing microflourescence detection
US5302509A (en) * 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
US5646264A (en) * 1990-03-14 1997-07-08 The Regents Of The University Of California DNA complexes with dyes designed for energy transfer as fluorescent markers
US5232075A (en) * 1991-06-25 1993-08-03 New Venture Gear, Inc. Viscous coupling apparatus with coined plates
US5405747A (en) * 1991-09-25 1995-04-11 The Regents Of The University Of California Office Of Technology Transfer Method for rapid base sequencing in DNA and RNA with two base labeling
US6048690A (en) * 1991-11-07 2000-04-11 Nanogen, Inc. Methods for electronic fluorescent perturbation for analysis and electronic perturbation catalysis for synthesis
US5403708A (en) * 1992-07-06 1995-04-04 Brennan; Thomas M. Methods and compositions for determining the sequence of nucleic acids
US5631134A (en) * 1992-11-06 1997-05-20 The Trustees Of Boston University Methods of preparing probe array by hybridation
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
US5620854A (en) * 1993-08-25 1997-04-15 Regents Of The University Of California Method for identifying biochemical and chemical reactions and micromechanical processes using nanomechanical and electronic signal identification
US5707804A (en) * 1994-02-01 1998-01-13 The Regents Of The University Of California Primers labeled with energy transfer coupled dyes for DNA sequencing
US6593148B1 (en) * 1994-03-01 2003-07-15 Li-Cor, Inc. Cyanine dye compounds and labeling methods
US5601982A (en) * 1995-02-07 1997-02-11 Sargent; Jeannine P. Method and apparatus for determining the sequence of polynucleotides
US5922591A (en) * 1995-06-29 1999-07-13 Affymetrix, Inc. Integrated nucleic acid diagnostic device
US5661028A (en) * 1995-09-29 1997-08-26 Lockheed Martin Energy Systems, Inc. Large scale DNA microsequencing device
US6027890A (en) * 1996-01-23 2000-02-22 Rapigene, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
US5723298A (en) * 1996-09-16 1998-03-03 Li-Cor, Inc. Cycle labeling and sequencing with thermostable polymerases
US5858671A (en) * 1996-11-01 1999-01-12 The University Of Iowa Research Foundation Iterative and regenerative DNA sequencing method
US6027709A (en) * 1997-01-10 2000-02-22 Li-Cor Inc. Fluorescent cyanine dyes
US6355420B1 (en) * 1997-02-12 2002-03-12 Us Genomics Methods and products for analyzing polymers
US6403311B1 (en) * 1997-02-12 2002-06-11 Us Genomics Methods of analyzing polymers using ordered label strategies
US7037687B2 (en) * 1998-05-01 2006-05-02 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US6210896B1 (en) * 1998-08-13 2001-04-03 Us Genomics Molecular motors
US6263286B1 (en) * 1998-08-13 2001-07-17 U.S. Genomics, Inc. Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer
US20010014850A1 (en) * 1998-08-13 2001-08-16 U.S. Genomics, Inc. Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer
US6280939B1 (en) * 1998-09-01 2001-08-28 Veeco Instruments, Inc. Method and apparatus for DNA sequencing using a local sensitive force detector
US6524829B1 (en) * 1998-09-30 2003-02-25 Molecular Machines & Industries Gmbh Method for DNA- or RNA-sequencing
US6221592B1 (en) * 1998-10-20 2001-04-24 Wisconsin Alumi Research Foundation Computer-based methods and systems for sequencing of individual nucleic acid molecules
US6044744A (en) * 1998-10-29 2000-04-04 At&T Corp. Fiber optic cable sheath removal tool
US6255083B1 (en) * 1998-12-14 2001-07-03 Li Cor Inc System and methods for nucleic acid sequencing of single molecules by polymerase synthesis
US7229799B2 (en) * 1998-12-14 2007-06-12 Li-Cor, Inc. System and method for nucleic acid sequencing by polymerase synthesis
US6762048B2 (en) * 1998-12-14 2004-07-13 Li-Cor, Inc. System and apparatus for nucleic acid sequencing of single molecules by polymerase synthesis
US6558945B1 (en) * 1999-03-08 2003-05-06 Aclara Biosciences, Inc. Method and device for rapid color detection
US20060160113A1 (en) * 1999-05-19 2006-07-20 Jonas Korlach Terminal-phosphate-labeled nucleotides
US20060057606A1 (en) * 1999-05-19 2006-03-16 Jonas Korlach Reagents containing terminal-phosphate-labeled nucleotides for nucleic acid sequencing
US7361466B2 (en) * 1999-05-19 2008-04-22 Cornell Research Foundation, Inc. Nucleic acid analysis using terminal-phosphate-labeled nucleotides
US7033764B2 (en) * 1999-05-19 2006-04-25 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7052847B2 (en) * 1999-05-19 2006-05-30 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US20030044781A1 (en) * 1999-05-19 2003-03-06 Jonas Korlach Method for sequencing nucleic acid molecules
US7056676B2 (en) * 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US20050158761A1 (en) * 1999-05-19 2005-07-21 Jonas Korlach Method for sequencing nucleic acid molecules
US20050164255A1 (en) * 1999-05-19 2005-07-28 Jonas Korlach Method for sequencing nucleic acid molecules
US20060078937A1 (en) * 1999-05-19 2006-04-13 Jonas Korlach Sequencing nucleic acid using tagged polymerase and/or tagged nucleotide
US7056661B2 (en) * 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US20060154288A1 (en) * 1999-05-19 2006-07-13 Jonas Korlach Methods for analyzing nucleic acid sequences
US20060134666A1 (en) * 1999-05-19 2006-06-22 Jonas Korlach Methods for detecting nucleic acid analyte
US20020025529A1 (en) * 1999-06-28 2002-02-28 Stephen Quake Methods and apparatus for analyzing polynucleotide sequences
US6911345B2 (en) * 1999-06-28 2005-06-28 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
US6982146B1 (en) * 1999-08-30 2006-01-03 The United States Of America As Represented By The Department Of Health And Human Services High speed parallel molecular nucleic acid sequencing
US6399335B1 (en) * 1999-11-16 2002-06-04 Advanced Research And Technology Institute, Inc. γ-phosphoester nucleoside triphosphates
US20070134716A1 (en) * 2000-05-17 2007-06-14 Levene Michael J Zero-mode metal clad waveguides for performing spectroscopy with confined effective observation volumes
US6869764B2 (en) * 2000-06-07 2005-03-22 L--Cor, Inc. Nucleic acid sequencing using charge-switch nucleotides
US20060063173A1 (en) * 2000-06-07 2006-03-23 Li-Cor, Inc. Charge switch nucleotides
US20070172865A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Sequence determination in confined regions
US20070172861A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Mutant polymerases
US20070172862A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Data stream determination
US20070172863A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions and methods for sequence determination
US20070172866A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination using depolymerizing agent
US20070172867A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination
US20070172868A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US20070172864A1 (en) * 2000-07-07 2007-07-26 Xiaolian Gao Composition for sequence determination
US20070172858A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination
US20060063247A1 (en) * 2000-09-19 2006-03-23 Li-Cor, Inc. Cyanine dyes
US6995274B2 (en) * 2000-09-19 2006-02-07 Li-Cor, Inc. Cyanine dyes
US20070172859A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: methods for direct detection of tagged monomers
US20070172860A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: compositions and methods
US20070172819A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: compositions including pyrophosphorolysis inhibitors
US20070172869A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: methods for inhibiting pyrophosphorolysis during sequencing synthesis
US20030134807A1 (en) * 2000-12-01 2003-07-17 Hardin Susan H. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
US20040015964A1 (en) * 2001-04-25 2004-01-22 Mccann Thomas Matthew Methods and systems for load sharing signaling messages among signaling links in networks utilizing international signaling protocols
US20030064400A1 (en) * 2001-08-24 2003-04-03 Li-Cor, Inc. Microfluidics system for single molecule DNA sequencing
US20070036502A1 (en) * 2001-09-27 2007-02-15 Levene Michael J Zero-mode waveguides
US7005518B2 (en) * 2002-10-25 2006-02-28 Li-Cor, Inc. Phthalocyanine dyes
US7393640B2 (en) * 2003-02-05 2008-07-01 Ge Healthcare Bio-Sciences Corp. Terminal-phosphate-labeled nucleotides with new linkers
US20050042633A1 (en) * 2003-04-08 2005-02-24 Li-Cor, Inc. Composition and method for nucleic acid sequencing
US6982186B2 (en) * 2003-09-25 2006-01-03 Dongbuanam Semiconductor Inc. CMOS image sensor and method for manufacturing the same
US20060063264A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for performing nucleic acid analysis
US20060061755A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for analysis of molecules
US20060060766A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and methods for optical analysis of molecules
US20060061754A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Arrays of optical confinements and uses thereof
US20060062531A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Fabrication of optical confinements
US20070048748A1 (en) * 2004-09-24 2007-03-01 Li-Cor, Inc. Mutant polymerases for sequencing and genotyping
US20070042398A1 (en) * 2005-06-30 2007-02-22 Li-Cor, Inc. Cyanine dyes and methods of use
US20070044538A1 (en) * 2005-09-01 2007-03-01 Li-Cor, Inc. Gas flux system chamber design and positioning method
US20070134128A1 (en) * 2005-11-28 2007-06-14 Pacific Biosciences Of California, Inc. Uniform surfaces for hybrid material substrate and methods for making and using same
US20070154921A1 (en) * 2005-12-16 2007-07-05 Applera Corporation Method and System for Phase-Locked Sequencing
US20080076189A1 (en) * 2006-03-30 2008-03-27 Visigen Biotechnologies, Inc. Modified surfaces for the detection of biomolecules at the single molecule level
US20080091005A1 (en) * 2006-07-20 2008-04-17 Visigen Biotechnologies, Inc. Modified nucleotides, methods for making and using same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"How many species of bacteria are there" (wisegeek.com; accessed 23 September 2011). *
"Viruses" (Wikipedia.com, accessed 18 April 2012). *

Cited By (334)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020085891A1 (en) * 1996-02-16 2002-07-04 Moore Richard A. Twist drill bit
US10214774B2 (en) 1998-05-01 2019-02-26 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9957561B2 (en) 1998-05-01 2018-05-01 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US10208341B2 (en) 1998-05-01 2019-02-19 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9096898B2 (en) 1998-05-01 2015-08-04 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7645596B2 (en) 1998-05-01 2010-01-12 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9212393B2 (en) 1998-05-01 2015-12-15 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9458500B2 (en) 1998-05-01 2016-10-04 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9725764B2 (en) 1998-05-01 2017-08-08 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9540689B2 (en) 1998-05-01 2017-01-10 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7485424B2 (en) 1999-05-19 2009-02-03 Cornell Research Foundation, Inc. Labeled nucleotide phosphate (NP) probes
US20060154288A1 (en) * 1999-05-19 2006-07-13 Jonas Korlach Methods for analyzing nucleic acid sequences
US20050164255A1 (en) * 1999-05-19 2005-07-28 Jonas Korlach Method for sequencing nucleic acid molecules
US20060188900A1 (en) * 1999-05-19 2006-08-24 Jonas Korlach High speed nucleic acid sequencing
US7943305B2 (en) 1999-05-19 2011-05-17 Cornell Research Foundation High speed nucleic acid sequencing
US7943307B2 (en) 1999-05-19 2011-05-17 Cornell Research Foundation Methods for analyzing nucleic acid sequences
US20080227654A1 (en) * 1999-05-19 2008-09-18 Jonas Korlach Method for sequencing nucleic acid molecules
US20110111401A1 (en) * 1999-05-19 2011-05-12 Cornell University Method for sequencing nucleic acid molecules
US7416844B2 (en) 1999-05-19 2008-08-26 Cornell Research Foundation, Inc. Composition for nucleic acid sequencing
US7361466B2 (en) 1999-05-19 2008-04-22 Cornell Research Foundation, Inc. Nucleic acid analysis using terminal-phosphate-labeled nucleotides
US7033764B2 (en) 1999-05-19 2006-04-25 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US20050158761A1 (en) * 1999-05-19 2005-07-21 Jonas Korlach Method for sequencing nucleic acid molecules
US7052847B2 (en) 1999-05-19 2006-05-30 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US20070026447A1 (en) * 1999-05-19 2007-02-01 Jonas Korlach Nucleotides
US7056661B2 (en) 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7056676B2 (en) 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US20020025529A1 (en) * 1999-06-28 2002-02-28 Stephen Quake Methods and apparatus for analyzing polynucleotide sequences
US8535881B2 (en) 1999-08-30 2013-09-17 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services High speed parallel molecular nucleic acid sequencing
US6982146B1 (en) 1999-08-30 2006-01-03 The United States Of America As Represented By The Department Of Health And Human Services High speed parallel molecular nucleic acid sequencing
US20090061447A1 (en) * 1999-08-30 2009-03-05 The Government of the United States of America as represented by the Secretary of the High speed parallel molecular nucleic acid sequencing
US20110008794A1 (en) * 1999-08-30 2011-01-13 The Government of USA represented by the Secretary of the Dept. of Health and Human Services High speed parallel molecular nucleic acid sequencing
US20070172863A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions and methods for sequence determination
US20070184475A1 (en) * 2000-07-07 2007-08-09 Susan Hardin Sequence determination by direct detection
US20070292867A1 (en) * 2000-07-07 2007-12-20 Susan Hardin Sequence determination using multiply tagged polymerizing agents
US20070172868A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US20070172858A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination
US20070172861A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Mutant polymerases
US20070172866A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination using depolymerizing agent
US20070275395A1 (en) * 2000-07-07 2007-11-29 Susan Hardin Tagged monomers for use in sequence determination
US20100255463A1 (en) * 2000-07-07 2010-10-07 Susan Harsin Compositions and methods for sequence determination
US20070172860A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: compositions and methods
US20110184163A1 (en) * 2000-12-01 2011-07-28 Life Technologies Corporation Enzymatic Nucleic Acid Synthesis: Compositions and Methods for Inhibiting Pyrophosphorolysis
US20100216122A1 (en) * 2000-12-01 2010-08-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: methods for direct detection of tagged monomers
US8648179B2 (en) 2000-12-01 2014-02-11 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US9243284B2 (en) 2000-12-01 2016-01-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US8314216B2 (en) 2000-12-01 2012-11-20 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US20070172819A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: compositions including pyrophosphorolysis inhibitors
US9845500B2 (en) 2000-12-01 2017-12-19 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US20020164629A1 (en) * 2001-03-12 2002-11-07 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension
US7052839B2 (en) * 2001-08-29 2006-05-30 Amersham Biosciences Corp Terminal-phosphate-labeled nucleotides and methods of use
US7041812B2 (en) * 2001-08-29 2006-05-09 Amersham Biosciences Corp Labeled nucleoside polyphosphates
US20030077610A1 (en) * 2001-08-29 2003-04-24 John Nelson Terminal-phosphate-labeled nucleotides and methods of use
US7033762B2 (en) * 2001-08-29 2006-04-25 Amersham Biosciences Corp Single nucleotide amplification and detection by polymerase
US20030162213A1 (en) * 2001-08-29 2003-08-28 Carl Fuller Terminal-phosphate-labeled nucleotides and methods of use
US20030096253A1 (en) * 2001-08-29 2003-05-22 John Nelson Single nucleotide amplification and detection by polymerase
US20030124576A1 (en) * 2001-08-29 2003-07-03 Shiv Kumar Labeled nucleoside polyphosphates
US20090081644A1 (en) * 2001-08-29 2009-03-26 General Electric Company Ligation amplification
US7223541B2 (en) * 2001-08-29 2007-05-29 Ge Healthcare Bio-Sciences Corp. Terminal-phosphate-labeled nucleotides and methods of use
US7727722B2 (en) * 2001-08-29 2010-06-01 General Electric Company Ligation amplification
US20040248186A1 (en) * 2001-09-24 2004-12-09 Intel Corporation Nucleic acid sequencing by Raman monitoring of uptake of precursors during molecular replication
US7364851B2 (en) 2001-09-24 2008-04-29 Intel Corporation Nucleic acid sequencing by Raman monitoring of uptake of precursors during molecular replication
US20040014096A1 (en) * 2002-04-12 2004-01-22 Stratagene Dual-labeled nucleotides
US20090186343A1 (en) * 2003-01-28 2009-07-23 Visigen Biotechnologies, Inc. Methods for preparing modified biomolecules, modified biomolecules and methods for using same
US7393640B2 (en) * 2003-02-05 2008-07-01 Ge Healthcare Bio-Sciences Corp. Terminal-phosphate-labeled nucleotides with new linkers
US20040241716A1 (en) * 2003-02-05 2004-12-02 Shiv Kumar Terminal-phosphate-labeled nucleotides with new linkers
JP2007524358A (ja) * 2003-02-05 2007-08-30 ジーイー・ヘルスケア・バイオサイエンス・コーポレイション 末端リン酸標識ヌクレオチド及び使用方法
WO2004072297A3 (en) * 2003-02-05 2007-01-18 Amersham Biosciences Corp Terminal-phosphate-labeled nucleotides and methods of use
US20040152104A1 (en) * 2003-02-05 2004-08-05 Anup Sood Nucleic acid amplification
US7125671B2 (en) * 2003-02-05 2006-10-24 Ge Healthcare Bio-Sciences Corp. Nucleic acid amplification with terminal-phosphate labeled nucleotides
US20090065471A1 (en) * 2003-02-10 2009-03-12 Faris Sadeg M Micro-nozzle, nano-nozzle, manufacturing methods therefor, applications therefor
US7939256B2 (en) * 2003-04-08 2011-05-10 Pacific Biosciences Of California, Inc. Composition and method for nucleic acid sequencing
US20090092970A1 (en) * 2003-04-08 2009-04-09 Pacific Biosciences Composition and method for nucleic acid sequencing
US9657344B2 (en) 2003-11-12 2017-05-23 Fluidigm Corporation Short cycle methods for sequencing polynucleotides
US7897345B2 (en) 2003-11-12 2011-03-01 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
US9012144B2 (en) 2003-11-12 2015-04-21 Fluidigm Corporation Short cycle methods for sequencing polynucleotides
US7981604B2 (en) 2004-02-19 2011-07-19 California Institute Of Technology Methods and kits for analyzing polynucleotide sequences
US9109251B2 (en) 2004-06-25 2015-08-18 University Of Hawaii Ultrasensitive biosensors
US10563252B2 (en) 2004-06-25 2020-02-18 University Of Hawaii Ultrasensitive biosensors
US7906284B2 (en) 2004-09-17 2011-03-15 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US7313308B2 (en) 2004-09-17 2007-12-25 Pacific Biosciences Of California, Inc. Optical analysis of molecules
US7170050B2 (en) 2004-09-17 2007-01-30 Pacific Biosciences Of California, Inc. Apparatus and methods for optical analysis of molecules
US20060062531A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Fabrication of optical confinements
US9709503B2 (en) 2004-09-17 2017-07-18 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US20060060766A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and methods for optical analysis of molecules
US7302146B2 (en) 2004-09-17 2007-11-27 Pacific Biosciences Of California, Inc. Apparatus and method for analysis of molecules
US20060063264A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for performing nucleic acid analysis
US20070206189A1 (en) * 2004-09-17 2007-09-06 Stephen Turner Optical analysis of molecules
US7476503B2 (en) 2004-09-17 2009-01-13 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US9588051B2 (en) 2004-09-17 2017-03-07 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US20060061754A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Arrays of optical confinements and uses thereof
US8709725B2 (en) 2004-09-17 2014-04-29 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US20060061755A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for analysis of molecules
US8753812B2 (en) 2004-11-12 2014-06-17 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection method for DNA and other molecules
US10822641B2 (en) 2004-11-12 2020-11-03 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for DNA and other molecules
US9228971B2 (en) 2004-11-12 2016-01-05 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for DNA and other molecules
US20100203504A1 (en) * 2005-07-07 2010-08-12 Yoichi Katsumoto Substance-Information Acquisition Method Using Evanescent Light Beam, Substance-Information Measurement Apparatus, Base-Sequence Determination Method and Base-Sequence Determination Apparatus
US10793904B2 (en) 2005-07-20 2020-10-06 Illumina Cambridge Limited Methods for sequencing a polynucleotide template
US9765391B2 (en) * 2005-07-20 2017-09-19 Illumina Cambridge Limited Methods for sequencing a polynucleotide template
US11542553B2 (en) 2005-07-20 2023-01-03 Illumina Cambridge Limited Methods for sequencing a polynucleotide template
US20100311597A1 (en) * 2005-07-20 2010-12-09 Harold Philip Swerdlow Methods for sequence a polynucleotide template
US9868978B2 (en) 2005-08-26 2018-01-16 Fluidigm Corporation Single molecule sequencing of captured nucleic acids
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
US8124343B2 (en) 2005-11-04 2012-02-28 Mannkind Corporation IRE-1α substrates
US8017331B2 (en) * 2005-11-04 2011-09-13 Mannkind Corporation IRE-1α substrates
US20070105123A1 (en) * 2005-11-04 2007-05-10 Mannkind Corporation IRE-1alpha substrates
US20080299565A1 (en) * 2005-12-12 2008-12-04 Schneider Thomas D Probe for Nucleic Acid Sequencing and Methods of Use
US20100227913A1 (en) * 2005-12-12 2010-09-09 The Govt. of the U.S.A. as represented by the Sec. of the Deparment of Health and Human Services Nanoprobes for detection or modification of molecules
US20110111975A1 (en) * 2005-12-12 2011-05-12 The Government of the U.S.A as represented by the Secretary of the Dept. of Health & Human Services Probe for nucleic acid sequencing and methods of use
US8344121B2 (en) 2005-12-12 2013-01-01 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Nanoprobes for detection or modification of molecules
US8703734B2 (en) 2005-12-12 2014-04-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Nanoprobes for detection or modification of molecules
US7871777B2 (en) 2005-12-12 2011-01-18 The United States Of America As Represented By The Department Of Health And Human Services Probe for nucleic acid sequencing and methods of use
US20070250274A1 (en) * 2006-02-06 2007-10-25 Visigen Biotechnologies, Inc. Method for analyzing dynamic detectable events at the single molecule level
US7668697B2 (en) 2006-02-06 2010-02-23 Andrei Volkov Method for analyzing dynamic detectable events at the single molecule level
US20070211467A1 (en) * 2006-03-08 2007-09-13 Helicos Biosciences Corporation Systems and methods for reducing detected intensity non-uniformity in a laser beam
US20080309926A1 (en) * 2006-03-08 2008-12-18 Aaron Weber Systems and methods for reducing detected intensity non uniformity in a laser beam
US20080091005A1 (en) * 2006-07-20 2008-04-17 Visigen Biotechnologies, Inc. Modified nucleotides, methods for making and using same
US20080241951A1 (en) * 2006-07-20 2008-10-02 Visigen Biotechnologies, Inc. Method and apparatus for moving stage detection of single molecular events
US20080241938A1 (en) * 2006-07-20 2008-10-02 Visigen Biotechnologies, Inc. Automated synthesis or sequencing apparatus and method for making and using same
US8764969B2 (en) 2006-12-14 2014-07-01 Life Technologies Corporation Methods for operating chemically sensitive sensors with sample and hold capacitors
US8535513B2 (en) 2006-12-14 2013-09-17 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US9404920B2 (en) 2006-12-14 2016-08-02 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8426899B2 (en) 2006-12-14 2013-04-23 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8426898B2 (en) 2006-12-14 2013-04-23 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US11435314B2 (en) 2006-12-14 2022-09-06 Life Technologies Corporation Chemically-sensitive sensor array device
US9269708B2 (en) 2006-12-14 2016-02-23 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8435395B2 (en) 2006-12-14 2013-05-07 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8441044B2 (en) 2006-12-14 2013-05-14 Life Technologies Corporation Methods for manufacturing low noise chemically-sensitive field effect transistors
US20110230375A1 (en) * 2006-12-14 2011-09-22 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale fet arrays
US8445945B2 (en) 2006-12-14 2013-05-21 Life Technologies Corporation Low noise chemically-sensitive field effect transistors
US8450781B2 (en) 2006-12-14 2013-05-28 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US11732297B2 (en) * 2006-12-14 2023-08-22 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US10203300B2 (en) 2006-12-14 2019-02-12 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8262900B2 (en) 2006-12-14 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8492800B2 (en) 2006-12-14 2013-07-23 Life Technologies Corporation Chemically sensitive sensors with sample and hold capacitors
US8492799B2 (en) 2006-12-14 2013-07-23 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8496802B2 (en) 2006-12-14 2013-07-30 Life Technologies Corporation Methods for operating chemically-sensitive sample and hold sensors
US8502278B2 (en) 2006-12-14 2013-08-06 Life Technologies Corporation Chemically-sensitive sample and hold sensors
US8264014B2 (en) 2006-12-14 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8519448B2 (en) 2006-12-14 2013-08-27 Life Technologies Corporation Chemically-sensitive array with active and reference sensors
US9039888B2 (en) 2006-12-14 2015-05-26 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8269261B2 (en) 2006-12-14 2012-09-18 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8530941B2 (en) 2006-12-14 2013-09-10 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US20220340965A1 (en) * 2006-12-14 2022-10-27 Life Technologies Corporation Methods and Apparatus for Measuring Analytes Using Large Scale FET Arrays
US9951382B2 (en) 2006-12-14 2018-04-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8540865B2 (en) 2006-12-14 2013-09-24 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8540866B2 (en) 2006-12-14 2013-09-24 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8540867B2 (en) 2006-12-14 2013-09-24 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8540868B2 (en) 2006-12-14 2013-09-24 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8558288B2 (en) 2006-12-14 2013-10-15 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8575664B2 (en) 2006-12-14 2013-11-05 Life Technologies Corporation Chemically-sensitive sensor array calibration circuitry
US10415079B2 (en) 2006-12-14 2019-09-17 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US9989489B2 (en) 2006-12-14 2018-06-05 Life Technnologies Corporation Methods for calibrating an array of chemically-sensitive sensors
US10502708B2 (en) 2006-12-14 2019-12-10 Life Technologies Corporation Chemically-sensitive sensor array calibration circuitry
US8415716B2 (en) 2006-12-14 2013-04-09 Life Technologies Corporation Chemically sensitive sensors with feedback circuits
US8293082B2 (en) 2006-12-14 2012-10-23 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8306757B2 (en) 2006-12-14 2012-11-06 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8658017B2 (en) 2006-12-14 2014-02-25 Life Technologies Corporation Methods for operating an array of chemically-sensitive sensors
US8313625B2 (en) 2006-12-14 2012-11-20 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8685230B2 (en) 2006-12-14 2014-04-01 Life Technologies Corporation Methods and apparatus for high-speed operation of a chemically-sensitive sensor array
US8349167B2 (en) 2006-12-14 2013-01-08 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US10633699B2 (en) 2006-12-14 2020-04-28 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US10816506B2 (en) 2006-12-14 2020-10-27 Life Technologies Corporation Method for measuring analytes using large scale chemfet arrays
US20090026082A1 (en) * 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20100197507A1 (en) * 2006-12-14 2010-08-05 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale fet arrays
US20100188073A1 (en) * 2006-12-14 2010-07-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale fet arrays
US8317999B2 (en) 2006-12-14 2012-11-27 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8313639B2 (en) 2006-12-14 2012-11-20 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US11002724B2 (en) 2007-05-08 2021-05-11 Trustees Of Boston University Chemical functionalization of solid-state nanopores and nanopore arrays and applications thereof
US9903820B2 (en) 2007-05-08 2018-02-27 The Trustees Of Boston University Chemical functionalization of solid-state nanopores and nanopore arrays and applications thereof
US10101315B2 (en) 2007-05-08 2018-10-16 Trustees Of Boston University Chemical functionalization of solid-state nanopores and nanopore arrays and applications thereof
WO2008154317A1 (en) * 2007-06-06 2008-12-18 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
US20090024331A1 (en) * 2007-06-06 2009-01-22 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
US10023911B2 (en) 2007-06-06 2018-07-17 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
US8703422B2 (en) 2007-06-06 2014-04-22 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
US8182993B2 (en) 2007-06-06 2012-05-22 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
US9175343B2 (en) 2007-06-06 2015-11-03 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
US11339430B2 (en) 2007-07-10 2022-05-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US11705217B2 (en) 2008-03-28 2023-07-18 Pacific Biosciences Of California, Inc. Sequencing using concatemers of copies of sense and antisense strands
US8470164B2 (en) 2008-06-25 2013-06-25 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8524057B2 (en) 2008-06-25 2013-09-03 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US11137369B2 (en) 2008-10-22 2021-10-05 Life Technologies Corporation Integrated sensor arrays for biological and chemical analysis
US8936763B2 (en) 2008-10-22 2015-01-20 Life Technologies Corporation Integrated sensor arrays for biological and chemical analysis
US9964515B2 (en) 2008-10-22 2018-05-08 Life Technologies Corporation Integrated sensor arrays for biological and chemical analysis
US20110281737A1 (en) * 2008-10-22 2011-11-17 Life Technologies Corporation Method and Apparatus for Rapid Nucleic Acid Sequencing
US9778188B2 (en) 2009-03-11 2017-10-03 Industrial Technology Research Institute Apparatus and method for detection and discrimination molecular object
US10996166B2 (en) 2009-03-11 2021-05-04 Industrial Technology Research Institute Apparatus and method for detection and discrimination molecular object
US9365838B2 (en) 2009-03-27 2016-06-14 Life Technologies Corporation Conjugates of biomolecules to nanoparticles
US10093974B2 (en) 2009-03-27 2018-10-09 Life Technologies Corporation Methods and apparatus for single molecule sequencing using energy transfer detection
US9365839B2 (en) 2009-03-27 2016-06-14 Life Technologies Corporation Polymerase compositions and methods
US11008612B2 (en) 2009-03-27 2021-05-18 Life Technologies Corporation Methods and apparatus for single molecule sequencing using energy transfer detection
US9695471B2 (en) 2009-03-27 2017-07-04 Life Technologies Corporation Methods and apparatus for single molecule sequencing using energy transfer detection
US9567629B2 (en) 2009-03-27 2017-02-14 Life Technologies Corporation Labeled enzyme compositions, methods and systems
US11453909B2 (en) 2009-03-27 2022-09-27 Life Technologies Corporation Polymerase compositions and methods
US11542549B2 (en) 2009-03-27 2023-01-03 Life Technologies Corporation Labeled enzyme compositions, methods and systems
US20110003343A1 (en) * 2009-03-27 2011-01-06 Life Technologies Corporation Conjugates of biomolecules to nanoparticles
US10093973B2 (en) 2009-03-27 2018-10-09 Life Technologies Corporation Polymerase compositions and methods
US10093972B2 (en) 2009-03-27 2018-10-09 Life Technologies Corporation Conjugates of biomolecules to nanoparticles
US11015220B2 (en) 2009-03-27 2021-05-25 Life Technologies Corporation Conjugates of biomolecules to nanoparticles
US8603792B2 (en) 2009-03-27 2013-12-10 Life Technologies Corporation Conjugates of biomolecules to nanoparticles
US9932573B2 (en) 2009-03-27 2018-04-03 Life Technologies Corporation Labeled enzyme compositions, methods and systems
AU2010245304B2 (en) * 2009-04-27 2015-06-04 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
US9200320B2 (en) 2009-04-27 2015-12-01 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
US8940507B2 (en) 2009-04-27 2015-01-27 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
US20100311061A1 (en) * 2009-04-27 2010-12-09 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
WO2010129019A3 (en) * 2009-04-27 2011-03-31 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
US8501405B2 (en) 2009-04-27 2013-08-06 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
US8776573B2 (en) 2009-05-29 2014-07-15 Life Technologies Corporation Methods and apparatus for measuring analytes
US10718733B2 (en) 2009-05-29 2020-07-21 Life Technologies Corporation Methods and apparatus for measuring analytes
US8592154B2 (en) 2009-05-29 2013-11-26 Life Technologies Corporation Methods and apparatus for high speed operation of a chemically-sensitive sensor array
US8592153B1 (en) 2009-05-29 2013-11-26 Life Technologies Corporation Methods for manufacturing high capacitance microwell structures of chemically-sensitive sensors
US11768171B2 (en) 2009-05-29 2023-09-26 Life Technologies Corporation Methods and apparatus for measuring analytes
US9927393B2 (en) 2009-05-29 2018-03-27 Life Technologies Corporation Methods and apparatus for measuring analytes
US8263336B2 (en) 2009-05-29 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes
US8994076B2 (en) 2009-05-29 2015-03-31 Life Technologies Corporation Chemically-sensitive field effect transistor based pixel array with protection diodes
US10451585B2 (en) 2009-05-29 2019-10-22 Life Technologies Corporation Methods and apparatus for measuring analytes
US8822205B2 (en) 2009-05-29 2014-09-02 Life Technologies Corporation Active chemically-sensitive sensors with source follower amplifier
US8673627B2 (en) 2009-05-29 2014-03-18 Life Technologies Corporation Apparatus and methods for performing electrochemical reactions
US8766327B2 (en) 2009-05-29 2014-07-01 Life Technologies Corporation Active chemically-sensitive sensors with in-sensor current sources
US8748947B2 (en) 2009-05-29 2014-06-10 Life Technologies Corporation Active chemically-sensitive sensors with reset switch
US8698212B2 (en) 2009-05-29 2014-04-15 Life Technologies Corporation Active chemically-sensitive sensors
US11692964B2 (en) 2009-05-29 2023-07-04 Life Technologies Corporation Methods and apparatus for measuring analytes
US8742469B2 (en) 2009-05-29 2014-06-03 Life Technologies Corporation Active chemically-sensitive sensors with correlated double sampling
US10809226B2 (en) 2009-05-29 2020-10-20 Life Technologies Corporation Methods and apparatus for measuring analytes
US9279153B2 (en) 2009-09-30 2016-03-08 Quantapore, Inc. Ultrafast sequencing of biological polymers using a labeled nanopore
US8771491B2 (en) 2009-09-30 2014-07-08 Quantapore, Inc. Ultrafast sequencing of biological polymers using a labeled nanopore
US20130345391A1 (en) * 2009-12-28 2013-12-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv Composite Probes and Use Thereof in Super Resolution Methods
US9273089B2 (en) * 2009-12-28 2016-03-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Composite probes and use thereof in super resolution methods
US9777321B2 (en) 2010-03-15 2017-10-03 Industrial Technology Research Institute Single molecule detection system and methods
US9482615B2 (en) 2010-03-15 2016-11-01 Industrial Technology Research Institute Single-molecule detection system and methods
US9670243B2 (en) 2010-06-02 2017-06-06 Industrial Technology Research Institute Compositions and methods for sequencing nucleic acids
US10112969B2 (en) 2010-06-02 2018-10-30 Industrial Technology Research Institute Compositions and methods for sequencing nucleic acids
US9995683B2 (en) 2010-06-11 2018-06-12 Industrial Technology Research Institute Apparatus for single-molecule detection
US8432149B2 (en) 2010-06-30 2013-04-30 Life Technologies Corporation Array column integrator
US9239313B2 (en) 2010-06-30 2016-01-19 Life Technologies Corporation Ion-sensing charge-accumulation circuits and methods
US8247849B2 (en) 2010-06-30 2012-08-21 Life Technologies Corporation Two-transistor pixel array
US8858782B2 (en) 2010-06-30 2014-10-14 Life Technologies Corporation Ion-sensing charge-accumulation circuits and methods
US8823380B2 (en) 2010-06-30 2014-09-02 Life Technologies Corporation Capacitive charge pump
US8415177B2 (en) 2010-06-30 2013-04-09 Life Technologies Corporation Two-transistor pixel array
US8415176B2 (en) 2010-06-30 2013-04-09 Life Technologies Corporation One-transistor pixel array
US8487790B2 (en) 2010-06-30 2013-07-16 Life Technologies Corporation Chemical detection circuit including a serializer circuit
US8524487B2 (en) 2010-06-30 2013-09-03 Life Technologies Corporation One-transistor pixel array with cascoded column circuit
US8217433B1 (en) 2010-06-30 2012-07-10 Life Technologies Corporation One-transistor pixel array
US8421437B2 (en) 2010-06-30 2013-04-16 Life Technologies Corporation Array column integrator
US9164070B2 (en) 2010-06-30 2015-10-20 Life Technologies Corporation Column adc
US8432150B2 (en) 2010-06-30 2013-04-30 Life Technologies Corporation Methods for operating an array column integrator
US10481123B2 (en) 2010-06-30 2019-11-19 Life Technologies Corporation Ion-sensing charge-accumulation circuits and methods
US11231451B2 (en) 2010-06-30 2022-01-25 Life Technologies Corporation Methods and apparatus for testing ISFET arrays
US10641729B2 (en) 2010-06-30 2020-05-05 Life Technologies Corporation Column ADC
US8731847B2 (en) 2010-06-30 2014-05-20 Life Technologies Corporation Array configuration and readout scheme
US8772698B2 (en) 2010-06-30 2014-07-08 Life Technologies Corporation CCD-based multi-transistor active pixel sensor array
US8455927B2 (en) 2010-06-30 2013-06-04 Life Technologies Corporation One-transistor pixel array with cascoded column circuit
US11307166B2 (en) 2010-07-01 2022-04-19 Life Technologies Corporation Column ADC
US9960253B2 (en) 2010-07-03 2018-05-01 Life Technologies Corporation Chemically sensitive sensor with lightly doped drains
US8653567B2 (en) 2010-07-03 2014-02-18 Life Technologies Corporation Chemically sensitive sensor with lightly doped drains
US20130004950A1 (en) * 2010-08-06 2013-01-03 Tandem Diagnostics, Inc. Assay systems for genetic analysis
US11031095B2 (en) 2010-08-06 2021-06-08 Ariosa Diagnostics, Inc. Assay systems for determination of fetal copy number variation
US9567639B2 (en) 2010-08-06 2017-02-14 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US11203786B2 (en) 2010-08-06 2021-12-21 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US9890421B2 (en) 2010-08-06 2018-02-13 Ariosa Diagnostics, Inc. Assay systems for genetic analysis
US10308981B2 (en) 2010-08-06 2019-06-04 Ariosa Diagnostics, Inc. Assay systems for determination of source contribution in a sample
US10131951B2 (en) 2010-08-06 2018-11-20 Ariosa Diagnostics, Inc. Assay systems for genetic analysis
US10131937B2 (en) * 2010-08-06 2018-11-20 Ariosa Diagnostics, Inc. Assay systems for genetic analysis
US10533223B2 (en) 2010-08-06 2020-01-14 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US10233496B2 (en) 2010-08-06 2019-03-19 Ariosa Diagnostics, Inc. Ligation-based detection of genetic variants
US9618475B2 (en) 2010-09-15 2017-04-11 Life Technologies Corporation Methods and apparatus for measuring analytes
US9958415B2 (en) 2010-09-15 2018-05-01 Life Technologies Corporation ChemFET sensor including floating gate
US9958414B2 (en) 2010-09-15 2018-05-01 Life Technologies Corporation Apparatus for measuring analytes including chemical sensor array
US8796036B2 (en) 2010-09-24 2014-08-05 Life Technologies Corporation Method and system for delta double sampling
US8685324B2 (en) 2010-09-24 2014-04-01 Life Technologies Corporation Matched pair transistor circuits
US9110015B2 (en) 2010-09-24 2015-08-18 Life Technologies Corporation Method and system for delta double sampling
US10718019B2 (en) 2011-01-25 2020-07-21 Ariosa Diagnostics, Inc. Risk calculation for evaluation of fetal aneuploidy
US10131947B2 (en) 2011-01-25 2018-11-20 Ariosa Diagnostics, Inc. Noninvasive detection of fetal aneuploidy in egg donor pregnancies
US11441185B2 (en) 2011-01-25 2022-09-13 Roche Molecular Systems, Inc. Noninvasive detection of fetal aneuploidy in egg donor pregnancies
US10718024B2 (en) 2011-01-25 2020-07-21 Ariosa Diagnostics, Inc. Risk calculation for evaluation of fetal aneuploidy
US11270781B2 (en) 2011-01-25 2022-03-08 Ariosa Diagnostics, Inc. Statistical analysis for non-invasive sex chromosome aneuploidy determination
US8756020B2 (en) 2011-01-25 2014-06-17 Ariosa Diagnostics, Inc. Enhanced risk probabilities using biomolecule estimations
US8700338B2 (en) 2011-01-25 2014-04-15 Ariosa Diagnosis, Inc. Risk calculation for evaluation of fetal aneuploidy
WO2012104851A1 (en) 2011-01-31 2012-08-09 Yeda Research And Development Co. Ltd. Methods of diagnosing disease using overlap extension pcr
US8712697B2 (en) 2011-09-07 2014-04-29 Ariosa Diagnostics, Inc. Determination of copy number variations using binomial probability calculations
US9164053B2 (en) * 2011-09-26 2015-10-20 The Regents Of The University Of California Electronic device for monitoring single molecule dynamics
US20130078622A1 (en) * 2011-09-26 2013-03-28 The Regents Of The University Of California Electronic device for monitoring single molecule dynamics
JP2013094149A (ja) * 2011-11-04 2013-05-20 Hitachi Ltd Ｄｎａ配列解読システム、ｄｎａ配列解読方法及びプログラム
US9970984B2 (en) 2011-12-01 2018-05-15 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array
US10365321B2 (en) 2011-12-01 2019-07-30 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array
US10598723B2 (en) 2011-12-01 2020-03-24 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array
US8821798B2 (en) 2012-01-19 2014-09-02 Life Technologies Corporation Titanium nitride as sensing layer for microwell structure
US8747748B2 (en) 2012-01-19 2014-06-10 Life Technologies Corporation Chemical sensor with conductive cup-shaped sensor surface
US10544454B2 (en) 2012-05-02 2020-01-28 Ibis Biosciences, Inc. DNA sequencing
EP3789502A1 (en) 2012-05-02 2021-03-10 Ibis Biosciences, Inc. Dna sequencing
EP3783111A1 (en) 2012-05-02 2021-02-24 Ibis Biosciences, Inc. Dna sequencing
EP3438286A1 (en) 2012-05-02 2019-02-06 Ibis Biosciences, Inc. Dna sequencing
US11359236B2 (en) 2012-05-02 2022-06-14 Ibis Biosciences, Inc. DNA sequencing
US10584377B2 (en) 2012-05-02 2020-03-10 Ibis Biosciences, Inc. DNA sequencing
EP3438285A1 (en) 2012-05-02 2019-02-06 Ibis Biosciences, Inc. Dna sequencing
US11404142B2 (en) 2012-05-21 2022-08-02 Roche Molecular Systems, Inc. Processes for calculating phased fetal genomic sequences
US10289800B2 (en) 2012-05-21 2019-05-14 Ariosa Diagnostics, Inc. Processes for calculating phased fetal genomic sequences
US10404249B2 (en) 2012-05-29 2019-09-03 Life Technologies Corporation System for reducing noise in a chemical sensor array
US9985624B2 (en) 2012-05-29 2018-05-29 Life Technologies Corporation System for reducing noise in a chemical sensor array
US9270264B2 (en) 2012-05-29 2016-02-23 Life Technologies Corporation System for reducing noise in a chemical sensor array
US9206417B2 (en) 2012-07-19 2015-12-08 Ariosa Diagnostics, Inc. Multiplexed sequential ligation-based detection of genetic variants
US9624490B2 (en) 2012-07-19 2017-04-18 Ariosa Diagnostics, Inc. Multiplexed sequential ligation-based detection of genetic variants
US9651539B2 (en) 2012-10-28 2017-05-16 Quantapore, Inc. Reducing background fluorescence in MEMS materials by low energy ion beam treatment
US9080968B2 (en) 2013-01-04 2015-07-14 Life Technologies Corporation Methods and systems for point of use removal of sacrificial material
US9852919B2 (en) 2013-01-04 2017-12-26 Life Technologies Corporation Methods and systems for point of use removal of sacrificial material
US10436742B2 (en) 2013-01-08 2019-10-08 Life Technologies Corporation Methods for manufacturing well structures for low-noise chemical sensors
US9841398B2 (en) 2013-01-08 2017-12-12 Life Technologies Corporation Methods for manufacturing well structures for low-noise chemical sensors
US9994897B2 (en) 2013-03-08 2018-06-12 Ariosa Diagnostics, Inc. Non-invasive fetal sex determination
US9995708B2 (en) 2013-03-13 2018-06-12 Life Technologies Corporation Chemical sensor with sidewall spacer sensor surface
US10481124B2 (en) 2013-03-15 2019-11-19 Life Technologies Corporation Chemical device with thin conductive element
US9823217B2 (en) 2013-03-15 2017-11-21 Life Technologies Corporation Chemical device with thin conductive element
US9671363B2 (en) 2013-03-15 2017-06-06 Life Technologies Corporation Chemical sensor with consistent sensor surface areas
US9835585B2 (en) 2013-03-15 2017-12-05 Life Technologies Corporation Chemical sensor with protruded sensor surface
US10422767B2 (en) 2013-03-15 2019-09-24 Life Technologies Corporation Chemical sensor with consistent sensor surface areas
US11028438B2 (en) 2013-05-09 2021-06-08 Life Technologies Corporation Windowed sequencing
US10100357B2 (en) 2013-05-09 2018-10-16 Life Technologies Corporation Windowed sequencing
US10655175B2 (en) 2013-05-09 2020-05-19 Life Technologies Corporation Windowed sequencing
US9862997B2 (en) 2013-05-24 2018-01-09 Quantapore, Inc. Nanopore-based nucleic acid analysis with mixed FRET detection
US11774401B2 (en) 2013-06-10 2023-10-03 Life Technologies Corporation Chemical sensor array having multiple sensors per well
US10816504B2 (en) 2013-06-10 2020-10-27 Life Technologies Corporation Chemical sensor array having multiple sensors per well
US10458942B2 (en) 2013-06-10 2019-10-29 Life Technologies Corporation Chemical sensor array having multiple sensors per well
US11499938B2 (en) 2013-06-10 2022-11-15 Life Technologies Corporation Chemical sensor array having multiple sensors per well
US10364469B2 (en) * 2014-01-16 2019-07-30 Illumina, Inc Gene expression panel for prognosis of prostate cancer recurrence
US11098372B2 (en) 2014-01-16 2021-08-24 Illumina, Inc. Gene expression panel for prognosis of prostate cancer recurrence
US10597712B2 (en) 2014-10-10 2020-03-24 Quantapore, Inc. Nanopore-based polymer analysis with mutually-quenching fluorescent labels
US9885079B2 (en) 2014-10-10 2018-02-06 Quantapore, Inc. Nanopore-based polymer analysis with mutually-quenching fluorescent labels
US11041197B2 (en) 2014-10-24 2021-06-22 Quantapore, Inc. Efficient optical analysis of polymers using arrays of nanostructures
US9624537B2 (en) 2014-10-24 2017-04-18 Quantapore, Inc. Efficient optical analysis of polymers using arrays of nanostructures
US11536688B2 (en) 2014-12-18 2022-12-27 Life Technologies Corporation High data rate integrated circuit with transmitter configuration
US10379079B2 (en) 2014-12-18 2019-08-13 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US10605767B2 (en) 2014-12-18 2020-03-31 Life Technologies Corporation High data rate integrated circuit with transmitter configuration
US10077472B2 (en) 2014-12-18 2018-09-18 Life Technologies Corporation High data rate integrated circuit with power management
US10767224B2 (en) 2014-12-18 2020-09-08 Life Technologies Corporation High data rate integrated circuit with power management
US10823721B2 (en) 2016-07-05 2020-11-03 Quantapore, Inc. Optically based nanopore sequencing

Also Published As

Publication number Publication date
WO2002004680A2 (en) 2002-01-17
CA2415897A1 (en) 2002-01-17
DE60131194T2 (de) 2008-08-07
EP1975251A2 (en) 2008-10-01
US20050266424A1 (en) 2005-12-01
JP2004513619A (ja) 2004-05-13
JP2009118847A (ja) 2009-06-04
EP1975251A3 (en) 2009-03-25
US20070172862A1 (en) 2007-07-26
US20070275395A1 (en) 2007-11-29
ATE377093T1 (de) 2007-11-15
EP1368460A2 (en) 2003-12-10
US20110059436A1 (en) 2011-03-10
US20100255463A1 (en) 2010-10-07
EP2100971A2 (en) 2009-09-16
CN100462433C (zh) 2009-02-18
US20070172861A1 (en) 2007-07-26
AU2001282881B2 (en) 2007-06-14
EP1368460B1 (en) 2007-10-31
WO2002004680A3 (en) 2003-10-16
US20070184475A1 (en) 2007-08-09
CN1553953A (zh) 2004-12-08
EP2100971A3 (en) 2009-11-25
US20110014604A1 (en) 2011-01-20
US20100317005A1 (en) 2010-12-16
US20100304367A1 (en) 2010-12-02
US20050260614A1 (en) 2005-11-24
US20070292867A1 (en) 2007-12-20
US20090275036A1 (en) 2009-11-05
US20070172864A1 (en) 2007-07-26
US20110021383A1 (en) 2011-01-27
US20070172865A1 (en) 2007-07-26
US20070172868A1 (en) 2007-07-26
JP2009240318A (ja) 2009-10-22
US20070172858A1 (en) 2007-07-26
AU8288101A (en) 2002-01-21
DE60131194D1 (de) 2007-12-13
US20090305278A1 (en) 2009-12-10
US7329492B2 (en) 2008-02-12
CN101525660A (zh) 2009-09-09
US20070172863A1 (en) 2007-07-26
US20070172867A1 (en) 2007-07-26

Similar Documents

Publication Publication Date Title
US7329492B2 (en) Methods for real-time single molecule sequence determination
US20070172866A1 (en) Methods for sequence determination using depolymerizing agent
AU2001282881A1 (en) Real-time sequence determination
US9382584B2 (en) Methods and systems for direct sequencing of single DNA molecules
US7939264B1 (en) DNA sequencing method
AU2007216737A1 (en) Real-time sequence determination
WO2023081653A1 (en) Nucleic acid polymerase for incorporating labeled nucleotides
EP2203568A1 (en) A method and system for obtaining ordered, segmented sequence fragments along a nucleic acid molecule

Legal Events

Date Code Title Description
AS Assignment

Owner name: VISIGEN, INC., TEXAS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HARDIN, SUSAN H.;BRIGGS, JAMES M.;TU, SHIAO-CHUN;AND OTHERS;REEL/FRAME:014941/0689;SIGNING DATES FROM 20040106 TO 20040107

AS Assignment

Owner name: LIFE TECHNOLOGIES CORPORATION, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:VISIGEN BIOTECHNOLOGIES, INC;REEL/FRAME:022073/0107

Effective date: 20090107

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION