Connect public, paid and private patent data with Google Patents Public Datasets

Real-time sequence determination

Download PDF

Info

Publication number
US20030064366A1
US20030064366A1 US09901782 US90178201A US2003064366A1 US 20030064366 A1 US20030064366 A1 US 20030064366A1 US 09901782 US09901782 US 09901782 US 90178201 A US90178201 A US 90178201A US 2003064366 A1 US2003064366 A1 US 2003064366A1
Authority
US
Grant status
Application
Patent type
Prior art keywords
polymerase
dna
tagged
taq
monomer
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
US09901782
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
GAO XIAOLIAN
Original Assignee
Susan Hardin
Xiaolian Gao
James Briggs
Richard Willson
Shiao-Chun Tu
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

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing

Abstract

A sequencing methodology is disclosed that allows a single DNA or RNA molecule or portion thereof to be sequenced directly and in substantially real time. The methodology involves engineering a polymerase and/or dNTPs with atomic and/or molecular tags that have a detectable property that is monitored by a detection system.

Description

    BACKGROUND OF THE INVENTION
  • [0001]
    1. Field of the Invention
  • [0002]
    The present invention relates to a single-molecule sequencing apparatus and methods.
  • [0003]
    More particularly, 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. In a preferred embodiment, the methods are carried out in real-time or near real-time.
  • [0004]
    2. Description of the Related Art
  • [0005]
    Overview of Conventional DNA Sequencing
  • [0006]
    The development of methods that allow one to quickly and reliably determine the order of bases or ‘sequence’ in a fragment of DNA is a key technical advance, the importance of which cannot be overstated. Knowledge of DNA sequence enables a greater understanding of the molecular basis of life. 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.
  • [0007]
    Knowledge of DNA sequence and the ability to manipulate these sequences has accelerated development of biotechnology and led to the development of molecular techniques that provide the tools to ask and answer important scientific questions. The polymerase chain reaction (PCR), an important biotechnique that facilitates sequence-specific detection of nucleic acid, relies on sequence information. DNA sequencing methods allow scientists to determine whether a change has been introduced into the DNA, and to assay the effect of the change on the biology of the organism, regardless of the type of organism that is being studied. Ultimately, DNA sequence information may provide a way to uniquely identify individuals.
  • [0008]
    In order to understand the DNA sequencing process, one must recall several facts about DNA. First, 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. The terms 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.
  • [0009]
    Sanger DNA Sequencing (Enzymatic Synthesis)
  • [0010]
    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. If a dideoxynucleotide (ddNTP; ddATP, ddTTP, ddGTP, ddCTP), a base analogue lacking a 3′ OH, is added into an enzymatic sequencing reaction, it is incorporated into the growing strand by the polymerase. However, once the ddNTP is incorporated, the polymerase is unable to add any additional bases to the end of the strand. Importantly, 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).
  • [0011]
    Fluorescent DNA Sequencing
  • [0012]
    A major advance in determining DNA sequence information occurred with the introduction of automated DNA sequencing machines (Smith et al., 1986). 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.
  • [0013]
    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 ddNTP; prematurely terminated products are not detected. Thus, reduced background noise typically results with this chemistry.
  • [0014]
    State-of-the-art dye-terminator chemistry uses four energy transfer fluorescent dyes (Rosenblum et al., 1997). These terminators include a fluorescein donor dye (6-FAM) linked to one of four different dichlororhodamine (dRhodamine) acceptor dyes. 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. Thus, energy transfer dye-labeled terminators produce brighter signals and improve spectral resolution. These improvements result in more accurate DNA sequence information.
  • [0015]
    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). Thus, 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. More specifically, 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. Thus, 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.
  • [0016]
    Genome Sequencing
  • [0017]
    Very often a researcher needs to determine the sequence of a DNA fragment that is larger than the 500-1,000 base average sequencing read length. Not surprisingly, strategies to accomplish this have been developed. These strategies are divided into two major classes, random or directed, and strategy choice is influenced by the size of the fragment to be sequenced.
  • [0018]
    In random or shotgun DNA sequencing, a large DNA fragment (typically one larger than 20,000 base pairs) is broken into smaller fragments that are inserted into a cloning vector. It is assumed that the sum of information contained within these smaller clones is equivalent to that contained within the original DNA fragment. Numerous smaller clones are randomly selected, DNA templates are prepared for sequencing reactions, and primers that will base-pair with the vector DNA sequence bordering the insert are used to begin the sequencing reaction (2-7 days for a 20 kbp insert). Subsequently, the quality of each base call is examined (manually or automatically via software (PHRED, Ewing et al., 1998); 1-10 minutes per sequence reaction), and the sequence of the original DNA fragment is reconstructed by computer assembly of the sequences obtained from the smaller DNA fragments. Based on the time estimates provided, if a shotgun sequencing strategy is used, a 20 kbp insert is expected to be completed in 3-10 days. This strategy was extensively used to determine the sequence of ordered fragments that represent the entire human genome (http://www.nhgri.nih.gov/HGP/). However, this random approach is typically not sufficient to complete sequence determination, since gaps in the sequence often remain after computer assembly. A directed strategy (described below) is usually used to complete the sequence project.
  • [0019]
    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. Thus, 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.
  • [0020]
    The necessity of designing and synthesizing new primers, coupled with the expense and the time required for their synthesis, has limited the routine application of primer-walking for sequencing large DNA fragments. Researchers have proposed using a library of short primers to eliminate the requirement for custom primer synthesis (Studier, 1989; Siemieniak and Slightom, 1990; Kieleczawa et al., 1992; Kotler et al., 1993; Burbelo and ladarola, 1994; Hardin et al., 1996; Raja et al., 1997; Jones and Hardin, 1998a,b; Ball et al., 1998; Mei and Hardin, 2000; Kraltcheva and Hardin, 2001). The availability of a primer library minimizes primer waste, since each primer is used to prime multiple reactions, and allows immediate access to the next sequencing primer.
  • [0021]
    One of the original goals of the Human Genome Project was to complete sequence determination of the entire human genomeby2005 (http://www.nhgri.nih.gov/HGP/). However, the plan is ahead of schedule and a ‘working draft’ of the human genome was published in February 2001 (Venter et al., 2001, “International Human Genome Sequencing Consortium 2001”). Due to technological advances in several disciplines, the completed genome sequence is expected in 2003, two years ahead of schedule. Progress in all aspects involving DNA manipulation (especially manipulation and propagation of large DNA fragments), evolution of faster and better DNA sequencing methods (http://www.abrf.org), development of computer hardware and software capable of manipulating and analyzing the data (bioinformatics), and automation of procedures associated with generating and analyzing DNA sequences (engineering) are responsible for this accelerated time frame.
  • [0022]
    Single-Molecule DNA Sequencing
  • [0023]
    Conventional DNA sequencing strategies and methods are reliable, but time, labor, and cost intensive. To address these issues, some researchers are investigating fluorescence-based, single-molecule sequencing methods that use enzymatic degradation, followed by single-dNMP detection and identification. The DNA polymer containing fluorescently-labeled nucleotides is digested by an exonuclease, and the labeled nucleotides are detected and identified by flow cytometry (Davis et al., 1991; Davis et al., 1992; Goodwin et al., 1997; Keller et al., 1996; Sauer et al., 1999; Werner et al., 1999). 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.
  • [0024]
    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 γ-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. Upon incorporation 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.
  • [0025]
    Although some single-molecular sequencing systems have been disclosed, many of them anticipate or require base modification. See, e.g., patent application Ser. Nos. WO 01/16375 A2,WO 01/23610A2,WO 1/25480,WO 00/06770,WO 99/05315,WO 00/60114,WO 00/36151, WO 00/36512, and WO 00/70073, incorporated herein by reference. Base modifications may distort DNA structure (which normally consists of A-form DNA nearest the enzyme active site; Li et al., 1998a). Since the dNTP and approximately 7 of the 3′-nearest bases in the newly synthesized strand contact internal regions of the polymerase (Li et al., 1998a), 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.
  • SUMMARY OF THE INVENTION Single-Moleccule Sequencing
  • [0026]
    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.
  • [0027]
    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.
  • [0028]
    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.
  • Single Site Tagged Polymerase
  • [0029]
    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.
  • [0030]
    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.
  • [0031]
    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.
  • [0032]
    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.
  • [0033]
    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.
  • [0034]
    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.
  • [0035]
    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. In a preferred embodiment, this change in detection propensity is cyclical occurring as each pyrophosphate group is released.
  • [0036]
    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. In a preferred embodiment, this change in detection propensity is cyclical occurring as each pyrophosphate group is released.
  • [0037]
    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. In a preferred embodiment, this change in detection propensity is cyclical occurring as each pyrophosphate group is released.
  • [0038]
    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.
  • Mutiple Site Tagged Polymerizing or Deolymerizing Agents
  • [0039]
    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.
  • [0040]
    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.
  • [0041]
    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.
  • [0042]
    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.
  • [0043]
    Preferably, the first and second states are different so that a change in the detected signal occurs. However, a no-change result may evidence other properties of the polymerizing media or depolymerizing media.
  • Mutiple Site Tagged Polymerase
  • [0044]
    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.
  • [0045]
    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.
  • [0046]
    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.
  • [0047]
    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.
  • [0048]
    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.
  • [0049]
    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.
  • [0050]
    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.
  • [0051]
    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.
  • [0052]
    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.
  • [0053]
    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.
  • [0054]
    It should be recognized that when a property changes from a first state to a second state and back again, then the property undergoes a cycle. Preferably, the first and second states are different so that a change in the detected signal occurs. However, a no-change result may evidence other properties of the polymerizing medium or depolymerizing medium.
  • Methods Using Tagged Polymerizing Agent
  • [0055]
    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.
  • [0056]
    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.
  • [0057]
    Preferably, the first and second states are different so that a change in the detected signal occurs. However, a no-change result may evidence other properties of the polymerizing medium.
  • Methods Using Tagged Polymerase
  • [0058]
    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.
  • [0059]
    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.
  • [0060]
    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.
  • [0061]
    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.
  • [0062]
    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.
  • [0063]
    Preferably, the first and second states are different so that a change in the detected signal occurs. However, a no-change result may evidence other properties of the polymerizing media.
  • Apparatuses Using Tagged Polymerizing Agent
  • [0064]
    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. Alternatively, 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. Alternatively, each polymerizing agent confinement structure can have its own monomer reservoir or sufficient monomer reservoirs so that each reservoir contains a specific monomer.
  • [0065]
    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. In this context, the term “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. Alternatively, 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. Alternatively, 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. Alternatively, each polymerizing agent structure can have its own monomer reservoir or sufficient monomer reservoirs, one reservoir of each specific monomer.
  • [0066]
    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.
  • Apparatus Using Tagged Polymerase
  • [0067]
    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. Alternatively, 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. Alternatively, each polymerase confinement structure can have its own monomer reservoir or four reservoirs, each reservoir containing a specific monomer.
  • [0068]
    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. In this context, the term “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. Alternatively, 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. Alternatively, 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. Alternatively, each polymerase structure can have its own monomer reservoir or four reservoirs, each reservoir containing a specific monomer.
  • [0069]
    The monomers for use in these apparatus including, without limitation, dNTPs, tagged dNTPs, ddNTPs, tagged ddNTPs, or mixtures or combinations thereof.
  • Methods Using the Single-Molecule Sequencing Apparatuses
  • [0070]
    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.
  • [0071]
    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.
  • [0072]
    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.
  • [0073]
    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.
  • Cooperatively Tagged Systems
  • [0074]
    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. In one preferred embodiment, the tag on the polymerase is positioned such that the tags interact before, during and/or after each monomer insertion. In the of case tags that are released from the monomers after monomer insert such as of β and/or γ phosphate tagged dNTPs, i.e., the tags reside on the β and/or γ 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.
  • [0075]
    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. Generally, the distance needed to cause a measurable change in the detectable property is within (less than or equal to) about 100 Å, preferably within about 50 Å, particularly within about 25 Å, especially within about 15 Å and most preferably within about 10 Å. Of course, one skilled in the art will recognize that 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.
  • [0076]
    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.
  • [0077]
    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.
  • [0078]
    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. Generally, the distance needed to cause a measurable change in the detectable property is within (less than or equal to) about 100 Å, preferably within about 50 Å, particularly within about 25 Å, especially within about 15 Å and most preferably within about 10 Å. Of course, one skilled in the art will recognize that 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.
  • [0079]
    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.
  • Cooperatively Tagged Systems Using a Polymerase
  • [0080]
    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. In the of case tags that are released from the monomers after monomer insert such as of β and/or γ phosphate tagged dNTPs, i.e., the tags reside on the β and/or γ 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. 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.
  • [0081]
    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. 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. Generally, the distance or close proximity is a distance between about 100 Å and about 10 Å. Alternatively, the distance is less than or equal to about 100 Å, preferably less than or equal to about 50 Å, particularly less than or equal to about 25 Å, especially less than or equal to about 15 Å and most preferably less than or equal to about 10 Å. Of course, one skilled in the art will recognize that 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.
  • [0082]
    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.
  • [0083]
    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. Generally, the distance or close proximity is a distance between about 100 Å and about 10 Å. Alternatively, the distance is less than or equal to about 100 Å, preferably less than or equal to about 50 Å, particularly less than or equal to about 25 Å, especially less than or equal to about 15 Å and most preferably less than or equal to about 10 Å. Of course, one skilled in the art will recognize that 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.
  • [0084]
    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.
  • Molecular Data Stream Reading Methods and Apparatus
  • [0085]
    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.
  • [0086]
    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.
  • [0087]
    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.
  • [0088]
    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.
  • [0089]
    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.
  • [0090]
    The term monomer as used herein means any compound that can be incorporated into a growing molecular chain by a given polymerase. Such 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. Additionally, amino acids (natural or synthetic) for protein or protein analog synthesis, mono saccharides for carbohydrate synthesis or other monomeric syntheses.
  • [0091]
    The term 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. The term 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.
  • [0092]
    Single Site Tagged Polymerizing or Depolymerizing Agents
  • [0093]
    The present invention provides 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.
  • [0094]
    The present invention provides 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.
  • [0095]
    The present invention provides a composition comprising 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.
  • [0096]
    The present invention provides 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.
  • [0097]
    Single Site Tagged Polymerase
  • [0098]
    The present invention provides 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.
  • [0099]
    The present invention provides 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.
  • [0100]
    The present invention provides 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.
  • [0101]
    The present invention provides a 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.
  • [0102]
    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.
  • [0103]
    The present invention provides 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.
  • [0104]
    The present invention provides 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.
  • [0105]
    The present invention provides 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.
  • [0106]
    The present invention provides 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.
  • [0107]
    Multiple Site Tagged Polymerizing and Depolymerizing Agents
  • [0108]
    The present invention provides 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.
  • [0109]
    The present invention provides 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.
  • [0110]
    The present invention provides a composition comprising 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.
  • [0111]
    The present invention provides a composition comprising 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.
  • [0112]
    Multiple Site Tagged Polymerase
  • [0113]
    The present invention provides 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.
  • [0114]
    The present invention provides 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.
  • [0115]
    The present invention provides 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.
  • [0116]
    The present invention provides 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.
  • [0117]
    The present invention provides 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.
  • [0118]
    The present invention provides 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.
  • [0119]
    The present invention provides 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.
  • [0120]
    The present invention provides 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.
  • [0121]
    Methods Using Tagged Polymerase
  • [0122]
    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.
  • [0123]
    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.
  • [0124]
    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.
  • [0125]
    Single-Molecule Sequencing Apparatus Using Tagged Polymerase
  • [0126]
    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.
  • [0127]
    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.
  • [0128]
    Cooperatively Tagged Monomers and Tagged Polymering Agent
  • [0129]
    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.
  • [0130]
    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.
  • [0131]
    Cooperatively Tagged Monomers and Tagged Polymerase
  • [0132]
    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.
  • [0133]
    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.
  • [0134]
    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.
  • [0135]
    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.
  • [0136]
    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.
  • [0137]
    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.
  • [0138]
    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.
  • [0139]
    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.
  • [0140]
    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.
  • [0141]
    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.
  • [0142]
    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.
  • [0143]
    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.
  • [0144]
    The present invention provides a method for synthesizing a γ-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 γ-phosphate tagged dNTP.
  • [0145]
    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 γ-phosphate of a γ-phosphate labeled ATP to the 5′ end of the biomolecule resulting in a covalently modified biomolecule.
  • [0146]
    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 γ-phosphate of a γ-phosphate labeled ATP to the 5′ end of the biomolecule resulting in a covalently modified biomolecule.
  • DESCRIPTION OF THE DRAWINGS
  • [0147]
    The invention can be better understood with reference to the following detailed description together with the appended illustrative drawings in which like elements are numbered the same:
  • [0148]
    [0148]FIG. 1 depicts FRET activity as a function of distance separating the fluorescent donor and acceptor;
  • [0149]
    [0149]FIG. 2 depicts the open and closed ternary complex forms of the large fragment of Taq DNA pol I (Klentaq 1);
  • [0150]
    FIGS. 3A-C depicts an overlay between 3 ktq (closed ‘black’) and 1 tau (open ‘light blue’), the large fragment of faq DNA polymerase I;
  • [0151]
    [0151]FIG. 4 depicts an image of a 20% denaturing polyacryamide gel containing size separated radiolabeled products from DNA extension experiments involving γ-ANS-phosphate-dATP;
  • [0152]
    [0152]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 γ-ANS-phosphate-dNTPs;
  • [0153]
    [0153]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 γ-ANS-phosphate-dNTPs;
  • DETAILED DESCRIPTION OF THE INVENTION
  • [0154]
    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. Moreover, 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.
  • [0155]
    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. Thus, 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.
  • [0156]
    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.
  • [0157]
    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.
  • [0158]
    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.
  • [0159]
    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. As the next dNTP is incorporated during the polymerization process, 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.
  • [0160]
    In another embodiment of the single-molecule sequencing technology of this invention, 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.
  • [0161]
    In another embodiment, 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. Concurrently, 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. One skilled in the art will recognize that 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.
  • [0162]
    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. Once 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. Once formed, the engineered polymerase can be contacted with one or more appropriate tags and used in the apparatuses and methods of this invention.
  • [0163]
    Engineering a polymerase to function as a direct molecular sensor of DNA base identity provides a route to a fast and potentially real-time enzymatic DNA sequencing system. 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.
  • [0164]
    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 in basic research, e.g., examination of polymerase incorporation rates in a variety of different sequence contexts; analysis of errors in different contexts; epigenotypic analysis; analysis of protein glycosylation; protein identification; (13) the systems allows the creation of new robust (rugged) single-molecule detection apparatus; (14) the systems allows the development of systems and procedures that are compatible with biomolecules; (15) the systems allows the development genetic nanomachines or nanotechnology; (16) the systems allows the construction of large genetic databases and (17) the system has high sensitivity for low mutation event detection.
  • BRIEF OVERVIEW OF SINGLE-MOLECULE DNA SEQUENCING
  • [0165]
    In one embodiment of the single-molecule DNA sequencing system of this invention, 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. As a tagged dNTP is incorporated into a growing DNA polymer, 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.
  • [0166]
    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 β and/or γ 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.
  • [0167]
    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. As an incoming, tagged dNTP is bound by the polymerase for DNA elongation, the detected fluorescent signal or spectrum is analyzed and the identity of the incorporated base is determined.
  • [0168]
    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.
  • REAGENTS
  • [0169]
    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.
  • [0170]
    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.
  • [0171]
    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.
  • [0172]
    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.
  • [0173]
    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.
  • [0174]
    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 acid, Coumarin 1, Coumarin 30, Coumarin 314, Coumarin 343, Coumarin 6 or the like; Cyanine Dye including 1,1′-diethyl-2,2′-cyanine iodide, Cryptocyanine, Indocarbocyanine (C3)dye, Indodicarbocyanine (C5)dye, Indotricarbocyanine (C7)dye, Oxacarbocyanine (C3)dye, Oxadicarbocyanine (C5)dye, Oxatricarbocyanine (C7)dye, Pinacyanol iodide, Stains all, Thiacarbocyanine (C3)dye, ethanol, Thiacarbocyanine (C3)dye, n-propanol, Thiadicarbocyanine (C5)dye, Thiatricarbocyanine (C7)dye, or the like; Dipyrrin dyes including N,N′-Difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)-dipyrrin, N,N′-Difluoroboryl-1,9-dimethyl-5-[(4-(2-trimethylsilylethynyl), N,N′-Difluoroboryl-1,9-dimethyl-5-phenydipyrrin, or the like; Merocyanines including 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM), acetonitrile, 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM), methanol, 4-Dimethylamino-4′-nitrostilbene, Merocyanine 540, or the like; Miscellaneous Dye including 4′,6-Diamidino-2-phenylindole (DAPI), 4′,6-Diamidino-2-phenylindole (DAPI), dimethylsulfoxide, 7-Benzylamino-4-nitrobenz-2-oxa-1,3-diazole, Dansyl glycine, H2O, Dansyl glycine, dioxane, Hoechst 33258, DMF, Hoechst 33258, H2O, Lucifer yellow CH, Piroxicam, Quinine sulfate, 0.05 M H2SO4, Quinine sulfate, 0.5 M H2SO4, Squarylium dye III, or the like; Oligophenylenes including 2,5-Diphenyloxazole (PPO), Biphenyl, POPOP, p-Quaterphenyl, p-Terphenyl, or the like; Oxazines including Cresyl violet perchlorate, Nile Blue, methanol, Nile Red, Nile blue, ethanol, Oxazine 1, Oxazine 170, or the like; Polycyclic Aromatic Hydrocarbons including 9,10-Bis(phenylethynyl)anthracene, 9,10-Diphenylanthracene, Anthracene, Naphthalene, Perylene, Pyrene, or the like; polyene/polyynes including 1,2-diphenylacetylene, 1,4-diphenylbutadiene, 1,4-diphenylbutadiyne, 1,6-Diphenylhexatriene, Beta-carotene, Stilbene, or the like; Redox-active Chromophores including Anthraquinone, Azobenzene, Benzoquinone, Ferrocene, Riboflavin, Tris(2,2′-bipyridyl)ruthenium(II), Tetrapyrrole, Bilirubin, Chlorophyll a, diethyl ether, Chlorophyll a, methanol, Chlorophyll b, Diprotonated-tetraphenylporphyrin, Hematin, Magnesium octaethylporphyrin, Magnesium octaethylporphyrin (MgOEP), Magnesium phthalocyanine (MgPc), PrOH, Magnesium phthalocyanine (MgPc), pyridine, Magnesium tetramesitylporphyrin (MgTMP), Magnesium tetraphenylporphyrin (MgTPP), Octaethylporphyrin, Phthalocyanine (Pc), Porphin, Tetra-t-butylazaporphine, Tetra-t-butylnaphthalocyanine, Tetrakis(2,6-dichlorophenyl)porphyrin, Tetrakis(o-aminophenyl)porphyrin, Tetramesitylporphyrin (TMP), Tetraphenylporphyrin (TPP), Vitamin B12, Zinc octaethylporphyrin (ZnOEP), Zinc phthalocyanine (ZnPc), pyridine, Zinc tetramesitylporphyrin (ZnTMP), Zinc tetramesitylporphyrin radical cation, Zinc tetraphenylporphyrin (ZnTPP), or the like; Xanthenes including Eosin Y, Fluorescein, basic ethanol, Fluorescein, ethanol, Rhodamine 123, Rhodamine 6G, Rhodamine B, Rose bengal, Sulforhodamine 101, or the like; or mixtures or combination thereof or synthetic derivatives thereof or FRET fluorophore-quencher pairs including DLO-FB 1 (5′-FAM/3′-BHQ-1) DLO-TEB 1 (5′-TET/3 ′-BHQ-1), DLO-JB1 (5′-JOE/3′-BHQ-1), DLO-HB1 (5′-HEX/3′-BHQ-1), DLO-C3B2 (5′-Cy3/3′-BHQ-2), DLO-TAB2 (5′-TAMRA/3′-BHQ-2), DLO-RB2 (5′-ROX/3′-BHQ-2), DLO-C5B3(5′-Cy5/3′-BHQ-3), DLO-C55B3 (5′-Cy5.5/3′-BHQ-3), MBO-FB1 (5′-FAM/3′-BHQ-1), MBO-TEB1 (5′-TET/3′-BHQ-1), MBO-JB1 (5′-JOE/3′-BHQ-1), MBO-HB1 (5′-HEX/3′-BHQ-1), MBO-C3B2 (5′-Cy3/3′-BHQ-2), MBO-TAB2 (5′-TAMRA/3′-BHQ-2), MBO-RB2 (5′-ROX/3′-BHQ-2); MBO-C5B3 (5′-Cy5/3′-BHQ-3), MBO-C55B3 (5′-Cy5.5/3′-BHQ-3) or similar FRET pairs available from Biosearch Technologies, Inc. of Novato, Calif., tags with nmr active groups, tags with spectral features that can be easily identified such as IR, far IR, visible UV, far UV or the like.
  • ENZYME CHOICE
  • [0175]
    The inventors have found that the DNA polymerase from Thermus aquaticus—Taq DNA polymerase I—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. Of course, ordinary artisans will recognize that other polymerases can be adapted for use in the single-molecule sequencing systems of this invention.
  • [0176]
    Since 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.
  • [0177]
    Crystal Structures Are Available for Tag DNA Polymerase
  • [0178]
    There are 13 structures solved for Taq DNA polymerase, with or without DNA template/primer, dNTP, or ddNTP, which allows sufficient information for the selection of amino acid sites within the polymerase to which an atomic and/or molecular tag such as a fluorescent tag can be attached without adversely affecting polymerase activity. See, e.g., Eom et al., 1996; Li et al, 1998a; Li et al., 1998b. Additionally, the inventors have a written program to aid in identifying optimal tag addition sites. 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.
  • [0179]
    Taq DNA Polymerase is Efficiently Expressed in E. coli
  • [0180]
    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.
  • [0181]
    No Cysteines are Present in the Protein Sequence
  • [0182]
    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.
  • [0183]
    The Processivity of the Enzyme Can Be Modified
  • [0184]
    Although 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. Alternatively, 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. See, e.g., Bedford et al., 1997; Bedford et al., 1999.
  • [0185]
    Taq DNA Polymerase Possesses a 5′ to 3′ Exonuclease Activity and is Thermostable
  • [0186]
    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.
  • [0187]
    To favor the single-stranded state, 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.
  • [0188]
    The Polymerase is Free from 3′ to 5′ Exonuclease Activity
  • [0189]
    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.
  • [0190]
    All polymerases make replication errors. The 3′ to 5′ exonuclease activity is used to proofread the newly replicated DNA strand. Since Taq DNA polymerase lacks this proofreading function, an error in base incorporation becomes an error in DNA replication. Error rates for Taq DNA polymerase are 1 error per ˜100,000 bases synthesized, which is sufficiently low to assure a relatively high fidelity. See, e.g., Eckert and Kunkel, 1990; Cline et al., 1996. It has been suggested and verified for a polymerase that the elimination of this exonuclease activity uncovers a decreased fidelity during incorporation. Thus, Taq polymerase must—by necessity—be more accurate during initial nucleotide selection and/or incorporation, and is therefore an excellent choice of use in the present inventions.
  • [0191]
    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. For example, the inventors have discovered that base context influences polymerase accuracy and reaction kinetics, and this information is used to assign confidence values to individual base calls. However, depending on the goal of a particular sequencing project, it maybe more important to generate a genome sequence as rapidly as possible. For example, it may be preferable to generate, or draft, the genome sequence of a pathogen at reduced accuracy for initial identification purposes or for fast screening of potential pathogens.
  • [0192]
    Taq DNA Polymerase is the Enzyme of Choice for Single-molecule DNA Sequencing
  • [0193]
    Engineering the polymerase to function as a direct molecular sensor of DNA base identity provides the fastest enzymatic DNA sequencing system possible. For the reasons detailed above, Taq DNA polymerase is the optimal enzyme to genetically modify and adapt for single-molecule DNA sequencing. Additionally, basic research questions concerning DNA polymerase structure and function during replication can be addressed using this technology advancing single-molecule detection systems and molecular models in other disciplines. The inventors have found that native Taq DNA polymerase incorporates gamma-tagged dNTPs, yielding extended DNA polymers. Importantly, incorporation of a modified nucleotide is not detrimental to polymerase activity and extension of primer strands by incorporation of a γ-tagged nucleotide conforms to Watson-Crick base pairing rules.
  • DETECTING TAGGED POLYMERASE-NUCLEOTIDE INTERACTIONS
  • [0194]
    One preferred method for detecting polymerase-nucleotide interactions involves a fluorescence resonance energy transfer-based (FRET-based) method to maximize signal and minimize noise. 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):
  • E=1/(1+[R/R 0]6)  (1)
  • [0195]
    where R0 is the Förster critical distance at E=0.5. R0 is calculated from equation (2):
  • R 0=(9.79×103)(κ2η−4 Q D J DA)  (2)
  • [0196]
    where η is the refractive index of the medium (η=1.4 for aqueous solution), κ2 is a geometric orientation factor related to the relative angle of the two transition dipoles (κ2 is generally assumed to be ⅔), JDA [M−1 cm3] is the overlap integral representing the normalized spectral overlap of the donor emission and acceptor absorption, and QD is the quantum yield. The overlap integral is computed from equation (3):
  • J DA =[∫F D(λ)εA(λ)λ4 dλ]/[∫F D(λ)dλ]  (3)
  • [0197]
    where FD is the donor emission, εA is the acceptor absorption. QD is calculated from equation (4):
  • Q D =Q RF(I D /I RF)(A RF /A D)  (4)
  • [0198]
    where ID and IRF are the fluorescence intensities of donor and a reference compound (fluorescein in 0.1 N NaOH), and ARF and AD are the absorbances of the reference compound and donor. QRF is the quantum yield of fluorescein in 0.1N NaOH and is taken to be 0.90.
  • [0199]
    R, the distance between the donor and acceptor, 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.
  • [0200]
    The distance between the tagged γ-phosphate and the selected amino acid sites for labeling in the open versus closed polymerase conformation delineates optimal dye combinations. If the distance (R) between the donor and acceptor is the same as R0 (R0 is the Förster critical distance), FRET efficiency (E) is 50%. If R is more than 1.5 R0, the energy transfer efficiency becomes negligible (E<0.02). Sites within the enzyme at which R/R0 differ by more than 1.6 in the open versus closed forms are identified and, if necessary, these distances and/or distance differences can be increased through genetic engineering. A plot of FRET efficiency verses distance is shown in FIG. 1.
  • [0201]
    Fluorescent Dye Selection Process
  • [0202]
    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. Given a molarity of each nucleotide in the reaction medium of no more than about 1 μM, an average distance between tagged nucleotides is calculated to be greater than or equal to about 250 Å. 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. Preferably, nucleotide concentrations are reduced below 1 μM. Reducing dNTP concentrations to levels of at least <10% of the Km 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. Additionally, 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.
  • [0203]
    In one preferred embodiment, 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. For example, 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. Preferably, the donor is selected such that the excitation light activates the donor, which then efficiency transfers the excitation energy to one of the acceptors. 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. However, the methods of this invention can also be performed using two, three or four unique fluorescence donor-acceptor pairs, by running parallel reactions.
  • [0204]
    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.
  • [0205]
    Although 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 β and/or γ 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. Once the tagged DNTP is incorporated into the growing DNA chain, the fluorescence acceptor, which is now attached to the pyrophosphate group, is released to the medium with the cleaved pyrophosphate group. In fact, the growing DNA chain includes no fluorescence acceptor molecules at all. In essence, 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 β or γ 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.
  • [0206]
    If the fluorescence from the tagged dNTPs in the polymerizing medium (background) is problematic, 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. Of course, the quenchers are also adapted to have insignificant contact with the donor on the polymerase. To minimize interaction between the collisional quenchers and 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.
  • [0207]
    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. Thus, the polymerase is tagged with a fluorophore, while each dNTP is labeled with a quencher for the fluorophore. Typically, 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. Preferably, 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. Although less preferred, if only 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.
  • SITE SELECTION FOR LABELING THE TAQ POLYMERASE AND dNTPs
  • [0208]
    Although 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.
  • [0209]
    Fluorescent Labeling of Polymerase and/or dNTPs
  • [0210]
    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.
  • [0211]
    In general, 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. Once a mutated DNA sequence is prepared, the mutant is inserted into E. coli for expression. After expression, the mutant polymerase is isolated and purified. The purified mutant polymerase is then tested for polymerase activity. After activity verification, the mutant polymerase is reacted with a slight molar excess of a desired tag to achieve near stoichiometric labeling. Alternatively, 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.
  • [0212]
    If the mutant polymerase includes several sites including the target residue that can undergo tagging with the desired molecular tag, then 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. If the target amino acid residue in the mutant polymerase is close to the active dNTP binding site, a saturating level of a protecting group or a 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. Finally, 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.
  • [0213]
    Alternatively, if the target residue is not near the active site, 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.
  • [0214]
    Amino Acide Site Selection for the Taq Polymerase
  • [0215]
    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.
  • [0216]
    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. In addition, 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.
  • [0217]
    Once tagged mutant polymerases are prepared and energy minimized in a full solvent environment, estimates of the effect on the structure of the polymerase due to the mutation and/or labeling are generated to provide information about relative tag positioning and separation. This data is then used to estimate FRET efficiencies prior to measurement. Of course, if the dNTPs are tagged with quenchers, then these considerations are not as important.
  • [0218]
    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.
  • [0219]
    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. These predicted ionization free energies are then recalculated so that shifts in ionization caused by the binding of a DNTP are taken into account. Unexpected ionization states are subject to further computational and experimental studies, leading to a set of partial charges for each residue in the protein, i.e., each ionizable residue in the protein can have a different charge state depending on the type of attached tag or amino acid substitution.
  • [0220]
    To further aid in amino acid site selection, 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.
  • [0221]
    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.
  • [0222]
    Molecular docking simulations are used to predict the docked orientation of the natural and fluorescently labeled dNTPs, within the polymerase binding pocket. The best-docked configurations are energy minimized in the presence of an explicit solvent environment. In conjunction with amino acid sites in the polymerase selected for labeling, the docking studies are used to analyze how the tags interact and to predict FRET efficiency for each selected amino acid site.
  • [0223]
    With the exception of the electrostatics calculations, all docking, quantum mechanics, molecular mechanics, and molecular dynamics calculations are and will be performed using the HyperChem (v6.0) computer program. The HyperChem software runs on PCs under a Windows operating system. A number of computer programs for data analysis or for FRET prediction (as described below) are and will be written on a PC using the Linux operating system and the UHBD program running under Linux.
  • [0224]
    Analysis of Polymerase Structures
  • [0225]
    Co-crystal structures solved for DNA polymerase I (DNA pol I) from E. coli, T. aquaticus, B. stearothermophilus, T7 bacteriophage, and human pol α 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. Comparison of these structures provides information about the conformational changes that occur within the polymerase during nucleotide incorporation. Specifically, in the closed conformation, the tip of the fingers domain is rotated inward by 46°, thereby positioning the dNTP at the 3′ end of the primer strand in the polymerase active site. The geometry of this terminal base pair is precisely matched with that of its binding pocket. The binding of the correct, complementary base facilitates formation of the closed conformation, whereas incorrect dNTP binding does not induce this conformational change. Reaction chemistry occurs when the enzyme is in the closed conformation.
  • [0226]
    Referring now to FIG. 2, 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 Cα 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.
  • [0227]
    Using a program to determine the change in position of amino acids in the open and closed conformation of the polymerase relative to the gamma phosphate of a bound ddGTP from two different crystal structures of the Taq polymerase containing the primer and bound ddGTP, lists of in 20 amino acid sites that undergo the largest change in position for mutation and labeling were identified. The distances were calculated for each amino acid between their alpha and beta carbon atoms and the gamma phosphate group of the bound ddGTP. Lists derived from the two different sets of crystallographic data for the Taq polymerase are given in Tables I, II, III and IV.
    TABLE I
    The 20 Amino Acid Sites Undergoing the Largest Positional Change in
    2ktq Data Between the Open Form of the Polymerase to the Closed
    Form of the Polymerase Relative to the Alpha Carbon of the Residue
    Residue Change in Residue Change in
    Loca- Residue Distance Loca- Residue Distance
    tion Identity (Å) tion Identity (Å)
    517 Alanine 9.10 491 Glutamic 2.90
    acid
    516 Alanine 6.86 486 Serine 2.78
    515 Serine 6.53 490 Leucine 2.62
    513 Serine 6.40 586 Valine 2.61
    518 Valine 5.12 492 Arginine 2.60
    514 Threonine 3.94 462 Glutamic 2.59
    acid
    488 Asparagine 3.73 483 Asparagine 2.47
    487 Arginine 3.50 685 Proline 2.46
    489 Glutamine 3.13 587 Arginine 2.44
    495 Phenylalanine 3.05 521 Alanine 2.38
  • [0228]
    [0228]
    TABLE II
    The 20 Amino Acid Sites Undergoing the Largest Positional Change in
    2ktq Data Between the Open Form of the Polymerase to the Closed Form
    of the Polymerase Relative to the Beta Carbon of the Residue
    Change Change
    in in
    Residue Residue Distance Residue Residue Distance
    Location Identity (Å) Location Identity (Å)
    517 Alanine 10.98 491 Glutamic 3.41
    Acid
    516 Alanine 9.05 587 Arginine 3.39
    515 Serine 8.02 521 Alanine 3.33
    513 Serine 7.46 498 Leucine 3.21
    518 Valine 5.47 489 Glutamine 3.08
    685 Proline 5.16 514 Threonine 2.97
    487 Arginine 4.24 581 Leucine 2.93
    495 Phenyl- 3.94 483 Asparagine 2.92
    alanine
    488 Aspartic 3.88 497 Glutamic 2.91
    Acid Acid
    520 Glutamic 3.66 462 Glutamic 2.83
    Acid Acid
  • [0229]
    [0229]
    TABLE III
    The 20 Amino Acid Sites Undergoing the Largest Positional Change in
    3ktq Data Between the Open Form of the Polymerase to the Closed
    Form of the Polymerase Relative to the Alpha Carbon of the Residue
    Residue
    Residue Residue Change in Loca- Residue Change in
    Location Identity Distance (Å) tion Identity Distance (Å)
    517 Alanine 8.95 515 Serine 6.36
    656 Proline 8.75 653 Alanine 6.16
    657 Leucine 8.59 661 Alanine 5.94
    655 Aspartic 8.05 652 Glutamic 5.44
    Acid Acid
    660 Arginine 7.35 647 Phenyl- 5.25
    alanine
    658 Metionine 7.06 649 Valine 5.22
    659 Arginine 6.69 518 Valine 5.15
    654 Valine 6.60 644 Serine 5.08
    513 Serine 6.59 643 Alanine 5.01
    516 Alanine 6.57 650 Proline 4.72
  • [0230]
    [0230]
    TABLE IV
    The 20 Amino Acid Sites Undergoing the Largest Positional Change
    in 3ktq Data Between the Open Form of the Polymerase to the Closed
    Form of the Polymerase Relative to the Beta Carbon of the Residue
    Residue
    Residue Residue Change in Loca- Residue Change in
    Location Identity Distance (Å) tion Identity Distance (Å)
    517 Alanine 10.85 654 Valine 6.25
    656 Proline 9.05 653 Alanine 6.14
    657 Leucine 8.75 661 Alanine 6.04
    516 Alanine 8.68 643 Alanine 5.74
    655 Aspartic 8.24 649 Valine 5.55
    Acid
    515 Serine 7.92 647 Phenyl- 5.45
    alanine
    660 Arginine 7.89 518 Valine 5.42
    513 Serine 7.60 652 Glutamic 5.13
    Acid
    659 Arginine 6.98 644 Serine 4.89
    658 Metionine 6.77 487 Arginine 4.77
  • [0231]
    The above listed 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.
  • [0232]
    To further refine the 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.
  • [0233]
    Referring now to FIGS. 3A-C, an overlay between 3 ktq (closed ‘black’) and 1 tau (open ‘light blue’), the large fragment of Taq DNA polymerase I is shown. Looking at FIG. 3A, 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. Addition of a larger side chain/tag is thought to diminish the ability of the polymerase to achieve a fully closed, active conformation. Conversely, Asp655 is entirely solvent exposed in both the closed and open conformations of the polymerase. Looking at 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. Looking at 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. In this representation, it is evident that 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.
  • MUTAGENESIS AND SEQUENCING OF POLYMERASE VARIANTS
  • [0234]
    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 Sequencher™ available from GeneCodes, Inc.
  • EXPRESSION AND PURIFICATION OF ENZYME VARIANTS
  • [0235]
    The inventors have demonstrated that the Taq polymerase is capable of incorporating γ-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.
  • [0236]
    Although 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. Of course, when optimized polymerases construct are prepared, then this large scale production will be used. During the development of optimized polymerases, the mutants are derived from E. coli cell masses grown in 10 L well-oxygenated batch cultures using a rich medium available from Amgen. For fast polymerase mutant screening, 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. coli 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.
  • [0237]
    Because the thermal stability of Taq polymerase is considerably greater than typical E. coli proteins, purification of Taq polymerase or its mutants from contaminating Taq polymerase proteins is achieved by a simple heat treatment of the crude polymerase at 75° C. for 60 minutes, which reduces E. coli protein contamination by approximately 100-fold. This reduction in E. coli protein contamination combined with the high initial expression level, produces nearly pure Taq polymerase or its mutants in a convenient initial step; provide, of course, that the mutant polymerase retains the thermal stability of the native polymerase.
  • [0238]
    For routine sequencing and PCR screening, further limited purification is generally required. 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. Preferably, 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.
  • SELECTION OF SITE IN dNTP TO ACCEPT FLUORESCENT TAG
  • [0239]
    Molecular docking simulations were carried out to predict the docked orientation of the natural and fluorescently labeled dNTPs using the AutoDock computer program (Morris et al., 1998; Soares et al., 1999). Conformational flexibility is permitted during the docking simulations making use of an efficient Lamarckian Genetic algorithm implemented in the AutoDock program. A subset of protein side chains is also allowed to move to accommodate the DNTP as it docks. The best docked configurations is then energy minimized in the presence of a solvent environment. Experimental data are available which identify amino acids in the polymerase active site that are involved in catalysis and in contact with the template/primer DNA strands or the dNTP to be incorporated. The computer-aided chemical modeling such as docking studies can be used identify and support sites in the dNTP that can be labeled and to predict the FRET efficiency of dNTPs carrying a specific label at a specific site.
  • [0240]
    In general, 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.
  • [0241]
    Chemical Modification of Nucleotides for DNA Polymerase Reactions
  • [0242]
    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.
  • [0243]
    Synthesis of γ-Phosphate Modified dNTPs
  • [0244]
    The inventors have found that 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 γ-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 β and/or γ phosphate groups.
  • [0245]
    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.
  • [0246]
    One generalized synthetic routine to synthesizing other γ-tagged dNTPs is given below:
  • [0247]
    where 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. Preferably, Z is Cl, Br, I, OH, SH, NH2, NHR, CO2H, CO2R, 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, CO2, SiO, GeO, where R is an alkyl, aryl, aralkyl, alkaryl, halogenated analogs thereof or hetero atom analogs thereof .
  • [0248]
    The synthesis involves reacting Z terminated fluorescent tag, FR-L-Z with a pyrophosphate group, P2O6X3H, in DCC and dichloromethane to produce a fluorescent tagged pyrophosphate. After the 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 γ-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.
  • [0249]
    A generalized synthesis of a the FR-L group is shown below:
  • [0250]
    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 γ-phosphate group or to tag amino acids. See, e.g., Ward et al., 1987; Engelhardt et al., 1993; Little et al., 2000; Hobbs, 1991.
  • [0251]
    By using different fluorescent tags on each dNTP, 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.
  • [0252]
    The synthetic scheme shown above for fluorescein is adaptable to other dyes as well such as tetrachlorofluorescein (JOE) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA). Typically, the gamma phosphate tagged reactions are carried out in basic aqueous solutions and a carbodiimide, such as DEC. Other fluorophore molecules and dNTPs can be similarly modified.
  • [0253]
    Synthesis of dNTP Tagged at on the Base
  • [0254]
    Although tagging the dNTPs at the beta and/or gamma phosphate is preferred, 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:
  • [0255]
    Polymerase Activity Assays Using a Fluorescently-tagged Enzyme
  • [0256]
    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.
  • [0257]
    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. To examine mutant Taq polymerase activity, the purified mutant Taq polymerase is incubated in polymerase reaction buffer with a 5′-32P 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 DNTβ and to confirm that fluorescent signatures are obtained during base incorporation. These assays use an end-labeled primer, the fluorescently-tagged dNTβ 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.
  • [0258]
    Primer Extension Assays
  • [0259]
    The ability of Taq DNA polymerase to incorporate a γ-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 γ-tagged dNTP, singly or collectively to produce long DNA chains.
  • [0260]
    To examine polymerase activity, the polymerase is incubated in polymerase reaction buffer such as Taq DNA polymerase buffer available from Promega Corporation of Madison, Wisconsin with either a 5′-32P 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.
    TABLE V
    Primer Strand:
    TOP 5′ GGT ACT AAG CGG CCG CAT G 3′
    Template Strands:
    BOT-T 3′ CCA TGA TTC GCC GGC GTA CTC 5′
    BOT-C 3′ CCA TGA TTC GCC GGC GTA CCC 5′
    BOT-G 3′ CCA TGA TTC GCC GGC GTA CGC 5′
    BOT-A 3′ CCA TGA TTC GCC GGC GTA CAC 5′
    BOT-3T 3′ CCA TGA TTC GCC GGC GTA CTT TC 5′
    BOT-Sau 3′ CCA TGA TTC GCC GGC GTA CCT AG 5′
  • [0261]
    In Table V, ‘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. For example, 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.
  • [0262]
    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. Similarly, 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.
  • [0263]
    Assay Fidelity of γ-phosphate Tagged Nucleotide Incorporation
  • [0264]
    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.
  • [0265]
    If the error rate for the incorporation of modified dNTPs exceeds a threshold level of about 1 error in 100, 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.
  • [0266]
    The BOT-variant templates are used to characterize the accuracy at which each γ-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.
  • [0267]
    Relative Fluorescence Intensity Assays
  • [0268]
    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 γ-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 γ-phosphate dNTPs, as was verified for ANS-pyrophosphate released by Taq and RNA polymerase and venom phosphodiesterase.
  • [0269]
    Experiments have been and are being performed by incubating γ-phosphate tagged dATP or TTP (Control: non-modified dATP and TTP) in an appropriate buffer (e.g., buffers available from Promega Corporation) in the presence of polymerase (Control: no enzyme) and DNA primer/template [poly(dA). poly(dT)] (Control: no primer/template DNA). When the polymerase incorporates a tagged dNTP, changes in fluorescence intensity and/or frequency, absorption and/or emission spectra, and DNA polymer concentration are detected. Changes in these measurables as a function of time and/or temperature for experimental versus control cuvettes allows for unambiguous determination of whether a polymerase is incorporating the γ-phosphate tagged dNTP. Excitation and fluorescence emission can be optimized for each tagged dNTP based on changes in these measurables.
  • [0270]
    Development of a Single-Molecule Detection System
  • [0271]
    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.
  • [0272]
    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. Although any type of light source can be used, 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.
  • [0273]
    Tag detection in practice, of course, 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. Moreover, single-molecule technology is a well-developed and commercially available technology. See, e.g., Peck et al., 1989; A mbrose et al., 1994; Goodwin et al., 1997; Brouwer et al., 1999; Castro and Williams, 1997; Davis et al., 1991; Davis et al., 1992; Goodwin et al., 1997; Keller et al., 1996; Michaelis et al., 2000; Orrit and Bernard, 1990; Orrit et al., 1994; Sauer et al., 1999; Unger et al., 1999; Zhuang et al., 2000.
  • [0274]
    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.
  • [0275]
    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.
  • [0276]
    In imaging the fluorescence of multiple single molecules, it is preferable to minimize the occurrence of multiple fluorescent emitters within 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.
  • [0277]
    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. First, 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).
  • [0278]
    Alternatively, 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.
  • [0279]
    Polymerase Activity Assays Using a Single-Molecule Detection System
  • [0280]
    These assays are performed essentially as described in for polymerase activity assays described herein. As stated above, 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. A variety of 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. These immobilization techniques are designed to provide specific areas for detection of the detectable property such as fluorescent, NMR, or the like, where the spacing is sufficient to decrease or minimize data collection channels having multiple emitters. Thus, 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.
  • [0281]
    Analysis of Fluorescent Signals from Single-molecule Sequencing System
  • [0282]
    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. Additionally, when required, 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.
  • INFORMATICS: ANALYSIS OF FLUORESCENT SIGNALS FROM THE SINGLE-MOLECULE DETECTION SYSTEM
  • [0283]
    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. At 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.
  • [0284]
    Additionally, in certain instances it is useful to perform reactions with reference controls, similar to microarray assays. Comparison of signal(s) between the reference sequence and the test sample are used to identify differences and similarities in sequences or sequence composition. Such reactions can be used for fast screening of DNA polymers to determine degrees of homololgy between the polymers, to determine polymorphisms in DNA polymers, or to identity pathogens.
  • EXAMPLES
  • [0285]
    Cloning and Mutagenesis of Taq Polymerase
  • [0286]
    Cloning
  • [0287]
    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:
  • [0288]
    Taq Pol I forward
  • [0289]
    5′-gc gaattc atgaggggga tgctgcccct ctttgagccc-3′
  • [0290]
    Taq Pol I reverse
  • [0291]
    5′-gc gaattc accctccttgg cggagcgc cagtcctccc-3′
  • [0292]
    The underlined segment of 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:
  • [0293]
    Taq Pol I_A293_trunk
  • [0294]
    5′-aatccatgggccctggaggaggc cccctggcccccgc-3′
  • [0295]
    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). Ideally, 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.
  • [0296]
    Mutagensis
  • [0297]
    Once a suitable construct is generated, individual cysteine mutations are introduced at preferred amino acid positions including positions 513-518, 643, 647, 649 and 653-661 of the native Taq polymerase. The following amino acid residues correspond to the amino acids between amino acid 643 and 661, where xxx represents intervening amino acid residues in the native polymerase: 643-Ala xxx xxx xxx Phe xxx Val xxx xxx Glu Ala Val Asp Pro Leu Met Arg Arg Ala-661
  • [0298]
    Overlapping primers are used to introduce point mutations into the native gene by PCR based mutagenesis (using Pfu DNA polymerase).
  • [0299]
    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. To remove the template DNA, the entire PCR product is treated with DpnI restriction enzyme (cuts at methylated guanosines in the sequence GATC). Following digestion of the template plasmid, the mutated plasmid is transformed and ligation occurs in vivo.
  • [0300]
    The following synthetic DNA oligonucleotide primers are used for mutagenesis as described below, where the letters designated in lowercase have been modified to yield the desired Cysteine substitution at the indicated position. Mutants are then screened via automated sequencing.
    Alanine 643 to Cysteine Replacement
    Taq Pol I_Ala643Cys_fwd
    5′-C CAC ACG GAG ACC tgC AGC TGG ATG TTC GGC G-3′
    Taq PolI_Ala643Cys_rev
    5′-C GCC GAA CAT CCA CGA Gca GGT CTC CGT GTG G-3′
    Phenylalanine 647 to Cysteine Replacement
    Taq Pol I_Phe647Cys_fwd
    5′-CC GCC AGC TGG ATG TgC GGC GTC CCC CGG GAG
    GCC-3′
    Taq Pol I_Phe647Cys_rev
    5′-GGC CTC CCG GGG GAC GCC GcA CAT CCA CGT GGC
    GG-3′
    Valine 649 to Cysteine Replacement
    Taq Pol I_Val649Cys_fwd
    5′-GCC AGC TGG ATG TTC GGC tgC CCC CGG GAG GCC
    GTG G-3′
    Taq Pol I_Val649Cys_rev
    5′-C CAC GGC CTC CCG GGG Gca GCC GAA CAT CCA GCT
    GGC-3′
    Glutamic Acid 652 to Cysteine Replacement
    Taq Pol I_Glu652Cys_fwd
    5′-GGC GTC CCC CGG tgc GCC GTG GAC CCC CTG ATG
    CGC-3′
    Taq PolI_Glu652Cys_rev
    5′-GCG CAT CAG GGG GTC CAC GGC gca CCG GGG GAC
    GCC-3′
    Alanine 653 to Cysteine Replacement
    Taq Pol I_Ala653Cys_fwd
    5′-GGC GTC CCC CGG GAG tgC GTG GAC CCC CTG ATG
    CGC-3′
    Taq Pol I_Ala653Cys_rev
    5′-GCG CAT CAG GGG GTC CAC Gca CTC CCG GGG GAC
    GCC-3′
    Valine 654 to Cysteine Replacement
    Taq Pol I_Val6S4Cys_fwd
    5′-GTC CCC CGG GAG GCC tgt GAC CCC CTG ATG CGC-3′
    Taq PolI_Val654Cys_rev
    5′-GCG CAT CAG GGG GTC aca GGC CTC CCG GGG GAC-3′
    Aspartic Acid 655 to Cysteine Replacement
    Taq Pol I_D655C_fwd
    5′-CCC CGG GAG GCC GTG tgC CCC CTG ATG CGC CGG-3′
    Taq Pol I_D655C_rev
    5′-CCG GCG CAT CAG GGG Gca CAC GGC CTC CCG GGG-3′
    Proline 656 to Cysteine Replacement
    Taq Pol I_Pro656Cys_fwd
    5′-CGG GAG GCC GTG GAC tgC CTG ATG CGC CGG GCG-3′
    Taq Pol I_Pro656Cys_rev
    5′-CGC CCG GCG CAT CAG Gca GTC CAC GGC CTC CCG-3′
    Leucine 657 to Cysteine Replacement
    Taq Pol I_Leu657Cys_fwd
    5′-GCC GTG GAC CCC tgc ATG CGC CGG GCG GCC-3′
    Taq Pol I_Leu657Cys_rev
    5′-GGC CGC CCG GCG CAT gca GGG GTC CAC GGC-3′
    Methionine 658 to Cysteine Replacement
    Taq Pol I_Met658Cys_fwd
    5′-GCC GTG GAC CCC CTG tgt CGC CGG GCG GCC-3′
    Taq Pol I_Met658Cys_rev
    5′-GGC CGC CCG GCG aca CAG GGG GTC CAC GGC-3′
    Arginine 659 to Cysteine Replacement
    Taq Pol I_Arg659Cys_fwd
    5′-GCC GTG GAC CCC CTG ATG tGC CGG GCG GCC AAG
    ACC-3′
    Taq Pol I_Arg659Cys_rev
    5′-GGT CTT GGC CGC CCG GCa CAT CAG GGG GTC CAC
    GGC-3′
    Arginine 660 to Cysteine Replacement
    Taq Pol I_Arg660Cys_fwd
    5′-GAC CCC CTG ATG CGC tGc GCG GCC AAG ACC ATC-3′
    Taq Pol I_Arg660Cys_rev
    5′-GAT GGT CTT GGC CGC gCa GCG CAT CAG GGG GTC-3′
    Alanine 661 to Cysteine Replacement
    Taq Pol I_Ala661Cys_fwd
    5′-CCC CTG ATG CGC CGG tgc GCC AAG ACC ATC AAC-3′
    Taq Pol I_Ala661Cys_rev
    5′-GTT GAT GGT CTT GGC gca CCG GCG CAT CAG GGG-3′
  • [0301]
    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. Thus, 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.
  • [0302]
    Synthesis of Modified dNTPs
  • [0303]
    Synthesis of (γ-AmNS)dATP
  • [0304]
    Nucleotide analogues which contain fluorophore 1-aminonaphalene-5-sulfonate attached to the γ-phosphate bond were synthesized (J. Biol. Chem.254,12069-12073,1979). dATP analog-(γ-AmNS) dATP was synthesized according to the procedures slightly altered from what was described by Yarbrough and co-workers for (γ-AmNS)ATP with some modifications.
  • [0305]
    This example illustrates the preparation of gamma ANS tagged dATP, shown graphically in FIG. 4.
  • [0306]
    1-Aminonaphthalene-5-sulphonic acid (447 mg, 2 mmol, 40 eq., from Lancaster) was added to 10 mL of H2O, and the pH was adjusted to 5.8 with 1 N NaOH. The insoluble material was removed by syringe filter, yielding a solution which was essentially saturated for this pH value (˜0.18 to 0.2 M). 4 mL of 12.5 mM 5′triphosphate-2′-deoxyadenosine disodium salt (0.05 mmol, 1 eq.) and 2 mL of 1 M 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (DEC, 2 mmol, 40eq., from Lancaster) were added to a reaction vessel at 22 ° C. The reaction was initiated by adding 10 mL of the 1-aminonaphthalene-5-sulfonate solution and allowed to continue for 2.5 h. The pH was kept between 5.65-5.75 by periodic addition of 0.1 N HC1. After 2.5 h, the reaction was diluted to 50 mL and adjusted to a solution of 0.05 M triethylammonium bicarbonate buffer (TEAB, pH ˜7.5). 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. Approximately 10 mL fractions were collected. Absorbance and fluorescence profiles (UV 366nm) of the fractions were obtained after appropriate dilution. The fluorescent fraction eluted at ˜0.7 M buffer after the peak of the unreacted DATβ and showed a brilliant blue fluorescence. The product-containing fractions were pooled, dried by lyophilizer and co-evaporated twice with H2O/ethanol (70/30). The residue was taken up in water and lyophilized. 31P NMR (1H decoupled, 600 MHz, D2O, Me3PO4 external reference, 293 K, pH 6.1) δ (ppm) −12.60, −14.10, −25.79. The reference compound dATP gave the following resonance peaks: 31P NMR (dATP, Na+) in D2O 293 K, δ (ppm) −11.53 (γ), −13.92 (α), −24.93 (β).
  • [0307]
    Using diode array UV detection HPLC, the fraction containing the desired product was easily identified by the distinct absorption of the ANS group at 366 nm. Additionally, 31P NMR spectra were recorded for the γ-phosphate tagged dATP and regular dATP in an aqueous solution. For each compound, three characteristic resonances were observed, confirming the triphosphate moiety in the γ-tagged dATP. The combined analyses—1H-NMR, HPLC, and UV spectra—provide supporting information for the formation of the correct compound.
  • [0308]
    The same synthetic procedure was used to prepare γ-ANS-phosphate modified dGTP, dTTP and dCTP.
  • [0309]
    γ-Phosphate-tagged dNTP Incorporation by Tag Polymerase
  • [0310]
    The following examples illustrate that commercially available Taq DNA polymerase efficiently incorporates the ANS-γ-phosphate dNTPs, the syntheses and characterization as described above.
  • [0311]
    In the first example, illustrates the incorporation of ANS-γ-phosphate DATP to produce extended DNA products from primer templates. The reactions were carried out in extension buffer and the resulting Radiolabeled products were size separated on a 20% denaturing polyacryamide gel. Data was collected using a phosphorimaging system. Referring now the FIG. 5, Lane 1 contains 5′ radiolabeled ‘TOP’ probe in extension buffer. Lane 2 contains Taq DNA polymerase, 50 μM dGTP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-Sau’). Lane 3 contains Taq DNA polymerase, 50 μM dATP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-Sau’). Lane 4 contains Taq DNA polymerase, 50 μM ANS-γ-dATP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-Sau’). Lane 5 contains Taq DNA polymerase, 50 μM 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 μM dATP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-T’). Lane 8 contains Taq DNA polymerase, 50 μM ANS-γ-dATP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-T’). Lane 9 contains Taq DNA polymerase, 50 μM dGTP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-3T’). Lane 10 contains Taq DNA polymerase, 50 μM dATP incubated with a DNA duplex (radiolabeled TOP with excess ‘BOT-3T’). Lane 11 contains Taq DNA polymerase, ANS-γ-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.
  • [0312]
    Quantitative comparison of lane 1 with lane 4 demonstrates that very little non-specific, single-base extension was detected when ANS-γ-dATP was included in the reaction, but the first incorporated base should be dGTP (which was not added to the reaction). Quantitative analysis of lanes 1 and 8 demonstrates that approximately 71% of the TOP primer are extended by a template-directed single base when ANS-γ-dATP was included in the reaction and the first incorporated base should be dATP. Thus, Taq DNA polymerase incorporates γ-tagged nucleotides. Equally important to the polymerase's ability to incorporate a γ-tagged nucleotide is its ability to extend the DNA polymer after the modified DATP was incorporated. Comparison of lane 1 with lane 11 demonstrated that a DNA strand was extended after a γ-tagged nucleotide was incorporated. Thus, incorporation of a modified nucleotide was not detrimental to polymerase activity. Note, too, that extension of the primer strand by incorporation of an ANS-γ-nucleotide depended upon Watson-Crick base-pairing rules. In fact, the fidelity of nucleotide incorporation was increased at least 15-fold by the addition of this tag to the γ-phosphate.
  • [0313]
    This next example illustrates the synthesis of extended DNA polymers using all four ANS tagged γ-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-32P dCTP. Lane 2 includes the same 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 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-32P-dCTP was cycled in parallel with lanes 5-8 reactions. Lane 5 includes the same 32P-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 32P-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 7 includes nonpurified, 10-base, 32P-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 8 includes nonpurified, 10-base, 32P-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.
  • [0314]
    This next example illustrates the synthesis of long DNA polymers using all four ANS tagged γ-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-32P dCTP. Lane 3 contains the same 32P -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 32P -labeled primer that was incubated with double-stranded DNA plasmid at 96° C. for 3 minutes, the reaction was brought to 37° C., Taq 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 5 includes the control, purified 10-base primer that was extended to 11 and 12 bases by the addition of alpha-32P -dCTP was cycled in parallel with lanes 5-8 reactions. Lane 6 includes the same 32P -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 32P -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,32P -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, 32P -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.
  • [0315]
    The majority of extension products in this reaction are several hundred bases long for both natural and γ-modified dNTPs, and a significant percentage of these products are too large to enter the gel. Thus, demonstrating the gamma phosphate tagged dNTPs are used by Taq polymerase to generate long DNA polymers that are non-tagged or native DNA polymer chains.
  • [0316]
    Different Polymerases React Differently to the Gamma-modified Nucleotides
  • [0317]
    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 μM 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.
  • [0318]
    Taq DNA polymerase efficiently uses the gamma-modified nucleotides to synthesize extended DNA polymers at increased accuracy as shown in FIGS. 4-6.
  • [0319]
    The Klenow fragment from E. coli DNA polymerase I efficiently uses the gamma-modified nucleotides, but does not exhibit the extreme fidelity improvements observed with other enzymes as shown in FIG. 8.
  • [0320]
    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.
  • [0321]
    HIV-1 reverse transcriptase efficiently uses the gamma-tagged nucleotides, and significant fidelity improvement results as shown in FIG. 10.
  • [0322]
    Polymerization activity is difficult to detect in the reaction products generated by native T7 DNA polymerase (due to the presence of the enzymes exonuclease activity). However, its genetically modified derivative, Sequenase, shows that the gamma-modified nucleotides are efficiently incorporated, and that incorporation fidelity is improved, relative to non-modified nucleotides. The experimental results for native T7 DNA polymerase and Sequenase are shown in FIG. 11.
  • [0323]
    Thus, for the Taq polymerase or the HIV1 reverse transcriptase, improved fidelity, due to the use of the gamma-modified dNTPs of this invention, enables single-molecule DNA sequencing. However, not all polymerases equally utilize the gamma-modified nucleotides of this invention, specifically, Klenow, Sequenase, HIV-1 reverse transcriptase and Taq polymerases incorporate the modified nucleotides of this invention, while the Pfu DNA polymerase does not appear to incorporate the modified nucleotides of this invention.
  • [0324]
    Improved PCR—Generation of Long DNA Sequences
  • [0325]
    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. Even low levels of nucleotide misincorporation may become critical for sequences longer than 10 kb. The data shown in FIG. 4 shows that the fidelity of DNA synthesis using gamma tagged dNTPs is improved for the native Taq polymerase making longer DNA extension possible without the need for adding polymerases with 3′-to5′ exonuclease, or “proofreading”, activity as required in the long-distance PCR method developed by Cheng et al., U.S. Pat. No. 5,512,462, incorporated herein by reference. Thus, 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.
  • [0326]
    Signal Intensity and Reaction Kinetics Provide Information Concerning Base Identity
  • [0327]
    Signal intensities for each nucleotide in the extended DNA strand are used to determine, confirm or support base identity data. Referring now to FIG. 12, the solid line corresponds to reaction products produced when the four natural nucleotides (dATP, dCTP, dGTP and dTTP) are included in the synthesis reaction. The dashed or broken line corresponds to reaction products produced when proprietary, base-modified nucleotides are included in the reaction. As is clearly demonstrated, sequence context and base modification(s) influence reaction product intensity and/or kinetics, and these identifying patterns are incorporated into proprietary base-calling software to provide a high confidence value for base identity at each sequenced position.
  • [0328]
    All references cited herein and listed in are incorporated by reference. While this invention has been described fully and completely, it should be understood that, within the scope of the appended claims, the invention maybe practiced otherwise than as specifically described. Although the invention has been disclosed with reference to its preferred embodiments, from reading this description those of skill in the art may appreciate changes and modification that may be made which do not depart from the scope and spirit of the invention as described above and claimed hereafter.
  • 1 48 1 38 DNA Thermus aquaticus primer (1)..(38) Synthetic DNA forward primer for amplifying full-length Taq Pol I coding sequence. 5′ to 3′ listing 1 gcgaattcat gagggggatg ctgcccctct ttgagccc 38 2 37 DNA Thermus aquaticus primer (1)..(37) Synthetic DNA Reverse primer for amplifying full-length Taq Pol I coding sequence. 5′ to 3′ listing. 2 gcgaattcac cctccttggc ggagcgccag tcctccc 37 3 37 DNA Thermus aquaticus primer (1)..(37) Synthetic DNA primer for truncated Taq Pol I coding sequence. 5′ to 3′ listing. 3 aatccatggg ccctggagga ggccccctgg cccccgc 37 4 32 DNA Thermus aquaticus mutation (14)..(16) Site 643 of Taq Pol I Alanine codon, gcc, to cysteine codon, tgc 5′ to 3′ listing 4 ccacacggag acctgcagct ggatgttcgg cg 32 5 32 DNA Thermus aquaticus Mutation (17)..(19) Site 643 of complement strand of Taq Pol I alanine antisense codon, ggc, to cysteine antisense codon, gca. 5′ to 3′ listing. 5 cgccgaacat ccacgagcag gtctccgtgt gg 32 6 35 DNA Thermus aquaticus Mutation (15)..(17) Mutant Taq Pol 1 site 647 phe to cys codon mutation ttc -> tgc. 5′ to 3′ listing 6 ccgccagctg gatgtgcggc gtcccccggg aggcc 35 7 35 DNA Thermus aquaticus Mutation (19)..(21) Taq Pol I Complement Strand Site 647 phe to cys mutation gaa -> gca. 5′ to 3′ listing. 7 ggcctcccgg gggacgccgc acatccacgt ggcgg 35 8 37 DNA Thermus aquaticus Mutation (19)..(21) Taq Pol I Mutation Site 649 val to cys gtc -> tgc. 5′ to 3′ listing. 8 gccagctgga tgttcggctg cccccgggag gccgtgg 37 9 37 DNA Thermus aquaticus Mutation (17)..(19) Taq Pol I Mutation Complementary strand Site 649 val to cys gac -> gca. 5′ to 3′ listing. 9 ccacggcctc ccgggggcag ccgaacatcc agctggc 37 10 36 DNA Thermus aquaticus Mutation (13)..(15) Taq Pol I Mutation Site 652 glu to cys Codon 652 gtc -> tgc. 5′ to 3′ listing. 10 ggcgtccccc ggtgcgccgt ggaccccctg atgcgc 36 11 36 DNA Thermus aquaticus Mutation (22)..(24) Taq Pol I Mutation Complementary Strand AA Site 652 glu to cys antisense codon ctc -> gca. 5′ to 3′ listing. 11 gcgcatcagg gggtccacgg cgcaccgggg gacgcc 36 12 36 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation AA Site 653 ala to cys codon gcc -> tgc. 5′ to 3′ listing. 12 ggcgtccccc gggagtgcgt ggaccccctg atgcgc 36 13 36 DNA Thermus aquaticus Mutation (19)..(21) Taq Pol I Mutation Complementary Strand AA Site 653 ala to cys antisense codon ggc -> gca. 5′ to 3′ listing. 13 gcgcatcagg gggtccacgc actcccgggg gacgcc 36 14 33 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation AA 654 val to cys codon gtg -> tgt. 5′ to 3′ listing. 14 gtcccccggg aggcctgtga ccccctgatg cgc 33 15 33 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation Complementary Strand AA Site 654 val to cys antisense codon cac -> aca. 5′ to 3′ listing. 15 gcgcatcagg gggtcacagg cctcccgggg gac 33 16 33 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation AA 655 asp to cys codon gac -> tgc 16 ccccgggagg ccgtgtgccc cctgatgcgc cgg 33 17 33 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation Complementary Strand AA Site 655 asp to cys antisense codon gtc -> gca. 5′ to 3′ listing. 17 ccggcgcatc agggggcaca cggcctcccg ggg 33 18 33 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation AA 656 pro to cys codon ccc -> tgc. 5′ to 3′ listing. 18 cgggaggccg tggactgcct gatgcgccgg gcg 33 19 33 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation Complementary Strand AA Site 656 pro to cys antisense codon ggg -> gca. 5′ to 3′ listing. 19 cgcccggcgc atcaggcagt ccacggcctc ccg 33 20 30 DNA Thermus aquaticus Mutation (13)..(15) Taq Pol I Mutation AA 657 leu to cys codon ctg -> tgc. 5′ to 3′ listing. 20 gccgtggacc cctgcatgcg ccgggcggcc 30 21 30 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation Complementary Strand AA Site 657 leu to cys antisense codon cag -> gca. 5′ to 3′ listing. 21 ggccgcccgg cgcatgcagg ggtccacggc 30 22 30 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation AA 658 met to cys codon atg -> tgt. 5′ to 3′ listing. 22 gccgtggacc ccctgtgtcg ccgggcggcc 30 23 30 DNA Thermus aquaticus Mutation (13)..(15) Taq Pol I Mutation Complementary Strand AA Site 658 met to cys antisense codon cat -> gca. 5′ to 3′ listing. 23 ggccgcccgg cgacacaggg ggtccacggc 30 24 36 DNA Thermus aquaticus Mutation (19)..(21) Taq Pol I Mutation AA 659 arg to cys codon cgc -> tgc. 5′ to 3′ lising. 24 gccgtggacc ccctgatgtg ccgggcggcc aagacc 36 25 36 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation Complementary Strand AA Site 659 arg to cys antisense codon gcg -> gca. 5′ to 3′ listing. 25 ggtcttggcc gcccggcaca tcagggggtc cacggc 36 26 33 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation AA 660 arg to cys codon cgg -> tgc. 5′ to 3′ lising. 26 gaccccctga tgcgctgcgc ggccaagacc atc 33 27 33 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation Complementary Strand AA Site 660 arg to cys antisense codon ccg -> gca. 5′ to 3′ listing. 27 gatggtcttg gccgcgcagc gcatcagggg gtc 33 28 33 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation AA 661 ala to cys codon gcg -> tgc. 5′ to 3′ lising. 28 cccctgatgc gccggtgcgc caagaccatc aac 33 29 33 DNA Thermus aquaticus Mutation (16)..(18) Taq Pol I Mutation Complementary Strand AA Site 661 ala to cys antisense codon cgc -> gca. 5′ to 3′ listing. 29 gttgatggtc ttggcgcacc ggcgcatcag ggg 33 30 19 PRT Thermus aquaticus Variant (1)..(1) Taq Pol I Variant AA Site 643 ala to cys replacement. 30 Cys Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met 1 5 10 15 Arg Arg Ala 31 19 PRT Thermus aquaticus Variant (5)..(5) Taq Pol I Variant AA Site 647 phe to cys replacement. 31 Ala Ser Trp Met Cys Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met 1 5 10 15 Arg Arg Ala 32 19 PRT Thermus aquaticus Variant (7)..(7) Taq Pol I Variant AA Site 649 val to cys replacement. 32 Ala Ser Trp Met Phe Gly Cys Pro Arg Glu Ala Val Asp Pro Leu Met 1 5 10 15 Arg Arg Ala 33 19 PRT Thermus aquaticus Variant (10)..(10) Taq Pol I Variant AA Site 652 glu to cys replacement. 33 Ala Ser Trp Met Phe Gly Val Pro Arg Cys Ala Val Asp Pro Leu Met 1 5 10 15 Arg Arg Ala 34 19 PRT Thermus aquaticus Variant (11)..(11) Taq Pol I Variant AA Site 653 ala to cys replacement. 34 Ala Ser Trp Met Phe Gly Val Pro Arg Glu Cys Val Asp Pro Leu Met 1 5 10 15 Arg Arg Ala 35 19 PRT Thermus aquaticus Variant (12)..(12) Taq Pol I Variant AA Site 654 val to cys replacement. 35 Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Cys Asp Pro Leu Met 1 5 10 15 Arg Arg Ala 36 19 PRT Thermus aquaticus Variant (13)..(13) Taq Pol I Variant AA Site 655 asp to cys replacement. 36 Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Cys Pro Leu Met 1 5 10 15 Arg Arg Ala 37 19 PRT Thermus aquaticus Variant (14)..(14) Taq Pol I Variant AA Site 656 pro to cys replacement. 37 Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Cys Leu Met 1 5 10 15 Arg Arg Ala 38 19 PRT Thermus aquaticus Variant (15)..(15) Taq Pol I Variant AA Site 657 leu to cys replacement. 38 Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro Cys Met 1 5 10 15 Arg Arg Ala 39 19 PRT Thermus aquaticus Variant (16)..(16) Taq Pol I Variant AA Site 658 met to cys replacement. 39 Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Cys 1 5 10 15 Arg Arg Ala 40 19 PRT Thermus aquaticus Variant (17)..(17) Taq Pol I Variant AA Site 659 arg to cys replacement. 40 Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met 1 5 10 15 Cys Arg Ala 41 19 PRT Thermus aquaticus Variant (18)..(18) Taq Pol I Variant Site 660 arg to cys replacement. 41 Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met 1 5 10 15 Arg Cys Ala 42 19 PRT Thermus aquaticus Variant (19)..(19) Taq Pol I Variant Site 661 ala to cys replacement. 42 Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met 1 5 10 15 Arg Arg Cys 43 6 PRT Thermus aquaticus Variant (1)..(1) Taq Pol I Variant Site 513 ser to cys replacement. 43 Cys Thr Ser Ala Ala Val 1 5 44 6 PRT Thermus aquaticus Variant (2)..(2) Taq Pol I Variant Site 514 thr to cys replacement. 44 Ser Cys Ser Ala Ala Val 1 5 45 6 PRT Thermus aquaticus Variant (3)..(3) Taq Pol I Variant Site 515 ser to cys replacement. 45 Ser Thr Cys Ala Ala Val 1 5 46 6 PRT Thermus aquaticus Variant (4)..(4) Taq Pol I Variant Site 516 ala to cys replacement. 46 Ser Thr Ser Cys Ala Val 1 5 47 6 PRT Thermus aquaticus Variant (5)..(5) Taq Pol I Variant Site 517 ala to cys replacement. 47 Ser Thr Ser Ala Cys Val 1 5 48 6 PRT Thermus aquaticus Variant (6)..(6) Taq Pol I Variant Site 518 val to cys replacement. 48 Ser Thr Ser Ala Ala Cys 1 5

Claims (34)

We claim:
1. 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 polymerizing agent, where a detectable property of the tag undergoes a change before, during and/or after monomer incorporation.
2. The composition of claim 1, wherein the detectable property has a first value when the polymerizing agent is in a first state and a second value when the polymerase is in a second state, and where the polymerizing agent changes from the first state to the second state and back again during each monomer incorporation.
3. The composition of claim 2, wherein the polymerizing agent is a polymerase or reverse transcriptase.
4. The composition of claim 3, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polyrmerase I.
5. The composition of claim 3, wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.
6. The composition of claim 3, wherein the polymerase comprises Taq DNA polymerase I having a tag attached at a site selected from the group consisting of 513-518, 643, 647, 649 and 653-661 and mixtures or combinations thereof of the Taq polymerase, where the tag comprises a fluorescent molecule.
7. A composition comprising a polymerase or reverse transcriptase 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, and where the polymerizing agent changes from the first state to the second state and back again during each monomer incorporation.
8. The composition of claim 7, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coliDNA polymerase I.
9. The composition of claim 7, wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.
10. A composition comprising a polymerizing agent including a molecular and/or atomic tag associated with or covalently bonded to a site on the polymerase and a monomer including a molecular and/or atomic tag, where at least one of the tags has a detectable property that undergoes a change before, during and/or after monomer incorporation due to an interaction between the polymerizing agent tag and the monomer tag.
11. The composition of claim 10, wherein the change in the detectable property results from a change in the conformation of the polymerase from a first conformational state to a second conformational state and back again during each monomer incorporation.
12. The composition of claim 10, wherein the detectable property has a first detection propensity when the polymerase is in the first conformational state and a second detection propensity when the polymerase is in the a second conformational state.
13. The composition of claim 12, wherein the polymerizing agent is a polymerase or reverse transcriptase.
14. The composition of claim 13, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coliDNA polymerase I.
15. The composition of claim 13, wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.
16. The composition of claim 12, wherein the monomer comprise a dNTP and the tag is covalently bonded to the β or γ phosphate group.
17. The composition of claim 10, wherein the tag comprises a fluorescent tag and the detectable property comprises an intensity and/or frequency of emitted light.
18. The composition of claim 16, wherein the detectable property is substantially active when the polymerase is in the first conformational state and substantially inactive when the polymerase is in the 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.
19. The composition of claim 14, wherein the polymerase comprises Taq DNA polymerase I having a tag attached at a site selected from the group consisting of 513-518, 643, 647, 649 and 653-661 and mixtures or combinations thereof of the Taq polymerase, where the tag comprises a fluorescent molecule.
20. A composition comprising a polymerase or reverse transcriptase including a pair of 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.
21. The composition of claim 20, wherein 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 where the polymerizing agent changes from the first state to the second state and back again during each monomer incorporation.
22. The composition of claim 21, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I.
23. The composition of claim 21, wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.
24. The composition of claim 22, wherein the polymerase comprises Taq DNA polymerase I having a tag attached at a site selected from the group consisting of 513-518, 643, 647, 649 and 653-661 and mixtures or combinations thereof of the Taq polymerase, where the tag comprises a fluorescent molecule.
24. A single molecule sequencing apparatus comprising a substrate having a first chamber in which at least one tagged polymerase is confined therein and a second chamber including tagged dNTPs and a channel interconnecting the chambers, where a detectable property of at least one tag undergoes a detectable change during a monomer incorporation cycle.
25. The apparatus of claims 24, further comprising a plurality of monomer chambers, one for each tagged dNTP.
26. A mutant Taq polymerase comprising native Taq polymerase with a cysteine residue replacement at a site selected from the group consisting of513-518, 643, 647, 649 and 653-661 and mixtures or combinations thereof.
27. The polymerase of claim 27, wherein the cysteine residue includes a tag covalently bonded thereto through the SH group.
28. A system for retrieving stored information comprising:
a unknown nucleotide sequence representing a data stream;
a single-molecule sequencer including a polymerase having a tag associated therewith and monomers for the polymerase, each monomer having a tag associated therewith;
an excitation source adapted to excite the at least one of the tags; and
a detector adapted to detect a response from at least one of the tag,
where the response changes during polymerization of a complementary sequence and the changes in response represent a content of the data stream.
29. A system for determining sequence information from a single molecule comprising:
a unknown nucleotide sequence;
a single-molecule sequencer comprising a polymerase having a tag associated therewith and monomers for the polymerase, each monomer having a tag associated therewith;
a excitation source adapted to excite at least one of the tags; and
a detector adapted to detect a response from at least one of the tags,
where the response changes during polymerization of a complementary sequence and the changes in the response represent the identity of each nucleotide in the unknown sequence.
30. A method for sequencing a molecular sequence comprising:
supplying an unknown sequence of nucleotides or nucleotide analogs to a single-molecule sequencer comprising a polymerase having a fluorescent donor covalently attached thereto and monomers for the polymerase, each monomer having a unique fluorescent acceptor covalently bonded thereto;
exciting the fluorescent donor with a light from an excitation light source;
detecting emitted fluorescent light from the acceptor during a monomer incorporation cycle via a fluorescent light detector, where an intensity and/or frequency of the emitted light for the acceptors changes during each monomer incorporation cycle; and
converting the changes into an identity of each nucleotide or nucleotide analog in the unknown sequence.
31. A method of sequencing an individual nucleic acid molecule or numerous individual molecules in parallel including the steps of:
immobilizing a member of the replication complex comprising a polymerase including a tag attached thereto, a primer or a template sufficiently spaced apart to allow resolution detection of each complex on a solid support;
incubating the replication complex with cooperatively-tagged nucleotides, each nucleotide including a unique tag at its gamma-phosphate, where each nucleotide can be individually detected;
detecting each nucleotide incorporated by the polymerase as the polymerase transitions between its open and closed form, which causes a change in a detectable property of at least one of the tags or as the pyrophosphate group is released by the polymerase; and
relating the changes in the detectable property to the sequence of nucleotides in an unknown nucleic acid sequence.
32. A γ-phosphate modified nucleoside comprising γ-phosphate modified dATP, dCTP, dGTP and dTTP.
33. A primer sequence or portion thereof selected from the group consisting of Sequence 1 through 29.
US09901782 2000-07-07 2001-07-09 Real-time sequence determination Abandoned US20030064366A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US21659400 true 2000-07-07 2000-07-07
US09901782 US20030064366A1 (en) 2000-07-07 2001-07-09 Real-time sequence determination

Applications Claiming Priority (23)

Application Number Priority Date Filing Date Title
US09901782 US20030064366A1 (en) 2000-07-07 2001-07-09 Real-time sequence determination
US11007642 US20050266424A1 (en) 2000-07-07 2004-12-08 Real-time sequence determination
US11007797 US7329492B2 (en) 2000-07-07 2004-12-08 Methods for real-time single molecule sequence determination
US11648722 US20070172868A1 (en) 2000-07-07 2006-12-29 Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US11648137 US20070292867A1 (en) 2000-07-07 2006-12-29 Sequence determination using multiply tagged polymerizing agents
US11648182 US20070172866A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination using depolymerizing agent
US11648191 US20070172867A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11648856 US20070275395A1 (en) 2000-07-07 2006-12-29 Tagged monomers for use in sequence determination
US11648174 US20070172865A1 (en) 2000-07-07 2006-12-29 Sequence determination in confined regions
US11648164 US20070172864A1 (en) 2000-07-07 2006-12-29 Composition for sequence determination
US11648713 US20070184475A1 (en) 2000-07-07 2006-12-29 Sequence determination by direct detection
US11648115 US20070172861A1 (en) 2000-07-07 2006-12-29 Mutant polymerases
US11648184 US20100255463A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US11648136 US20070172862A1 (en) 2000-07-07 2006-12-29 Data stream determination
US11648106 US20070172858A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11648138 US20070172863A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US12410370 US20110014604A1 (en) 2000-07-07 2009-03-24 Methods for sequence determination
US12412208 US20100304367A1 (en) 2000-07-07 2009-03-26 Compositions for real-time, single molecule sequence determination
US12411997 US20110021383A1 (en) 2000-07-07 2009-03-26 Apparatuses for real-time, single molecule sequence determination
US12414417 US20090275036A1 (en) 2000-07-07 2009-03-30 Systems and methods for real time single molecule sequence determination
US12419214 US20090305278A1 (en) 2000-07-07 2009-04-06 Sequence determination in confined regions
US12419660 US20110059436A1 (en) 2000-07-07 2009-04-07 Methods for sequence determination
US12724392 US20100317005A1 (en) 2000-07-07 2010-03-15 Modified Nucleotides and Methods for Making and Use Same

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11007797 Division US7329492B2 (en) 2000-07-07 2004-12-08 Methods for real-time single molecule sequence determination

Publications (1)

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

Family

ID=22807697

Family Applications (22)

Application Number Title Priority Date Filing Date
US09901782 Abandoned US20030064366A1 (en) 2000-07-07 2001-07-09 Real-time sequence determination
US11007797 Active 2022-07-12 US7329492B2 (en) 2000-07-07 2004-12-08 Methods for real-time single molecule sequence determination
US11007642 Abandoned US20050266424A1 (en) 2000-07-07 2004-12-08 Real-time sequence determination
US11648174 Abandoned US20070172865A1 (en) 2000-07-07 2006-12-29 Sequence determination in confined regions
US11648138 Abandoned US20070172863A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US11648713 Abandoned US20070184475A1 (en) 2000-07-07 2006-12-29 Sequence determination by direct detection
US11648136 Abandoned US20070172862A1 (en) 2000-07-07 2006-12-29 Data stream determination
US11648191 Abandoned US20070172867A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11648856 Abandoned US20070275395A1 (en) 2000-07-07 2006-12-29 Tagged monomers for use in sequence determination
US11648137 Abandoned US20070292867A1 (en) 2000-07-07 2006-12-29 Sequence determination using multiply tagged polymerizing agents
US11648722 Abandoned US20070172868A1 (en) 2000-07-07 2006-12-29 Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US11648184 Abandoned US20100255463A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US11648164 Abandoned US20070172864A1 (en) 2000-07-07 2006-12-29 Composition for sequence determination
US11648106 Abandoned US20070172858A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11648115 Abandoned US20070172861A1 (en) 2000-07-07 2006-12-29 Mutant polymerases
US12410370 Abandoned US20110014604A1 (en) 2000-07-07 2009-03-24 Methods for sequence determination
US12411997 Abandoned US20110021383A1 (en) 2000-07-07 2009-03-26 Apparatuses for real-time, single molecule sequence determination
US12412208 Abandoned US20100304367A1 (en) 2000-07-07 2009-03-26 Compositions for real-time, single molecule sequence determination
US12414417 Abandoned US20090275036A1 (en) 2000-07-07 2009-03-30 Systems and methods for real time single molecule sequence determination
US12419214 Abandoned US20090305278A1 (en) 2000-07-07 2009-04-06 Sequence determination in confined regions
US12419660 Abandoned US20110059436A1 (en) 2000-07-07 2009-04-07 Methods for sequence determination
US12724392 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
US11007797 Active 2022-07-12 US7329492B2 (en) 2000-07-07 2004-12-08 Methods for real-time single molecule sequence determination
US11007642 Abandoned US20050266424A1 (en) 2000-07-07 2004-12-08 Real-time sequence determination
US11648174 Abandoned US20070172865A1 (en) 2000-07-07 2006-12-29 Sequence determination in confined regions
US11648138 Abandoned US20070172863A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US11648713 Abandoned US20070184475A1 (en) 2000-07-07 2006-12-29 Sequence determination by direct detection
US11648136 Abandoned US20070172862A1 (en) 2000-07-07 2006-12-29 Data stream determination
US11648191 Abandoned US20070172867A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11648856 Abandoned US20070275395A1 (en) 2000-07-07 2006-12-29 Tagged monomers for use in sequence determination
US11648137 Abandoned US20070292867A1 (en) 2000-07-07 2006-12-29 Sequence determination using multiply tagged polymerizing agents
US11648722 Abandoned US20070172868A1 (en) 2000-07-07 2006-12-29 Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US11648184 Abandoned US20100255463A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
US11648164 Abandoned US20070172864A1 (en) 2000-07-07 2006-12-29 Composition for sequence determination
US11648106 Abandoned US20070172858A1 (en) 2000-07-07 2006-12-29 Methods for sequence determination
US11648115 Abandoned US20070172861A1 (en) 2000-07-07 2006-12-29 Mutant polymerases
US12410370 Abandoned US20110014604A1 (en) 2000-07-07 2009-03-24 Methods for sequence determination
US12411997 Abandoned US20110021383A1 (en) 2000-07-07 2009-03-26 Apparatuses for real-time, single molecule sequence determination
US12412208 Abandoned US20100304367A1 (en) 2000-07-07 2009-03-26 Compositions for real-time, single molecule sequence determination
US12414417 Abandoned US20090275036A1 (en) 2000-07-07 2009-03-30 Systems and methods for real time single molecule sequence determination
US12419214 Abandoned US20090305278A1 (en) 2000-07-07 2009-04-06 Sequence determination in confined regions
US12419660 Abandoned US20110059436A1 (en) 2000-07-07 2009-04-07 Methods for sequence determination
US12724392 Abandoned US20100317005A1 (en) 2000-07-07 2010-03-15 Modified Nucleotides and Methods for Making and Use Same

Country Status (7)

Country Link
US (22) US20030064366A1 (en)
JP (3) JP2004513619A (en)
CN (2) CN101525660A (en)
CA (1) CA2415897A1 (en)
DE (2) DE60131194T2 (en)
EP (3) EP2100971A3 (en)
WO (1) WO2002004680A3 (en)

Cited By (82)

* 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
US20070172863A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions and methods for sequence determination
US20070172860A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: compositions and methods
US20070172866A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination using depolymerizing agent
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
US20080241938A1 (en) * 2006-07-20 2008-10-02 Visigen Biotechnologies, Inc. Automated synthesis or sequencing apparatus and method 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
US20080299565A1 (en) * 2005-12-12 2008-12-04 Schneider Thomas D Probe for Nucleic Acid Sequencing and Methods of Use
US20080309926A1 (en) * 2006-03-08 2008-12-18 Aaron Weber Systems and methods for reducing detected intensity non uniformity in a laser beam
WO2008154317A1 (en) * 2007-06-06 2008-12-18 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
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
US20100311061A1 (en) * 2009-04-27 2010-12-09 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
US20100311597A1 (en) * 2005-07-20 2010-12-09 Harold Philip Swerdlow Methods for sequence a polynucleotide template
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 (en) * 2011-11-04 2013-05-20 Hitachi Ltd Dna sequence decoding system, dna sequence decoding method, and program
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
US9670243B2 (en) 2010-06-02 2017-06-06 Industrial Technology Research Institute Compositions and methods for sequencing nucleic acids
US9671363B2 (en) 2013-03-15 2017-06-06 Life Technologies Corporation Chemical sensor with consistent sensor surface areas
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

Families Citing this family (162)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1141409B2 (en) 1998-12-14 2009-05-27 Pacific Biosciences of California, Inc. A kit and methods for nucleic acid sequencing of single molecules by polymerase synthesis
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
EP2363500A1 (en) * 2002-11-14 2011-09-07 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
US7405434B2 (en) * 2004-11-16 2008-07-29 Cornell Research Foundation, Inc. Quantum dot conjugates in a sub-micrometer fluidic channel
JP2006197832A (en) * 2005-01-19 2006-08-03 Tohoku Univ Multi-purpose method for detecting substrate affinity of abc transporter having cysteine residue induced by variation by using environment-sensitive fluorescent probe
US7767394B2 (en) * 2005-02-09 2010-08-03 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
US7825037B2 (en) * 2005-10-17 2010-11-02 Stc.Unm Fabrication of enclosed nanochannels using silica nanoparticles
US9156004B2 (en) 2005-10-17 2015-10-13 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
EP2126072A4 (en) * 2007-02-21 2011-07-13 Life Technologies Corp 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
US9598724B2 (en) 2007-06-01 2017-03-21 Ibis Biosciences, Inc. Methods and compositions for multiple displacement amplification of nucleic acids
WO2009046094A1 (en) 2007-10-01 2009-04-09 Nabsys, Inc. Biopolymer sequencing by hybridization of probes to form ternary complexes and variable range alignment
JP2011505119A (en) * 2007-10-22 2011-02-24 ライフ テクノロジーズ コーポレーション Method and system for obtaining fragmented sequence fragments ordered along the 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 (en) 2008-03-10 2013-05-22 周国华 Method for quantitatively measuring nucleic acid with sequence tags
EP3170904B1 (en) * 2008-03-28 2017-08-16 Pacific Biosciences Of California, Inc. Compositions and methods for nucleic acid sequencing
JP4586081B2 (en) * 2008-03-31 2010-11-24 株式会社日立ハイテクノロジーズ Fluorescence analyzer
US20090269746A1 (en) * 2008-04-25 2009-10-29 Gil Atzmon Microsequencer-whole genome sequencer
JP5143953B2 (en) 2008-06-11 2013-02-13 レーザーゲン インコーポレイテッド The method of use thereof in the nucleotide and nucleoside and dna sequencing
WO2010002939A3 (en) * 2008-06-30 2010-04-22 Life Technologies Corporation Methods for real time single molecule sequencing
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
US20100035252A1 (en) 2008-08-08 2010-02-11 Ion Torrent Systems Incorporated Methods for sequencing individual nucleic acids under tension
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
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
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
JP5717634B2 (en) * 2008-09-03 2015-05-13 ナブシス, インコーポレイテッド For voltage sensing of biomolecules and other analytes in the fluid channel, the use of nanoscale electrodes are displaced in the longitudinal direction
WO2010039189A3 (en) * 2008-09-23 2011-03-03 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
WO2010075188A3 (en) 2008-12-23 2010-11-04 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
EP2411536B1 (en) * 2009-03-27 2014-09-17 Nabsys, Inc. Methods for analyzing biomolecules and probes bound thereto
WO2011078897A1 (en) * 2009-09-15 2011-06-30 Life Technologies Corporation Improved sequencing methods, compositions, systems, kits and apparatuses
EP2427572B1 (en) * 2009-05-01 2013-08-28 Illumina, Inc. Sequencing methods
US8246799B2 (en) * 2009-05-28 2012-08-21 Nabsys, Inc. Devices and methods for analyzing biomolecules and probes bound thereto
WO2010141390A3 (en) 2009-06-05 2011-08-11 Life Technologies Corporation Nucleotide transient binding for sequencing methods
US8609421B2 (en) * 2009-06-12 2013-12-17 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
ES2573277T3 (en) 2009-08-14 2016-06-07 Epicentre Technologies Corporation Methods, compositions and kits generation impoverished samples rRNA or rRNA isolation samples
US20110059864A1 (en) * 2009-09-07 2011-03-10 Caerus Molecular Diagnostics Incorporated Sequence Determination By Use Of Opposing Forces
EP2957641B1 (en) 2009-10-15 2017-05-17 Ibis Biosciences, Inc. Multiple displacement amplification
EP2507387B1 (en) 2009-12-01 2017-01-25 Oxford Nanopore Technologies Limited Biochemical analysis instrument and method
US9315857B2 (en) 2009-12-15 2016-04-19 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse label-tags
US8835358B2 (en) 2009-12-15 2014-09-16 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
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
CN202281746U (en) * 2010-03-06 2012-06-20 伊鲁米那股份有限公司 Measuring equipment for detecting optical signal from sample as well as optical module and optical system for measuring equipment
CA2798635A1 (en) 2010-05-06 2011-11-10 Ibis Biosciences, Inc. Integrated sample preparation systems and stabilized enzyme mixtures
US8865078B2 (en) 2010-06-11 2014-10-21 Industrial Technology Research Institute Apparatus for single-molecule detection
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
US8906612B2 (en) 2010-08-25 2014-12-09 Pacific Biosciences Of California, Inc. Scaffold-based polymerase enzyme substrates
WO2012037394A1 (en) 2010-09-16 2012-03-22 Ibis Biosciences, Inc. Stabilization of ozone-labile fluorescent dyes by thiourea
CA2811333A1 (en) 2010-09-16 2012-03-22 Gen-Probe Incorporated Capture probes immobilizable via l-nucleotide tail
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
WO2012061412A1 (en) 2010-11-01 2012-05-10 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
JP5998148B2 (en) 2010-11-16 2016-09-28 ナブシス 2.0 エルエルシー The method for sequencing of biomolecules by detecting the relative position of the hybridized probe
WO2012074855A3 (en) 2010-11-22 2013-04-04 The Regents Of The University Of California Methods of identifying a cellular nascent rna transcript
CN103502463B (en) 2010-12-27 2016-03-16 艾比斯生物科学公司 The method of nucleic acid sample preparation 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
EP2670864B1 (en) 2011-01-31 2017-03-08 Illumina, Inc. Methods for reducing nucleic acid damage
WO2012109315A1 (en) * 2011-02-08 2012-08-16 Life Technologies Corporation Linking methods, compositions, systems, kits and apparatuses
WO2012109574A3 (en) 2011-02-11 2012-12-20 Nabsys, Inc. Assay methods using dna binding proteins
EP2505641B1 (en) * 2011-04-01 2015-04-15 F. Hoffmann-La Roche AG T7 RNA polymerase variants with Cysteine-Serine substitutions
EP2694709B1 (en) 2011-04-08 2016-09-14 Prognosys Biosciences, Inc. Peptide constructs and assay systems
US8778848B2 (en) 2011-06-09 2014-07-15 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
US20130040365A1 (en) 2011-08-10 2013-02-14 Life Technologies Corporation Polymerase compositions, methods of making and using same
WO2013040257A1 (en) 2011-09-13 2013-03-21 Lasergen, Inc. 5-methoxy. 3'-oh unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing
US20140235461A1 (en) 2011-09-26 2014-08-21 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
US8778849B2 (en) 2011-10-28 2014-07-15 Illumina, Inc. Microarray fabrication system and method
US8637242B2 (en) 2011-11-07 2014-01-28 Illumina, Inc. Integrated sequencing apparatuses and methods of use
EP2788499B1 (en) 2011-12-09 2016-01-13 Illumina, Inc. Expanded radix for polymeric tags
WO2013096692A1 (en) * 2011-12-21 2013-06-27 Illumina, Inc. Apparatus and methods for kinetic analysis and determination of nucleic acid sequences
WO2013096799A1 (en) 2011-12-22 2013-06-27 Ibis Biosciences, Inc. Systems and methods for isolating nucleic acids from cellular samples
EP2794927B1 (en) 2011-12-22 2017-04-12 Ibis Biosciences, Inc. Amplification primers and methods
EP3269820A1 (en) 2011-12-22 2018-01-17 Ibis Biosciences, Inc. Kit for the amplification of a sequence from a ribonucleic acid
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
US9222115B2 (en) 2011-12-30 2015-12-29 Abbott Molecular, Inc. Channels with cross-sectional thermal gradients
EP2802673A1 (en) 2012-01-09 2014-11-19 Oslo Universitetssykehus HF Methods and biomarkers for analysis of colorectal cancer
EP2809807A1 (en) 2012-02-01 2014-12-10 Gen-Probe Incorporated Asymmetric hairpin target capture oligomers
EP2820174A4 (en) * 2012-02-27 2016-01-13 Univ North Carolina Methods and uses for molecular tags
CA2864276A1 (en) 2012-03-06 2013-09-12 Illumina Cambridge Limited Improved methods of nucleic acid sequencing
US20130261984A1 (en) 2012-03-30 2013-10-03 Illumina, Inc. Methods and systems for determining fetal chromosomal abnormalities
WO2013152114A1 (en) 2012-04-03 2013-10-10 The Regents Of The University Of Michigan Biomarker associated with irritable bowel syndrome and crohn's disease
CN204832037U (en) 2012-04-03 2015-12-02 伊鲁米那股份有限公司 Detection apparatus
WO2013166302A1 (en) 2012-05-02 2013-11-07 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
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
WO2014005076A9 (en) 2012-06-29 2014-04-03 The Regents Of The University Of Michigan Methods and biomarkers for detection of kidney disorders
US20150167084A1 (en) 2012-07-03 2015-06-18 Sloan Kettering Institute For Cancer Research Quantitative Assessment of Human T-Cell Repertoire Recovery After Allogeneic Hematopoietic Stem Cell Transplantation
US9322060B2 (en) 2012-10-16 2016-04-26 Abbott Molecular, Inc. Methods and apparatus 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
US9683230B2 (en) 2013-01-09 2017-06-20 Illumina Cambridge Limited Sample preparation on a solid support
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
WO2014142850A1 (en) 2013-03-13 2014-09-18 Illumina, Inc. Methods and compositions for nucleic acid sequencing
WO2014142841A1 (en) 2013-03-13 2014-09-18 Illumina, Inc. Multilayer fluidic devices and methods for their fabrication
WO2014160117A1 (en) 2013-03-14 2014-10-02 Abbott Molecular Inc. Multiplex methylation-specific amplification systems and methods
EP2971070A1 (en) 2013-03-14 2016-01-20 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues
US9193998B2 (en) 2013-03-15 2015-11-24 Illumina, Inc. Super resolution imaging
KR20160027065A (en) 2013-07-01 2016-03-09 일루미나, 인코포레이티드 Catalyst-free surface functionalization and polymer grafting
US9193999B2 (en) 2013-07-03 2015-11-24 Illumina, Inc. Sequencing by orthogonal synthesis
CN103333680B (en) * 2013-07-16 2015-05-27 北京化工大学 Dinyl oxazole eutectic material with multi-color fluorescence characteristic and preparation method of dinyl oxazole eutectic material
KR20160040514A (en) 2013-08-08 2016-04-14 일루미나, 인코포레이티드 Fluidic system for reagent delivery to a flow cell
WO2015031691A1 (en) 2013-08-28 2015-03-05 Cellular Research, Inc. Massively parallel single cell analysis
EP3038834A1 (en) 2013-08-30 2016-07-06 Illumina, Inc. Manipulation of droplets on hydrophilic or variegated-hydrophilic surfaces
CN105745528A (en) 2013-10-07 2016-07-06 赛卢拉研究公司 Methods and systems for digitally counting features on arrays
US20150125053A1 (en) * 2013-11-01 2015-05-07 Illumina, Inc. Image analysis useful for patterned objects
EP3077943A4 (en) 2013-12-03 2017-06-28 Illumina Inc Methods and systems for analyzing image data
WO2015095226A3 (en) 2013-12-20 2015-09-03 Illumina, Inc. Preserving genomic connectivity information in fragmented genomic dna samples
WO2015100373A3 (en) 2013-12-23 2015-08-13 Illumina, Inc. Structured substrates for improving detection of light emissions and methods relating to the same
US20150197798A1 (en) 2014-01-16 2015-07-16 Illumina, Inc. Amplicon preparation and sequencing on solid supports
WO2015107430A3 (en) 2014-01-16 2015-11-26 Oslo Universitetssykehus Hf Methods and biomarkers for detection and prognosis of cervical cancer
US9677132B2 (en) 2014-01-16 2017-06-13 Illumina, Inc. Polynucleotide modification on solid support
WO2015175832A1 (en) 2014-05-16 2015-11-19 Illumina, Inc. Nucleic acid synthesis techniques
CN106536055A (en) 2014-05-27 2017-03-22 伊鲁米那股份有限公司 Systems and methods for biochemical analysis including a base instrument and a removable cartridge
EP3152320A2 (en) 2014-06-03 2017-04-12 Illumina, Inc. Compositions, systems, and methods for detecting events using tethers anchored to or adjacent to nanopores
US20150376608A1 (en) 2014-06-26 2015-12-31 IIIumina, Inc. Library preparation of tagged nucleic acid using single tube add-on protocol
JP2017518750A (en) 2014-06-27 2017-07-13 イルミナ インコーポレイテッド Modified polymerase to improve the nucleotide analog of incorporation
US9777340B2 (en) 2014-06-27 2017-10-03 Abbott Laboratories Compositions and methods for detecting human Pegivirus 2 (HPgV-2)
CN106661561A (en) 2014-06-30 2017-05-10 亿明达股份有限公司 Methods and compositions using one-sided transposition
US20160017396A1 (en) 2014-07-21 2016-01-21 Illumina, Inc. Polynucleotide enrichment using crispr-cas systems
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
EP3191606A1 (en) 2014-09-12 2017-07-19 Illumina, Inc. Compositions, systems, and methods for detecting the presence of polymer subunits using chemiluminescence
EP3201355A1 (en) 2014-09-30 2017-08-09 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues
WO2016153999A1 (en) 2015-03-25 2016-09-29 Life Technologies Corporation Modified nucleotides and uses thereof
US20160109693A1 (en) 2014-10-16 2016-04-21 Illumina, Inc. Optical scanning systems for in situ genetic analysis
CA2965578A1 (en) 2014-10-31 2016-05-06 Illumina Cambridge Limited Novel polymers and dna copolymer coatings
EP3215616A1 (en) 2014-11-05 2017-09-13 Illumina Cambridge Limited Reducing dna damage during sample preparation and sequencing using siderophore chelators
RU2582198C1 (en) * 2014-11-20 2016-04-20 Федеральное государственное бюджетное учреждение науки Лимнологический институт Сибирского отделения Российской академии наук (ЛИН СО РАН) Analogues of natural deoxyribonucleoside triphosphates and ribonucleoside triphosphates containing reporter fluorescent groups, for use in analytical bioorganic chemistry
CN104458686B (en) * 2014-12-02 2017-01-18 公安部第研究所 Fluorescence spectra acquisition method of dna wherein the molecular weight internal standard quantitative analysis
US20160177373A1 (en) 2014-12-16 2016-06-23 Life Technologies Corporation Polymerase compositions and methods of making and using same
CN107208158A (en) 2015-02-27 2017-09-26 赛卢拉研究公司 Spatially addressable molecular barcoding
CA2982146A1 (en) 2015-04-10 2016-10-13 Spatial Transcriptomics Ab Spatially distinguished, multiplex nucleic acid analysis of biological specimens
WO2016168386A1 (en) 2015-04-14 2016-10-20 Illumina, Inc. Structured substrates for improving detection of light emissions and methods relating to the same
WO2016196358A1 (en) 2015-05-29 2016-12-08 Epicentre Technologies Corporation Methods of analyzing nucleic acids
WO2017007757A1 (en) 2015-07-06 2017-01-12 Illumina, Inc. Balanced ac modulation for driving droplet operations electrodes
WO2017015018A1 (en) 2015-07-17 2017-01-26 Illumina, Inc. Polymer sheets for sequencing applications
WO2017014762A1 (en) * 2015-07-21 2017-01-26 Omniome, Inc. Nucleic acid sequencing methods and systems
WO2017019456A3 (en) 2015-07-27 2017-05-04 Illumina, Inc. Spatial mapping of nucleic acid sequence information
CA2984702A1 (en) 2015-07-30 2017-02-02 Illumina, Inc. Orthogonal deblocking of nucleotides
WO2017058810A3 (en) 2015-10-01 2017-06-22 Life Technologies Corporation Polymerase compositions and kits, and methods of using and making the same
WO2017095917A1 (en) 2015-12-01 2017-06-08 Illumina, Inc. Digital microfluidic system for single-cell isolation and characterization of analytes
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
WO2017165703A1 (en) 2016-03-24 2017-09-28 Illumina, Inc. Photonic superlattice-based devices and compositions for use in luminescent imaging, and methods of using the same
WO2017177017A1 (en) 2016-04-07 2017-10-12 Omniome, Inc. Methods of quantifying target nucleic acids and identifying sequence variants
WO2017176896A1 (en) 2016-04-07 2017-10-12 Illumina, Inc. Methods and systems for construction of normalized nucleic acid libraries
WO2017184997A1 (en) 2016-04-22 2017-10-26 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

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
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
US6982186B2 (en) * 2003-09-25 2006-01-03 Dongbuanam Semiconductor Inc. CMOS image sensor and method for manufacturing the same
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
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
US20060061754A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Arrays of optical confinements and uses thereof
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
US20070172866A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination using depolymerizing agent
US20070172858A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination
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 (en) * 1991-12-25 2001-11-12 キヤノン株式会社 An ink jet recording apparatus having a device and the device comprising a flexible cable
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
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
US5961923A (en) * 1995-04-25 1999-10-05 Irori Matrices with memories and uses thereof
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
EP2202312B1 (en) * 1997-03-12 2016-01-06 Applied Biosystems, LLC DNA polymerases having improved labelled nucleotide incorporation properties
CN1152140C (en) 1997-07-28 2004-06-02 医疗生物系统有限公司 Nucleic acid sequence analysis
CA2339121A1 (en) 1998-07-30 2000-02-10 Shankar Balasubramanian Arrayed biomolecules and their use in sequencing
US6556505B1 (en) 1998-12-15 2003-04-29 Matsushita Electric Industrial Co., Ltd. Clock phase adjustment method, and integrated circuit and design method therefor
DK1159453T3 (en) * 1999-03-10 2008-10-06 Asm Scient Inc A method of direct nucleic acid sequencing
GB9907812D0 (en) 1999-04-06 1999-06-02 Medical Biosystems Ltd Sequencing
WO2001016375A3 (en) 1999-08-30 2001-10-04 Denise Rubens High speed parallel molecular nucleic acid sequencing
EP1218543A2 (en) 1999-09-29 2002-07-03 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
US7033762B2 (en) * 2001-08-29 2006-04-25 Amersham Biosciences Corp Single nucleotide amplification and detection by polymerase
US7223541B2 (en) * 2001-08-29 2007-05-29 Ge Healthcare Bio-Sciences Corp. Terminal-phosphate-labeled nucleotides and methods of use
US7041812B2 (en) * 2001-08-29 2006-05-09 Amersham Biosciences Corp Labeled nucleoside polyphosphates
US7052839B2 (en) * 2001-08-29 2006-05-30 Amersham Biosciences Corp Terminal-phosphate-labeled nucleotides and methods of use
WO2004072304A1 (en) * 2003-02-05 2004-08-26 Amersham Biosciences 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
US7767394B2 (en) * 2005-02-09 2010-08-03 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
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
US5230781A (en) * 1984-03-29 1993-07-27 Li-Cor, Inc. Sequencing near infrared and infrared fluorescence labeled DNA for detecting using laser diodes
US5639874A (en) * 1984-03-29 1997-06-17 Li-Cor, Inc. Method for preparing fluorescent-labeled DNA
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
US6207421B1 (en) * 1984-03-29 2001-03-27 Li-Cor, Inc. DNA sequencing and DNA terminators
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
US6403311B1 (en) * 1997-02-12 2002-06-11 Us Genomics Methods of analyzing polymers using ordered label strategies
US6355420B1 (en) * 1997-02-12 2002-03-12 Us Genomics Methods and products for analyzing polymers
US7037687B2 (en) * 1998-05-01 2006-05-02 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
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
US6210896B1 (en) * 1998-08-13 2001-04-03 Us Genomics Molecular motors
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
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
US6255083B1 (en) * 1998-12-14 2001-07-03 Li-Cor Inc System and methods 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
US7033764B2 (en) * 1999-05-19 2006-04-25 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7361466B2 (en) * 1999-05-19 2008-04-22 Cornell Research Foundation, Inc. Nucleic acid analysis using terminal-phosphate-labeled nucleotides
US20030044781A1 (en) * 1999-05-19 2003-03-06 Jonas Korlach 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
US20060078937A1 (en) * 1999-05-19 2006-04-13 Jonas Korlach Sequencing nucleic acid using tagged polymerase and/or tagged nucleotide
US20050158761A1 (en) * 1999-05-19 2005-07-21 Jonas Korlach Method for sequencing nucleic acid molecules
US20060057606A1 (en) * 1999-05-19 2006-03-16 Jonas Korlach Reagents containing terminal-phosphate-labeled nucleotides for nucleic acid sequencing
US7052847B2 (en) * 1999-05-19 2006-05-30 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
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
US20050164255A1 (en) * 1999-05-19 2005-07-28 Jonas Korlach Method for sequencing nucleic acid molecules
US6911345B2 (en) * 1999-06-28 2005-06-28 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
US20020025529A1 (en) * 1999-06-28 2002-02-28 Stephen Quake 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
US20070172862A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Data stream determination
US20070172861A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Mutant polymerases
US20070172867A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination
US20070172863A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions and methods for sequence determination
US20070172865A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Sequence determination in confined regions
US20070172864A1 (en) * 2000-07-07 2007-07-26 Xiaolian Gao Composition for sequence determination
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
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
US20070172869A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: methods for inhibiting pyrophosphorolysis during sequencing synthesis
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
US20030134807A1 (en) * 2000-12-01 2003-07-17 Hardin Susan H. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
US20070172819A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: compositions including pyrophosphorolysis inhibitors
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
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
US20060061755A1 (en) * 2004-09-17 2006-03-23 Stephen Turner 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
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 (234)

* 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
US7645596B2 (en) 1998-05-01 2010-01-12 Arizona Board Of Regents 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
US9540689B2 (en) 1998-05-01 2017-01-10 Life Technologies Corporation 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
US9725764B2 (en) 1998-05-01 2017-08-08 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
US20060188900A1 (en) * 1999-05-19 2006-08-24 Jonas Korlach High speed nucleic acid sequencing
US20110111401A1 (en) * 1999-05-19 2011-05-12 Cornell University Method for sequencing nucleic acid molecules
US7056676B2 (en) 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US20070026447A1 (en) * 1999-05-19 2007-02-01 Jonas Korlach Nucleotides
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
US7056661B2 (en) 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7416844B2 (en) 1999-05-19 2008-08-26 Cornell Research Foundation, Inc. Composition for nucleic acid sequencing
US7943305B2 (en) 1999-05-19 2011-05-17 Cornell Research Foundation High speed nucleic acid sequencing
US7361466B2 (en) 1999-05-19 2008-04-22 Cornell Research Foundation, Inc. Nucleic acid analysis using terminal-phosphate-labeled nucleotides
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
US7033764B2 (en) 1999-05-19 2006-04-25 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US20080227654A1 (en) * 1999-05-19 2008-09-18 Jonas Korlach Method for sequencing nucleic acid molecules
US7943307B2 (en) 1999-05-19 2011-05-17 Cornell Research Foundation Methods for analyzing nucleic acid sequences
US7052847B2 (en) 1999-05-19 2006-05-30 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
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
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
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
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
US20070172868A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US20070292867A1 (en) * 2000-07-07 2007-12-20 Susan Hardin Sequence determination using multiply tagged polymerizing agents
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
US20070184475A1 (en) * 2000-07-07 2007-08-09 Susan Hardin Sequence determination by direct detection
US20070172861A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Mutant polymerases
US20070172863A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions and methods for sequence determination
US20070172858A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination
US20070172866A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination using depolymerizing agent
US20110184163A1 (en) * 2000-12-01 2011-07-28 Life Technologies Corporation Enzymatic Nucleic Acid Synthesis: Compositions and Methods for Inhibiting Pyrophosphorolysis
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
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
US9845500B2 (en) 2000-12-01 2017-12-19 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US8648179B2 (en) 2000-12-01 2014-02-11 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
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
US7223541B2 (en) * 2001-08-29 2007-05-29 Ge Healthcare Bio-Sciences Corp. Terminal-phosphate-labeled nucleotides and methods of use
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
US7727722B2 (en) * 2001-08-29 2010-06-01 General Electric Company Ligation amplification
US7033762B2 (en) * 2001-08-29 2006-04-25 Amersham Biosciences Corp Single nucleotide amplification and detection by polymerase
US20030077610A1 (en) * 2001-08-29 2003-04-24 John Nelson Terminal-phosphate-labeled nucleotides and methods of use
US7041812B2 (en) * 2001-08-29 2006-05-09 Amersham Biosciences Corp Labeled nucleoside polyphosphates
US7364851B2 (en) 2001-09-24 2008-04-29 Intel Corporation Nucleic acid sequencing by Raman monitoring of uptake of precursors during molecular replication
US20040248186A1 (en) * 2001-09-24 2004-12-09 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
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
WO2004072297A3 (en) * 2003-02-05 2007-01-18 Amersham Biosciences Corp Terminal-phosphate-labeled nucleotides and methods of use
JP2007524358A (en) * 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 Terminal-phosphate-labeled nucleotides and methods of use
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
US20090065471A1 (en) * 2003-02-10 2009-03-12 Faris Sadeg M Micro-nozzle, nano-nozzle, manufacturing methods therefor, applications therefor
US20090092970A1 (en) * 2003-04-08 2009-04-09 Pacific Biosciences Composition and method for nucleic acid sequencing
US7939256B2 (en) * 2003-04-08 2011-05-10 Pacific Biosciences Of California, Inc. Composition and method for nucleic acid sequencing
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
US9657344B2 (en) 2003-11-12 2017-05-23 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
US20060063264A1 (en) * 2004-09-17 2006-03-23 Stephen Turner 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
US7313308B2 (en) 2004-09-17 2007-12-25 Pacific Biosciences Of California, Inc. Optical analysis of molecules
US7302146B2 (en) 2004-09-17 2007-11-27 Pacific Biosciences Of California, Inc. Apparatus and method for analysis of molecules
US7476503B2 (en) 2004-09-17 2009-01-13 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US20060062531A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Fabrication of optical confinements
US7170050B2 (en) 2004-09-17 2007-01-30 Pacific Biosciences Of California, Inc. Apparatus and methods for optical analysis of molecules
US20060060766A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and methods for optical analysis of molecules
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US20060061754A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Arrays of optical confinements and uses thereof
US20060061755A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for analysis of molecules
US20070206189A1 (en) * 2004-09-17 2007-09-06 Stephen Turner Optical analysis of molecules
US8709725B2 (en) 2004-09-17 2014-04-29 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US9709503B2 (en) 2004-09-17 2017-07-18 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US7906284B2 (en) 2004-09-17 2011-03-15 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
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
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
US9765391B2 (en) * 2005-07-20 2017-09-19 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
US8017331B2 (en) * 2005-11-04 2011-09-13 Mannkind Corporation IRE-1α substrates
US8124343B2 (en) 2005-11-04 2012-02-28 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
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
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
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
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
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
US7668697B2 (en) 2006-02-06 2010-02-23 Andrei Volkov Method for analyzing dynamic detectable events at the single molecule level
US20070250274A1 (en) * 2006-02-06 2007-10-25 Visigen Biotechnologies, Inc. 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
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
US20080091005A1 (en) * 2006-07-20 2008-04-17 Visigen Biotechnologies, Inc. Modified nucleotides, methods for making and using same
US8426899B2 (en) 2006-12-14 2013-04-23 Life Technologies Corporation 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
US20110230375A1 (en) * 2006-12-14 2011-09-22 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale fet arrays
US8540866B2 (en) 2006-12-14 2013-09-24 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8349167B2 (en) 2006-12-14 2013-01-08 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US9039888B2 (en) 2006-12-14 2015-05-26 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8658017B2 (en) 2006-12-14 2014-02-25 Life Technologies Corporation Methods for operating an array of chemically-sensitive sensors
US20100197507A1 (en) * 2006-12-14 2010-08-05 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale fet arrays
US8415716B2 (en) 2006-12-14 2013-04-09 Life Technologies Corporation Chemically sensitive sensors with feedback circuits
US20100188073A1 (en) * 2006-12-14 2010-07-29 Ion Torrent Systems Incorporated 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
US8426898B2 (en) 2006-12-14 2013-04-23 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
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
US8435395B2 (en) 2006-12-14 2013-05-07 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
US8764969B2 (en) 2006-12-14 2014-07-01 Life Technologies Corporation Methods for operating chemically sensitive sensors with sample and hold capacitors
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
US9404920B2 (en) 2006-12-14 2016-08-02 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8441044B2 (en) 2006-12-14 2013-05-14 Life Technologies Corporation Methods for manufacturing low noise chemically-sensitive field effect transistors
US8293082B2 (en) 2006-12-14 2012-10-23 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
US8540868B2 (en) 2006-12-14 2013-09-24 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8519448B2 (en) 2006-12-14 2013-08-27 Life Technologies Corporation Chemically-sensitive array with active and reference sensors
US9269708B2 (en) 2006-12-14 2016-02-23 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
US8530941B2 (en) 2006-12-14 2013-09-10 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8269261B2 (en) 2006-12-14 2012-09-18 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8535513B2 (en) 2006-12-14 2013-09-17 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
US8264014B2 (en) 2006-12-14 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8540867B2 (en) 2006-12-14 2013-09-24 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8262900B2 (en) 2006-12-14 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US20090026082A1 (en) * 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US8558288B2 (en) 2006-12-14 2013-10-15 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US9175343B2 (en) 2007-06-06 2015-11-03 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
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
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
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
US20110281737A1 (en) * 2008-10-22 2011-11-17 Life Technologies Corporation Method and Apparatus for Rapid Nucleic Acid Sequencing
US8936763B2 (en) 2008-10-22 2015-01-20 Life Technologies Corporation Integrated sensor arrays for biological and chemical analysis
US9778188B2 (en) 2009-03-11 2017-10-03 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
US9567629B2 (en) 2009-03-27 2017-02-14 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
US9365839B2 (en) 2009-03-27 2016-06-14 Life Technologies Corporation Polymerase compositions and methods
US9695471B2 (en) 2009-03-27 2017-07-04 Life Technologies Corporation Methods and apparatus for single molecule sequencing using energy transfer detection
US8603792B2 (en) 2009-03-27 2013-12-10 Life Technologies Corporation Conjugates of biomolecules to nanoparticles
US8501405B2 (en) 2009-04-27 2013-08-06 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
US9200320B2 (en) 2009-04-27 2015-12-01 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
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
US8592154B2 (en) 2009-05-29 2013-11-26 Life Technologies Corporation Methods and apparatus for high speed operation of a chemically-sensitive sensor array
US8742469B2 (en) 2009-05-29 2014-06-03 Life Technologies Corporation Active chemically-sensitive sensors with correlated double sampling
US8776573B2 (en) 2009-05-29 2014-07-15 Life Technologies Corporation Methods and apparatus for measuring analytes
US8592153B1 (en) 2009-05-29 2013-11-26 Life Technologies Corporation Methods for manufacturing high capacitance microwell structures of chemically-sensitive sensors
US8263336B2 (en) 2009-05-29 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes
US8698212B2 (en) 2009-05-29 2014-04-15 Life Technologies Corporation Active chemically-sensitive sensors
US8822205B2 (en) 2009-05-29 2014-09-02 Life Technologies Corporation Active chemically-sensitive sensors with source follower amplifier
US8994076B2 (en) 2009-05-29 2015-03-31 Life Technologies Corporation Chemically-sensitive field effect transistor based pixel array with protection diodes
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
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
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
US9482615B2 (en) 2010-03-15 2016-11-01 Industrial Technology Research Institute Single-molecule detection system and methods
US9777321B2 (en) 2010-03-15 2017-10-03 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
US8415177B2 (en) 2010-06-30 2013-04-09 Life Technologies Corporation Two-transistor pixel array
US8421437B2 (en) 2010-06-30 2013-04-16 Life Technologies Corporation Array column integrator
US8432150B2 (en) 2010-06-30 2013-04-30 Life Technologies Corporation Methods for operating an array column integrator
US9164070B2 (en) 2010-06-30 2015-10-20 Life Technologies Corporation Column adc
US8415176B2 (en) 2010-06-30 2013-04-09 Life Technologies Corporation One-transistor pixel array
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
US8455927B2 (en) 2010-06-30 2013-06-04 Life Technologies Corporation One-transistor pixel array with cascoded column circuit
US8823380B2 (en) 2010-06-30 2014-09-02 Life Technologies Corporation Capacitive charge pump
US9239313B2 (en) 2010-06-30 2016-01-19 Life Technologies Corporation Ion-sensing charge-accumulation circuits and methods
US8217433B1 (en) 2010-06-30 2012-07-10 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
US8772698B2 (en) 2010-06-30 2014-07-08 Life Technologies Corporation CCD-based multi-transistor active pixel sensor array
US8524487B2 (en) 2010-06-30 2013-09-03 Life Technologies Corporation One-transistor pixel array with cascoded column circuit
US8432149B2 (en) 2010-06-30 2013-04-30 Life Technologies Corporation Array column integrator
US8731847B2 (en) 2010-06-30 2014-05-20 Life Technologies Corporation Array configuration and readout scheme
US8653567B2 (en) 2010-07-03 2014-02-18 Life Technologies Corporation Chemically sensitive sensor with lightly doped drains
US9567639B2 (en) 2010-08-06 2017-02-14 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US20130004950A1 (en) * 2010-08-06 2013-01-03 Tandem Diagnostics, Inc. Assay systems for genetic analysis
US9618475B2 (en) 2010-09-15 2017-04-11 Life Technologies Corporation Methods and apparatus for measuring analytes
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
US8796036B2 (en) 2010-09-24 2014-08-05 Life Technologies Corporation Method and system for delta double sampling
US8700338B2 (en) 2011-01-25 2014-04-15 Ariosa Diagnosis, Inc. Risk calculation for evaluation of fetal aneuploidy
US8756020B2 (en) 2011-01-25 2014-06-17 Ariosa Diagnostics, Inc. Enhanced risk probabilities using biomolecule estimations
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 (en) * 2011-11-04 2013-05-20 Hitachi Ltd Dna sequence decoding system, dna sequence decoding method, and program
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
US9270264B2 (en) 2012-05-29 2016-02-23 Life Technologies Corporation System for reducing noise in a chemical sensor array
US9624490B2 (en) 2012-07-19 2017-04-18 Ariosa Diagnostics, Inc. Multiplexed sequential ligation-based detection of genetic variants
US9206417B2 (en) 2012-07-19 2015-12-08 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
US9841398B2 (en) 2013-01-08 2017-12-12 Life Technologies Corporation Methods for manufacturing well structures for low-noise chemical sensors
US9835585B2 (en) 2013-03-15 2017-12-05 Life Technologies Corporation Chemical sensor with protruded sensor surface
US9671363B2 (en) 2013-03-15 2017-06-06 Life Technologies Corporation Chemical sensor with consistent sensor surface areas
US9823217B2 (en) 2013-03-15 2017-11-21 Life Technologies Corporation Chemical device with thin conductive element
US9862997B2 (en) 2013-05-24 2018-01-09 Quantapore, Inc. Nanopore-based nucleic acid analysis with mixed FRET detection
US9624537B2 (en) 2014-10-24 2017-04-18 Quantapore, Inc. Efficient optical analysis of polymers using arrays of nanostructures

Also Published As

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

Similar Documents

Publication Publication Date Title
US7777013B2 (en) Labeled nucleotide analogs and uses therefor
US6723509B2 (en) Method for 3′ end-labeling ribonucleic acids
US8133672B2 (en) Two slow-step polymerase enzyme systems and methods
US5641633A (en) Fluorescence polarization detection of nucleic acids
US6297018B1 (en) Methods and apparatus for detecting nucleic acid polymorphisms
US20060166203A1 (en) New sequencing method for sequencing rna molecules
US6268149B1 (en) Nucleic acid mediated electron transfer
US20100255487A1 (en) Methods and apparatus for single molecule sequencing using energy transfer detection
US20050260640A1 (en) Encoding and decoding reactions for determining target molecules
US6046005A (en) Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group
US20110165652A1 (en) Compositions, methods and systems for single molecule sequencing
US5800989A (en) Method for detection of nucleic acid targets by amplification and fluorescence polarization
US7973146B2 (en) Engineered fluorescent dye labeled nucleotide analogs for DNA sequencing
US6720148B1 (en) Methods and systems for identifying nucleotides by primer extension
US20030044779A1 (en) Nucleic acid typing by polymerase extension of oligonucleotides using terminator mixtures
US6297016B1 (en) Template-dependent ligation with PNA-DNA chimeric probes
Chan Advances in sequencing technology
US20030180769A1 (en) Substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene compounds for 8-color DNA sequencing
US7160997B2 (en) Methods of using FET labeled oligonucleotides that include a 3′→5′ exonuclease resistant quencher domain and compositions for practicing the same
US5804386A (en) Sets of labeled energy transfer fluorescent primers and their use in multi component analysis
US7153658B2 (en) Methods and compositions for detecting targets
US20090035777A1 (en) High throughput nucleic acid sequencing by expansion
US20060024711A1 (en) Methods for nucleic acid amplification and sequence determination
US7211414B2 (en) Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
US20050042633A1 (en) Composition and method for nucleic acid sequencing

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