Sequence determination in confined regions   

This application is a continuation of U.S. patent application Ser. No. 11/648,174, filed Dec. 29, 2006 and published as United States Patent Application Publication No. 2007/0172865 A1 on Jul. 26, 2007, which is a divisional of U.S. application Ser. No. 09/901,782, filed Jul. 9, 2001 and published as United States Patent Application Publication No. 2003/0064366 A1 on Apr. 3, 2003, which claims provisional priority to U.S. Provisional Patent Application No. 60/216,594, filed Jul. 7, 2000, all of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Background of the Invention

The present invention relates to a single-molecule sequencing apparatus and methods.

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.

2. Description of the Related Art

Overview of Conventional DNA Sequencing

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.

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.

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.

Sanger DNA Sequencing (Enzymatic Synthesis)

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 analog 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).

Fluorescent DNA Sequencing

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.

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.

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.

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.

Genome Sequencing

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.

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 (see the United States Government website nhgri.nih, the HGP sublink at 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.

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.

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 Iadarola, 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.

One of the original goals of the Human Genome Project was to complete sequence determination of the entire human genome by 2005 (see the United States Government website nhgri.nih, the HGP sublink at 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 er 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 (see the abrg.org at 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.

Single-Molecule DNA Sequencing

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 fluorescently-labeled base(s). This requirement limits the length of sequence that can be determined, and increases the number of manipulations that must be performed before any sequence data is obtained. A related approach proposes to sequentially separate single (unlabeled) nucleotides from a strand of DNA, confine them in their original order in a solid matrix, and detect the spectroscopic emission of the separated nucleotides to reconstruct DNA sequence information (Ulmer, 1997; Mitsis and Kwagh, 1999; Dapprich, 1999). This is the approach that is being developed by Praelux, Inc., a company with a goal to develop single-molecule DNA sequencing. Theoretically, this latter method should not be as susceptible to length limitations as the former enzymatic degradation method, but it does require numerous manipulations before any sequence information can be obtained.

Li-cor, Inc. is developing an enzyme synthesis based strategy for single-molecule sequencing as set forth in PCT application WO 00/36151. The Li-cor method involves multiply modifying each dNTP by attaching a fluorescent tag to the γ-phosphate and a quenching moiety to 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.

Although some single-molecular sequencing systems have been disclosed, many of them anticipate or require base modification. See, e.g., Patent Application Serial Numbers WO 01/16375 A2, WO 01/23610A2, WO 01/25480, WO 00/06770, WO 99/05315, WO00/60114, 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 may be 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

The present invention provides a polymerizing agent modified with at least one molecular or atomic tag located at or near, associated with or covalently bonded to a site on the polymerizing agent, where a detectable property of the tag undergoes a change before, during and/or after monomer incorporation. The monomers can be organic, inorganic or bio-organic monomers such as nucleotides for DNA, RNA, mixed DNA/RNA sequences, amino acids, monosaccharides, synthetic analogs of naturally occurring nucleotides, synthetic analogs of naturally occurring amino acids or synthetic analogs of naturally occurring monosaccharides, synthetic organic or inorganic monomers, or the like.

The present invention provides a depolymerizing agent modified with at least one molecular or atomic tag located at or near, associated with or covalently bonded to a site on the depolymerizing agent, where a detectable property of the tag undergoes a change before, during and/or after monomer removal. The polymers can be DNA, RNA, mixed DNA/RNA sequences containing only naturally occurring nucleotides or a mixture of naturally occurring nucleotides and synthetic analogs thereof, polypeptide sequences containing only naturally occurring amino acids or a mixture of naturally occurring amino acids and synthetic analogs thereof, polysaccharide or carbohydrate sequences containing only naturally occurring monosaccharides or a mixture of naturally occurring monosaccharides and synthetic analogs thereof, or polymers containing synthetic organic or inorganic monomers, or the like.

The present invention also provides a system that enables detecting a signal corresponding to a detectable property evidencing changes in interactions between a synthesizing/polymerizing agent or a depolymerizing agent (molecule) and its substrates (monomers or depolymerizable polymers) and decoding the signal into monomer order specific information or monomer sequence information, preferably in real-time or near real-time.

Single Site Tagged Polymerase

The present invention provides a polymerase modified with at least one molecular or atomic tag located at or near, associated with, or covalently bonded to a site on the polymerase, where a detectable property of the tag undergoes a change before, during and/or after monomer incorporation. The monomers can be nucleotides for DNA, RNA or mixed DNA/RNA monomers or synthetic analogs polymerizable by the polymerase.

The present invention provides an exonuclease modified with at least one molecular or atomic tag located at or near, associated with, or covalently bonded to a site on the exonuclease, where a detectable property of the tag undergoes a change before, during and/or after monomer release. The polymers can be DNA, RNA or mixed DNA/RNA sequences comprised of naturally occurring monomers or synthetic analogs depolymerizable by the exonuclease.

The present invention provides a polymerase modified with at least one molecular or atomic tag located at or near, associated with, or covalently bonded to a site that undergoes a conformational change before, during and/or after monomer incorporation, where the tag has a first detection propensity when the polymerase is in a first conformational state and a second detection propensity when the polymerase is in a second conformational state.

The present invention provides a polymerase modified with at least one chromophore located at or near, associated with, or covalently bonded to a site that undergoes a conformational change before, during and/or after monomer incorporation, where an intensity and/or frequency of emitted light of the chromophore has a first value when the polymerase is in a first conformational state and a second value when the polymerase is in a second conformational state.

The present invention provides a polymerase modified with at least one fluorescently active molecular tag located at or near, associated with, or covalently bonded to a site that undergoes a conformational change before, during and/or after monomer incorporation, where the tag has a first fluorescence propensity when the polymerase is in a first conformational state and a second fluorescence propensity when the polymerase is in a second conformational state.

The present invention provides a polymerase modified with a molecular tag located at or near, associated with, or covalently bonded to a site that undergoes a conformational change before, during and/or after monomer incorporation, where the tag is substantially detectable when the polymerase is in a first conformational state and substantially non-detectable when the polymerase is in a second conformational state or substantially non-detectable when the polymerase is in the first conformational state and substantially detectable when the polymerase is in the second conformational state.

The present invention provides a polymerase modified with at least one molecular or atomic tag located at or near, associated with, or covalently bonded to a site that interacts with a tag on the released pyrophosphate group, where the polymerase tag has a first detection propensity before interacting with the tag on the released pyrophosphate group and a second detection propensity when interacting with the tag on the released pyrophosphate group. In a preferred embodiment, this change in detection propensity is cyclical occurring as each pyrophosphate group is released.

The present invention provides a polymerase modified with at least one chromophore located at or near, associated with, or covalently bonded to a site that interacts with a tag on the released pyrophosphate group, where an intensity and/or frequency of light emitted by the chromophore has a first value before the chromophore interacts with the tag on the released pyrophosphate and a second value when interacting with the tag on the released pyrophosphate group. In a preferred embodiment, this change in detection propensity is cyclical occurring as each pyrophosphate group is released.

The present invention provides a polymerase modified with at least one fluorescently active molecular tag located at or near, associated with, or covalently bonded to a site that interacts with a tag on the released pyrophosphate group, where the polymerase tag changes from a first state prior to release of the pyrophosphate group and a second state as the pyrophosphate group diffuses away from the site of release. In a preferred embodiment, this change in detection propensity is cyclical occurring as each pyrophosphate group is released.

The present invention provides a polymerase modified with a molecular tag located at or near, associated with, or covalently bonded to a site that interacts with a tag on the released pyrophosphate group, where the polymerase tag changes from a substantially detectable state prior to pyrophosphate release to a substantially non-detectable state when the polymerase tag interacts with the tag on the pyrophosphate group after group release, or changes from a substantially non-detectable state prior to pyrophosphate release to a substantially detectable state when the polymerase tag interacts with the tag on the pyrophosphate group after group release.

Multiple Site Tagged Polymerizing or Depolymerizing Agents

The present invention provides a monomer polymerizing agent modified with at least one pair of molecular and/or atomic tags located at or near, associated with, or covalently bonded to sites on the polymerizing agent, where a detectable property of at least one tag of the pair undergoes a change before, during and/or after monomer incorporation or where a detectable property of at least one tag of the pair undergoes a change before, during and/or after monomer incorporation due to a change in inter-tag interaction.

The present invention provides a depolymerizing agent modified with at least one pair of molecular and/or atomic tags located at or near, associated with, or covalently bonded to sites on the depolymerizing agent, where a detectable property of at least one tag of the pair undergoes a change before, during and/or after monomer release or where a detectable property of at least one tag of the pair undergoes a change before, during and/or after monomer release due to a change in inter-tag interaction.

The present invention provides a monomer polymerizing agent modified with at least one pair of molecular and/or atomic tags located at or near, associated with, or covalently bonded to sites on the polymerizing agent, where a detectable property of at least one tag of the pair has a first value when the polymerizing agent is in a first state and a second value when the polymerizing agent is in a second state, where the polymerizing agent changes from the first state to the second state and back to the first state during a monomer incorporation cycle.

The present invention provides a depolymerizing agent modified with at least one pair of molecular and/or atomic tags located at or near, associated with or covalently bonded to sites on the polymerizing agent, where a detectable property of at least one tag of the pair has a first value when the depolymerizing agent is in a first state and a second value when the depolymerizing agent is in a second state, where the depolymerizing agent changes from the first state to the second state and back to the first state during a monomer release cycle.

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.

Multiple Site Tagged Polymerase

The present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with, or covalently bonded to sites at least one of the tags undergoes a change during monomer incorporation, where a detectable property of the pair has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state, where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.

The present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites at least one of the tags undergoes conformational change during monomer incorporation, where the detectably property of the pair has a first value when the polymerase is in a first conformational state and a second value when the polymerase is in a second conformational state, where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.

The present invention provides a polymerase modified with at least one pair of molecules or atoms located at or near, associated with or covalently bonded to sites at least one of the tags undergoes conformational change during monomer incorporation, where the pair interact to form a chromophore when the polymerase is in a first conformational state or a second conformational state, where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.

The present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites at least one of the tags undergoes conformational change during monomer incorporation, where the tags have a first fluorescence propensity when the polymerase is in a first conformational state and a second fluorescence propensity when the polymerase is in a second conformational state, where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.

The present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites at least one of the tags undergoes conformational change during monomer incorporation, where the pair is substantially active when the polymerase is in a first conformational state and substantially inactive when the polymerase is in a second conformational state or substantially inactive when the polymerase is in the first conformational state and substantially active when the polymerase is in the second conformational state, where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.

The present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with, or covalently bonded to sites at least one of the tags undergoes a change during and/or after pyrophosphate release during the monomer incorporation process, where a detectable property of the pair has a first value when the tag is in a first state prior to pyrophosphate release and a second value when the tag is in a second state during and/or after pyrophosphate release, where the tag changes from its first state to its second state and back to its first state during a monomer incorporation cycle.

The present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites at least one of the tags undergoes a change in position due to a conformational change in the polymerase during the pyrophosphate release process, where the detectably property of the pair has a first value when the tag is in its first position and a second value when the tag is in its second position, where the tag changes from its first position to its second position and back to its first position during a release cycle.

The present invention provides a polymerase modified with at least one pair of molecules or atoms located at or near, associated with or covalently bonded to sites, where the tags change relative separation due to a conformational change in the polymerase during pyrophosphate release, where the tags interact to form a chromophore having a first emission profile when the tags are a first distance apart and a second profile when the tags are a second distance apart, where the separation distance changes from its first state to its second state and back to its first state during a pyrophosphate release cycle.

The present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites, where the tags change relative separation due to a conformational change in the polymerase during pyrophosphate release, where the tags have a first fluorescence propensity when the polymerase is in a first conformational state and a second fluorescence propensity when the polymerase is in a second conformational state, where the propensity changes from its the first value to its second value and back again during a pyrophosphate release cycle.

The present invention provides a polymerase modified with at least one pair of molecular tags located at or near, associated with or covalently bonded to sites, where the tags change relative separation due to a conformational change in the polymerase during pyrophosphate release, where the pair is substantially fluorescently active when the tags have a first separation and substantially fluorescently inactive when the tags have a second separation or substantially fluorescently inactive when the tags have the first separation and substantially fluorescently active when the tags have the second separation, where the fluorescence activity undergoes one cycle during a pyrophosphate release cycle.

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

The present invention provides a method for determining when a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of an atomic or molecular tag, where the tag is located at or near, associated with, or covalently bonded to a site on a polymerizing agent, where the detectable property of the tag undergoes a change before, during and/or after monomer incorporation.

The present invention provides a method for determining when a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of an atomic or molecular tag, where the tag is located at or near, associated with, or covalently bonded to a site on a polymerizing agent, where the detectable property has a first value when the agent is in a first state and a second value when the agent is in a second state, where the agent changes from the first state to the second state and back to the first state during a monomer incorporation cycle.

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

The present invention provides a method for determining when or whether a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of a tag, where the tag is located at or near, associated with, or covalently bonded to a site on a polymerase, where the site undergoes a change during monomer incorporation and where the detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state, where the values signify that the site has undergone the change and where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.

The present invention provides a method for determining when or whether a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of a tag, where the tag is located at or near, associated with, or covalently bonded to a site on a polymerase, where the site undergoes a conformational change during monomer incorporation and where the detectable property has a first value when the polymerase is in a first conformational state and a second value when the polymerase is in a second conformational state, where the values signify that the site has undergone the change and where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.

The present invention provides a method for determining when or whether a monomer is incorporated into a growing molecular chain comprising the steps of exposing a tagged polymerase to light, monitoring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase, where the tagged polymerase comprises a polymerase including a tag located at or near, associated with, or covalently bonded to a site that undergoes conformational change during monomer incorporation and where the tag emits fluorescent light at a first intensity and/or frequency when the polymerase is in a first conformational state and a second intensity and/or frequency when the polymerase is in a second conformational state, where the change in intensities and/or frequencies signifies that the site has undergone the change and where the polymerase changes from the first state to the second state and back to the first state during a monomer incorporation cycle.

The present invention also provides the above methods using a plurality of tagged polymerases permitting parallel and/or massively parallel sequencing simultaneously. Such parallelism can be used to ensure confidence. Such parallelism can also be used to quickly detect the degree of homology in DNA sequences for a given gene across species or to quickly screen patient DNA for specific genetic traits or to quickly screen DNA sequences for polymorphisms.

The present invention also provides a method for determining if or when a monomer is incorporated into a growing DNA chain associated with a polymerase, where a tag is located on the polymerase so that as the pyrophosphate group is released after base incorporation and prior to its diffusion away from the polymerase, the polymerase tag interacts with the tag on the pyrophosphate causing a change in a detectable property of one of the tags or a detectable property associated with both tags in the case of a fluorescent pair.

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

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.

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, apolar bonding, attractive electrostatic interactions, dipole interactions, or any other electrical or quantum mechanical interaction sufficient in toto 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.

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

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.

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, apolar 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.

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

The present invention provides a method for single-molecule sequencing comprising the step of supplying a plurality of monomers to a tagged polymerizing agent confined on or tethered to a substrate and monitoring a detectable property of the tag over time. The method can also include a step of relating changes in the detectable property to the occurrence (timing) of monomer addition and/or to the identity of each incorporated monomer and/or to the near simultaneous determination of the sequence of incorporated monomers.

The present invention provides a method for single-molecule sequencing comprising the step of supplying a plurality of monomers to a tagged polymerizing agent confined on or tethered to a substrate, exposing the tagged polymerizing agent to light either continuously or periodically and measuring an intensity and/or frequency of fluorescent light emitted by the tag over time. The method can further comprise relating the changes in the measured intensity and/or frequency of emitted fluorescent light from the tag over time to the occurrence (timing) of monomer addition and/or to the identity of each incorporated monomer and/or to the near simultaneous determination of the sequence of the incorporated monomers.

The present invention provides a method for single-molecule sequencing comprising the step of supplying a plurality of monomers to a tagged polymerase confined on or tethered to a substrate and monitoring a detectable property of the tag over time. The method can also include a step of relating changes in the detectable property over time to the occurrence (timing) of monomer addition and/or to the identity of each incorporated monomer and/or to the near simultaneous determination of the sequence of the incorporated monomers.

The present invention provides a method for single-molecule sequencing comprising the step of supplying a plurality of monomers to a tagged polymerase confined on a substrate, exposing the tagged polymerase to light continuously or periodically and measuring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase over time. The method can further comprise relating changes in the measured intensity and/or frequency of emitted fluorescent light from the tag over time to the occurrence (timing) of monomer addition and/or to the identity of each incorporated monomer and/or to the near simultaneous determination of the sequence of the incorporated monomers.

Cooperatively Tagged Systems

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.

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.

The present invention provides a tagged polymerizing agent and tagged monomer precursor(s), where an intensity and/or frequency of fluorescence light emitted by at least one tag changes when the tags interact before, during and/or after monomer insertion.

The present invention provides cooperatively tagged depolymerizing agents and tagged depolymerizable polymer, where a detectable property of at least one of the tags changes when the tags interact before, during and/or after monomer release. The tag on the depolymerizing agent can be designed so that the tags interact before, during and/or after each monomer release.

The present invention provides cooperatively tagged depolymerizing agents and tagged polymers, where a detectable property of at least one of the tags changes when the tags are within a distance sufficient to cause a change in measurable change in the detectable property. If the detectable property is fluorescence induced in one tag by energy transfer to the other tag or due to one tag quenching the fluorescence of the other tag or causing a measurable change in the fluorescence intensity and/or frequency, the measurable change is caused by bringing two 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.

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

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.

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 two 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 15A 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.

The present invention provides a tagged polymerase and tagged monomer precursors, where the tags form a fluorescently active pair such as a donor-acceptor pair and an intensity and/or frequency of fluorescence light emitted by at least one tag (generally the acceptor tag in donor-acceptor pairs) changes when the tags interact.

The present invention provides a tagged polymerase and a tagged monomer precursors, where the tags form a fluorescently active pair such as a donor-acceptor pair and an intensity and/or frequency of fluorescence light emitted by at least one tag (generally the acceptor tag in donor-acceptor pairs) changes when the tags are a distance sufficient or in close proximity to change either the intensity and/or frequency of the fluorescent light. 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.

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

The present invention provides a method for single-molecule sequencing comprising the step of supplying a plurality of tagged monomers to a tagged polymerase confined on an interior surface of a container, exposing the tagged polymerase to light and measuring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase during each successive monomer addition or insertion into a growing polymer chain. The method can further comprise relating the measured intensity and/or frequency of emitted fluorescent light to incorporation events and/or to the identification of each inserted or added monomer resulting in a near real-time or real-time readout of the sequence of the a growing nucleic acid sequence—DNA sequence, RNA sequence or mixed DNA/RNA sequences.

The present invention also provides a system for retrieving stored information comprising a molecule having a sequence of known elements representing a data stream, a single-molecule sequencer comprising a polymerase having at least one tag associated therewith, an excitation source adapted to excite at least one tag on the polymerase, and a detector adapted to detect a response from the excited tag on the polymerase, where the response from the at least one tag changes during polymerization of a complementary sequence of elements and the change in response represents a content of the data stream.

The present invention also provides a system for determining sequence information from a single-molecule comprising a molecule having a sequence of known elements, a single-molecule sequencer comprising a polymerase having at least one tag associated therewith, a excitation source adapted to excite at least one tag on the polymerase, and a detector adapted to detect a response from the excited tag on the polymerase, where the response from at least one tag changes during polymerization of a complementary sequence of elements representing the element sequence of the molecule.

The present invention also provides a system for determining sequence information from a single-molecule comprising a molecule having a sequence of known elements, a single-molecule sequencer comprising a polymerase having at least one fluorescent tag associated therewith, an excitation light source adapted to excite at least one fluorescent tag on the polymerase and/or monomer and a fluorescent light detector adapted to detect at least an intensity of emitted fluorescent light from at least one fluorescent tag on the polymerase and/or monomer, where the signal intensity changes each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.

The present invention also provides a system for storing and retrieving data comprising a sequence of nucleotides or nucleotide analogs representing a given data stream; a single-molecule sequencer comprising a polymerase having at least one fluorescent tag covalently attached thereto; an excitation light source adapted to excite the at least one fluorescent tag on the polymerase and/or monomer; and a fluorescent light detector adapted to detect emitted fluorescent light from at least one fluorescent tag on the polymerase and/or monomer, where at least one fluorescent tag emits or fails to emit fluorescent light each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.

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.

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.

Single Site Tagged Polymerizing or Depolymerizing Agents

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.

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.

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.

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.

Single Site Tagged Polymerase

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.

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.

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.

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 has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state during monomer removal.

The present invention provides a composition comprising an enzyme modified to produce a detectable response prior to, during and/or after interaction with an appropriately modified monomer, where the monomers are nucleotides, nucleotide analogs, amino acids, amino acid analogs, monosaccharides, monosaccharide analogs or mixtures or combinations thereof.

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.

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.

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.

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.

Multiple Site Tagged Polymerizing and Depolymerizing Agents

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.

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.

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.

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.

Multiple Site Tagged Polymerase

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.

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.

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.

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.

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.

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.

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.

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.

Methods Using Tagged Polymerase

The present invention provides a method for determining when a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of a tag, where the tag is located at or near, associated with or covalently bonded to a site on a polymerase or associated with or covalently bonded to a site on the monomer, where the site undergoes a change during monomer incorporation and where the detectable property has a first value when the polymerase is in a first state and a second value when the polymerase is in a second state and cycles from the first value to the second value during each monomer addition.

The present invention provides a method for determining when a monomer is incorporated into a growing molecular chain comprising the steps of monitoring a detectable property of a tag, where the tag is located at or near, associated with or covalently bonded to a site on a polymerase or associated with or covalently bonded to a site on the monomer, where the site undergoes a conformational change during monomer incorporation and where the detectable property has a first value when the polymerase is in a first conformational state and a second value when the polymerase is in a second conformational state and cycles from the first value to the second value during each monomer addition.

The present invention provides a method for determining when a monomer is incorporated into a growing molecular chain comprising the steps of exposing a tagged polymerase to light, monitoring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase and/or monomer, where the tagged polymerase comprises a polymerase including a tag located at or near, associated with or covalently bonded to a site that undergoes conformational change during monomer incorporation or associated with or covalently bonded to a site on the monomer and where the tag emits fluorescent light at a first intensity and/or frequency when the polymerase is in a first conformational state and a second intensity and/or frequency when the polymerase is in a second conformational state and cycles from the first value to the second value during each monomer addition.

Single-Molecule Sequencing Apparatus Using Tagged Polymerase

The present invention provides a composition comprising a single-molecule sequencing apparatus comprising a substrate having a chamber or chip surface in which at least one tagged polymerase is confined therein and a plurality of chambers, each of which includes a specific monomer and a plurality of channels interconnecting the chambers, where each replication complex is sufficiently distant to enable data collection from each complex individually.

The present invention provides a method for single-molecule sequencing comprising the steps of supplying a plurality of monomers to a tagged polymerase confined on a substrate, exposing the tagged polymerase to light and measuring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase. The method can further comprise the step of relating the measured intensity and/or frequency of emitted fluorescent light to incorporation of a specific monomer into a growing DNA chain.

Cooperatively Tagged Monomers and Tagged Polymerizing Agent

The present invention provides a composition comprising a cooperatively tagged polymerizing agent and tagged monomers, where a detectable property of at least one of the tags changes when the tags interact.

The present invention provides a composition comprising a cooperatively tagged depolymerizing agent and tagged depolymerizable monomers, where a detectable property of at least one of the tags changes when the tags interact.

Cooperatively Tagged Monomers and Tagged Polymerase

The present invention provides a composition comprising a cooperatively tagged polymerase and tagged monomers, where a detectable property of at least one of the tags changes when the tags interact.

The present invention provides a composition comprising a cooperatively tagged polymerase and tagged monomers, where a detectable property of at least one of the tags changes when the tag are within a distance sufficient to cause a change in the intensity and/or frequency of emitted fluorescent light.

The present invention provides a composition comprising a tagged polymerase and tagged monomer precursors, where an intensity and/or frequency of fluorescence light emitted by at least one tag changes when the tags interact.

The present invention provides a composition comprising a tagged polymerase and a tagged monomer precursors, where an intensity and/or frequency of fluorescence light emitted by at least one tag changes when the tags are within a distance sufficient to cause a change in the intensity and/or frequency of emitted fluorescent light.

The present invention provides a single-molecule sequencing apparatus comprising a container having at least one tagged polymerase confined on an interior surface thereof and having a solution containing a plurality of tagged monomers in contact with the interior surface or a subset of tagged monomers and a subset of untagged monomers which together provide all monomers precursor for polymerization.

The present invention provides a method for single-molecule sequencing comprising the steps of supplying a plurality of tagged monomers to a tagged polymerase confined on an interior surface of a container, exposing the tagged polymerase to light and measuring an intensity and/or frequency of fluorescent light emitted by the tagged polymerase. The method can further comprise relating the measured intensity and/or frequency of emitted fluorescent light to incorporation of a specific monomer into a growing DNA chain.

The present invention provides a system for retrieving stored information comprising: (a) a molecule having a sequence of elements representing a data stream; (b) a single-molecule sequencer comprising a polymerase having at least one tag associated therewith; (c) an excitation source adapted to excite the at least one tag on the polymerase; and (d) a detector adapted to detect a response from the tag on the polymerase or on the monomers; where the response from at least one tag changes during polymerization of a complementary sequence of elements and the change in response represents a data stream content.

The present invention provides a system for determining sequence information from a single-molecule comprising: (a) a molecule having a sequence of elements; (b) a single-molecule sequencer comprising a polymerase having at least one tag associated therewith; (c) an excitation source adapted to excite at least one tag on the polymerase or on the monomers; and (d) a detector adapted to detect a response from the tag on the polymerase; where the response from at least one tag changes during polymerization of a complementary sequence of elements representing the element sequence of the molecule.

The present invention provides a system for determining sequence information from an individual molecule comprising: (a) a molecule having a sequence of elements; (b) a single-molecule sequencer comprising a polymerase having at least one fluorescent tag associated therewith; (c) an excitation light source adapted to excite the at least one fluorescent tag on the polymerase or on the monomers; and (d) a fluorescent light detector adapted to detect at least an intensity of emitted fluorescent light from the at least one fluorescent tag on the polymerase; where the intensity change of at least one fluorescent tag emits or fails to emit fluorescent light each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.

The present invention provides a system for storing and retrieving data comprising: (a) a sequence of nucleotides or nucleotide analogs representing a given data stream; (b) a single-molecule sequencer comprising a polymerase having at least one fluorescent tag covalently attached thereto; (c) an excitation light source adapted to excite at least one fluorescent tag on the polymerase; and (d) a fluorescent light detector adapted to detect emitted fluorescent light from at least one fluorescent tag on the polymerase; where at least one fluorescent tag emits or fails to emit fluorescent light each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.

The present invention provides a system for storing and retrieving data comprising: (a) a sequence of nucleotides or nucleotide analogs representing a given data stream; (b) a single-molecule sequencer comprising a polymerase having at least one fluorescent tag covalently attached thereto; (c) an excitation light source adapted to excite the at least one fluorescent tag on the polymerase or the monomers; and (d) a fluorescent light detector adapted to detect emitted fluorescent light from at least one fluorescent tag on the polymerase or the monomers; where at least one fluorescent tag emits or fails to emit fluorescent light each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.

The present invention provides a method for sequencing a molecular sequence comprising the steps of: (a) a sequenced of nucleotides or nucleotide analogs representing a given data stream; (b) a single-molecule sequencer comprising a polymerase having at least one fluorescent tag covalently attached thereto; (c) an excitation light source adapted to excite at least one fluorescent tag on the polymerase or the monomers; and (d) a fluorescent light detector adapted to detect emitted fluorescent light from at least one fluorescent tag on the polymerase; where at least one fluorescent tag emits or fails to emit fluorescent light each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.

The present invention provides a method for synthesizing a 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.

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.

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.

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:

FIG. 1 depicts FRET activity as a function of distance separating the fluorescent donor and acceptor;

FIG. 2 depicts the open and closed ternary complex forms of the large fragment of Taq DNA pol I (Klentaq 1);

FIGS. 3A-C depicts an overlay between 3ktq (closed ‘black’) and 1tau (‘open light blue’), the large fragment of Taq DNA polymerase I;

FIG. 4 depicts an image of a 20% denaturing polyacrylamide gel containing size separated radiolabeled products from DNA extension experiments involving γ-ANS-phosphate-dATP;

FIG. 5 depicts an image of (A) the actual gel, (B) a lightened phosphorimage and (C) an enhanced phosphorimage of products generated in DNA extension reactions using γ-ANS-phosphate-dNTPs;

FIG. 6 depicts an image of (A) 6% denaturing polyacrylamide gel, (B) a lightened phosphorimage of the actual gel, and (C) an enhanced phosphorimage of the actual gel containing products generated in DNA extension reactions using γ-ANS-phosphate-dNTPs;

FIG. 7 depicts an image of (A) the actual gel, (B) a lightened phosphorimage of the actual gel, and (C) an enhanced phosphorimage of the actual gel;

FIG. 8 depicts data for the Klenow fragment from E. coli DNA polymerase I incorporation of gamma-modified nucleotides;

FIG. 9 depicts data for the Pfu DNA polymerase incorporation of gamma-modified nucleotides;

FIG. 10 depicts data for the HIV-1 reverse transcriptase incorporation of gamma-tagged nucleotides;

FIG. 11 depicts experimental results for the native T7 DNA polymerase and SEQUENASE® incorporation of gamma-tagged nucleotides; and

FIG. 12 depicts reaction products produced when the four natural nucleotides (dATP, dCTP, dGTP and dTTP) are used in the synthesis reaction (solid line) and reaction products produced when base-modified nucleotides are used in the synthesis reaction.

DETAILED DESCRIPTION

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.

The pattern of emission signals is collected, either directly, such as by an Intensified 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.

The single-molecule DNA sequencing systems of this invention have the potential to replace current DNA sequencing technologies, because the methodology can decrease time, labor, and costs associated with the sequencing process, and can lead to highly scalable sequencing systems, improving the DNA sequence discovery process by at least one to two orders of magnitude per reaction.

The single-molecule DNA sequencing technology of this invention can: (1) make it easier to classify an organism or identify variations within an organism by simply sequencing the genome or a portion thereof; (2) make rapid identification of a pathogen or a genetically-modified pathogen easier, especially in extreme circumstances such as in pathogens used in warfare; and (3) make rapid identification of persons for either law enforcement and military applications easier.

One embodiment of the single-molecule sequencing technology of this invention involves strategically positioning a pair of tags on a DNA polymerase so that as a dNTP is incorporated during the polymerization reaction, the tags change relative separation. This relative change causes a change in a detectable property, such as the intensity and/or frequency of fluorescence from one or both of the tags. A time profile of these changes in the detectable property evidences each monomer incorporation event and provides evidence about which particular dNTP is being incorporated at each incorporation event. The pair of tags do not have to be covalently attached to the polymerase, but can be attached to molecules that associate with the polymerase in such a way that the relative separation of the tags change during base incorporation.

Another embodiment of the single-molecule sequencing technology of this invention involves a single tag strategically positioned on a DNA polymerase that interacts with a tag on a dNTP or separate tags on each dNTP. The tags could be different for each dNTP such as color-coded tags which emit a different color of fluorescent light. 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.

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.

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.

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.

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.

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

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.

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.

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.

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

Suitable polymerizing agents for use in this invention include, without limitation, any polymerizing agent that polymerizes monomers relative to a specific template such as a DNA or RNA polymerase, reverse transcriptase, or the like or that polymerizes monomers in a step-wise fashion.

Suitable polymerases for use in this invention include, without limitation, any polymerase that can be isolated from its host in sufficient amounts for purification and use and/or genetically engineered into other organisms for expression, isolation and purification in amounts sufficient for use in this invention such as DNA or RNA polymerases that polymerize DNA, RNA or mixed sequences, into extended nucleic acid polymers. Preferred polymerases for use in this invention include mutants or mutated variants of native polymerases where the mutants have one or more amino acids replaced by amino acids amenable to attaching an atomic or molecular tag, which have a detectable property. Exemplary DNA polymerases include, without limitation, HIV1-Reverse Transcriptase using either RNA or DNA templates, DNA pol I from T. aquaticus or E. coli, Bateriophage T4 DNA pol, T7 DNA pol or the like. Exemplary RNA polymerases include, without limitation, T7 RNA polymerase or the like.

Suitable depolymerizing agents for use in this invention include, without limitation, any depolymerizing agent that depolymerizes monomers in a step-wise fashion such as exonucleases in the case of DNA, RNA or mixed DNA/RNA polymers, proteases in the case of polypeptides and enzymes or enzyme systems that sequentially depolymerize polysaccharides.

Suitable monomers for use in this invention include, without limitation, any monomer that can be step-wise polymerized into a polymer using a polymerizing agent. Suitable nucleotides for use in this invention include, without limitation, naturally occurring nucleotides, synthetic analogs thereof, analog having atomic and/or molecular tags attached thereto, or mixtures or combinations thereof.

Suitable atomic tag for use in this invention include, without limitation, any atomic element amenable to attachment to a specific site in a polymerizing agent or dNTP, especially Europium shift agents, nmr active atoms or the like.

Suitable atomic tag for use in this invention include, without limitation, any atomic element amenable to attachment to a specific site in a polymerizing agent or dNTP, especially fluorescent dyes such as d-Rhodamine acceptor dyes including dichloro[R110], dichloro[R6G], dichloro[TAMRA], dichloro[ROX] or the like, fluorescein donor dye including fluorescein, 6-FAM, or the like; Acridine including Acridine orange, Acridine yellow, Proflavin, pH 7, or the like; Aromatic Hydrocarbon including 2-Methylbenzoxazole, Ethyl p-dimethylaminobenzoate, Phenol, Pyrrole, benzene, toluene, or the like; Arylmethine Dyes including Auramine O, Crystal violet, H2O, Crystal violet, glycerol, Malachite Green or the like; Coumarin dyes including 7-Methoxycoumarin-4-acetic 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-diphenylbutadiene, 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-FB1 (5′-FAM/3′-BHQ-1) DLO-TEB1 (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-g), DLO-TAB2 (5′-TAMRA/3′-BHQ-g), DLO-RB2 (5′-ROX/3′-BHQ-g), 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-g), MBO-TAB2 (5′-TAMRA/3′-BHQ-g), MBO-RB2 (5′-ROX/3′-BHQ-g); 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

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.

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.

Crystal Structures are Available for Taq DNA Polymerase

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.

Taq DNA Polymerase is Efficiently Expressed in E. Coli

The Taq DNA polymerase is efficiently expressed in E. coli allowing efficient production and purification of the nascent polymerase and variants thereof for rapid identification, characterization and optimization of an engineered Taq DNA polymerase for use in the single-molecule DNA sequencing systems of this invention.

No Cysteines are Present in the Protein Sequence

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.

The Processivity of the Enzyme can be Modified

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 H1 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.

Taq DNA Polymerase Possesses a 5′ to 3′ Exonuclease Activity and is Thermostable

Single-stranded M13 DNA and synthetic oligonucleotides are used in the initial studies. After polymerase activity is optimized, the sequencing system can be used to directly determine sequence information from an isolated chromosome—a double-stranded DNA molecule. Generally, heating a sample of double-stranded DNA is sufficient to produce or maintain the double-stranded DNA in stranded DNA form for sequencing.

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 renature downstream from the replication site using a nick-translation reaction mechanism. Synthesis from the engineered polymerase is initiated either by a synthetic oligonucleotide primer (if a specific reaction start is necessary) or by a nick in the DNA molecule (if multiple reactions are processed) to determine the sequence of an entire DNA molecule.

The Polymerase is Free from 3′ to 5′ Exonuclease Activity

The Taq DNA polymerase is does not contain 3′ to 5′ exonuclease activity, which means that the polymerase cannot replace a base, for which fluorescent signal was detected, with another base which would produce another signature fluorescent signal.

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.

The error rate of engineered polymerases of this invention is 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 may be 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.

Taq DNA Polymerase is the Enzyme of Choice for Single-Molecule DNA Sequencing

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_-tagged nucleotide conforms to Watson-Crick base pairing rules.

Detecting Tagged Polymerase-Nucleotide Interactions

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 equation (1) as follows:

E=1/(1+[R/R0]6)  (1)

where R0 is the Forster critical distance at E=0.5. R0 is calculated from equation (2):

R0=(9.79×103)(κ2η−4QDJDA)1/6  (2)

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):

JDA=[∫FD(λ)εA(λ)λ4dλ]/[∫FD(λ)dλ]  (3)

where FD is the donor emission, εA is the acceptor absorption. QD is calculated from equation (4):

QD=QRF(ID/IRF)(ARF/AD)  (4)