WO2011078897A1

WO2011078897A1 - Improved sequencing methods, compositions, systems, kits and apparatuses

Info

Publication number: WO2011078897A1
Application number: PCT/US2010/050406
Authority: WO
Inventors: Joseph Beechem; Michael Previte; Rigo Pantoja; Tommie Llyod Lincecum; Vladimir I. Bashkirov; Charles W. Tweedy; Xinzhan Peng; Anelia Kraltcheva; Kirk M. Hirano
Original assignee: Life Technologies Corporation
Priority date: 2009-09-15
Filing date: 2010-09-27
Publication date: 2011-06-30

Abstract

Disclosed herein are sequencing methods, compositions, systems and apparatuses for sequencing of polymer templates, wherein a first portion of the template can be sequenced using a first set of reagents (e.g., polymerases, nucleotides and/pr polymerization initiation sites) and a second portion of the template can be sequenced using a second set of reagents (e.g., polymerases, nucleotides and/pr polymerization initiation sites). In some embodiments, the first and second portions of the template are non-overlapping; for example, the first and second portions are contiguous. In some embodiments, the first and second portions of the template overlap partly or completely. In some embodiments, different polymerases are used to sequence each of the two portions. In some embodiments, different nucleotide combinations are used to sequence each of the two portions. In some embodiments, different polymerization initiation sites are used to sequence each of the two portions.

Description

IMPROVED SEQUENCING METHODS, COMPOSITIONS, SYSTEMS,

KITS AND APPARATUSES

[0001] This application is a continuation-in-part of PCT/US2010/028952, filed on March 26, 2010, and is also a continuation-in-part of U.S. Application No. 12/748,168, filed on March 26, 2010, both of which applications claim the benefit of U.S. Provisional Application Nos.:

61/164,324, filed on March 27, 2009; 61/184,770, filed on June 5, 2009; 61/242,771, filed on September 15, 2009; 61/245,457, filed on September 24, 2009; 61/263,974, filed on November 24, 2009; 61/289,388; filed on December 22, 2009; 61/293,618, filed on January 8, 2010; 61/293,616, filed on January 8, 2010; 61/299,919, filed on January 29, 2010; 61/299,917, filed on January 29, 2010; 61/307,356, filed on February 23, 2010. All of the aforementioned patent applications are incorporated by reference in their entireties.

BACKGROUND

[0002] Nucleic acid sequencing is the process of obtaining information concerning the order and identity of the individual nucleotides within a target nucleic acid molecule of interest. Obtaining such sequence information is an important starting point for medical and academic research endeavors. Such information facilitates medical studies of active disease and genetic disease predispositions, and assists in rational design of drugs targeting specific diseases. Sequence information is also the basis for genomic and evolutionary studies, and many genetic engineering applications. Reliable sequence information is critical for paternity tests, criminal investigations, and forensic studies.

[0003] A variety of different nucleic acid sequencing methods are known in the art. Many of these methods exploit the ability of a sequencing enzyme, for example a polymerase, exonuclease or helicase, to replicate a target nucleic acid molecule of interest by synthesizing a nascent nucleic acid molecule via template-dependent nucleotide incorporation. During such nucleic acid synthesis, sequence- specific signals indicating one or more template-dependent nucleotide incorporations are detected and can be analyzed to determine the identity of one or more incorporated nucleotides. In conventional population-based sequencing methods, nucleic acid sequence information is typically obtained from a population of identical nucleic acid molecules. Such methods can include use of chain termination and/or size separation procedures, such as those described by Sanger, et al., (1977 Proc. Nat. Acad. Sci. USA 74:5463-5467). Prior to gel separation, the nucleic acid target molecules of interest are cloned, amplified, and isolated.

Sequencing reactions are normally conducted in four separate reaction vessels, one for each nucleotide: A, G, C and T. These sequencing methods are adequate for read lengths of 500-10000 nucleotides. However, they are time-consuming and require relatively large amounts of target molecules. Additionally, these methods can be expensive, especially with respect to methods requiring reagents for four reaction vessels. Any amplification steps are error-prone, which can jeopardize acquiring reliable sequence information. Furthermore, these methods suffer from sequence-dependent artifacts including band compression during size separation. Technological advances in automated sequencing machines, fluorescently-labeled nucleotides, and detector systems permit massively parallel sequencing runs for high throughput methods. But these procedures typically take on the order of days for large projects such as sequencing the human genome, which contains approximately three billion bases of DNA sequence.

[0004] Recently, a host of technologies have been developed to permit sequencing with increased speed and throughput. These new technologies include methods for single molecule sequencing, wherein sequence information is obtained from a single nucleic acid template as opposed to a bulk population. The single nucleic acid template can be sequenced in isolation; alternatively, the single nucleic acid template can be sequenced in parallel with other single nucleic acid templates in a multiplexed arrangement. In many single molecule techniques, incorporation of labeled nucleotides into an extending nucleic acid molecule is detected and analyzed in real time. For example, a labeled polymerase can be used to perform template- dependent nucleic acid synthesis using labeled nucleotides, resulting in the generation of a sequence-specific signal during a template-dependent nucleotide incorporation as the labeled polymerase undergoes FRET with an incorporating labeled nucleotide; the resulting FRET signal can be detected and analyzed to determine the identity of the incorporating nucleotide. In another example, labeled nucleotide incorporation can be monitored using an optical waveguide structure, such as a zero mode waveguide. The operating principles of some exemplary single molecule sequencing methods are discussed, for example, in U.S. Pat. Nos. 6,210,896, issued April 3, 2001 to Chan; 6,982,146, issued Jan. 3, 2006 to Schneider; 7,056,661, issued June 6, 2006 to Korlach et al.; and 7,329,492, issued February 12, 2008 to Hardin et al.

[0005] All sequencing methods involving polymerase-based nucleic acid synthesis can be complicated by various aspects of polymerase behavior. For example, the length of the average sequencing "read" within a given system is typically limited by various factors such as the polymerase processivity, duration of polymerase detectability and/or active lifetime of the polymerase. Similarly, the accuracy of any polymerase-based sequencing system can be limited by factors such as the intrinsic error rate of nucleic acid synthesis by a given polymerase. It is therefore desirable to develop nucleic acid sequencing methods, compositions, systems, kits and apparatuses that will permit sequencing the human genome (or the genome of any organism) in a relatively short time span and at a reduced cost, along with increased accuracies and/or read lengths as compared to conventional sequencing methods.

SUMMARY

[0006] In some embodiments the disclosure provides methods and related compositions, kits, systems and apparatuses useful for sequencing two or more portions of a template strand of a target nucleic acid molecule, optionally using different polymerases to sequence each portion.

[0007] In some embodiments, the disclosure relates generally to methods, (as well as related compositions, systems and apparatuses) for obtaining sequence information from a target nucleic acid molecule, comprising: providing a target nucleic acid molecule including a template strand; sequencing a first portion of the template strand using a first polymerase; and sequencing a second portion of the template strand using a second polymerase.

[0008] Optionally, the first and second portions are contiguous with each other. In some embodiments, the first and second portions overlap with each other. The first and second portions can overlap partially or completely. When the first and second portions overlap completely, they share the same nucleic acid sequence.

[0009] In some embodiments, sequencing the first portion includes: synthesizing a first nascent nucleic acid molecule by contacting the template strand with the first polymerase in the presence of nucleotides. Optionally, the contacting is performed under conditions where the first polymerase binds to the template strand. The first polymerase can optionally catalyze one or more template-dependent nucleotide incorporations. In some embodiments, sequencing the first portion further includes detecting a sequence-specific signal indicating a template-dependent nucleotide incorporation by the first polymerase. The detecting can optionally be performed during the synthesizing.

[0010] In some embodiments, sequencing the second portion includes: synthesizing a second nascent nucleic acid molecule by contacting the template strand with the second polymerase in the presence of nucleotides. Optionally, the contacting is performed under conditions where the second polymerase binds to the template strand. The second polymerase can optionally catalyze one or more template-dependent nucleotide incorporations. In some embodiments, sequencing the second portion further includes detecting a sequence-specific signal indicating a template- dependent nucleotide incorporation by the second polymerase. The detecting can optionally be performed during the synthesizing.

[0011] In some embodiments, the target nucleic acid molecule is linked to a solid or semi-solid substrate.

[0012] In some embodiments, the second polymerase synthesizes the second nascent nucleic acid molecule by extending the first nascent nucleic acid molecule synthesized by the first polymerase. Optionally, the second polymerase continues the extension of the first nascent nucleic acid molecule.

[0013] In some embodiments, the disclosed methods (as well as the related compositions, systems and apparatuses) can further include exchanging the first polymerase with the second polymerase following sequencing the first portion and prior to sequencing the second portion. The exchanging can optionally further include removing the first polymerase from the template strand after sequencing the first portion and prior to sequencing the second portion using the second polymerase. Optionally, the exchanging can further include binding the second polymerase to the template strand. The removing can be performed in a manner such that where the first nascent nucleic acid molecule can be extended by the second polymerase after the removing.

[0014] In some embodiments, the first nascent nucleic acid molecule hybridizes to the template strand to form a synthesized nucleic acid duplex.

[0015] In some embodiments, the disclosed methods (as well as the related compositions, systems and apparatuses) can further include removing the first polymerase from the template strand after sequencing the first portion. The removing can be performed without completely denaturing the synthesized nucleic acid duplex.

[0016] Optionally, none of the labeled nucleotides includes a blocking group.

[0017] In some embodiments, at least one of the first and second polymerases includes a polymerase label, at least one nucleotide includes a nucleotide label, and the sequence specific signal includes a FRET signal between a polymerase label and a nucleotide label.

[0018] In some embodiments, the first nascent nucleic acid molecule hybridizes to the template strand to form a synthesized nucleic acid duplex. [0019] Optionally, the disclosed methods (and related compositions, systems and apparatuses) further include denaturing the synthesized nucleic acid duplex after sequencing the first portion and prior to sequencing the second portion.

[0020] Optionally, the first portion includes at least a third portion and a fourth portion, the third portion and fourth portion being contiguous with each other, and sequencing the first portion further includes: sequencing the third portion using a third polymerase, removing the third polymerase from the template strand, and sequencing the fourth portion using a fourth polymerase.

[0021] In some embodiments, sequencing the third portion further includes synthesizing a third nascent nucleic acid molecule by contacting the template strand with the third polymerase in the presence of nucleotides under conditions where the third polymerase binds to the template strand and catalyzes one or more template-dependent nucleotide incorporations; and detecting, during the synthesizing, a sequence- specific signal indicating a template-dependent nucleotide incorporation catalyzed by the third polymerase.

[0022] In some embodiments, sequencing the fourth portion further includes synthesizing a fourth nascent nucleic acid molecule by contacting the template strand with the fourth polymerase in the presence of nucleotides under conditions where the fourth polymerase binds to the template strand and catalyzes one or more template-dependent nucleotide incorporations; and detecting, during the synthesizing, a sequence-specific signal indicating a template-dependent nucleotide incorporation catalyzed by the fourth polymerase.

[0023] Optionally, the removing of the third polymerase includes exchanging the third polymerase for the fourth polymerase. The fourth polymerase can synthesize the fourth nascent nucleic acid molecule by extending the third nascent nucleic acid molecule.

[0024] In some embodiments, the first, second, third and fourth polymerases are different. The different polymerases can be derived from the same organism and can have the same structure and/or amino acid sequence.

[0025] In some embodiments, at least one polymerase includes a polymerase label, at least one nucleotide includes a nucleotide label, and the sequence specific signal includes a FRET signal between a polymerase label and a nucleotide label.

[0026] In some embodiments, the disclosure relates generally to methods (as well as related compositions, systems and apparatuses) for obtaining sequence information from a target nucleic acid molecule, comprising: providing a target nucleic acid molecule including a template strand; sequencing a portion of the template strand using a first polymerase, wherein the sequencing includes synthesizing a nascent nucleic acid molecule via template-dependent nucleotide incorporation and forming a synthesized nucleic acid duplex through hybridization of the nascent nucleic acid molecule and the template strand and denaturing the synthesized nucleic acid duplex, and resequencing at least some of the portion of the template strand using another polymerase.

[0027] In some embodiments, the disclosed methods (and related compositions, systems and apparatuses) further include repeating the denaturing and resequencing at least once.

[0028] In some embodiments, the denaturing includes contacting the synthesized nucleic acid duplex with a denaturing agent. The denaturing agent can optionally be alkali (e.g., NaOH, KOH and the like) or heat.

[0029] In some embodiments, the portion of the template strand includes at least a first subportion and a second subportion that are contiguous with each other, and sequencing the portion includes: sequencing the first subportion using a first polymerase, removing the first polymerase from the template strand, and sequencing the second subportion of the nucleic acid template using a second polymerase.

[0030] In some embodiments, sequencing the first subportion includes contacting the template strand with the first polymerase and a plurality of labeled nucleotides under conditions where the polymerase binds to the template strand and polymerizes one or more labeled nucleotides in a template-dependent fashion to form a synthesized nucleic acid duplex, and identifying, during the polymerizing, at least one labeled nucleotide polymerized by the first polymerase.

[0031] In some embodiments, sequencing the second subportion includes contacting the template strand with the first polymerase and a plurality of labeled nucleotides under conditions where the polymerase binds to the template strand and polymerizes one or more labeled nucleotides in a template-dependent fashion to form a synthesized nucleic acid duplex, and identifying, during the polymerizing, at least one labeled nucleotide polymerized by the first polymerase.

[0032] In some embodiments, the method further includes removing the first polymerase from the template strand after sequencing the first subportion and prior to sequencing the second subportion.

[0033] In some embodiments, the removing includes contacting the first polymerase with a removing agent. The removing agent can include chaotropic salts. In some embodiments, the removing agent includes guanidine hydrochloride and/or sodium hydroxide. [0034] In some embodiments, the nascent nucleic acid molecule synthesized by the first polymerase hybridizes to the template strand to form a synthesized nucleic acid duplex, which is further extended by the second polymerase after the removing.

[0035] In some embodiments, none of the labeled nucleotides includes a blocking group.

[0036] In some embodiments, at least one polymerase includes a polymerase label, at least one nucleotide includes a nucleotide label, and the sequence specific signal includes a FRET signal between a polymerase label and a nucleotide label.

[0037] In some embodiments, the target nucleic acid molecule is linked to a solid or semi-solid substrate.

[0038] In some embodiments, sequence information is obtained from two or more target nucleic acid molecules in parallel by simultaneously sequencing a plurality of target nucleic acid molecules in parallel according to any one or more of the methods of the disclosure.

DESCRIPTIONS OF THE DRAWINGS

[0039] FIGURE 1 depicts one exemplary embodiment of the reagent exchange-based sequencing methods of the disclosure, in which two or more overlapping portions of a single immobilized template are sequenced using different polymerases to sequence each portion. The direction of sequencing is away from the surface on which the template is immobilized.

[0040] FIGURE 2 depicts another exemplary embodiment of the reagent exchange-based sequencing methods of the disclosure, in which two or more overlapping portions of a single immobilized template are sequenced using different polymerases to sequence each portion. The template is first tailed using a terminal transferase enzyme and then ligated to an immobilized capture molecule using a splinter oligonucleotide.

[0041] FIGURE 3 depicts another exemplary embodiment of the reagent exchange-based sequencing methods of the disclosure, in which two or more overlapping portions of a single immobilized template strand of a target molecule are sequenced using different polymerases to sequence each portion. The template strand is ligated to an immobilized hairpin capture molecule and then sequenced.

[0042] FIGURE 4 depicts another exemplary embodiment of the reagent exchange-based sequencing methods of the disclosure, in which two or more overlapping portions of a single immobilized template strand of a target molecule are sequenced using different polymerases to sequence each portion. FIGURE 4A depicts the ligation of the template to an adaptor, followed by tailing of the template. FIGURE 4B depicts the capture of the tailed template using an immobilized oligonucleotide and the subsequent sequencing reaction.

[0043] FIGURE 5 depicts another exemplary embodiment of the reagent exchange-based sequencing methods of the disclosure, in which two or more overlapping portions of a single immobilized template are sequenced using different polymerases to sequence each portion.

FIGURE 5A depicts tailing of the template, which is then captured by an immobilized

oligonucleotide. FIGURE 5B depicts the stages in the subsequent sequencing reaction.

[0044] FIGURE 6 depicts another exemplary embodiment of the reagent exchange-based sequencing methods of the disclosure, in which two or more overlapping portions of a single circularized template are sequenced.

[0045] FIGURE 7 depicts an exemplary embodiment wherein stem-loop adaptor molecules are ligated to both ends of a double- stranded target molecule using T4 ligase.

[0046] FIGURE 8 depicts an exemplary embodiment of nucleic acid sequencing involving polymerase exchange, wherein contiguous portions of a nucleic acid template strand are sequenced using different polymerases according to the procedure of Example 8.

[0047] FIGURE 9 depicts results from exemplary mapping assays showing the mapping of quantum dot-labeled polymerases (referred to as "conjugates") to a labeled template-primer duplex over 6 successive rounds of polymerase exchange according to the procedure of Example

8. Left: schematic of the experimental setup; Right: Bar graphs depicting the resulting mapping data.

[0048] FIGURE 10 depicts the results from exemplary polymerase exchange assays conducted according to the procedure of Example 8. Figure 10A depicts the observed conjugate removal efficiency (i.e., efficiency of removal of labeled polymerases) using different wash buffers comprising different removing agents. FIGURE 10B depicts conjugate mapping profiles to labeled duplexes after the first binding/first conjugate removal wash with the removing agent guanidine hydrochloride (GndHCl), and second round of conjugate binding. The top panel depicts the scheme for the experimental design and data collection; the bottom panel depicts average images of the donor and acceptor channels along with the % mapped duplexes for each; FIGURE IOC depicts a histogram showing the percentage of labeled polymerase conjugates mapped to labeled duplexes (Y axis) after each successive wash.

[0049] FIGURE 11 depicts the effect of the removing agent Guanidine Hydrochloride (GndHCl) on the distribution of a labeled duplex after successive washes with buffer including 8M GndHCl according to the procedure of Example 8. FIGURE 11A depicts a LIP plot showing the number of Tuples (Y axis) for each wash (X) axis, each bright or detected duplex tuple is represented by a black bar and a white bar represents a dark or photo-bleached or washed duplex; FIGURE 1 IB depicts average images of the acceptor channel (duplex) representative results from an exemplary field of view (FOV).

[0050] FIGURE 12 depicts exemplary assays comparing the observed sequencing reaction efficiency and read length with and without treatment with the removing agent Guanidine

Hydrochloride (GndHCl) according to the procedure of Example 8.

[0051] FIGURE 13 depicts an exemplary Venn Diagram showing the total number of mapped donor-labeled polymerases ("Donors mapped in Rl"), the number of mapped donor-labeled polymerases that yielded decipherable leader sequence ("Mapped Donors with leader sequence") during the first cycle of polymerase exchange, and the number of mapped donors that yielded decipherable sequence in the second and third cycles of polymerase exchange, in an assay according to the procedure of Example 8.

[0052] FIGURE 14 depicts a histogram plotting the average length of the sequencing "read" (Y axis) in assays using a single polymerase (left bar) and assays involving three successive polymerase exchanges (right bar) in an assay according to the procedure of Example 8.

[0053] FIGURE 15 depicts exemplary embodiments of a sequencing method involving exchange of polymerization initiation sites according to the disclosure. FIGURE 15 A depicts an

embodiment where multiple target nucleic acid molecules are isolated and sequencing in parallel using polymerization initiation sites comprising nicks; FIGURE 15B depicts the various stages of a sequencing method involving nick exchange.

[0054] FIGURE 16 depicts one the configuration of one exemplary flow cell cartridge that can be used in sequencing apparatuses according to the disclosure.

[0055] FIGURE 17 depicts a plot showing the relationships between the pressure drop, flow rate, and channel height for an exemplary flow cell cartridge that can be used in sequencing apparatuses according to the disclosure.

[0056] FIGURE 18 depicts some exemplary flow cell geometries that can be used in sequencing apparatuses according to the disclosure.

[0057] FIGURE 19 depicts an exemplary embodiment of a flow cell that can be used in sequencing apparatuses according to the disclosure. [0058] FIGURE 20 depicts some views of an exemplary electro wetting flow cell; FIGURE 20A depicts a cross section an exemplary electro wetting flow cell; FIGURE 20B depicts the top view of this exemplary electro wetting flow cell.

[0059] FIGURE 21 depicts some exemplary embodiments of a fluid handling system to be used in conjunction with the sequencing apparatuses of the disclosure; FIGURE 21A depicts an exemplary fluid handling system including a syringe-type pump; FIGURE 2 IB depicts an exemplary fluid handling system including a pressure-driven pump.

[0060] FIGURE 22 depicts some exemplary embodiments of a temperature control system to be used in conjunction with the sequencing apparatuses of the disclosure; FIGURE 22A depicts one exemplary temperature control system; FIGURE 22B depicts another exemplary temperature control system.

[0061] FIGURE 23 depicts the structure of the ends of an exemplary lambda genomic DNA molecule used according to the procedure of Example 9.

DETAILED DESCRIPTION

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which these inventions belong. All patents, patent applications, published applications, treatises and other publications referred to herein, both supra and infra, are incorporated by reference in their entirety. If a definition and/or description is explicitly or implicitly set forth herein that is contrary to or otherwise inconsistent with any definition set forth in the patents, patent applications, published applications, and other publications that are herein incorporated by reference, the definition and/or description set forth herein prevails over the definition that is incorporated by reference.

[0063] The practice of the disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology and recombinant DNA techniques, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook, J., and Russell, D.W., 2001, Molecular Cloning: A Laboratory Manual, Third Edition; Ausubel, F.M., et al., eds., 2002, Short Protocols In Molecular Biology, Fifth Edition.

[0064] As used herein, the terms "a," "an," and "the" and similar referents used herein are to be construed to cover both the singular and the plural unless their usage in context indicates otherwise. Accordingly, the use of the word "a" or "an" when used in the claims or specification, including when used in conjunction with the term "comprising", may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0065] As used herein, the terms "link", "linked", "linkage" and variants thereof comprise any type of fusion, bond, adherence or association that is of sufficient stability to withstand use in the particular biological application of interest. Such linkage can comprise, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like. Optionally, such linkage can occur between a combination of different molecules, including but not limited to: between a nanoparticle and a protein; between a protein and a label; between a linker and a functionalized nanoparticle; between a linker and a protein; between a nucleotide and a label; and the like. Some examples of linkages can be found, for example, in Hermanson, G., Bioconjugate Techniques, Second Edition (2008); Aslam, M., Dent, A., Bio conjugation: Protein Coupling Techniques for the Biomedical Sciences, London: Macmillan (1998); Aslam, M., Dent, A., Bio conjugation: Protein Coupling Techniques for the Biomedical Sciences, London: Macmillan (1998).

[0066] As used herein, the term "polymerase" and its variants comprise any enzyme that can catalyze the polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically but not necessarily such nucleotide polymerization can occur in a template-dependent fashion. Such polymerases can include without limitation naturally-occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof that retain the ability to catalyze such polymerization. Optionally, the polymerase can be a mutant polymerase comprising one or more mutations involving the replacement of one or more amino acids with other amino acids, the insertion or deletion of one or more amino acids, or the linkage of parts of two or more polymerases. Typically, the polymerase comprises one or more active sites at which nucleotide binding and/or catalysis of nucleotide polymerization can occur. Some exemplary polymerases include without limitation DNA polymerases (such as for example Phi-29 DNA polymerase, reverse transcriptases and E. coli DNA polymerase) and RNA polymerases. The term

"polymerase" and its variants, as used herein, also refers to fusion proteins comprising at least two portions linked to each other, where the first portion comprises a peptide that can catalyze the polymerization of nucleotides into a nucleic acid strand and is linked to a second portion that comprises a second polypeptide, such as, for example, a reporter enzyme or a processivity- enhancing domain. One exemplary embodiment of such a polymerase is Phusion® DNA polymerase (New England Biolabs), which comprises a Pyrococcus-Yke, polymerase fused to a processivity-enhancing domain as described, for example, in U.S. Patent No. 6,627,424.

[0067] As used herein, the term "polymerase activity" and its variants, when used in reference to a given polymerase, comprises any in vivo or in vitro enzymatic activity characteristic of a given polymerase that relates to catalyzing the polymerization of nucleotides into a nucleic acid strand, e.g., primer extension activity, and the like. Typically, but not necessarily such nucleotide polymerization occurs in a template-dependent fashion. In addition to such polymerase activity, the polymerase can typically possess other enzymatic activities, for example, 3' to 5' or 5' to 3' exonuclease activity.

[0068] As used herein, the term "nucleotide" and its variants comprises any compound that can bind selectively to, or can be polymerized by, a polymerase. Typically, but not necessarily, selective binding of the nucleotide to the polymerase is followed by polymerization of the nucleotide into a nucleic acid strand by the polymerase; occasionally however the nucleotide may dissociate from the polymerase without becoming incorporated into the nucleic acid strand, an event referred to herein as a "non-productive" event. Such nucleotides include not only naturally- occurring nucleotides but also any analogs, regardless of their structure, that can bind selectively to, or can be polymerized by, a polymerase. While naturally- occurring nucleotides typically comprise base, sugar and phosphate moieties, the nucleotides of the disclosure can include compounds lacking any one, some or all of such moieties. In some embodiments, the nucleotide can optionally include a chain of phosphorus atoms comprising three, four, five, six, seven, eight, nine, ten or more phosphorus atoms. In some embodiments, the phosphorus chain can be attached to any carbon of a sugar ring, such as the 5' carbon. The phosphorus chain can be linked to the sugar with an intervening O or S. In one embodiment, one or more phosphorus atoms in the chain can be part of a phosphate group having P and O. In another embodiment, the phosphorus atoms in the chain can be linked together with intervening O, NH, S, methylene, substituted methylene, ethylene, substituted ethylene, CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where R can be a 4-pyridine or 1 -imidazole). In one embodiment, the phosphorus atoms in the chain can have side groups having O, BH₃, or S. In the phosphorus chain, a phosphorus atom with a side group other than O can be a substituted phosphate group. Some examples of nucleotide analogs are described in Xu, U.S. Patent No. 7,405,281. In some embodiments, the nucleotide comprises a label (e.g., reporter moiety) and referred to herein as a "labeled nucleotide"; the label of the labeled nucleotide is referred to herein as a "nucleotide label". In some embodiments, the label can be in the form of a fluorescent dye attached to the terminal phosphate group, i.e., the phosphate group or substitute phosphate group most distal from the sugar. Some examples of nucleotides that can be used in the disclosed methods and compositions include, but are not limited to, ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, ribonucleotide polyphosphates, deoxyribonucleotide polyphosphates, modified ribonucleotide polyphosphates, modified deoxyribonucleotide polyphosphates, peptide nucleotides, metallonucleosides, phosphonate nucleosides, and modified phosphate- sugar backbone nucleotides, analogs, derivatives, or variants of the foregoing compounds, and the like. In some embodiments, the nucleotide can comprise non-oxygen moieties such as, for example, thio- or borano- moieties, in place of the oxygen moiety bridging the alpha phosphate and the sugar of the nucleotide, or the alpha and beta phosphates of the nucleotide, or the beta and gamma phosphates of the nucleotide, or between any other two phosphates of the nucleotide, or any combination thereof.

[0069] As used herein, the term "nucleotide incorporation" and its variants comprise

polymerization of one or more nucleotides into a nucleic acid strand.

[0070] In some embodiments, the disclosure relates generally methods and related compositions, systems, kits and apparatuses for obtaining sequence information from a target nucleic acid molecule with increased average sequencing read lengths and/or accuracies. For example, the disclosed methods, compositions, systems, kits and apparatuses relate to exchanging (e.g., substituting) one or more components of the sequencing reaction, for example reagents for nucleotide incorporation reactions, with fresh reagents in such a manner that fresh reagents can act upon the same target nucleic acid molecule or template strand, a process termed "reagent exchange". In some embodiments, the one or more exchanged reagents can including one or more nucleotides, which may be labeled or unlabeled. In some embodiments, the one or more exchanged reactions can include one or more polymerases. In some embodiments, the one or more exchanged reactions can include one or more polymerization initiation sites (e.g., primers, nicks, gaps, etc).

[0071] In some embodiments, the disclosure provides methods and related compositions, systems, kits and apparatuses for obtaining sequence information from a target nucleic acid molecule comprising sequencing two or more portions of a template strand of a target nucleic acid molecule. Optionally, each portion is sequenced using a different polymerase. [0072] In some embodiments, the disclosure provides methods and related compositions, systems, kits and apparatuses that relate to obtaining sequence information from a target nucleic acid molecule, comprising: providing a target nucleic acid molecule including a template strand, sequencing a first portion of the template strand using a first polymerase, and sequencing a second portion of the template strand using a second polymerase. In some embodiments, the first and second portions of the template strand are contiguous with each other; in some embodiments, they are overlapping.

[0073] In some embodiments, sequencing a portion of the template strand (e.g., first portion, second portion, etc.) using a polymerase (e.g., first polymerase, second polymerase, etc.) includes synthesizing a nascent nucleic acid molecule. The nascent nucleic acid molecule (also referred to as the synthesized strand) can be complementary to the template strand and can optionally hybridize to a portion of the template strand to form a synthesized nucleic acid duplex.

[0074] Optionally, one or more components of the sequencing reaction are exchanged between the sequencing of the first portion using the first polymerase and the sequencing of the second portion using the second polymerase. The exchanged component of the sequencing reaction can be selected from the group consisting of: polymerase, template strand, nascent nucleic acid molecule, primer, nucleotide, label, energy transfer moiety.

[0075] In embodiments where the first and second portions of the template strand are contiguous, the use of different polymerases to sequence contiguous portions of the template strand can optionally increase the average read length of the sequencing reaction. Contiguous portions include any two portions of the template strand that are adjacent to each other such that there are no intervening nucleotides separating the two portions within the template strand. In some embodiments, a terminal nucleotide of one contiguous portion of the template strand is covalently linked to a terminal nucleotide of the other contiguous portion, for example via a phosphodiester linkage. Any two contiguous portions of the template strand can include an upstream portion (more proximal to the 5' end of the template strand) and a downstream portion (more distal from the 5' end of the template strand). In one example, a phosphodiester bond links the 3' hydroxyl group of a terminal nucleotide of the upstream contiguous portion with the 5' phosphate group of a terminal nucleotide of the downstream contiguous portion. Typically, contiguous portions do not overlap, i.e., the contiguous portions do not share any nucleotides in common. In some embodiments, sequencing contiguous portions can include removal of the first polymerase from the template strand following the sequencing of the first portion and prior to the sequencing of the second portion using the second polymerase.

[0076] In some embodiments, the first and second portions of the template are overlapping, i.e., share at least one nucleotide in common. The use of different polymerases to sequence overlapping portions of the template strand can optionally increase the accuracy of the sequencing reaction; for example, through successively re-sequencing the template strand. Optionally, sequencing the first portion results in the formation of a synthesized nucleic acid duplex through hybridization of the synthesized strand with the template strand. In some embodiments, the synthesized nucleic acid duplex is denatured prior to sequencing the second portion using the second polymerase. For example, provided are methods and related compositions, systems, kits and apparatuses, for obtaining sequence information from a target nucleic acid molecule, comprising: providing a target nucleic acid molecule including a template strand; sequencing a first portion of the template strand, wherein the sequencing of the first portion results in formation of a synthesized nucleic acid duplex; denaturing the synthesized nucleic acid duplex, and sequencing a second portion of the template strand using a second polymerase, wherein the first portion and the second portion are overlapping.

[0077] In some embodiments, any desired number of additional portions of the template strand (for example, third, fourth, fifth, sixth, seventh, eighth portions, etc.) are sequenced after the first and second portions using a corresponding number of additional polymerases. For example, a series of polymerases (for example, first, second, third, fourth, fifth polymerases, etc.) can be used to sequence a series of portions (for example, first, second, third, fourth, fifth portions) of the template strand, with each portion optionally being sequenced in succession. Each portion in the series of portions can be contiguous with both the preceding portion and the next portion along the template strand. In one exemplary embodiment, a first polymerase can be used to sequence a first portion; a second polymerase is used to sequence a second portion; a third polymerase is used to sequence a third portion, a fourth polymerase is used to sequence a fourth portion, and so forth. Each polymerase in the series can catalyze the synthesis of a nascent nucleic acid molecule that is complementary to a corresponding portion of the template strand.

[0078] In some embodiments, both variants of reagent exchange sequencing, (i.e., sequencing of contiguous portions, and sequencing of overlapping portions) are performed upon the same template strand in succession. For example, a first portion of the template strand is sequenced using a first polymerase; a second portion of the template strand is sequenced using a second polymerase; and a third portion of the template strand is sequenced using a third polymerase. In some embodiments, the first and second portions are contiguous and where the third portion overlaps with the first portion and/or the second portion. The first polymerase can optionally form a synthesized nucleic acid duplex, the second polymerase can optionally extend the synthesized nucleic acid duplex, and the synthesized nucleic acid duplex can optionally be denatured prior to sequencing the third portion using the third polymerase. In another embodiment, the first and second portions are overlapping, the third portion is contiguous with the second portion, and the synthesized nucleic acid duplex is optionally denatured after sequencing the first portion and prior to synthesizing the second and third portions. The use of different polymerases to sequence both overlapping and contiguous portions of the same template strand can optionally increase both the accuracy and the average read length of the sequencing reaction.

[0079] Sequencing a portion (e.g., first portion, second portion, etc.) of the template strand using a polymerase (e.g., first polymerase, second polymerase, etc.) can optionally be performed using any suitable method for obtaining sequence information from the target nucleic acid molecule. Sequence information includes any information relating to the position and/or the identity of a given nucleotide within the target nucleic acid molecule.

[0080] In some embodiments, sequencing a portion of the template strand (e.g., first portion, second portion, etc.) includes contacting the template strand with a polymerase, optionally in the presence of nucleotides. The nucleotides can optionally be labeled with at least one detectable label, which can be referred to as a "nucleotide label". The contacting can be performed under conditions where the polymerase binds to the template strand; optionally the polymerase synthesizes a nascent nucleic acid molecule. The synthesizing can proceed continuously, without significant pause or interruption between successive nucleotide incorporations; alternatively, the synthesizing can proceed discontinuously, wherein the synthesis is paused or halted between at least two successive nucleotide incorporations.

[0081] In some embodiments, synthesizing a nascent nucleic acid molecule includes catalyzing one or more nucleotide incorporations. In some embodiments, at least one of the nucleotide incorporations is a template-dependent nucleotide incorporation. A template-dependent nucleotide incorporation includes any nucleotide incorporation where the identity of the incorporated nucleotide correlates with the identity of the opposing nucleotide in the template strand according to a fixed set of consistent and predictable rules; such rules collectively being referred to as a "pairing paradigm". The incorporated nucleotide can optionally be selected by the polymerase (for example, via an "induced-fit" mechanism) to specifically pair with the opposing nucleotide in the template strand according to the rules of the controlling pairing paradigm. In a typical embodiment, nucleic acid synthesis from a single template strand consistently proceeds according to a single pairing paradigm, such that the identity of every nucleotide incorporated into the nascent nucleic acid molecule correlates with the identity of the opposing nucleotide within the template strand according to the rules of the same pairing paradigm. In some embodiments, nucleic acid synthesis from a single template strand is governed by two or more pairing paradigms, where synthesis of the nascent nucleic acid molecule from a first portion of the template is controlled by one pairing paradigm, and for a second portion of the template by another pairing paradigm.

[0082] Typically, the identity of a given nucleotide is defined by the structure of its base moiety (or, in the case of nucleotide analogs lacking a base moiety, by any structurally or functionally equivalent moiety). In some embodiments, the order of nucleotide incorporation proceeds according to the Watson-Crick base pairing paradigm whereby the five common naturally occurring base types (adenine, cytosine, guanine, thymine and uracil, referred to as A, C, G, T and U, respectively), will pair with each other through the formation of hydrogen bonds according to the following rules: A typically pairs with T or U, and G pairs with C. Typically, the base of the incoming nucleotide and base of the opposing nucleotide in the template strand will pair according to the Watson-Crick base pairing paradigm. In some embodiments, the polymerase selects the incoming nucleotide according to Watson-Crick base-pairing rules. However, in some embodiments, the identity of the incoming nucleotide can be selected using other types of pairing paradigms, for example pairing paradigms for engineered base moieties that are specifically designed to pair with other base moieties according to non Watson-Crick rules. Template- dependent nucleotide incorporation according to any particular base pairing paradigm typically results in the synthesis of a complementary nascent nucleic acid molecule. The order of nucleotide incorporation into the nascent nucleic acid molecule is guided by, and therefore correlates with, the order of nucleotides within the template according to the controlling pairing paradigm. Once the order of incorporation of nucleotides into the nascent nucleic acid molecule is determined, the complementary sequence of the template strand can be deduced by applying the rules of the controlling pairing paradigm.

[0083] During the sequencing reaction, the polymerase can bind to at least one strand of the target nucleic acid molecule and synthesize a nascent nucleic acid molecule (also referred to as the "synthesized" strand) via template-dependent nucleotide incorporation. The nascent nucleic acid molecule can optionally be complementary to the template strand; in such embodiments, the nascent nucleic acid molecule can be referred to as "the complementary strand." The strand that is bound and processed by the polymerase to synthesize a nascent nucleic acid molecule is referred to as the template strand. During template-dependent nucleic acid synthesis, the polymerase typically synthesizes a complementary nucleic acid strand according to the controlling pairing paradigm. In some embodiments, the target nucleic acid molecule includes only one strand, which serves as the template strand. In some embodiments, the target nucleic acid molecule includes more than one strand, and any one of the strands can serve as the template. In some embodiments, multiple strands of a single target nucleic acid molecule can each serve as a template in a given sequencing reaction.

[0084] In some embodiments, sequencing a first portion of the template strand can optionally include synthesizing a first nascent nucleic acid molecule, and sequencing a second portion of the template strand can optionally include synthesizing a second nascent nucleic acid molecule. In some embodiments, the first polymerase synthesizes the first nascent nucleic acid molecule (also referred to as a first synthesized strand) by catalyzing one or more template-dependent nucleotide incorporations. The first nascent nucleic acid molecule can be complementary to the first portion of the template strand and in some embodiments can hybridize to the first portion of the template strand, thereby forming a first synthesized nucleic acid duplex. The second polymerase can synthesize the second nascent nucleic acid molecule by catalyzing one or more template- dependent nucleotide incorporations. The second nascent nucleic acid molecule can be complementary to the second portion and in some embodiments can hybridize to the second portion of the template strand, thereby forming a second synthesized nucleic acid duplex. In some embodiments, the first nascent nucleic acid molecule is covalently linked to a nucleotide of the second nascent nucleic acid molecule. For example, in some embodiments, the second nascent nucleic acid molecule is formed via extension of the first nascent nucleic acid molecule by the second polymerase. Such extension can optionally include one or more template-dependent nucleotide incorporations, optionally onto the 3' end, of the first nascent nucleic acid molecule by the second polymerase.

[0085] In some embodiments, each template-dependent nucleotide incorporation includes any one, some or more of the following events: approach of a nucleotide (the "incoming" nucleotide) to the polymerase-template complex; binding of the incoming nucleotide to the polymerase, optionally to the polymerase active site; catalysis of bond formation between the incoming nucleotide and another nucleotide at the terminal end of the polymerization initiation site by the polymerase; nucleophilic attack by the terminal 3' OH of the other nucleotide on the bond between the a and β phosphates (or between any other two phosphate groups of the polyphosphate chain of the nucleotide, or between the structural or functional equivalents of such phosphate groups) of the incoming nucleotide; a nucleotidyl transfer reaction resulting in phosphodiester bond formation between a terminal nucleotide of the nascent nucleic acid molecule and the incoming nucleotide, cleavage of the incoming nucleotide to form one or more cleavage products, and/or diffusion of the cleavage product out of the zone of detection. In some embodiments, the polymerase can liberate a cleavage product. For example, when the polymerase incorporates a nucleotide having phosphate groups, the cleavage product can include one or more phosphate groups. Where the polymerase incorporates a nucleotide having substituted phosphate groups, the cleavage product can optionally include one or more substituted phosphate groups.

[0086] In some embodiments, nucleotide incorporation is not template-dependent, and the incorporated nucleotide may not be complementary to the opposing template nucleotide on the target molecule.

[0087] In some embodiments, the nucleotide can dissociate from the polymerase without becoming incorporated. If the nucleotide dissociates from the polymerase, it can be liberated and typically carries intact polyphosphate groups. When the nucleotide dissociates from the polymerase, the event is known as a "non-productive binding" event. The dissociating nucleotide may or may not be complementary to the template nucleotide on the target molecule. When the incoming nucleotide becomes incorporated into the nascent nucleic acid molecule, the event is referred to as a "productive binding" event. For template-dependent nucleotide incorporations, the incorporated nucleotide is typically complementary to the opposing nucleotide on the template strand, although a non-complementary nucleotide can also occasionally be incorporated during template-dependent synthesis.

[0088] In some embodiments, the length of time, frequency, or duration of the binding of a complementary incoming nucleotide to the polymerase can differ from that of a non- complementary incoming nucleotide. This time difference can be used to distinguish between the complementary and non-complementary nucleotides, and/or can be used to identify the incorporated nucleotide, and/or can be used to deduce the sequence of the target molecule. [0089] Nucleotide incorporation can include incorporation of any types of nucleotide, for example ribonucleotides (in the case of RNA synthesis) and deoxyribonucleotides (in the case of DNA synthesis).

[0090] In some embodiments, the target nucleic acid molecule, or the template strand, is linked to a solid or semi-solid substrate. Optionally, multiple target nucleic acid molecules (or multiple template strands) can be linked to the same surface. Optionally, multiple target nucleic acid molecules (or multiple template strands) can be contacted with the same sequencing reaction mixture simultaneously.

[0091] Typically, the first polymerase and second polymerases are different polymerases, i.e., are not the same polymerase molecule; however, they can be two identical polymerases. The second polymerase can be a mutant, variant, recombinant or modified version of the first polymerase. In some embodiments, the first and second polymerases are from different organisms.

[0092] In some embodiments, the sequencing of the first portion and/or the sequencing of the second portion of the template strand includes contacting the polymerase with the template strand in the presence of at least two different types of nucleotides. In some embodiments, the contacting can be performed in the presence of at least three different types of nucleotides. Sequencing a portion of the template strand can optionally include polymerizing at least two labeled nucleotides in a template-dependent fashion.

[0093] In some embodiments, the first polymerase and the second polymerase are derived from different organisms. Alternatively, they can be derived from the same organism, and can optionally include the same amino acid sequence. In some embodiments, the first and second polymerases include amino acid sequences that are at least 90% identical to each other. In one example, the first and second polymerases are two identical polymerases.

[0094] In some embodiments, the first polymerase synthesizes a nucleic acid strand that is hybridized to the first portion of the nucleic acid template.

[0095] In some embodiments, provided herein are reagent exchange methods, where the existing target molecule, synthesized strand, primer, sequencing enzyme (e.g., polymerase, helicase or exonuclease), nucleotides, and/or other reagents, can be exchanged with fresh reagents in a manner that allows continued sequencing of the template strand following the addition of fresh reagents in place of the old ones. For example, fresh polymerase, nucleotides, and/or reagents can be added to an immobilized target/template strand so as to permit continued nucleotide

incorporation into the synthesized strand (also referred to as the nascent nucleic acid molecule). [0096] Such reagent exchange during the sequencing reaction can be useful in a variety of ways. For example, exchanging of one or more sequencing reagents with fresh reagents in this manner avoids the problems associated with loss of reagent activity in sequencing systems over time. For example, use of polymerases in single molecule sequencing systems can frequently be

complicated by the loss of polymerase activity, which eventually terminates the sequence "read" from a given nucleic acid strand. Exchanging in the new polymerase will obviate this problem, thus increasing the length of the sequencing read obtained from the nucleic acid strand. The resulting sequence information can be further increased by performing multiple exchanges of old polymerases for new ones in the same assay, further increasing the read length for each exchange. Put another way, such reagent exchange can allow replacement any 'spent' or otherwise dysfunctional component of the sequencing reaction with fresh, functional components to permit continuing the sequencing reaction on the same target nucleic acid molecule or template strand.

[0097] Such exchange of sequencing reagents can greatly increase the total amount of sequence information obtained from a single sequencing assay, including both single molecule sequencing assays, in which multiple different nucleic acid strands are read simultaneously and

asynchronously, as well as ensemble or population-based sequencing methods, in which multiple identical nucleic acid molecules are sequenced synchronously.

[0098] Such exchange of sequencing reagents can also allow the performance of sequencing reactions exhibiting particular nucleotide binding or nucleotide incorporation properties followed by switching to sequencing reactions having different nucleotide binding and/or nucleotide incorporation reaction properties, where both types of sequencing reactions act on the same template strand of the target nucleic acid molecule. For example, a first portion of the template strand can optionally be sequenced using a polymerase, nucleotides, and other reagents, which exhibit certain properties, such as: nucleotide fidelity; rate of nucleotide incorporation;

processivity; strand displacement; kinetics of nucleotide binding, catalysis, release of the cleavage product, and/or polymerase translocation; exonuclease activity; and/or activity at certain temperatures. One, some or all of the sequencing reagents (e.g., polymerases and/or nucleotides) can be exchanged with different reagents to then sequence a second portion of the template strand using a second set of reagents that exhibit different nucleotide incorporation properties.

[0099] In some embodiments, the disclosure relates to reagent exchange methods (and related compositions, systems, kits and apparatuses) where the sequencing enzyme (e.g., polymerase, helicase or exonuclease) is exchanged for a fresh enzyme. The average length of the sequencing reads can optionally be increased by using different sequencing enzymes, for example different polymerases, to sequence different portions, typically two or more contiguous portions, of the same nucleic acid template during a single sequencing reaction. Such exchange can, for example, allow the replacement of old, "spent" polymerases with new, active polymerases during the sequencing of the same template strand, thus additively increasing the length of the resulting sequence read with each additional polymerase exchanged into the reaction. In some

embodiments, a first sequencing enzyme can optionally be exchanged for a second sequencing enzyme during the sequencing assay. One exemplary sequencing method that includes such reagent exchange comprises: providing a target nucleic acid molecule including a template strand, sequencing a first portion of the template strand using a first sequencing enzyme (e.g., a polymerase, helicase or exonuclease), exchanging the first sequencing enzyme with a second sequencing enzyme, and sequencing a second portion of the template strand using the second sequencing enzyme, where the first and second portions are contiguous with each other. Such enzyme exchange can include substitution or replacement of a first polymerase that is synthesizing the nascent nucleic acid molecule with another, fresh, polymerase ("second" polymerase). In some embodiments, the exchanging can occur after the first polymerase synthesizes a nascent nucleic acid molecule and prior to synthesis of a second nascent nucleic acid molecule by the second polymerase. When different polymerases are used to sequence contiguous portions of the template strand, the new polymerase can optionally continue synthesis of the nascent nucleic acid molecule from the point where the old polymerase left off.

[00100] In some embodiments, the first polymerase can be exchanged for the second polymerase, such that the second polymerase continues synthesis of the nascent nucleic acid molecule from the point where the first polymerase left off. For example, in embodiments where the first and second portions of the template strand are contiguous with each other, the second polymerase can be exchanged with the first polymerase in such a manner that the second polymerase continues replication of the template strand from the point where the first polymerase left off. For example, the second polymerase can extend the first nascent nucleic acid molecule synthesized by the first polymerase, optionally by polymerizing nucleotides onto an end of the first nascent nucleic acid molecule. In some embodiments, the second polymerase continues the incorporation of nucleotides into the nascent nucleic acid molecule beginning from the point where nucleotide incorporation by the first polymerase ended; optionally, such incorporation can be performed in a template-dependent fashion. The second polymerase can extend the first nascent nucleic acid molecule by polymerizing nucleotides onto an end of the first nascent nucleic acid molecule. In one exemplary embodiment, the second polymerase catalyzes one or more template-dependent nucleotide additions to the nascent nucleic acid molecule synthesized by the first polymerase. The second polymerase can optionally catalyze the formation of a covalent bond between a 3' hydroxyl group of a terminal nucleotide of the first nascent nucleic acid molecule and a 5' phosphate group of an incoming nucleotide. The 5' phosphate group of the first nucleotide incorporated by the second polymerase can optionally be covalently bonded to the 3' hydroxyl group of the last nucleotide incorporated by the preceding polymerase. The second nascent nucleic acid molecule synthesized by the second polymerase can optionally be an extended version of the first nascent nucleic acid molecule synthesized by the first polymerase.

[00101] In some embodiments, the exchanging comprises removing a polymerase (e.g., first polymerase, second polymerase, etc.) from the template strand. For example, once a first polymerase binds to a template strand, it may need to be removed from the template strand before the second polymerase can bind to the template strand and continue synthesis of a complementary strand from the point where synthesis by the first polymerase ended. In some embodiments, exchanging further includes removing the first polymerase from the template strand and then binding the second polymerase to the template strand. Optionally, the second polymerase binds to the template strand at the same position vacated by the first polymerase.

[00102] Removing the polymerase can optionally be accomplished using any suitable means; actual physical displacement of the polymerase from the template strand is not necessary as long as the removing process renders the nascent nucleic acid molecule synthesized by the polymerase accessible to further extension by another polymerase. In some embodiments, removing a polymerase from a template strand can include any treatment that terminates nucleotide incorporation into the nascent nucleic acid molecule by the polymerase, and allows another polymerase to catalyze one or more nucleotide incorporations into the nascent nucleic acid molecule.

[00103] The removing can be performed using any suitable conditions, including physical, chemical or enzymatic conditions, and can optionally include physical displacement of the polymerase from the template strand, inactivation, degradation (including photodegradation, enzymatic degradation or chemical degradation), denaturation, treatment with a reversible or irreversible inhibitor, physical or mechanical separation (such as, for example, using

chromatography, gel filtration or other suitable technique). [00104] In some embodiments, the polymerase can be removed from the template strand using any suitable physical, chemical, and/or enzymatic methods, in any combination and in any order. For example, the polymerase can be deactivated using elevated temperatures, such as 45-80 °C, for about 30 seconds to 10 minutes. In another example, the polymerase can be removed from the target molecule or synthesized strand by treatment with a suitable removing agent. Removing agents that do not disrupt the ability of the synthesized strand to be further extended are preferred. In some embodiments, the removing agent is selected such that it avoids completely denaturing the double- stranded duplex formed by hybridization of the nascent nucleic acid molecule to the template strand, so that when the duplex is contacted with a fresh polymerase it will be further extended by the fresh polymerase. In some embodiments, the removing agent is a protein- degrading enzyme, such as proteinase-K. In another example, the polymerase can be removed from the target molecule or synthesized strand using removing agents known to disrupt protein complexes, such as for example, high salt concentrations (e.g., NaCl); alkali (e.g., NaOH, KOH and the like); detergents (e.g., N-lauroyl sarcosine, SDS), chaotropic salt (e.g., guanidine or guanidium hydrochloride), lithium sulfate, and EDTA, etc.

[00105] In an exemplary embodiment, removing the polymerase and/or nucleotides includes adding a removing agent comprising a chaotropic salt, such as guanidine hydrochloride, to the sequencing reaction. The surface and associated template is then washed several times before addition of the replacement sequencing reaction that includes fresh polymerase and/or nucleotides.

[00106] In some embodiments, removing the first polymerase from the template strand can be performed under conditions where the nascent nucleic acid molecule synthesized by the first polymerase remains at least partially hybridized to the nucleic acid template. In some

embodiments, the removing is performed under conditions that do not completely denature the synthesized nucleic acid duplex. Complete denaturation of the synthesized nucleic acid duplex includes denaturation of the two strands of the synthesized nucleic acid duplex to such an extent that the synthesized nucleic acid duplex can no longer support template-dependent extension of the nascent nucleic acid molecule by the second polymerase. In some embodiments, exchanging can be performed using conditions that partially denature the synthesized nucleic acid duplex. Partial denaturation occurs when some portions of the template strand are hybridized to the nascent nucleic acid molecule (also known as the synthesized strand) but at least one portion of the template strand is not hybridized to the nascent nucleic acid molecule even though the sequences of the two strands are complementary over at least 99% of their length. The at least one portion that is not hybridized to the synthesized strand can be at least 1, 2, 5, 10, 15, 20, 25, 50 or 100 nucleotides in length. Exchanging can be performed under conditions that denature significant portions of the synthesized nucleic acid duplex; typically, further extension of the nascent nucleic acid molecule by the second polymerase can remain possible as long as the 3' end of the nascent nucleic acid molecule remains hybridized to the template strand.

[00107] In some embodiments, exchanging can further include adding a fresh reagent to the sequencing reaction following removal of the preexisting reagent. For example, a polymerase can optionally be removed from the template strand prior to the addition of another, fresh, polymerase to the sequencing reaction. The removing can optionally be followed by binding of the fresh polymerase to the template strand. In some embodiments, different portions of a nucleic acid template are sequenced by using a first sequencing enzyme, e.g., a polymerase, helicase or exonuclease, to sequence a first portion of a template strand of a target nucleic acid molecule, removing the first sequencing enzyme from the template and then sequencing a second portion of the template using a second sequencing enzyme.

[00108] In some embodiments, reagent exchange can be performed without the need for removing the reagent from the sequencing reaction. For example, exchange can optionally include continuous partial replacement of a given reagent, rather than discrete cycles involving complete removal of a reagent following by addition of new reagent. For example, fresh reagent (e.g., polymerase, nucleotide) may be continuously flowed into the sequencing reaction mixture, for example through an inlet port; optionally, a corresponding volume of the sequencing reaction mixture can continuously be withdrawn from the chamber, for example through an outlet port. As spent reagent is removed from the mixture, fresh reagent can replace the spent reagent and participate in the sequencing reaction without any disruption to the overall progress of the sequencing reaction. In this manner, some fraction of the total population of a particular sequencing reaction is continuously being replenished at any given time. For example, as some amount of new reagent is continuously being added and some amount of old reagent is continuously being discharged.

[00109] In some embodiments, such continuous replacement-type exchange can include the continuous replacement of sequencing enzyme within the sequencing reaction. For example, polymerase activity can constantly decrease as a result of photo-damage, etc. The polymerase can spontaneously dissociate from the template strand, thus obviating the need for removal and rendering the nascent nucleic acid molecule synthesized by the polymerase accessible for further extension by another polymerase. Continuous introduction of fresh polymerase into the sequencing reaction mixture allows the continuous replacement of "dead" or inactive polymerase, which can spontaneously dissociate from the template strand, with fresh, active polymerase. Since the rate of binding of active polymerase to the template can be very fast, fresh polymerase can bind to the position vacated by the spent polymerase quickly, typically in seconds, thus resulting in minimal interruption of the sequencing reaction and allowing the continuous generation of sequencing signals. The benefit of this approach is to allow fresh polymerases to continuously re- occupying positions in the template strand vacated by spent polymerases. Even when only a fraction of the spent polymerases are exchanged at any given time, this method can still increase the average read length of the sequencing system. Optionally, a less processive polymerase, which tends to dissociate frequently from the template strand, can be used, thus increasing the frequency of polymerase exchange during the sequencing reaction. Another benefit of such continuous-replacement exchange is to allow fresh polymerases to continuously associate with unbound templates in the sample, thus allowing the sequencing of template strands not initially bound by polymerases at the outset of the sequencing reaction.

[00110] In some embodiments, the disclosure relates generally to methods (and related compositions, systems and apparatuses) relating to sequencing of a template strand of a target nucleic acid molecule, wherein one or more nucleotides are exchanged during the sequencing reaction, such that a first portion of the template strand is sequenced using one set of nucleotides, and a second portion of the template strand is sequenced using a different set of nucleotides. For example, in some embodiments the disclosed methods (and related compositions, systems and apparatuses) relate to sequencing a target nucleic acid molecule, or a template strand of a target nucleic acid molecule, comprising: sequencing a first portion of the target nucleic acid molecule (or template strand) by contacting the target nucleic acid molecule (or template strand) with a polymerase in the presence of a first set of nucleotides; exchanging the first set of nucleotides for a second set of nucleotides; and sequencing a second portion of the target nucleic acid molecule (or template strand) by contacting the target nucleic acid molecule (or template strand) with polymerase in the presence of a second set of nucleotides. In some embodiments, the sequencing includes substitution or replacement of one particular combination of nucleotides with another fresh combination, which can be the same or different combination. In one exemplary

embodiment of sequencing using nucleotide exchange, a first portion of the template strand is sequenced by synthesizing a nascent nucleic acid molecule using a first polymerase and a first set of nucleotides, the first set of nucleotides including four different nucleotide types, three types (e.g., A, G, and C) each being labeled with a different energy transfer acceptor, and the fourth type (e.g., T) being unlabeled. For example, for the first round of nucleic acid synthesis, the A nucleotides can be labeled with label 1, G nucleotides can be labeled with label 2, and C nucleotides can be labeled with label 3. Following synthesis of a first nascent nucleic acid molecule by the polymerase, the first set of nucleotides is exchanged with a second set of nucleotides, the second set including the four different types, where a different nucleotide type is unlabeled, and a second round of nucleic acid synthesis can be conducted. For example, in the second round, the sequencing reaction can be conducted using three types of nucleotides (e.g., G, C, and T) labeled with a different type of energy transfer acceptor dye, and another different type of nucleotide (e.g., A) can be unlabeled. In a third round, the reagent exchange reaction can be conducted using three types of nucleotides (e.g., C, T, and A) labeled with a different type of energy transfer acceptor dye, and another different type of nucleotide (e.g., G) can be unlabeled. In a fourth round, the reagent exchange reaction can be conducted using three types of nucleotides (e.g., T, A, and G) labeled with a different type of energy transfer acceptor dye, and another different type of nucleotide (e.g., C) can be unlabeled. The first, second, third, and fourth rounds of reagent exchange reactions can be conducted in any order, and in any combination. In any of the rounds of reagent exchange reactions, the different types of nucleotides can be linked to the same or different type of energy transfer dye.

[00111] In another example, multiple rounds of reagent exchange reactions can be conducted using four types of nucleotides (e.g., A, G, C, and T) each labeled with a different type of energy transfer acceptor dye in each round. In one embodiment, in round one, the A nucleotides can be labeled with dye type 1, G labeled with dye type 2, C labeled with dye type 3, and T labeled with dye type 4. In a subsequent round, the reagent exchange reaction can be conducted using A labeled with dye type 2, G labeled with dye type 3, C labeled with dye type 4, and T labeled with dye type 1. One skilled in the art will readily recognize that many combinations are possible.

[00112] In another embodiment, only a subset of the nucleotides within the sequencing reaction are labeled, while some remain unlabeled. For example, the sequencing reaction can include four different nucleotide types (A, C, G and T), with at least one type remaining unlabeled. The template strand can be subjected to multiple rounds of sequencing, wherein overlapping portions of the template strand are sequenced, with one of the overlapping portions being sequenced per round. Each round of sequencing can optionally include contacting the target nucleic acid molecule (or template strand) with a polymerase in the presence of different combinations of labeled and unlabeled nucleotides. The sequence of the template strand can then be determined by combining the reads from all cycles. Such approaches can be advantageous by, for example, reducing the number of different and distinguishable labels required for sequencing.

[00113] In some embodiments, the disclosure relates generally to methods (and related compositions, systems and apparatuses) relating to sequencing of a template strand of a target nucleic acid molecule, comprising: sequencing a first portion of the target nucleic acid molecule (or template strand) by contacting the target nucleic acid molecule (or template strand) with a polymerase in the presence of nucleotides; and sequencing a second portion of the target nucleic acid molecule (or template strand) by contacting the target nucleic acid molecule (or template strand) with a polymerase in the presence of a second set of nucleotides, wherein the first portion and the second portion overlap with each other. The first and portions of the template can overlap partly or completely, and sequencing of the second portion typically involves resequencing at least some of the first portion. Sequencing of overlapping portions is referred to herein as "recursive" sequencing to indicate the repeated sequencing of the overlapping section of the template strand. In some embodiments, the first and second portions can overlap completely and may even consist of the same region of the template. One exemplary embodiment relates to completely

resequencing the entire template strand repeatedly (i.e., iteratively), using either the same polymerase or a different polymerase to perform each iteration. Sequencing of overlapping portions of the same template can be advantageous in increasing the accuracy of the sequencing reaction. Since any given sequencing reaction typically has an intrinsic error rate, resequencing a given portion more than once will allow identification of errors and the generation of a high- accuracy consensus read.

[00114] In some embodiments wherein two overlapping portions of a template strand are sequenced, a denaturing step can optionally be performed between the sequencing of any two portions. Such denaturing can be useful in situations where the two portions are overlapping. For example, sequencing the first portion of the template strand can include synthesis of a first nascent nucleic acid molecule by the first polymerase; this synthesized strand is typically complementary to the first portion of the template strand and can hybridize to the first portion to form a first synthesized nucleic acid duplex. When the first portion overlaps with the second portion of the template strand to be sequenced, at least some part of the second portion of the template strand may fall within the first synthesized nucleic acid duplex and thus may not be accessible for sequencing by the second polymerase. Denaturation of the synthesized nucleic acid duplex can liberate the second portion of the template strand and render it accessible for sequencing by the second polymerase.

[00115] Frequently, the hybridization between the two strands of the synthesized nucleic acid duplex can be partial or incomplete depending on the reaction conditions, the polymerase fidelity and/or nucleotides or nucleotide analogs included in the sequencing reaction. In some

embodiments, less than all the bases of first nascent nucleic acid molecule may be hybridized to complementary bases of the template strand within the synthesized nucleic acid duplex. In some embodiments, the number of bases of the synthesized strand that are hybridized to a

complementary base of the template strand can be less than or equal to 97%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%. However, even in such situations involving synthesized nucleic acid duplex wherein the two strands are not completely hybridized, a denaturation step may still be required before resequencing any portion of the template strand can be performed.

[00116] Suitable conditions for denaturing the synthesized nucleic acid duplex include any conditions that liberate the second portion of the template strand from the duplex sufficiently to permit sequencing of the second portion by the second polymerase, and that preserve the ability of the second polymerase to catalyze one or more nucleotide incorporations in a template-dependent fashion. In one example, denaturation can include transiently subjecting the synthesized nucleic acid duplex to strong acidic or alkaline conditions, for example via treatment with alkaline reagents such as sodium hydroxide (NaOH) or acidic reagents such as HC1; to heat (melting); inorganic salts, organic solvents (e.g., chloroform, etc.) and the like. For example, the synthesized nucleic acid duplex can be denatured using elevated temperatures, such as about 75-100 °C (e.g., without formamide) or about 45-90 °C (e.g., with formamide). In another example, the target molecule or synthesized strand can be degraded using a nucleic acid degrading enzyme, such as a 5'→ 3' or 3'→ 5' exonuclease (e.g., exonuclease III, T7 gene 6 exonuclease, exonuclease I). In yet another example, the target nucleic acid molecule or synthesized strand can be denatured using any compound known to dissociate double-stranded nucleic acid molecules, such as any combination of: formamide, urea, DMSO, alkali conditions (e.g., NaOH at about 0.01 - 0.3 M, or about 0.05 - 0.1 M; e.g., elevated pH of about 7-12), or low salt or very-low salt conditions (e.g., about less than 0.001 - 0.3 mM cationic conditions), or water. [00117] In some embodiments of the exchange-based sequencing methods (and related compositions, systems and apparatuses) of the disclosure, sequencing of two or more overlapping portions, including a first and second portions that overlap at least partly or completely, can be performed by providing a target nucleic acid molecule including a template strand; sequencing a first portion of the template strand using a first polymerase, wherein sequencing the first portion includes: and sequencing a second portion of the template strand using a second polymerase, wherein the first and second portions of the template strand overlap with each other. Optionally, the target nucleic acid molecule is linked to a solid or semi-solid substrate. Sequencing a given portion (e.g., first portion, second portion, and the like) can optionally include synthesizing a nascent nucleic acid molecule by contacting the template strand with a polymerase in the presence of nucleotides under conditions where the polymerase binds to the template strand and catalyzes one or more template-dependent nucleotide incorporations; and detecting, during the synthesizing, a sequence- specific signal indicating a template-dependent nucleotide incorporation catalyzed by the polymerase. The nascent nucleic acid molecule can hybridize to the template strand to form a synthesized nucleic acid duplex. The synthesized nucleic acid duplex can be denatured after sequencing the first portion and prior to sequencing the second portion.

[00118] Reagent exchange methods involving sequencing of two or more overlapping portions of a template strand can be useful for: re- sequencing at least a portion of the template strand, thereby increasing the accuracy of the sequencing reaction. For example, sequencing methods involving exchanging of various reaction components can be used to sequence the same target nucleic acid molecule (or same template strand) 1, 2, 3, 4, or 5 times, or up to 10 times, or up to 25 times, or up to 50 times, or more than 50 times. Sequencing the same target molecule multiple times, and/or sequencing the same synthesized strand multiple times, can provide multiple data sets of sequence information which can be aligned and compared. In one embodiment, the alignment can be used to deduce a consensus sequence of the target molecule or the synthesized strand. The alignment can be used to provide multi-fold coverage of the nucleotides contained within the target molecule or synthesized strand.

[00119] In some embodiments, the disclosure relates generally to methods (and related compositions, systems and apparatuses) relating to sequencing of a template strand of a target nucleic acid molecule, wherein the sequencing includes substituting or replacing one or more polymerization initiation sites with one or more different polymerization initiation sites. A polymerization initiation site includes any site to which one or more nucleotides can be added in a template-dependent fashion. In some embodiments, sequencing can include the addition of nucleotides to a polymerization initiation site, optionally via polymerization of nucleotides onto the polymerization initiation site by a polymerase. The template strand can optionally be base- paired with the polymerization initiation site. The polymerization initiation site can be used by the polymerase (e.g., DNA or RNA polymerase) to initiate synthesis of the nascent nucleic acid molecule; once synthesis is initiated, the polymerase can extend the nascent nucleic acid molecule by successively incorporating nucleotides into the nascent nucleic acid molecule, optionally in a template-dependent fashion. In some embodiments, the polymerization initiation site can include a terminal 3' OH group. For example, the polymerization initiation site can include a terminal 3ΌΗ of a primer molecule or of a self-primed target molecule; or can include a 3ΌΗ group within a gap or nick of a nucleic acid strand. The 3' OH group can serve as a substrate for the polymerase for nucleotide polymerization. The 3' OH group can serve as a substrate for the polymerase to form a phosphodiester bond between the terminal 3' OH group and the 5' phosphate group of an incoming nucleotide. The 3' OH group can be provided by: the terminal end of a primer molecule; a nick or gap within a nucleic acid molecule (e.g., oligonucleotide) that is base-paired with the target molecule; the terminal end of a secondary structure (e.g., the end of a hairpin-like structure); or an origin of replication. The polymerization initiation site can be provided by an accessory protein (e.g., RNA polymerase or helicase/primase). The

polymerization initiation site can be provided by a terminal protein that can be bound (covalently or non-covalently) to the end of the target nucleic acid, including terminal protein (e.g., TP) found in phage (e.g., TP from phi29 phage). Thus, the polymerization initiation site may be at a terminal end or within a base-paired nucleic acid molecule. In other embodiments, the polymerization initiation site used by some polymerases (e.g., RNA polymerase) may not include a 3ΌΗ group.

[00120] In some embodiments, the portion of the target molecule that is base paired with the primer or with the oligonucleotide, or the self-primed portion of the target molecule, can form hydrogen bonds by Watson-Crick or Hoogstein binding to form a duplex nucleic acid structure. The primer, oligonucleotide, and self -priming sequence may be complementary, or partially complementary, to the nucleotide sequence of the target molecule. The complementary base pairing can be the standard A-T or C-G base pairing, or can be other forms of base-pairing interactions.

[00121] In some embodiments, at least one strand of the target nucleic acid molecule is linked to a solid or semi-solid surface. In some embodiments, the polymerization initiation site can be in a position on the target nucleic acid molecule to permit nucleotide incorporation so as to extend the nascent nucleic acid molecule in a direction away from, or towards, the solid or semi-solid surface.

[00122] Some polymerases exhibit a preference for binding single- stranded nucleic acid molecules. For example, multiple polymerases may preferentially bind the single- stranded portion of a target nucleic acid molecule that is base-paired with a primer. Initiating nucleotide

polymerization using a gap can improve the number of target nucleic acid molecules that can undergo polymerization. In one embodiment, an unexpected procedure for improving a nucleotide polymerization can include initiating the polymerization reaction with the terminal 3ΌΗ within a gap, rather than from a primer that is base-paired with the target molecule. In one embodiment, the polymerases that can initiate nucleotide polymerization from a gap include strand-displacing polymerases. For example, the strand-displacing polymerase can be a phi29-like polymerases including: phi29, B103, or GA-1. In one embodiment, the gap can be the length of a

polynucleotide molecule that is about 2-15 nucleotides in length, or about 3-14 in length, or about 4-13 in length, or about 5-12 in length, or about 6-11 in length, or about 7-10 in length. The gap can be formed by annealing a target nucleic acid molecule to two primer nucleic acid molecule, or via any other suitable technique.

[00123] The methods, compositions, systems, kits and apparatuses disclosed herein can include sequencing using a primer molecule that can hybridize with the target nucleic acid molecule, optionally under high-stringency conditions. The sequence of the primer molecule can be complementary or non-complementary with the sequence of the sequence of the target molecule. The terminal 3' OH of the primer molecule can provide the polymerization initiation site.

[00124] The primers can be modified with a chemical moiety to protect the primer from serving as a polymerization initiation site or as a restriction enzyme recognition site. The chemical moiety can be a natural or synthetic amino acid linked through an amide bond to the primer.

[00125] The primer, oligonucleotide, or self-priming portion, may be naturally-occurring, or may be produced using enzymatic or chemical synthesis methods. The primer, oligonucleotide, or self- priming portion may be any suitable length including 5, 10, 15, 20, 25, 30, 40, 50, 75, or 100 nucleotides or longer in length. The primer, oligonucleotide, or self-priming portion may be linked to an energy transfer moiety (e.g., donor or acceptor) or to a label (e.g., a dye) using methods well known in the art.

[00126] The primer molecule, oligonucleotide, and self -priming portion of the target molecule, may comprise ribonucleotides, deoxyribonucleotides, ribonucleotides, deoxyribonucleotides, peptide nucleotides, modified phosphate-sugar backbone nucleotides including phosphorothioate and phosphoramidate, metallonucleosides, phosphonate nucleosides, and any variants thereof, or combinations thereof.

[00127] In one embodiment, the primer molecule can be a recombinant DNA molecule. The primer can be linked at the 5' or 3' end, or internally, with at least one binding partner, such as biotin. The biotin can be used to immobilize the primer molecule to the surface (via an avidin-like molecule), or for attaching the primer to a label. The primer can be linked to at least one energy transfer moiety, such as a fluorescent dye or a nanoparticle, or to a label. The primer molecule can hybridize to the target nucleic acid molecule. The primer molecule can be used as a capture probe to immobilize the target molecule.

[00128] In some embodiments, the disclosure relates generally to methods (and related compositions, systems and apparatuses) relating to sequencing a template strand of a target nucleic acid molecule, wherein one or more polymerization initiation sites are exchanged with one or more polymerization initiation sites during the sequencing. The exchange can include

replacement or substitution of the one or more polymerization initiation sites with one or more identical or different polymerization initiation sites.

[00129] For example, in some embodiments, the disclosure relates to methods (and related compositions, systems and apparatuses) for sequencing a template strand of a target nucleic acid molecule, comprising: sequencing a first portion of the template strand using a first

polymerization initiation site (or a first set of polymerization initiation sites), and sequencing a second portion of the template strand using a second polymerization initiation site (or a second set of polymerization initiation sites). The first and second portions of the template strand can be contiguous. In some embodiments, the first and second portions of the template strand can overlap partly or completely with each other. In some embodiments, any one or more

polymerization initiation sites can be provided by a terminal 3ΌΗ group of a primer, e.g., an oligonucleotide primer. In some embodiments, any one or more polymerization initiation sites can be provided by a terminal 3ΌΗ group of a nick or gap within a nucleic acid molecule.

[00130] In some embodiments, the sequencing includes exchanging the first polymerization initiation site (or the first set of polymerization initiation sites) for a second polymerization initiation site (or a second set of polymerization initiation sites).

[00131] Exchange of one or more polymerization initiation site during a sequencing reaction can provide various advantages. For example, in some embodiments, exchange of one or more polymerization initiation sites can allow repeated or "iterative" sequencing of a template strand, or "reverse" sequencing of the template strand (through sequencing of the complementary strand), either of which can increase the accuracy of the sequencing reaction.

[00132] In some embodiments, primer exchange is used to "reverse" sequence a portion of the template strand via sequencing of the complementary nascent nucleic acid molecule synthesized by the polymerase (i.e., synthesized strand) during a first sequencing reaction. Typically, the nascent nucleic acid molecule can be hybridized to the template strand to form a synthesized nucleic acid duplex. The synthesized nucleic acid duplex can optionally be denatured following synthesis of the nascent nucleic acid molecule. Fresh primers can then be added that can hybridize to the nascent nucleic acid molecule. The primers annealed to the nascent nucleic acid molecule can then be extended via template-dependent nucleic acid synthesis. Using such methods, the synthesized strand can be sequenced 1, 2, 3, 4, or 5 times, or up to 10 times, or up to 25 times, or up to 50 times, or more than 50 times, to provide redundant nucleotide sequence information.

[00133] In some embodiments, exchange of polymerization initiation sites comprises introducing one or more nicks or breaks into the template strand (or into the complementary strand), at least one introduced nick or break including a terminal group (e.g., a 3'-OH group) that is capable of serving as a polymerization initiation site that acts to prime a new sequencing read. Such nicks or breaks can be introduced using any suitable method. In some embodiments, nicks are generated in the template strand randomly; alternatively, nicks can be generated in a site-specific manner. In some embodiments, random nicks are generated by limited DNase I digestion, by nicking by Ser- His, or chemical treatment of the template strand, and the like. In some embodiments, site-specific nicks are generated by treatment of the template strand with nicking endonucleases, with specific chemicals, and the like.

[00134] Prior to nick generation, any pre-existing nicks present in template strand (e.g., formed as a by-product of the template isolation procedure, or during normal cellular metabolism) can optionally be repaired. Such repair can be performed using any suitable method, for example by treatment with DNA ligase (i.e., T4 DNA ligase).

[00135] In some embodiments, the disclosure relates generally to methods (and related compositions, systems and apparatuses) relating to sequencing a template strand of a target nucleic acid molecule, comprising: sequencing a first portion of the template strand by adding one or more nucleotides onto a first polymerization initiation site using a polymerase, and sequencing a second portion of the template strand by adding one or more nucleotides onto a second polymerization initiation site using a polymerase. In some embodiments, the first and/or second polymerization sites comprise a nick within the template strand or within a nucleic acid molecule that is complementary to the template strand. In some embodiments, sequencing the first portion further includes creating the first polymerization initiation site by introducing a first nick (or first gap) into the template strand or into the complementary nucleic acid molecule. In some embodiments, sequencing the second portion further includes creating the second polymerization initiation site by introducing a second nick (or second gap) into the template strand or into complementary nucleic acid molecule.

[00136] In some embodiments, the disclosure relates generally to methods (and related

compositions, systems and apparatuses) relating to sequencing a template strand of a target nucleic acid molecule, comprising: sequencing a first set of portions of the template strand by adding one or more nucleotides onto a first set of polymerization initiation sites using a polymerase, and sequencing a second set of portions of the template strand by adding one or more nucleotides onto a second set of polymerization initiation sites using a polymerase. In some embodiments, the first set and/or second set of polymerization sites comprise a first set and/or second set of nicks within a template strand or within a nucleic acid molecule that is complementary to the template strand. In some embodiments, sequencing the first set of portions further includes creating the first set of polymerization initiation sites by introducing a first set of nicks (or first set of gaps) into the template strand and/or into the complementary nucleic acid molecule. In some embodiments, sequencing the second set of portions further includes creating the second set of polymerization initiation sites by introducing a second set of nicks (or second set of gaps) into the template strand and/or into the complementary nucleic acid molecule. Optionally, the first set and/or second set of nicks are introduced into both the template strand and into the complementary nucleic acid molecule, such that priming and consequent nucleic acid synthesis can occur in both directions. Typically, when the polymerization initiation site is a nick or gap within a nucleic acid molecule, the polymerase is a strand-displacing polymerase, for example, Phi29 polymerase or any other suitable strand-displacing polymerase.

[00137] In some embodiments, following sequencing of the first set of portions of the template strand, the polymerization initiation sites can reset or exchanged prior to sequencing the second set of portions of the template strand. In some embodiments, resetting the polymerization initiation sites includes introducing a second set of nicks into the template strand and/or the complementary nucleic acid molecule. The process can be repeated until multiple sequencing coverage of template is achieved.

[00138] In some embodiments, the first and second sets of polymerization initiation sites are generated using site-specific nicking enzymes in conjunction with random nicking enzymes.

[00139] One exemplary embodiment of a sequencing method involving exchange of nick-based polymerization initiation sites is depicted in Figure 15. Figure 15A depicts sequencing a first set of portions of a template strand of target nucleic acid molecules isolated and displayed within nanochannels, where nick-based priming occurs at several points along each nucleic acid molecule and in both directions (i.e., nicks are present both in the template strand and the complementary strand). The sequencing reads initiated from each nick can be assembled to form a consensus sequence of the template strand.

[00140] Figure 15B depicts various optional steps in an exemplary embodiment of a sequencing method involving nick exchange, including introducing the first set of nicks, sequencing using the first set of nicks, repairing the template strand and/or complementary strand, introducing the second set of nicks, and sequencing using the second set of nicks.

[00141] Introducing the first set of nicks can include introducing one or more random nicks and/or one or more site- specific nicks into the template strand and/or the complementary strand of the target nucleic acid molecule. For random nicking (e.g., by DNase I) the enzyme can optionally be titrated to generate on average 1 nick per several kilobases of DNA (the optimal nick density will depend on the desired sequencing read-length). For site-specific nicking, site-specific nicking endonucleases can optionally be used in consecutive manner. The total number of nicking enzymes can determine the number of iterative reads of the template strand obtained (either via direct sequencing of the template strand or via reverse sequencing of the complementary strand), and thus the degree of sequencing coverage obtained.

[00142] Repairing the template strand and/or complementary strand can be performed to repair the target nucleic acid molecule prior to introducing the second set of nicks. Repairing can be accomplished via treatment with, e.g., T4 DNA ligase or with any other suitable repairing agent. In embodiments wherein a strand-displacing polymerase was used to sequence using the first set of nicks, it may be necessary to remove the single strands of the 3'-flap structures formed during strand displacement synthesis prior to repairing the template strand and/or complementary strand. Such removal can be achieved by any suitable treatment. For example, the template strand and/or complementary strand can first be treated with S 1 nuclease and then with T4 polymerase (to fill potential gaps without further strand displacement) to generate nicks, and finally with T4 DNA ligase (to ligate nicks and restore DNA template integrity).

[00143] In some embodiments, the nicks can be elongated into gaps using any suitable digesting agents or treatments that can degrade the template strand or the complementary strand from a nick in the target nucleic acid molecule. In some embodiments, the digesting agent is T7 exonuclease. The presence of gaps can in some embodiments increase the efficiency of priming at each gap site relative to the priming efficiency observed using nicks. In some embodiments, a particular polymerase will initiate nucleic acid synthesis at nicks with low frequency, but will initiate nucleic acid synthesis at gaps at a much higher frequency.

[00144] In some embodiments, the polymerase used to sequence the first set of portions of the template strand following can optionally be removed prior to sequencing the second set of portions using a second polymerase. In some embodiments, the removing can include washing away the polymerase from an immobilized template strand, and/or heat inactivation of the polymerase. Such removal can be important when the first polymerase possesses intrinsic exonuclease activity, which can lead to the generation of unwanted gaps in the template strand and/or complementary strand that can complicate the results.

[00145] One exemplary embodiment involving the generation of nicks or gaps within a long double stranded target DNA molecule, and the binding of labeled polymerases thereto, is described in Example 9, below.

[00146] In some embodiments, both overlapping and contiguous portions of the same template strand can be sequenced, thus allowing both increased read lengths (an advantage provided by "relay" sequencing of contiguous portions) as well as higher sequencing accuracies (an advantage provided by "recursive" sequencing of overlapping portions). For example, in some

embodiments, two contiguous portions of a template strand are sequenced in succession using different polymerases, wherein the second polymerase extends the nascent nucleic acid molecule synthesized by the first polymerase, thereby forming a synthesized nucleic acid duplex that includes both the first and second portions of the template strand and a synthesized strand that includes portions complementary to both the first and second portions of the template strand. The synthesized nucleic acid duplex is then denatured and the first and second portions can then be re- sequenced using different polymerases.

[00147] Accordingly, in some embodiments the disclosure relates generally to methods (as well as related compositions, systems and apparatuses) for obtaining sequence information from a target nucleic acid molecule, comprising: providing a target nucleic acid molecule including a template strand; sequencing a first portion of the template strand using a first polymerase, wherein sequencing the first portion includes synthesizing a nascent nucleic acid molecule via template- dependent nucleotide incorporation and forming a synthesized nucleic acid duplex through hybridization of the nascent nucleic acid molecule and the template strand; denaturing the synthesized nucleic acid duplex, and resequencing at least some of the first portion of the template strand using another polymerase.

[00148] Optionally, the first portion of the template strand includes at least a first subportion and a second subportion that are contiguous with each other, and sequencing the first portion includes: sequencing the first subportion using a first polymerase, removing the first polymerase from the template strand, and sequencing the second subportion of the nucleic acid template using a second polymerase.

[00149] Optionally, the second portion of the template strand includes at least a first subportion and a second subportion that are contiguous with each other, and sequencing the second portion includes: sequencing the first subportion using a first polymerase, removing the first polymerase from the template strand, and sequencing the second subportion of the nucleic acid template using a second polymerase.

[00150] In some embodiments, any of the sequencing methods described herein can be coupled with the generation of physical maps of long templates. Such physical maps can be generated using any suitable method, including, for example, fluorescence staining, chromosome "painting" or nanocoding. Nanocoding methods are described, for example, in Jo et al., A single molecule barcoding system using nanoslits for DNA analysis, Proc Natl Acad Sci USA 104: 2673-2678 (2007). In one exemplary embodiment, a first set of portions of a template strand in a nucleic acid molecule are sequenced from random nicks, and then a physical map of the template strand is generated by nanocoding. Such coupling of two technologies, where the physical map serves as a scaffold for sequence assembly, allows for the correct detection of structural rearrangements (or variations in normal genomes). In some embodiments, the resulting sequencing data can be directly assembled on the physical scaffold of a reference genome. Such a combination of sequencing and optical mapping can be particularly useful for sequencing of genomes with large number of gross chromosomal rearrangements, e.g., cancer genomes.

[00151] In some embodiments, the sequencing can also include a detecting step. In some embodiments, the detecting includes detecting a nucleotide incorporation, which can optionally be a template-dependent nucleotide incorporation. In some embodiments, a nucleotide incorporation is detected by detecting a signal emitted from the sequencing reaction. Optionally the signal can be monitored over time. The signal can be an optically detectable signal, for example a fluorescent signal. In some embodiments, the signal can be emitted by a labeled component of the sequencing reaction. The signal can be a sequence- specific signal. The labeled component can optionally be selected from the group consisting of: nucleotide, polymerase, primer, template and reaction chamber surface. In some embodiments, the signal is a fluorescent signal emitted by a fluorescent label attached to a component of the sequencing reaction and the detecting includes detecting one or more fluorescence signals emitted by the fluorescent label. The fluorescent label can optionally be attached to the nucleotide and/or to the polymerase. In some embodiments, the signal is emitted upon exposure to suitable excitation radiation, such as electromagnetic radiation, for example, light.

[00152] In some embodiments, the detecting can include detecting a sequence-specific signal. The sequence- specific signal can be any signal that is associated with the incorporation of a nucleotide into a nascent nucleic acid molecule and that indicates the identity of the nucleotide. Optionally, the sequence- specific signal also indicates the location of the nucleotide, i.e., its position within nascent nucleic acid molecule following incorporation. The sequence-specific signal can be generated as a result of any one or more events associated with the incorporation of the nucleotide into the nascent nucleic acid molecule, for example without limitation: approach of an incoming nucleotide to the polymerase-template complex; binding of the incoming nucleotide to the polymerase active site; catalysis of covalent bond formation between the incoming nucleotide and the terminal nucleotide of the nascent nucleic acid molecule being synthesized by the polymerase; cleavage of the incoming nucleotide within the polymerase active site, release of one or more cleavage products and/or diffusion of a cleavage product out of the zone of detection. The sequence- specific signal can optionally be emitted during the nucleotide incorporation, shortly prior to the nucleotide incorporation and/or shortly following the nucleotide incorporation. Since the sequence- specific signal indicates the identity of the incorporated nucleotide, it can optionally be analyzed to determine the identity of the nucleotide.

[00153] In some embodiments, the methods, systems, compositions and kits disclosed herein can involve the use of one or more labels, which can optionally be linked to the solid surfaces, nanoparticles, polymerases, nucleotides, target nucleic acid molecules, primers, and/or

oligonucleotides. For example, the detecting can include detecting a sequence- specific signal emitted by the label of a labeled component of the sequencing reaction. In a typical example, the sequencing reaction includes one or more labeled nucleotides, each comprising a nucleotide label, and the sequence- specific signal is emitted by a nucleotide label. Optionally, the labeled nucleotide includes a label whose identity correlates with the identity of the nucleotide; for example, each nucleotide type can optionally be associated with a different type of label, such that the sequence- specific signal emitted by the label can be analyzed to determine the identity of the nucleotide.

[00154] In some embodiments, the sequence-specific signal is emitted directly by the nucleotide label. In one example, the nucleotide label can emit a fluorescent signal upon excitation, and the fluorescent signal can be the sequence- specific signal. Each different type of nucleotide can optionally be labeled with a different label. The identity of the label can correlate with the identity of the nucleotide, such that the signal emitted by the label can indicate the identity of the nucleotide. In one exemplary embodiment, the sequencing reaction includes four different types of nucleotides A, C, G and T (or optionally U substituted for T), each type being linked to a different fluorescent label that can be spectrally distinguished from the label of the other three types. Identification of the incorporated nucleotide is accomplished by exciting the nucleotide labels and detecting a fluorescent signal from a nucleotide label indicating incorporation of the underlying nucleotide by the polymerase. The wavelength of the fluorescent signal can indicate the base identity of the incorporated nucleotide.

[00155] In some embodiments, the sequence-specific signal is emitted as a result of the interaction between at least two different labels, each label being attached to two different components of the sequencing reaction. Put another way, at least two components of the sequencing reaction can include labels that can interact with each other to produce a signal, which can optionally be a sequence- specific signal. In some embodiments, the label can include one or more energy transfer moieties that are capable of undergoing energy transfer, for example Forster Resonance Energy Transfer ("FRET") with each other. FRET is a distance-dependent

radiationless transmission of excitation energy from a first moiety, referred to as a donor moiety, to a second moiety, referred to as an acceptor moiety. The donor and acceptor together make up an energy transfer pair. An energy transfer pair can include any two energy transfer moieties that can under energy transfer, for example, resonance energy transfer, with each other. In some embodiments, the energy transfer donor absorbs electromagnetic energy (e.g., light) at a first wavelength and emits excitation energy in response. The energy acceptor can optionally absorb excitation energy emitted by the donor and fluoresce at a second wavelength in response.

[00156] Typically, the efficiency of FRET energy transmission is dependent on the inverse sixth- power of the separation distance between the donor and acceptor, r. For a typical donor-acceptor pair, r can vary between approximately 10-100 Angstroms. FRET is useful for investigating changes in proximity between and/or within biological molecules. In some embodiments, FRET efficiency may depend on donor-acceptor distance r as 1/r⁶ or 1/r⁴. The efficiency of FRET energy transfer can sometimes be dependent on energy transfer from a point to a plane that varies by the fourth power of distance separation (E. Jares-Erijman, et al., 2003 Nat. Biotechnol.

21: 1387). The distance where FRET efficiency is 50% is termed R₀, also known as the Forster distance. Ro is unique for each donor- acceptor combination and may be about 1-20 nm, or about 1-10 nm, or about 1-5 nm, or about 5-10 nm. A change in fluorescence from a donor or acceptor during a FRET event (e.g., increase or decrease in the signal) can be an indication of proximity between the donor and acceptor.

[00157] In biological applications, FRET can provide an on-off type signal indicating when the donor and acceptor moieties are proximal (e.g., within Ro) of each other. Additional factors affecting FRET efficiency include the quantum yield of the donor, the extinction coefficient of the acceptor, and the degree of spectral overlap between the donor and acceptor. Procedures are well known for maximizing the FRET signal and detection by selecting high yielding donors and high absorbing acceptors with the greatest possible spectral overlap between the two (D. W. Piston and G.J. Kremers 2007 Trends Biochem. Sci. 32:407). Resonance energy transfer may be either an intermolecular or intramolecular event. Thus, the spectral properties of the energy transfer pair as a whole, change in some measurable way if the distance and/or orientation between the moieties are altered.

[00158] The production of signals from FRET donors and acceptors can be sensitive to the distance between donor and acceptor moieties, the orientation of the donor and acceptor moieties, and/or a change in the environment of one of the moieties (Deuschle et al. 2005 Protein Science 14: 2304-2314; Smith et al. 2005 Protein Science 14:64-73). For example, a nucleotide linked with a FRET moiety (e.g., acceptor) may produce a detectable signal when it approaches,

associates with, or binds a polymerase linked to a FRET moiety (e.g., donor). In another example, a FRET donor and acceptor linked to one protein can emit a FRET signal upon conformational change of the protein. [00159] In some embodiments, the energy transfer moieties can be linked to any suitable component of the sequencing reaction, including the solid surfaces, nanoparticles, polymerases, nucleotides, target nucleic acid molecules, primers, and/or oligonucleotides. In some

embodiments, a first component of the sequencing reaction is linked to a first energy transfer moiety, and a second component is linked to a second energy transfer moiety, where the first and second energy transfer moieties form an energy transfer pair. In some embodiments, the sequence-specific signal results from energy transfer between a donor and acceptor moiety within the sequencing reaction.

[00160] In some embodiments, the energy transfer moieties may not undergo FRET, but may undergo other types of energy transfer with each other, including luminescence resonance energy transfer, bioluminescence resonance energy transfer, chemiluminescence resonance energy transfer, and similar types of energy transfer not strictly following the Forster's theory, such as the non-overlapping energy transfer when non- overlapping acceptors are utilized (Laitala and

Hemmila 2005 Anal. Chem. 77: 1483-1487). For example, the donor and acceptor moieties can transfer energy in various modes, including: fluorescence resonance energy transfer (FRET) (L. Stryer 1978 Ann. Rev. Biochem. 47: 819-846; Schneider, U.S. Patent No. 6,982,146; Hardin, U.S. Patent No. 7,329,492; Hanzel U.S. published patent application No. 2007/0196846), scintillation proximity assays (SPA) (Hart and Greenwald 1979 Molecular Immunology 16:265- 267; U.S. Pat. No. 4,658,649), luminescence resonance energy transfer (LRET) (G. Mathis 1995 Clin. Chem. 41: 1391-1397), direct quenching (Tyagi et al, 1998 Nature Biotechnology 16:49-53), chemiluminescence energy transfer (CRET) (Campbell and Patel 1983 Biochem. Journal 216: 185- 194), bioluminescence resonance energy transfer (BRET) (Y. Xu, et al., 1999 Proc. Natl. Acad. Sci. 96: 151-156), and excimer formation (J. R. Lakowicz 1999 "Principles of Fluorescence Spectroscopy", Kluwer Academic/Plenum Press, New York).

[00161] In one exemplary embodiment, the energy transfer moieties can include a FRET donor/acceptor pair. The FRET donor/acceptor pair can optionally exhibit one or more changes in absorbance or emission in response to changes in their environment, such as changes in pH, ionic strength, ionic type (N0₂. Ca⁺², Mg⁺², Zn⁺², Na⁺, CI^", K⁺), oxygen saturation, and solvation polarity. The FRET donor and/or acceptor of the pair can be a fluorophore, luminophore, chemiluminophore, bioluminophore, or quencher (P. Selvin 1995 Methods Enzymol 246:300-334; C. G. dos Remedios 1995 J. Struct. Biol. 115: 175-185; P. Wu and L. Brand 1994 Anal Biochem 218: 1-13). [00162] The energy transfer moiety can optionally be an energy transfer donor or an energy transfer acceptor. In some embodiments, the energy transfer moiety is a nanoparticle or a fluorescent dye. In one embodiment, the energy transfer moiety can be a quencher moiety.

[00163] In some embodiments, both members of an energy transfer pair, i.e., donor and acceptor, can be linked to the same molecule. For example, the energy transfer donor and acceptor pair can be linked to a single polymerase, which can provide detection of conformational changes in the polymerase. In other embodiments, the donor and acceptor can be linked to different molecules in any combination. For example, the donor can be linked to the polymerase, target molecule, or primer molecule, and/or the acceptor can be linked to the nucleotide, the target molecule, or the primer molecule.

[00164] In some embodiments, the donor and acceptor moieties can interact with each other physically or optically in a manner that produces a detectable signal when the two moieties are in proximity with each other. In some embodiments, the detectable signal can be an energy transfer signal, which can optionally be a sequence- specific signal. The detectable signal can indicate a proximity event that includes two different moieties (e.g., energy transfer donor and acceptor) approaching each other, or associating with each other, or binding each other.

[00165] In some embodiments, the polymerase can be linked to an energy transfer donor moiety. In some embodiments, the nucleotide can be linked to an energy transfer acceptor moiety. In one example, the nucleotide comprises an energy transfer moiety. The energy transfer moiety of the nucleotide can optionally be linked to the polyphosphate chain of the nucleotide, for example to the terminal phosphate group of the polyphosphate chain (or to any structural or functional equivalent thereof). A change in a fluorescent signal emitted by the energy transfer moiety of the nucleotide can occur when the labeled nucleotide is proximal to another labeled component of the sequencing reaction, for example a labeled polymerase. For examples of embodiments involving FRET-based sequencing using a donor-labeled polymerase and acceptor-labeled nucleotides, see U.S. Patent Application Ser. No. 12/748,168, filed March 26, 2010, incorporated by reference in its entirety.

[00166] In some embodiments, the nucleotide includes an acceptor label and the polymerase includes a donor label, and the signal occurs when the acceptor-labeled nucleotide is proximal to the donor-labeled polymerase. Optionally, the signal emitted by the donor moiety decreases and/or the signal emitted by the acceptor moiety increases. A decrease in donor signal and/or an increase in acceptor signal can correlate with nucleotide binding to the polymerase and/or with polymerase-dependent nucleotide incorporation.

[00167] In an exemplary embodiment, the polymerase is labeled with a FRET donor and each type of nucleotide is labeled with a different acceptor. The sequence- specific signal is generated as a result of FRET between the donor of the polymerase and the acceptor of the incoming nucleotide during a nucleotide incorporation. Optionally, the acceptor of the incoming nucleotide fluoresces as a result of the FRET, and the acceptor fluorescence indicates the base identity of the incoming nucleotide. The acceptor fluorescence can be detected and analyzed to identify the incoming nucleotide.

[00168] In some embodiments, detecting can be performed using confocal laser scanning microscopy, Total Internal Reflection (TIR), Total Internal Reflection Fluorescence (TIRF), near- field scanning microscopy, far-field confocal microscopy, wide-field epi-illumination, light scattering, dark field microscopy, photoconversion, wide field fluorescence, single and/or multi- photon excitation, spectral wavelength discrimination, evanescent wave illumination, scanning two-photon, scanning wide field two-photon, Nipkow spinning disc, and/or multi-foci multi- photon.

[00169] In practicing the nucleotide binding and/or nucleotide incorporation methods, non- desirable fluorescent signals can come from sources including background and/or noise. In one embodiment, the energy transfer signals can be distinguished from the non-desirable fluorescent signals by measuring, analyzing and characterizing attributes of all fluorescent signals generated by the nucleotide incorporation reaction. In one embodiment, attributes of the energy transfer signal that can permit distinction from the non-desirable fluorescent signals can include: duration; wavelength; amplitude; photon count; and/or the rate of change of the duration, wavelength, amplitude; and/or photon count. In one embodiment, the identifying the energy transfer signal, includes measuring, analyzing and characterizing attributes of: duration; wavelength; amplitude; photon count and/or the rate of change of the duration, wavelength, amplitude; and/or photon count. In one embodiment, identifying the energy transfer signal can be used to identify the incorporated nucleotide.

[00170] In some embodiments, the disclosed methods, compositions, systems and apparatuses involve identifying one or more nucleotides incorporated by the polymerase during nucleic acid synthesis. In some embodiments, the disclosure relates to obtaining sequence information from a target nucleic acid molecule, comprising: contacting a polymerase with a template strand of a target nucleic acid molecule in the presence of nucleotides under conditions where the polymerase catalyzes one or more template-dependent nucleotide incorporations; and identifying one or more nucleotides incorporated in a template-dependent fashion by the polymerase. In some

embodiments, the identifying can include detecting a sequence- specific signal, and analyzing the sequence-specific signal to determine the identity of an incorporated nucleotide. In some embodiments, the sequence- specific signal can be analyzed to determine the position of the incorporated nucleotide within the template strand.

[00171] In some embodiments, the disclosure provides a method for obtaining sequence information from a target nucleic acid molecule, comprising: providing a target nucleic acid molecule including a template strand; sequencing a first portion of the template strand using a first polymerase, wherein the sequencing includes identifying a nucleotide incorporated in a template- dependent fashion into a nascent nucleic acid molecule by the first polymerase; and sequencing a second portion of the template using a second polymerase, wherein the sequencing includes identifying a nucleotide incorporated in a template-dependent fashion into a nascent nucleic acid molecule by the second polymerase. In some embodiments, the first and second portions are contiguous; in other embodiments, they are overlapping. The identifying can optionally include detecting a sequence- specific signal. The identifying can include analyzing the sequence- specific signal to determine the identity of the incorporated nucleotide.

[00172] In some embodiments, for example methods, compositions and systems for single molecule sequencing, identifying the sequence- specific signal can include mapping the sequence- specific signal to a given target nucleic acid molecule, or to a given template strand. The mapping can include any suitable method for correlating a sequence- specific signal with a particular target nucleic acid molecule (or a particular template strand). In an exemplary embodiment, multiple labeled target nucleic acid molecules (or template strands) are immobilized on a surface in an array, and a region of the array is visualized. The signals emitted from a given portion of the array are ascribed to a particular target nucleic acid molecule (or template strand). Alternatively, multiple polymerases can be immobilized to form an enzyme array; each polymerase then associates with a single template, and signals emitted by the polymerase-template complex are ascribed to the template of the complex. Methods for making nucleic acid or protein arrays are known in the art.

[00173] In another exemplary embodiment, the detection system includes one or more cameras (or detectors) with at least two spectrally separate channels, and each channel is used to detect the template and the polymerase, respectively. For example, the polymerase can be labeled with a first label that is detected in the first detection channel, and the template can be labeled with a second label that is detected in the second detection channel. The images obtained in each channel can be overlaid to determine regions of "overlap" and thus detect templates to which polymerases have bound. Some examples of such techniques are provided in Example 8.

[00174] Optionally, the sequencing method can include a mapping step wherein all of the target nucleic acid molecules (or template strands) in the nucleic acid array, or all of the polymerases in the protein array, are simultaneously visualized and their location marked prior to initiation of the sequencing reaction. Signals emitted from a given location are then ascribed to the target nucleic acid molecule, template strand or polymerase known to reside in that location. In an exemplary embodiment, every target nucleic acid molecule (or template strand) in a nucleotide array, or every polymerase within a protein array, can be labeled with a mapping label prior to its attachment to the surface. The location of each member of the array (nucleic acid molecule, template strand or polymerase) is then identified through a mapping step wherein all of the array members are visualized simultaneously, and the location of each member in the array is determined. The mapping labels are then inactivated, e.g., via photobleaching, prior to initiation of the sequencing reaction. Sequencing signals emitted from a particular location in the array are ascribed to the member known to reside at that location.

[00175] In another exemplary embodiment, target nucleic acid molecules are distributed into wells or cavities filled with sequencing reaction mixture. Suitable methods, e.g., endpoint dilution or limiting dilution, are used to ensure that an average of one target nucleic acid molecule (or template strand) is distributed into each well or cavity. Signals emitted from a single well or cavity are then ascribed to a single target nucleic acid molecule (or single template strand).

Optionally, the members of the array can be immobilized in a suitable well or cavity that confines each member to a given location on the surface of the array. For example, the target nucleic acid molecule, template strand or polymerase can be confined within an optical waveguide. Signals emitted by the confined components can be detected and analyzed to obtain sequence information about the target nucleic acid molecule. In one exemplary embodiment, a template strand or a polymerase is immobilized within a waveguide, for example a zero mode waveguide, and the identifying includes detecting a signal emitted by a labeled nucleotide while the labeled nucleotide is incorporated into the synthesized nucleic acid strand by the polymerase. [00176] In one example, one or more components of the sequencing reaction are labeled, and identifying the incorporated nucleotide includes detecting a signal emitted by a labeled component of the sequencing reaction. In some embodiments, the nucleotide is labeled with a nucleotide label. In some embodiments, both the polymerase and nucleotide are labeled. Identifying the incorporated nucleotide can include detecting a signal emitted by the polymerase label, by the nucleotide label, or by both the polymerase and nucleotide labels.

[00177] In some embodiments, the sequencing reaction includes one or more labeled nucleotides. Identifying the incorporated nucleotide can include detecting a sequence-specific signal emitted by the label of the nucleotide shortly before, during and/or after incorporation of the nucleotide by a polymerase.

[00178] In some embodiments, identification is performed by detecting a signal resulting from an interaction between the nucleotide label and a label on the polymerase. The interaction can include FRET. The interaction can occur shortly before, after, or during the incorporation of the nucleotide by the polymerase.

[00179] Optionally, the identifying includes exciting the nucleotide label with an excitation source; (b) detecting a signal or a change in signal from nucleotide label; and (c) identifying the signal or the change in signal from the nucleotide label. In some embodiments, the signal is emitted directly by the nucleotide label upon excitation. The signal can be emitted as a result of interaction between the nucleotide label and any other label in the sequencing reaction.

Optionally, the signal is emitted as a result of FRET between the nucleotide label and a label attached to the polymerase.

[00180] In some embodiments, sequencing a portion of the target nucleic acid molecule (or a template strand of the target nucleic acid molecule) can include catalyzing one or more nucleotide incorporations using a polymerase. The sequencing conditions can include any conditions suitable for: forming the polymerase-target nucleic acid complex; binding the nucleotide to the

polymerase; incorporating the nucleotide; detecting the sequence- specific signal; and/or translocation of the polymerase to the next position on the target molecule.

[00181] Methods for performing template-dependent nucleotide incorporations using a polymerase are well known in the art. Typically, the template strand is contacted with a polymerase in the presence of nucleotides under polymerizing conditions, optionally in the presence of suitable buffers and divalent cations. Depending on the reagents used, the suitable conditions for catalyzing nucleotide incorporation can vary according to various parameters such as time, temperature, pH, reagents, buffers, reagents, salts, co-factors, nucleotides, target DNA, primer DNA, enzymes such as nucleic acid-dependent polymerase, amounts and/or ratios of the components in the reactions, and the like. The reagents or buffers can include a source of monovalent cations, such as KC1, K-acetate, NH₄-acetate, K-glutamate, NH₄C1, or ammonium sulfate. The reagents or buffers can include a source of divalent cations, such as Mg²⁺ and/or Mn²⁺, MgCl₂, or Mg-acetate. The buffer can include Tris, Tricine, HEPES, MOPS, ACES, or MES, which can provide a pH range of about 5.0 to about 9.5. The buffer can include chelating agents such as EDTA and EGTA, and the like. The suitable conditions can also include compounds that reduce photo-damage.

[00182] In some embodiments, the sequencing can be performed in the presence of one or more different types of divalent cations. The divalent cations can include any cation that permits nucleotide binding and/or nucleotide incorporation, including for example: manganese, magnesium, cobalt, strontium, or barium. The divalent cations can include any cation that promotes the formation and/or stability of the closed complex (polymerase/target/nucleotide), including magnesium, manganese, and chromium. The divalent cations can include any cation that permits nucleotide binding to the polymerase but inhibits nucleotide incorporation (e.g., calcium). The divalent cations can include chloride or acetate forms, including MnCl₂, Mn- acetate, MgCl₂, Mg-acetate, and the like.

[00183] In practicing the sequencing methods, some polymerases exhibit improved nucleotide binding and/or nucleotide incorporation kinetics when used with (i) manganese and/or

magnesium, and/or with (ii) tri-, tetra-, penta-, hexa-, or hepta-phosphate nucleotides. In one embodiment, the disclosed nucleotide incorporation methods can be practiced using manganese or magnesium, or a combination of manganese and magnesium. For example, the methods can include manganese at about 0.1-5 mM, or about 0.2-4 mM, or about 0.3-3 mM, or about 0.4-2 mM, or about 0.5-2 mM, or about 1-2 mM.

[00184] In another example, the methods can include magnesium at about 0.01-0.3 mM, or about 0.025-0.2 mM, or about 0.05-0.1 mM, or about 0.075-0.1 mM, or about 0.1 mM.

[00185] In yet another example, the methods can include a combination of manganese and magnesium at about 0.25-1 mM of manganese and 0.025-0.2 mM of magnesium, or about 0.5- 0.75 mM of manganese and 0.05-0.075 mM of magnesium, or about 0.5 mM manganese and 0.1 mM magnesium. [00186] In another example, the nucleotide incorporation reaction includes a polymerase derived from a bacteriophage, for example Phi29 or B 103 polymerase, and labeled hexa-phosphate nucleotides, with about 0.5-2 mM MnCl₂, or with a combination of about 0.5 mM MnCl₂ and about 0.1 mM MgCl₂.

[00187] In some embodiments, the disclosed methods, systems, kits and compositions relate to obtaining sequence information from a target nucleic acid molecule. The target nucleic acid molecule can be any nucleic acid molecule of interest. The target nucleic acid molecule can be DNA or RNA or a DNA/RNA hybrid. It can include naturally occurring nucleotides (for example, A, G, C, T or U) and/or non-natural or synthetic analogs of naturally occurring nucleotides.

[00188] In some embodiments, the methods, compositions, systems and kits disclosed herein can involve the use of one or more target nucleic acid molecules. The target nucleic acid molecule can optionally be comprised of one, two or more nucleic acid strands. In some embodiments, the target nucleic acid molecule can be single stranded or double-stranded. The two strands of a double-stranded nucleic acid molecule can optionally be paired with each other according to any particular pairing paradigm; in one example, the strands are paired with each other according to the Watson-Crick base pairing paradigm. The target nucleic acid molecule can be linear or circular. The target nucleic acid molecules may be DNA, RNA or hybrid DNA-RNA molecules, DNA hairpins, DNA/RNA hybrids, or RNA hairpins. The target nucleic acid molecules may be isolated in any form including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotide, or any type of nucleic acid library. The target nucleic acid molecules may be isolated from any source including from: organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, and viruses; cells; tissues; body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, and semen; environmental samples; culture samples; or synthesized nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods.

[00189] The target nucleic acid molecules comprise naturally-occurring nucleotides, nucleotide variants, or any combination thereof. For example, the target molecules comprise alternate backbones, including: phosphoramidate; phosphorothioate; phosphorodithioate; O- methylphosphoroamidite linkages; and peptide nucleic acid backbones and linkages. Other nucleic acids include those with bicyclic structures including locked nucleic acids; positive backbones; non-ionic backbones; and non-ribose backbones. [00190] The target nucleic acid molecules can carry a tag (e.g., His-tag), a polynucleotide tail (e.g., polynucleotide tail of A, G, C, T, or U), or can be methylated. The target nucleic acid molecules may be nicked, sheared, or treated with an enzyme such as a restriction endonuclease or a nuclease. The target nucleic acid molecules can be about 10-50 nucleotides, about 50-100 nucleotides, about 100-250 nucleotides, about 250-500 nucleotides, or about 500-1000 nucleotides in length, or longer. The target nucleic acid molecules may be linked to an energy transfer moiety (e.g., donor or acceptor) or to a label (e.g., dye) using methods well known in the art.

[00191] The target nucleic acid molecules can have a nucleotide sequence that has been previously determined or is unknown (e.g., de novo sequencing). The target molecule can be fragmented into shorter pieces and/or modified for immobilization. Selection of the fragmentation and modification technique may depend upon the desired fragment sizes and subsequent preparation steps. Any combination of fragmentation and/or modification techniques may be practiced in any order.

[00192] In some embodiments, multiple target nucleic acid molecules (or multiple isolated template strands) can be linked to a solid or semi-solid surface. The target nucleic acid molecules (or template strands) can be linked to the surface using any suitable linking methodology and/or in any arrangement. Optionally, some or all of the target nucleic acid molecules (or template strands) can be contacted with the same sequencing reaction mixture simultaneously, such that multiple target nucleic acid molecules (or multiple template strands) are sequenced simultaneously in parallel within the same sequencing reaction. The multiple target nucleic acid molecules (or multiple template strands) can optionally include the same sequence, or include overlapping sequences, or include completely different sequences. In one exemplary embodiment, the sequencing reaction mixture is flowed through a reaction chamber, e.g., a flow cell, where the chamber includes a surface that is linked to one or more target nucleic acid molecules (or template strands), typically to multiple target nucleic acid molecules, even more typically to multiple template strands.

[00193] In some embodiments, the target nucleic acid molecule can be linked to a solid or semisolid surface. Such linkage can be helpful to anchor the target nucleic acid molecule and/or to localize the template to a particular region of the surface. Localization of the template can be especially useful in, e.g., single molecule sequencing reactions wherein multiple single target nucleic acid molecules, each having a unique sequence, are simultaneously sequenced in parallel, as discussed further below. [00194] The surface can be chemically or enzymatically modified to have one or more reactive groups, including amines, aldehyde, hydroxyl, sulfate or carboxylate groups, which can be used to attach the surface to the nanoparticles, polymerases, nucleotides, target nucleic acid molecules, primers, and/or oligonucleotides.

[00195] The linkage of the target nucleic acid molecule to the surface can be achieved using any suitable method, including the exemplary methods described herein. In some embodiments, the target nucleic acid molecule is linked directly to the surface; in other embodiments, the target nucleic acid molecule is paired (for example, through Watson-Crick bonding or hybridization) to another nucleic acid molecule, for example a primer that is directly linked to the surface. The target nucleic acid molecules, primers, and/or oligonucleotides can be modified at their 5' or 3' end, or internally, to carry a reactive group that can bind to a reactive group on the surface.

Typically, the surface is treated or untreated to provide reactive groups such as silanol, carboxyl, amino, epoxide, and methacryl groups. The nucleic acid molecules can be treated or untreated to provide reactive groups including: amino, hydroxyl, thiol, and disulfide. The nucleic acid molecules can include non-natural nucleotides having reactive group that will attach to a surface reactive group. For example, the non-natural nucleotides include peptide nucleic acids, locked nucleic acids, oligonucleotide N3'→ P5' phosphoramidates, and oligo-2'-0-alkylribonucleotides.

[00196] In one aspect, nucleic acid molecules (for example, the target nucleic acid molecule, the template strand and/or an oligonucleotide primer) can be modified with one or more amino groups at the 5' or 3' end, or internally, can be attached to modified surfaces.

[00197] In another aspect, the nucleic acid molecules can be attached at their 5' ends with one or more amino groups, including: a simple amino group; a short or long tethering arm having one or more terminal amino groups; or amino-modified thymidine or cytosine. The tethering arms can be linear or branched, have various lengths, charged or uncharged, hydrophobic, flexible, cleavable, or have one or multiple terminal amino groups. The number of plural valent atoms in a tethering arm may be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30 or a larger number up to 40 or more.

[00198] In another aspect, the 3' end of nucleic acid molecules can be modified to carry an amino group. Typically, the amino group is initially protected by a fluorenylmethylcarbamoyl (FMOC) group. To expose the amino group, the protecting group can be removed and acylated with an appropriate succinimidyl ester, such as an N-hydroxy succinimidyl ester (NHS ester). [00199] In another aspect, the nucleic acid molecules can carry internal amino groups for binding to the solid surface. For example, 2' amino modified nucleic acid molecules can be produce by methoxyoxalamido (MOX) or succinyl (SUC) chemistry to produce nucleotides having amino linkers attached at the 2' C of the sugar moiety.

[00200] In another aspect, the surface can be modified to bind the amino modified nucleic acid molecules. For example, 5' amino-modified nucleic acid molecules can be attached to surfaces modified with silane, such as epoxy silane derivatives (J. B. Lamture, et al., 1994 Nucleic Acids Res. 22:2121-2125; W. G. Beattie et al., 1995 Mol. Biotechnol. 4:213-225) or isothiocyanate (Z. Guo, et al., 1994 Nucleic Acids Res. 22:5456-5465). Acylating reagents can be used to modify the surface for attaching the amino-modified nucleic acid molecules. The acylating reagents include: isothiocyanates, succinimidyl ester, and sulfonyl chloride. The amino-modified nucleic acid molecules can attach to surface amino groups that have been converted to amino reactive phenylisothiocyanate groups by treating the surface with p-phenylene 1,4 diisothiocyanate (PDC). In other methods, the surface amino groups can be reacted with homobifunctional crosslinking agents, such as disuccinimidylcaronate (DCS), disuccinimidyloxalate (DSO),

phenylenediisothiocyanate (PDITC) or dimethylsuberimidate (DMS) for attachment to the amino- modified nucleic acid molecules. In another example, metal and metal oxide surfaces can be modified with an alkoxysilane, such as 3-aminopropyltriethoxysilane (APTES) or

glycidoxypropyltrimethoxysilane (GOPMS).

[00201] In another aspect, succinylated nucleic acid molecules can be attached to aminophenyl- or aminopropyl-modified surfaces (B. Joos et al., 1997 Anal. Biochem. 247: 96-101).

[00202] In yet another aspect, a thiol group can be placed at the 5' or 3' end of the nucleic acid molecules. The thiol group can form reversible or irreversible disulfide bonds with the surface. The thiol attached to the 5' or 3' end of the nucleic acid molecule can be a phosphoramidate. The phosphoramidate can be attached to the 5' end using S-trityl-6-mercaptohexyl derivatives.

[00203] In another aspect, the thiol-modified nucleic acid molecules can be attached to a surface using heterobifunctional reagents (e.g. cross linkers). For example, the surface can be treated with an alkylating agent such as iodoacetamide or maleimide for linking with thiol modified nucleic acid molecules. In another example, silane-treated surfaces (e.g., glass) can be attached with thiol-modified nucleic acid molecules using succinimidyl 4-(malemidophenyl)butyrate (SMPB). [00204] In another aspect, the nucleic acid molecule can be modified to carry disulfide groups can be attached to thiol-modified surfaces (Y. H. Rogers et al., 1999 Anal. Biochem. 266:23-30).

[00205] Still other aspects include methods that employ modifying reagents such as:

carbodiimides (e.g., dicyclohexylcarbodiimide, DCC), carbonyldiimidazoles (e.g.,

carbonyldiimidazole, CDI_Z), and potassium periodate. The nucleic acid molecules can have protective photoprotective caps (Fodor, U.S. Patent No. 5,510,270) capped with a photoremovable protective group. DMT-protected nucleic acid molecules can be immobilized to the surface via a carboxyl bond to the 3' hydroxyl of the nucleoside moiety (Pease, U.S. Pat. No. 5,599,695; Pease et al., 1994 Proc. Natl. Acad. Sci. USA 91(l l):5022-5026). The nucleic acid molecules can be functionalized at their 5' ends with activated 1-O-mimethoxytrityl hexyl disulfide l'-[(2- cyanoethyl)-N,N-diisopropyl)] phosphoramidate (Rogers et al., 1999 Anal. Biochem. 266:23). Exemplary methods of attaching nucleic acid molecules to suitable substrates are disclosed, for example, in Schwartz, U.S. Patent Nos. 6,221,592, 6,294,136 and U.S. Published App. Nos.

2006/0275806 and 2007/0161028 (Schwartz et al.). Linking agents, can be symmetrical bifunctional reagents, such as bis succinimide (e.g., bis-N-hydroxy succinimide) and maleimide (bis-N-hydroxy maleimide) esters, or toluene diisocyanate can be used. Heterobifunctional cross- linkers include: m-maleimido benzoyl-N-hydroxy succinimidyl ester (MBS); succinimidyl-4-(p- maleimido phenyl) -Butyrate (SMPB); and succinimidyl-4-(N-Maleimidomethyl)Cyclohexane-l- Carboxylate (SMCC) (L. A. Chrisey et al., 1996 Nucleic Acids Res. 24:3031-3039). In one example, a glass surface can be layered with a gold (e.g., about 2 nm layer) that is reacted with mercaptohexanoic acid. The mercaptohexanoic acid can be placed in a patterned array. The mercaptohexanoic acid can be reacted with PEG. The PEG can be reacted to bind nucleic acid molecules such as the target nucleic acid molecules.

[00206] In another aspect, the target nucleic acid molecule can be linked to an amine- functionalized solid surface. In one embodiment, the amine-functionalized solid surface can be a spot surrounded by PEG molecules, where the target molecule preferentially binds the amine- functionalized spots (see Fry, et al., U.S. Serial No. 61/245,248, filed on September 23, 2009).

[00207] In some embodiments, the target nucleic acid molecule is not linked to any surface, but some other component of the sequencing reaction is linked to a surface. For example, the polymerase can be linked to the surface using any suitable method. In some embodiments, the polymerase is linked to a first member of a binding pair, the surface is linked to a second member of the binding pair, and the polymerase and surface are contacted with each other under conditions where the first and second members bind to each other, thereby linking the polymerase to the surface. The binding pair can include avidin and biotin.

[00208] In some embodiments, the labels can be selected so that each absorbs excitation radiation and/or emits fluorescence at a wavelength distinguishable from the other labels to permit monitoring the presence of different labels in the same reaction. Two or more different labels can be selected having spectrally distinct emission profiles, or having minimal overlapping spectral emission profiles.

[00209] In one aspect, the signals (e.g., sequence- specific signals) from the different labels do not significantly overlap or interfere, by quenching, colorimetric interference, or spectral interference.

[00210] In some embodiments, the label can include any one or more of the following types of moieties: a chromophore moiety, a chemiluminescent moiety, a fluorigenic moiety and a fluorescent moiety.

[00211] The chromophore moiety may be 5-bromo-4-chloro-3-indolyl phosphate, 3-indoxyl phosphate, p-nitrophenyl phosphate, β-lactamase, peroxidase-based chemistry, and derivatives thereof.

[00212] The chemiluminescent moiety may be a phosphatase-activated 1,2-dioxetane compound. The 1,2-dioxetane compound includes disodium 2-chloro-5-(4-methoxyspiro[l,2-dioxetane-3,2'- (5-chloro-)tricyclo[3,3,l-l^3,7 ]-decan]-l-yl)-l -phenyl phosphate (e.g., CDP-STAR) ,

chloroadamant-2'-ylidenemethoxyphenoxy phosphorylated dioxetane (e.g., CSPD) , and 3-(2'- spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (e.g., AMPPD).

[00213] In some embodiments, the fluorescent moiety can optionally include: rhodols; resorufins; coumarins; xanthenes; acridines; fluoresceins; rhodamines; erythrins; cyanins; phthalaldehydes; naphthylamines; fluorescamines; benzoxadiazoles; stilbenes; pyrenes; indoles;

borapolyazaindacenes; quinazolinones; eosin; erythrosin; Malachite green; CY dyes (GE

Biosciences), including Cy3 (and its derivatives) and Cy5 (and its derivatives); DYOMICS and DYLIGHT dyes (Dyomics) including DY-547, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680, DY-682, DY-701, DY-734, DY-752, DY- 777 and DY-782; Lucifer Yellow; CASCADE BLUE ; TEXAS RED; BODIPY (boron- dipyrromethene) (Molecular Probes) dyes including BODIPY 630/650 and BODIPY 650/670; ATTO dyes (Atto-Tec) including ATTO 390, ATTO 425, ATTO 465, ATTO 610 61 IX, ATTO 610 (N-succinimidyl ester), ATTO 635 (NHS ester); ALEXA FLUORS including ALEXA FLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750, and ALEXA FLUOR 680 (Molecular Probes); DDAO (7-hydroxy-9H-(l,3-dichloro- 9,9-dimethylacridin-2-one or any derivatives thereof) (Molecular Probes); QUASAR dyes (Biosearch); IRDYES dyes (LiCor) including IRDYE 700DX (NHS ester), IRDYE 800RS (NHS ester) and IRDYE 800CW (NHS ester); EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes (Applied Biosystems); HILYTE dyes (AnaSpec); MR121 and MR200 dyes (Roche); Hoechst dyes 33258 and 33242 (Invitrogen); FAIR OAKS RED (Molecular Devices); SUNNYVALE RED (Molecular Devices); LIGHT CYCLER RED (Roche); EPOCH (Glen Research) dyes including EPOCH REDMOND RED (phosphoramidate), EPOCH YAKIMA YELLOW (phosphoramidate), EPOCH GIG HARBOR GREEN (phosphoramidate); Tokyo green (M. Kamiya, et al, 2005 Angew. Chem. Int. Ed. 44:5439-5441); and CF dyes including CF 647 and CF555 (Biotium).

[00214] Quencher dyes may include: ATTO 540Q, ATTO 580Q, and ATTO 612Q (Atto-Tec); QSY dyes including QSY 7, QSY 9, QSY 21, and QSY 35 (Molecular Probes); and EPOCH ECLIPSE QUENCHER (phosphoramidate) (Glen Research). The fluorescent moiety can be a 7- hydroxycoumarin-hemicyanine hybrid molecule that is a far-red emitting dye (Richard 2008 Org. Lett. 10:4175-4178).

[00215] The fluorescent moiety may be a fluorescence-emitting metal such as a lanthanide complex, including those of Europium and Terbium.

[00216] A number of examples of fluorescent moieties are found in PCT publication

WO/2008/030115, and in Haugland, Molecular Probes Handbook, (Eugene, Oregon) 6th Edition; The Synthegen catalog (Houston, Tex.), Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999).

[00217] In some embodiments, one or more components of the sequencing reaction is labeled with an energy transfer moiety, for example a FRET donor or a FRET acceptor. In some embodiments, at least one energy transfer moiety includes a FRET quencher. Typically, quenchers have an absorption spectrum with large extinction coefficients, however the quantum yield for quenchers is reduced, such that the quencher emits little to no light upon excitation. Quenching can be used to reduce the background fluorescence, thereby enhancing the signal-to- noise ratio. In one embodiment, energy transferred from the donor may be absorbed by the quencher that emits moderated (e.g., reduced) fluorescence. For example, the acceptor can be a non-fluorescent chromophore that absorbs the energy transferred from the donor and emits heat (e.g., the energy acceptor is a dark quencher). In another example, a quencher can be used as an energy acceptor with a nanoparticle donor in a FRET system, see I.L. Medintz, et al., 2003 Nature Materials 2:630. Yet another example involves the use of quenchers in conjunction with reporters comprising fluorescent labels. In this example, certain nucleotides in the sequencing reaction are labeled with a reporter comprising a fluorescent label, while the remaining nucleotides are labeled with one or more quenchers. Alternatively, each of the nucleotides in the reaction mixture can be labeled with one or more quenchers. Discrimination of the nucleotide bases is based on the wavelength and/or intensity of light emitted from the FRET acceptor, as well as the intensity of light emitted from the FRET donor. If no signal is detected from the FRET acceptor, a corresponding reduction in light emission from the FRET donor indicates incorporation of a nucleotide labeled with a quencher. The degree of intensity reduction may be used to distinguish between different quenchers.

[00218] Examples of fluorescent donors and non-fluorescent acceptor (e.g., quencher) combinations have been developed for detection of proteolysis (Matayoshi 1990 Science 247:954- 958) and nucleic acid hybridization (L. Morrison, in: Nonisotopic DNA Probe Techniques, ed., L. Kricka, Academic Press, San Diego, (1992) pp. 31 1-352; S. Tyagi 1998 Nat. Biotechnol. 16:49- 53; S. Tyagi 1996 Nat. Biotechnol. 14:947-8). FRET donors, acceptors and quenchers can be moieties that absorb electromagnetic energy (e.g., light) at about 300-900 nm, or about 350-800 nm, or about 390-800 nm.

[00219] Energy transfer donor and acceptor moieties can be made from materials that typically fall into four general categories (see the review in: K. E. Sapford, et al., 2006 Angew. Chem. Int. Ed. 45:4562-4588), including: (1) organic fluorescent dyes, dark quenchers and polymers (e.g., dendrimers); (2) inorganic material such as metals, metal chelates and semiconductors

nanoparticles; (3) biomolecules such as proteins and amino acids (e.g., green fluorescent protein and derivatives thereof); and (4) enzymatically catalyzed bioluminescent molecules. The material for making the energy transfer donor and acceptor moieties can be selected from the same or different categories.

[00220] The FRET donor and acceptor moieties can include traditional dyes that emit in the UV, visible, or near-infrared region. The UV emitting dyes include coumarin-, pyrene-, and naphthalene-related compounds. The visible and near-infrared dyes include

xanthene-, fluorescein-, rhodol-, rhodamine-, and cyanine-related compounds. The fluorescent dyes also includes DDAO ((7-hydroxy-9H-(l,3-dichloro-9,9-dimethylacridin-2-one)), resorufin, ALEXA FLUOR and BODIPY dyes (both Molecular Probes), HILYTE Fluors (AnaSpec), ATTO dyes (Atto-Tec), DY dyes (Dyomics GmbH), TAMRA (Perkin Elmer), tetramethylrhodamine (TMR), TEXAS RED, DYLIGHT (Thermo Fisher Scientific), FAM (AnaSpec), JOE and ROX (both Applied Biosystems), and Tokyo Green.

[00221] Additional fluorescent dyes that can be used as quenchers includes: DNP, DABSYL, QSY (Molecular Probes), ATTO (Atto-Tec), BHQ (Biosearch Technologies), QXL (AnaSpec), BBQ (Berry and Associates) and CY5Q/7Q (Amersham Biosciences).

[00222] The FRET donor and acceptor moieties that comprise inorganic materials include gold (e.g., quencher), silver, copper, silicon, semiconductor nanoparticles, and fluorescence-emitting metal such as a lanthanide complex, including those of Europium and Terbium.

[00223] Suitable FRET donor/acceptor pairs include: FAM as the donor and JOE, TAMRA, and ROX as the acceptor dyes. Other suitable pairs include: CYA as the donor and R6G, TAMRA, and ROX as the donor dyes. Other suitable donor/acceptor pairs include: a nanoparticle as the donor, and ALEXA FLUORS dyes (e.g., 610, 647, 660, 680, 700). DYOMICS dyes, such as 634 and 734 can be used as energy transfer acceptor dyes.

[00224] In some embodiments, both the polymerase and the nucleotide are labeled, and the label of the incorporating (or soon to be incorporated) labeled nucleotide interacts with the polymerase label, for example via RET, to produce the sequence-specific signal, while the labels of unincorporated nucleotides do not interact significantly with the polymerase label.

[00225] In some embodiments, the sequence-specific signal results from an interaction, for example resonance energy transfer or RET, between two or more labels in the sequencing reaction. The two or more labels can include a donor/acceptor pair, where the donor/acceptor pair includes an energy transfer donor and an energy transfer acceptor. The interaction can include FRET. The interaction can occur between a nucleotide label and a polymerase label. The signal resulting from the interaction can be emitted shortly before, during, and/or after the incorporation of the nucleotide by the polymerase. In some embodiments, the sequencing reaction includes nucleotides and polymerases labeled with energy transfer donor and acceptor moieties. The energy transfer donor and acceptor moieties can fluoresce in response to exposure to an excitation source, such as electromagnetic radiation. The energy transfer acceptor moiety can fluoresce in response to energy transferred from a proximal excited energy transfer donor moiety. The energy transfer can occur via RET, for example via FRET. The proximal distance between the donor and acceptor moieties that accommodates energy transfer can be dependent upon the particular donor/acceptor pair. The proximal distance between the donor and acceptor moieties can be about 1-20 nm, or about 1-10 nm, or about 1-5 nm, or about 5-10 nm. In another embodiment, the energy transfer signal generated by proximity of the donor moiety to the acceptor moiety can remain unchanged. In another embodiment, the proximity of the donor moiety to the acceptor moiety results in changes in the sequence- specific signal. In another embodiment, the changes in the sequence specific signals from the donor or acceptor moiety can include changes in the:

intensity of the signal; duration of the signal; wavelength of the signal; amplitude of the signal; polarization state of the signal; duration between the signals; and/or rate of the change in intensity, duration, wavelength or amplitude. In another embodiment, the change in the sequence- specific signal can include a change in the ratio of the change of the energy transfer donor signal relative to change of the energy transfer acceptor signals. In another embodiment, the signal from the donor can increase or decrease. In another embodiment, the signal from the acceptor can increase or decrease. In another embodiment, the sequence-specific signal associated with nucleotide incorporation includes: a decrease in the donor signal when the donor is proximal to the acceptor; an increase in the acceptor signal when the acceptor is proximal to the donor; an increase in the donor signal when the distance between the donor and acceptor increases; and/or a decrease in the acceptor signal when the distance between the donor and acceptor increases.

[00226] The nature of the method used to detect the sequence-specific signal can vary depending on the nature of the sequencing reaction itself. Typically, the sequence-specific signal results from a nucleotide incorporation within a sequencing reaction that includes a population of template strands and a population of nucleotides. Multiple nucleotide incorporations can occur within the sequencing reaction simultaneously, and each such incorporation can result in the emission of a sequence-specific signal. The detection requirements for single molecule sequencing methods, wherein multiple different template strands are sequenced in isolation or in parallel, can be quite different from the detection requirements for "bulk" or population-based sequencing methods wherein a population of identical template strands are sequenced collectively. For example, population-based sequencing methods typically involve template-dependent replication of a population of identical template strands, where the sequencing reaction is synchronized such that identical nucleotides are incorporated simultaneously into a population of identical nascent nucleic acid molecules. Each such identical nucleotide incorporation gives rise to an identical sequence- specific signal. Since the sequence- specific signals are identical to each other, detection of the sequence-specific signal can be accomplished simply by detecting the aggregate signal emitted from the sequencing reaction as a whole. Furthermore, it is not necessary to correlate the signal with any particular template strand in the population, since all of the template strands are identical. Because the sequencing reaction must be synchronized between different template strands, synthesis of the nascent nucleic acid molecule by each polymerase within the population of typically proceeds discontinuously, where synthesis is paused or halted after each nucleotide incorporation in order to ensure that the reaction is driven to completion and to allow detection and identification of the incorporated nucleotide prior to addition of the next nucleotide. The detection methods associated with such population-based sequencing methods typically cannot distinguish between incorporated and unincorporated nucleotides within a sequencing reaction, or between nucleotides incorporated into different template molecules. Such processes therefore typically require wash steps after each template-dependent nucleotide incorporation to remove unincorporated background signal prior to the detection of the sequence- specific signal.

Polymerization or nascent nucleic acid molecule synthesis is typically performed prior to and separately from the detection of the sequence-specific signal, and detection is performed only after the polymerizing or nascent nucleic acid molecule synthesis is complete.

[00227] Alternatively, when the sequencing reaction is a single molecule sequencing reaction, the sequence-specific signal indicating a template-dependent nucleotide incorporation can result from the action of a single polymerase on a single template strand. Such single molecule methods can proceed asynchronously, as each template strand within a sample population can be monitored separately. Because there is no need to synchronize the population, synthesis of the nascent nucleic acid molecule by each polymerase typically proceeds continuously. The detection methods associated with such single molecule sequencing methods can typically distinguish between incorporated and unincorporated nucleotides within a sequencing reaction, or between two nucleotides incorporated into different template molecules. Such processes therefore do not typically require washes between each template-dependent nucleotide incorporation because there is no need to remove unincorporated background signal prior to the detection of the sequence- specific signal. Detection of the sequence- specific signal can be performed while polymerization or synthesis of the nascent nucleic acid molecule is ongoing.

[00228] In some embodiments of the disclosed methods, sequencing includes separately catalyzing nucleotide incorporation and detecting a sequence-specific signal, such that catalysis does not occur during detection, and detection does not occur during nucleotide incorporation. In some embodiments, the detecting and synthesizing of the nascent nucleic acid molecule are performed separately. For example, many sequencing methods can involve wash steps wherein unincorporated nucleotides (which can contribute to background signal) are removed from the reaction, following which the nucleotide incorporated at the end of the nascent nucleic acid molecule is detected.

[00229] Other sequencing methods wherein detection typically occurs separately from nucleotide incorporation, or nucleic acid synthesis, can include methods using nucleotides that comprise blocking groups. Blocking groups include any chemical group or moiety that can delay, slow, inhibit, impede or altogether prevent extension of a nucleic acid. Typically, incorporation of a nucleotide including a blocking group into the nascent nucleic acid molecule can delay, slow, inhibit, impede or altogether prevent further incorporation of nucleotides into the nascent nucleic acid molecule. In some embodiments, the blocking group is a chain terminating group, for example a reversible or irreversible terminator. Alternatively, the blocking group can simply slow or delay the incorporation of the next nucleotide, such slowing or delay being measured relative to a nucleotide lacking the blocking group, Such delay can be on the order of milliseconds or seconds. The blocking group can be attached to any suitable position of the nucleotide. For example, the blocking group can be attached to the 2', 3' or 5' position of the sugar moiety of the nucleotide. In some embodiments, the blocking group can be attached to the base moiety of the nucleotide, or the phosphate chain of the nucleotide.

[00230] Once the nucleotide including the blocking group is incorporated, further incorporation of the nucleotides can optionally be delayed until the blocking effect of the blocking group is disrupted or otherwise removed. In some embodiments, the blocking effect can be removed by cleavage and removal of the blocking group from the incorporated nucleotide. For example, some blocking groups can be removed via photocleavage, enzymatic cleavage, or chemical cleavage. Such removal can optionally be performed after the incorporated nucleotide including the blocking group is detected and/or identified. The nucleotide including the blocking group can optionally also include one or more detectable labels; in some embodiments, the blocking group itself can serve as the detectable label.

[00231] In some embodiments of the disclosed methods, sequencing includes detecting the sequence-specific signal while nucleic acid synthesis is ongoing. Such detecting can include detecting a signal emitted by a labeled nucleotide in the sequencing reaction. Such detection methods typically include discriminating between the incorporated (or soon-to-be-incorporated) labeled nucleotide and the unincorporated labeled nucleotides in the reaction mixture. In one exemplary embodiment, the polymerase-template complex is confined in such a manner that the detection is exclusively focused on a small region around the site of polymerization. For example, use of a zero-mode waveguide can enable the detection of sequence-specific signals emitted by labeled nucleotides as they are polymerized into a nascent nucleic acid molecule within the waveguide.

[00232] In some embodiments, detecting the sequence- specific signal can be performed using any suitable detection technique, for example, confocal laser scanning microscopy, Total Internal Reflection (TIR), Total Internal Reflection Fluorescence (TIRF), near-field scanning microscopy, far-field confocal microscopy, wide-field epi-illumination, light scattering, dark field microscopy, photoconversion, wide field fluorescence, single and/or multi-photon excitation, spectral wavelength discrimination, evanescent wave illumination, scanning two-photon, scanning wide field two-photon, Nipkow spinning disc, and/or multi-foci multi-photon.

[00233] Optionally, the detecting includes eliminating or reducing background, or "noise", that is not indicative of a nucleotide incorporation. In some embodiments, the sequence- specific signal can be distinguished from background signals by measuring, analyzing and characterizing attributes of all signals emitted by the sequencing reaction. In one embodiment, attributes of the sequence-specific signal that can permit distinction from the background signals can include: duration; wavelength; amplitude; photon count; and/or the rate of change of the duration, wavelength, amplitude; and/or photon count. In one embodiment, detecting the sequence-specific signal can include measuring, analyzing and characterizing attributes of: duration; wavelength; amplitude; photon count and/or the rate of change of the duration, wavelength, amplitude; and/or photon count.

[00234] In some embodiments, the methods, compositions, systems and kits disclosed herein can involve the use of one or more labels comprising at least one nanoparticle. The nanoparticle can be any suitable nanoparticle capable of contributing to the generation of a sequence-specific signal within the sequencing reaction. For example, the nanoparticle can optionally act as a donor fluorophore in an energy transfer reaction such as FRET.

[00235] The nanoparticle can be attached to the solid surface or to any component of the nucleotide incorporation or nucleotide polymerization reactions in any combination (e.g., polymerases, nucleotides, target nucleic acid molecules, primers, and/or oligonucleotides).

[00236] "Nanoparticle" may refer to any particle with at least one major dimension in the nanosize range. In general, nanoparticles can be made from any suitable metal (e.g., noble metals, semiconductors, etc.) and/or non-metal atoms. Nanoparticles can have different shapes, each of which can have distinctive properties including spatial distribution of the surface charge; orientation dependence of polarization of the incident light wave; and spatial extent of the electric field. The shapes include, but are not limited to: spheres, rods, discs, triangles, nanorings, nanoshells, tetrapods, nanowires, etc.

[00237] In one embodiment, the nanoparticle can be a core/shell nanoparticle that typically comprises a core nanoparticle surrounded by at least one shell. For example, the core/shell nanoparticle can be surrounded by an inner and outer shell. In another embodiment, the nanoparticle is a core nanoparticle that has a core but no surrounding shell. The outmost surface of the core or shell can be coated with tightly associated ligands that are not removed by ordinary solvation.

[00238] Examples of a nanoparticle include a nanocrystal, such as a core/shell nanocrystal, plus any associated organic ligands (which are not removed by ordinary solvation) or other materials which may coat the surface of the nanocrystal. In one embodiment, a nanoparticle has at least one major dimension ranging from about 1 to about 1000 nm. In other embodiments, a nanoparticle has at least one major dimension ranging from about 1 to about 20 nm, about 1 to about 15 nm, about 1 to about 10 nm or about 1 to 5 nm.

[00239] In some embodiments, a nanoparticle can have a layer of ligands on its surface which can further be cross-linked to each other. In some embodiments, a nanoparticle can have other or additional surface coatings which can modify the properties of the particle, for example, increasing or decreasing solubility in water or other solvents. Such layers on the surface are included in the term 'nanoparticle.'

[00240] In one embodiment, nanoparticle can refer to a nanocrystal having a crystalline core, or to a core/shell nanocrystal, and may be about 1 nm to about 100 nm in its largest dimension, about 1 nm to about 20 nm, about 1 nm to about 15 nm, about 1 nm to about lOnm or preferably about 5 nm to about lOnm in its largest dimension. Small nanoparticles are typically less than about 20nm in their largest dimension.

[00241] "Nanocrystal" as used herein can refer to a nanoparticle made out of an inorganic substance that typically has an ordered crystalline structure. It can refer to a nanocrystal having a crystalline core (core nanocrystal) or to a core/shell nanocrystal.

[00242] A core nanocrystal is a nanocrystal to which no shell has been applied. Typically, it is a semiconductor nanocrystal that includes a single semiconductor material. It can have a homogeneous composition or its composition can vary with depth inside the nanocrystal. [00243] A core/shell nanocrystal is a nanocrystal that includes a core nanocrystal and a shell disposed over the core nanocrystal. Typically, the shell is a semiconductor shell that includes a single semiconductor material. In some embodiments, the core and the shell of a core/shell nanocrystal are composed of different semiconductor materials, meaning that at least one atom type of a binary semiconductor material of the core of a core/shell is different from the atom types in the shell of the core/shell nanocrystal.

[00244] The semiconductor nanocrystal core can be composed of a semiconductor material (including binary, ternary and quaternary mixtures thereof), from: Groups II- VI of the periodic table, including ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe; Groups III-V, including GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, A1P, AlSb, A1S; and/or Group IV, including Ge, Si, Pb.

[00245] The semiconductor nanocrystal shell can be composed of materials (including binary, ternary and quaternary mixtures thereof) comprising: ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe, GaAs, GaN, GaP, GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP, InSb, AlAs, AIN, A1P, or AlSb.

[00246] Many types of nanocrystals are known, and any suitable method for making a nanocrystal core and applying a shell to the core may be employed. Nanocrystals can have a surface layer of ligands to protect the nanocrystal from degradation in use or during storage.

[00247] "Quantum dot" as used herein refers to a crystalline nanoparticle made from a material which in the bulk is a semiconductor or insulating material, which has a tunable photophysical property in the near ultraviolet (UV) to far infrared (IR) range.

[00248] "Water-soluble" or "water-dispersible" is used herein to mean the item can be soluble or suspendable in an aqueous-based solution, such as in water or water-based solutions or buffer solutions, including those used in biological or molecular detection systems as known by those skilled in the art. While water-soluble nanoparticles are not truly 'dissolved' in the sense that term is used to describe individually solvated small molecules, they are solvated (via hydrogen, electrostatic or other suitable physical/chemical bonding) and suspended in solvents that are compatible with their outer surface layer, thus a nanoparticle that is readily dispersed in water is considered water-soluble or water-dispersible. A water-soluble nanoparticle can also be considered hydrophilic, since its surface is compatible with water and with water solubility. [00249] "Hydrophobic nanoparticle" as used herein refers to a nanoparticle that is readily dispersed in or dissolved in a water-immiscible solvent like hexanes, toluene, and the like. Such nanoparticles are generally not readily dispersed in water.

[00250] "Hydrophilic" as used herein refers to a surface property of a solid, or a bulk property of a liquid, where the solid or liquid exhibits greater miscibility or solubility in a high-dielectric medium than it does in a lower dielectric medium. By way of example, a material that is more soluble in methanol than in a hydrocarbon solvent such as decane would be considered hydrophilic.

[00251] "Coordinating solvents" as used herein refers to a solvent such as TDPA, OP, TOP, TOPO, carboxylic acids, and amines, which are effective to coordinate to the surface of a nanocrystal. 'Coordinating solvents' also include phosphines, phosphine oxides, phosphonic acids, phosphinic acids, amines, and carboxylic acids, which are often used in growth media for nanocrystals, and which form a coating or layer on the nanocrystal surface. Coordinating solvents can exclude hydrocarbon solvents such as hexanes, toluene, hexadecane, octadecene and the like, which do not have heteroatoms that provide bonding pairs of electrons to coordinate with the nanocrystal surface. Hydrocarbon solvents which do not contain heteroatoms such as O, S, N or P to coordinate to a nanocrystal surface are referred to herein as non-coordinating solvents. Note that the term 'solvent' is used in its ordinary way in these terms: it refers to a medium which supports, dissolves or disperses materials and reactions between them, but which does not ordinarily participate in or become modified by the reactions of the reactant materials. However, in certain instances, the solvent can be modified by the reaction conditions. For example, TOP may be oxidized to TOPO, or a carboxylic acid can be reduced to an alcohol.

[00252] As used herein, the term "population" refers to a plurality of nanoparticles having similar physical and/or optical properties. 'Population' can refer to a solution or structure with more than one nanoparticle at a concentration suitable for single molecule analysis. In some embodiments, the population can be monodisperse and can exhibit less than at least 15% rms deviation in diameter of the nanoparticles, and spectral emissions in a narrow range of no greater than about 75 nm full width at half max (FWHM). In the context of a solution, suspension, gel, plastic, or colloidal dispersion of nanoparticles, the nature of the population can be further characterized by the number of nanoparticles present, on average, within a particular volume of the liquid or solid, or the concentration. In a two-dimensional format such as an array of nanoparticles adhered to a solid substrate, the concept of concentration is less convenient than the related measure of particle density, or the number of individual particles per two-dimensional area. In this case, the maximum density would typically be that obtained by packing particles "shoulder-to-shoulder" in an array. The actual number of particles in this case would vary due to the size of the particles - a given array could contain a large number of small particles or a small number of larger particles.

[00253] As used herein, the terms "moderate to high excitation" refers to monochromatic illumination or excitation (e.g., laser illumination) having a high power intensity sufficiently high such that the absorbed photons per second for a given sample is between about 200,000 and about 1,600,000.

[00254] In one aspect, the nanoparticle is a semiconductor nanoparticle having size-dependent optical and electronic properties. For example, the nanoparticle can emit a fluorescent signal in response to excitation energy. The spectral emission of the nanoparticle can be tunable to a desired energy by selecting the particle size, size distribution, and/or composition of the semiconductor nanoparticle. For example, depending on the dimensions, the semiconductor nanoparticle can be a fluorescent nanoparticle which emits light in the UV-visible-IR spectrum. The shell material can have a bandgap greater than the bandgap of the core material.

[00255] In one aspect, the nanoparticle is an energy transfer donor. The nanoparticle can be excited by an electromagnetic source such as a laser beam, multi-photon excitation, or electrical excitation. The excitation wavelength can range between about 190 to about 800 nm including all values and ranges there in between. In some embodiments, the nanoparticle can be excited by an energy source having a wavelength of about 405 nm. In other embodiments, in response to excitation, the nanoparticle can emit a fluorescent signal at about 400-800 nm, or about 605 nm.

[00256] In one aspect, the nanoparticle can undergo Raman scattering when subjected to an electromagnetic source (incident photon source) such as a laser beam. The scattered photons have a frequency that is different from the frequency of the incident photons. As result, the wavelength of the scattered photons is different than the incident photon source. In one embodiment, the nanoparticle can be attached to a suitable tag or label to enhance the detectability of the nanoparticle via Raman spectroscopy. The associated tag can be fluorescent or nonfluorescent. Such approaches can be advantageous in avoiding problems that can arise in the context of fluorescent nanoparticles, such as photobleaching and blinking. See, e.g. , Sun et al., "Surface- Enhanced Raman Scattering Based Nonfluorescent Probe for Multiplex DNA Detection", Anal. Chem. 79(l l):3981-3988 (2007) [00257] In one aspect, the nanoparticle is comprised of a multi- shell layered core which is achieved by a sequential shell material deposition process, where one shell material is added at a time, to provide a nanoparticle having a substantially uniform shell of desired thickness which is substantially free of defects. The nanoparticle can be prepared by sequential, controlled addition of materials to build and/or applying layers of shell material to the core. See e.g., U.S. PCT Application Serial No. PCT/US09/061951 which is incorporated herein by reference as if set forth in full.

[00258] In another aspect, a method is provided for making a nanoparticle comprising a core and a layered shell, where the shell comprises at least one inner shell layer and at least one outer shell layer. The method comprises the steps: (a) providing a mixture comprising a core, at least one coordinating solvent; (b) heating the mixture to a temperature suitable for formation of an inner shell layer; (c) adding a first inner shell precursor alternately with a second inner shell precursor in layer additions, to form an inner shell layer which is a desired number of layers thick; (d) heating the mixture to a temperature suitable for formation of an outer shell layer; and (e) adding a first outer shell precursor alternately with a second outer shell precursor in layer additions, to form an outer shell layer which is a desired number of layers thick. In one embodiment, if the coordinating solvent of (a) is not amine, the method further comprises an amine in (a).

[00259] In one aspect, at least one coordinating solvent comprises a trialkylphosphine, a trialkylphosphine oxide, phosphonic acid, or a mixture of these. In another aspect, at least one coordinating solvent comprises trioctylphosphine (TOP), trioctylphosphine oxide (TOPO), tetradecylphosphonic acid (TDPA), or a mixture of these. In yet another aspect, the coordinating solvent comprises a primary or secondary amine, for example, decylamine, hexadecylamine, or dioctylamine.

[00260] In one aspect, the nanoparticle comprises a core comprising CdSe. In another aspect, the nanoparticle shell can comprise YZ wherein Y is Cd or Zn, and Z is S, or Se. In one embodiment, at least one inner shell layer comprises CdS, and the at least one outer shell layer comprises ZnS.

[00261] In one aspect, the first inner shell precursor is Cd(OAc)₂ and the second inner shell precursor is bis(trimethylsilyl)sulfide (TMS₂S). In other aspects, the first and second inner shell precursors are added as a solution in trioctylphosphine (TOP). In other aspects, the first outer shell precursor is diethylzinc (Et₂Zn) and the second inner shell precursor is dimethyl zinc (TMS₂S). Sometimes, the first and second outer shell precursors are added as a solution in trioctylphosphine (TOP). [00262] In one aspect, the nanoparticle can have ligands which coat the surface. The ligand coating can comprise any suitable compound(s) which provide surface functionality (e.g. , changing physicochemical properties, permitting binding and/or other interaction with a biomolecule, etc.). In some embodiments, the disclosed nanoparticle has a surface ligand coating (in direct contact with the external shell layer) that adds various functionalities which facilitate it being water-dispersible or soluble in aqueous solutions. There are a number of suitable surface coatings which can be employed to permit aqueous dispersibility of the described nanoparticle. For example, the nanoparticle(s) disclosed herein can comprise a core/shell nanocrystal which is coated directly or indirectly with lipids, phospholipids, fatty acids, polynucleic acids, polyethylene glycol (PEG), primary antibodies, secondary antibodies, antibody fragments, protein or nucleic acid based aptamers, biotin, streptavidin, proteins, peptides, small organic molecules (e.g. , ligands), organic or inorganic dyes, precious or noble metal clusters. Specific examples of ligand coatings can include, but are not limited to, amphiphilic polymer (AMP), bidentate thiols (i.e. , DHLA), tridentate thiols, dipeptides, functionalized organophosphorous compounds (e.g., phosphonic acids, phosphinic acids), etc.

Non-Blinking Nanoparticles

[00263] Provided herein are nanoparticles which exhibit modulated, reduced, or no intermittent (e.g., continuous, non-blinking) fluorescence.

[00264] In one aspect, the nanoparticle or populations thereof exhibit modulated, reduced or non- detectable intermittent (e.g. , continuous, etc.) fluorescence properties. The nanoparticles can have a stochastic blinking profile in a timescale which is shifted to very rapid blinking or very slow or infrequent blinking relative to a nanoparticle previously described in the art (conventional nanoparticles are described in the art as having on-time fractions of <0.2 in the best of conditions examined). For example, the nanoparticles may blink on and off on a timescale which is too rapid to be detected under the methods employed to study this behavior.

[00265] In one aspect the nanoparticle or populations thereof are photostable. The nanoparticles can exhibit a reduced or no photobleaching with long exposure to moderate to high intensity excitation source while maintaining a consistent spectral emission pattern.

[00266] In one aspect, the nanoparticle or populations thereof have a consistently high quantum yield. For example, the nanoparticles can have a quantum yield greater than: about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70% or about 80%. [00267] As used herein, fluorescence (or Forster) resonance energy transfer (FRET) is a process by which a fluorophore (the donor) in an excited state transfers its energy to a proximal molecule (the acceptor) by nonradiative dipole-dipole interaction (Forster, T. "Intermolecular Energy Migration and Fluorescence", Ann. Phys., 2:55-75, 1948; Lakowicz, J.R., Principles of

Fluorescence Spectroscopy, 2nd ed. Plenum, New York. 367-394., 1999).

[00268] FRET efficiency (E) can be defined as the quantum yield of the energy transfer transition, i.e. the fraction of energy transfer event occurring per donor excitation event. It is a direct measure of the fraction of photon energy absorbed by the donor which is transferred to an acceptor, as expressed in Equation 1 : E = fc_ET / k_f +

+∑¾ where fc_ET is the rate of energy transfer, k_f the radiative decay rate and the ¾ are the rate constants of any other de-excitation pathway.

[00269] FRET efficiency E generally depends on the inverse of the sixth power of the distance r (nm) between the two fluorophores (i.e., donor and acceptor pair), as expressed in Equation 2: E = 1 / 1 + (r/Ro)⁶.

[00270] The distance where FRET efficiency is at 50% is termed Ro, also known as the Forster distance. Ro can be unique for each donor- acceptor combination and can range from between about 5nm to about lOnm. Therefore, the FRET efficiency of a donor (i.e., nanoparticle) describes the maximum theoretical fraction of photon energy which is absorbed by the donor (i.e., nanoparticle) and which can then be transferred to a typical organic dye (e.g., fluoresceins, rhodamines, cyanines, etc.).

[00271] In some embodiments, the disclosed nanoparticles are relatively small (i.e., <15nm) and thus may be particularly well suited to be used as a donor or an acceptor in a FRET reaction. That is, some embodiments of the disclosed nanoparticles exhibit higher FRET efficiency than conventional nanoparticles and thus are excellent partners (e.g., donors or acceptors) in a FRET reaction.

[00272] "Quantum yield" as used herein refers to the emission efficiency of a given fluorophore assessed by the number of times which a defined event, e.g., light emission, occurs per photon absorbed by the system. In other words, a higher quantum yield indicates greater efficiency and thus greater brightness of the described nanoparticle or populations thereof.

[00273] Any suitable method can be used to measure quantum yield. In one example, quantum yield can be obtained using standard methods such as those described in Casper et al (Casper, J. V.; Meyer, T. J. J. Am. Chem. Soc. 1983, 105, 5583) and can be analyzed relative to known fluorophores chosen as appropriate for maximal overlap between standard emission and sample emission (e.g., fluorescein, Rhodamine 6G, Rhodamine 101). Dilute solutions of the standard and sample can be matched or nearly matched in optical density prior to acquisition of absorbance and emission spectra for both. The emission quantum yield ( _em) then can be determined according to

Equation 3: _αη =

[00274] where A and A' are the absorbances at the excitation wavelength for the sample and the standard respectively and I and Γ are the integrated emission intensities for the sample and standard respectively. In this case '_em can be the agreed upon quantum yield for the standard.

[00275] Disclosed herein are fluorescent nanoparticles with superior and robust properties which significantly expand the applications in which nanoparticles are useful. These nanoparticles are superior and surprisingly robust in that they are simultaneously stable, bright, and sensitive to environmental stimuli. Moreover, the disclosed nanoparticles have limited or no detectable blinking (i.e. , where the nanoparticle emits light non-intermittently when subject to excitation), are highly photostable, have a consistently high quantum yield, are small (e.g. , <20 nm) and can act as a donor which undergoes FRET with a suitable acceptor moiety (e.g. , fluorescent dyes, etc.). The photo stability of these nanoparticles is reflected in their exhibiting reduced or no photobleaching (i.e. , fading) behavior when subjected to moderate to high intensity excitation for at least about 20 minutes. Additionally, the particles can remain substantially free from photo-induced color shifting.

[00276] Put another way, the nanoparticles can maintain a consistent spectral emission pattern (i.e., maintain the ability to fluoresce) even when exposed to a large quantity of photons (i.e. , moderate to high intensity excitation) for a long period of time. This unique combination of characteristics makes these types of nanoparticles sensitive tools for single molecule analysis and other sensitive high throughput applications. Moreover, these properties make the

nanoparticles particularly well suited for use as highly efficient donor fluorophores in energy transfer reactions such as FRET reactions (i.e., high FRET efficiency) or other reactions as well as applications which require or are enhanced by greater response to the environment.

[00277] Without being bound to a particular theory, blinking or fluorescence intermittency may arise during the nanoparticle charging process when an electron is temporarily lost to the surrounding matrix (Auger ejection or charge tunneling) or captured to surface-related trap states. The nanoparticle is "on" or fluorescing when all of the electrons are intact and the particle is "neutral" and the particle is "off or dark when the electron is lost and the particle is temporarily (or in some cases permanently) charged. It is important to note that the complete suppression of blinking may not necessarily be required and in some instances may not be desirable. Blinking which occurs on a timescale much shorter or much longer than the interrogation period for a particular assay has relatively little impact on the performance of the system. Thus, nanoparticles and nanoparticle populations having modulated blinking properties, where blinking occurs on a very short or very fast timescale relative to the assay interrogation periods are also useful and fall within the scope of the disclosure. Localization of timescale or simply pushing timescale to one side (e.g., to where the blinking is undetectable within the assay system) can provide substantial benefit in application development.

[00278] The blinking behavior of the nanoparticles described herein can be analyzed and characterized by any suitable number of parameters using suitable methodologies. The probability distribution function of the "on" and "off" blinking time durations (i.e., blinking behavior) can be determined using the form of an inverse power law. A value, alpha ( ) can be calculated, wherein represents an exponent in the power law. As the percentage of the population which is non- blinking increases, the value of _on theoretically approaches zero. In conventional nanoparticle populations previously described, _on typically ranges from about 1.5 to about 2.5, under moderate to high excitation energy.

[00279] Most alpha calculations can use a predetermined threshold to determine the "on" and "off ' values of alpha-on and alpha-off (i.e., _OD and _0ff)- Typically, an alpha estimator which calculates the on/off threshold for each dot individually can be employed. The data can be represented by a plot of signal versus frequency, and typically appears as a series of Gaussian distributions around the "off state" and one or more "on states." A log-log plot of frequency versus time for each period of time that the dot is "on" provides a straight line having a slope of on- The value of alpha-off ( _Qff) can be similarly determined.

[00280] In a specific example (the "TIRF example"), the fluorescent intermittency measurements can be made using a Total Internal Reflection Fluorescence (TIRF) microscope fitted with a 60x oil immersion objective lens, using a dual view with a longpass filter on the acceptor side and a bandpass filter on the donor side. Using the TIRF setup, the nanoparticles were imaged at 30 Hz (33 ms), typically for 5 minutes, to produce a movie showing the time and intensity of the emitted light for each individual spot (corresponding to a single particle) within a binned frame which was 33 ms long; the intensity for each binned frame can be integrated. Each data set can be manually analyzed dot-by-dot, and aggregates and other artifacts were excluded. From the edited results, the following parameters can be calculated: alpha-on (" _on"); alpha-off (" _0ff"); the percent on; longest on/longest off; overlap scores; and the median values for each of these parameters.

[00281] In some aspects, provided herein is a nanoparticle or population thereof which has an _on of less than about 1.5, _on of less than about 1.4, _on of less than about 1.3, _on of less than about 1.2, or an _on of less than about 1.1, under moderate to high excitation energy. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the population has an _on of less than about 1.5, _on of less than about 1.4, _on of less than about 1.3, o_n of less than about 1.2, or _on of less than about 1.1 for the time observed, under moderate to high excitation energy. The observation time can be at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 90 minutes, at least about 120 minutes or more under moderate to high excitation energy. Compositions comprising such a nanoparticle and populations thereof also are contemplated.

[00282] In some aspects, provided herein is a nanoparticle or a population thereof having a stochastic blinking profile which is either undetectable or rare (e.g., no more than 1-2 events during the interrogation period) over an observed timescale. In this case, "undetectable" encompasses the situation in which evidence might exist for ultra-fast blinking on a timescale which is faster than the binning timescale (e.g., dimming and brightening from bin to bin) but there are no "off events persisting for longer than the bin time. Therefore, in some embodiments, a nanoparticle or population thereof has a stochastic blinking profile which is undetectable for at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the time observed, under moderate to high excitation energy. In other embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the individual nanoparticles in a population have a stochastic blinking on a timescale which is undetectable for the time observed, under moderate to high excitation energy. The timescale can be at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 90 minutes, at least about 120 minutes or more under moderate to high excitation energy. [00283] In some aspects, the longest on and longest off values can relate to the longest period of time a nanoparticle is observed to be in either the "on" or the "off state. In particular, the longest on value can be important to determining the length of time and amount of data which may be measured in a particular assay.

[00284] Thus, the blinking characteristics of the nanoparticles herein can also be characterized by their on-time fraction, which represents the (total on-time)/(total experiment time). Under the TIRF example disclosed herein, the total on time can be determined by the total number of frames "on" multiplied by 33 ms, and the total experiment time is 5 minutes. For example, the blinking properties of the disclosed nanoparticles or populations thereof can be determined under continuous irradiation conditions using a 405 nm laser with an intensity of about 1 watt per cm during an experimental window of at least 5 minutes.

[00285] On-time fractions can be used to characterize the blinking behavior of a single nanoparticle or of a population of nanoparticles. It is important to note that the on-time fraction for a particular nanoparticle or population of nanoparticles is a function of the specific conditions under which the percent of blinking or "non-blinking" nanoparticles is determined.

[00286] In some aspects, provided herein is a nanoparticle or population thereof having an on- time fraction of at least about 0.50, at least about 0.60, at least about 0.70, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.90, at least about 0.95, at least about 0.96, at least about 0.97, at least about 0.98, or at least about 0.99 or more, under moderate to high excitation energy. In some embodiments, a nanoparticle or populations thereof having a percent on-time of about 98%, about 99% (i.e., on-time fraction of about 0.99) can be considered to be "non-blinking," under moderate to high excitation energy. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more of the individual nanoparticles in a population of nanoparticles can have an on-time fraction of at least about 0.50, at least about 0.60, at least about 0.70, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.90, at least about 0.95, at least about 0.96, at least about 0.97, at least about 0.98, or at least about 0.99 or more, under moderate to high excitation energy. The on-times of the nanoparticles are typically for at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, at least about 120 minutes under moderate to high intensity excitation of the nanoparticle or nanoparticle population. Under one set of conditions, continuous irradiation with 405 nm laser with an approximate intensity of 1 watt per cm was used to determine the stochastic blinking profile.

[00287] In some embodiments, nanoparticles which have a stochastic (i.e., random) blinking profile in a timescale which shifts from very rapid blinking or very slow/ infrequent blinking (relative to a nanoparticle previously described in the art) can be considered to have modulated blinking properties. In some embodiments, these nanoparticles may blink on and off on a timescale which is too rapid to be detected under the methods employed to study this behavior. Thus, certain nanoparticles can effectively appear to be "always on" or to have on-time fractions of about 0.99, when in fact they flicker on and off at a rate too fast or too slow to be detected. Such flickering has relatively little impact on the performance of a system, and for practical purposes such nanoparticles can be considered to be non-blinking.

[00288] In some instances, the disclosed nanoparticles and populations thereof are not observed to blink off under the analysis conditions, and such particles can be assessed as "always on" (e.g., non-blinking). The percent of usable dots which are "always on" can be a useful way to compare nanoparticles or populations of nanoparticles. However, a determination of "always on" may mean that the "off time was insufficient to provide enough a signal gap for accurate

determination and thus the value in the regime of particles is insufficient to calculate. Even these "non-blinking" nanoparticles may flicker on and off on a timescale which is not detected under the conditions used to assess blinking. For example, certain particles may blink on a timescale which is too fast to be detected, or they may blink very rarely, and, in some embodiments, such particles may also be considered to be "always-on" or non-blinking, as the terms are used herein.

[00289] In one aspect, provided herein is a nanoparticle or population thereof which demonstrate some fluctuation in fluorescence intensity. In some embodiments, the change in fluorescence intensity for the nanoparticle is less than about 5%, less than about 10%, less than about 20%, or less than about 25% of the nanoparticle or populations thereof at its greatest intensity, under moderate to high excitation energy. In some embodiments, such changes in fluorescence intensity of less than about 5%, less than about 10%, less than about 20%, or less than about 25% of the highest intensity can occur in at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% of the nanoparticles in the population, under moderate to high excitation energy.

[00290] In some aspects, the nanoparticles with modulated, reduced or no intermittent (e.g., continuous, non-blinking) fluorescence provided herein can comprise of a core and a layered gradient shell. In some embodiments, the nanoparticle(s) disclosed herein can be comprised of a nanocrystal core (e.g., CdSe, etc.), at least one inner (intermediate) shell layer (e.g., CdS, etc.), and at least one outer (external) shell layer (e.g., ZnS, etc.). In some embodiments, the inner and/or outer shell layers are each comprised of two or more discrete monolayers of the same material. In some embodiments, the largest dimension of the disclosed nanoparticle(s) is less than about 15 nm. See for example, PCT Application Serial No. PCT US/09/61951. See also

PCT/US09/061951 and PCT/US09/061953 both filed on October 23, 2009.

[00291] As discussed previously, the disclosed nanoparticles may be particularly well suited for use as a donor or acceptor which undergoes FRET with a suitable complementary partner (donor or acceptor). A "FRET capable" nanoparticle refers to a nanoparticle which can undergo a measurable FRET energy transfer event with a donor or an acceptor moiety. In some

embodiments, a FRET capable nanoparticle is one which has at least about 25% efficiency in a FRET reaction.

[00292] Thus, in one aspect, a FRET capable fluorescent nanoparticle or population thereof with modulated, reduced or non intermittent (e.g. , continuous, etc.) fluorescence is provided. In some embodiments, the nanoparticle is the donor in a FRET reaction. In some embodiments, the nanoparticle is the acceptor in the FRET reaction.

[00293] In some embodiments, the FRET capable non-blinking fluorescent nanoparticle(s) disclosed herein can comprise a core and a layered gradient shell. In some embodiments, the FRET capable non-blinking nanoparticle(s) disclosed herein can be comprised of a nanocrystal core (e.g., CdSe, etc.), at least one inner (intermediate) shell layer (e.g., CdS, etc.), and at least one outer (external) shell layer (e.g., ZnS, etc.). In some embodiments, the inner and/or outer shell layers are each comprised of two or more discrete monolayers of the same material. In some embodiments, the largest dimension of the disclosed FRET capable nanoparticle(s) is less than about 15 nm.

[00294] In some embodiments, the nanoparticle or population thereof has a FRET efficiency of at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or greater.

[00295] In some embodiments, at least about 30%, at least about 40%, at least about 50%, at least about 60% at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the individual nanoparticles in the population have a FRET efficiency of at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more.

[00296] In some embodiments, the FRET efficiency of the disclosed nanoparticle or population thereof can be maintained for at least about the first 10%, at least about the first 20%, at least about the first 30%, at least about the first 40%, at least about the first 50%, at least about the first 60%, at least about the first 70%, at least about the first 80%, at least about the first 90% or more of the total emitted photons under conditions of moderate to high excitation.

[00297] As discussed above, the nanoparticle(s) provided herein can be considered to be surprisingly photostable. In particular, the nanoparticle and populations described herein can be photostable over an extended period of time while maintaining the ability to effectively participate in energy transfer (i.e., FRET) reactions. The disclosed nanoparticles can be stable under high intensity conditions involving prolonged or continuous irradiation over an extended period of time from a moderate to high excitation source.

[00298] Thus, in one aspect, provided herein is a non-blinking fluorescent nanoparticle and population thereof which is photostable.

[00299] In some embodiments, the disclosed photostable nanoparticle and population thereof can have an emitted light or energy intensity sustained for at least about 10 minutes and does not decrease by more than about 20% of maximal intensity achieved during that time. Further, these nanoparticles and populations thereof can have a wavelength spectrum of emitted light which does not change more than about 10% upon prolonged or continuous exposure to an appropriate energy source (e.g. irradiation).

[00300] In one embodiment, the photostable nanoparticles disclosed herein can remain photostable under moderate to high intensity excitation from at least about 10 minutes to about 2 hours. In another embodiment, the photostable nanoparticles disclosed herein can remain photostable under moderate to high intensity excitation from at least about 10 minutes to about 10 hours. In still another embodiment, the photostable nanoparticles disclosed herein can remain photostable under moderate to high from about 10 minutes to about 48 hours. However, it should be appreciated, that these are just example photostable times for the disclosed nanoparticles, in practice the nanoparticles can remain photostable for longer periods of time depending on the particular application. [00301] It should be appreciated that nanoparticles which are photostable over longer timescales in combination with moderate to high excitation energy sources are well suited for more sensitive and broad-ranging applications such as the real-time monitoring of single molecules involving FRET. That is, the nanoparticle and population thereof described herein can be photostable over an extended period of time while maintaining the ability to effectively participate in energy transfer (i.e., FRET) reactions, which makes the subject nanoparticles particularly useful for many applications involving the real-time monitoring of single molecules. As such, in some

embodiments the photostable nanoparticles disclosed herein have FRET efficiencies of at least about 20%.

[00302] In some embodiments, the disclosed nanoparticles are stable upon prolonged or continuous irradiation (under moderate to high excitation rate) in which they do not exhibit significant photo-bleaching on the timescales indicated. Photobleaching can result from the photochemical destruction of a fluorophore (and can be characterized by the nanoparticles losing the ability to produce a fluorescent signal) by the light exposure or excitation source used to stimulate the fluorescence. Photobleaching can complicate the observation of fluorescent molecules in microscopy and the interpretation of energy transfer reactions because the signals can be destroyed or diminished increasingly as timescales for the experiment increase or the energy intensity increases.

[00303] Photobleaching can be assessed by measuring the intensity of the emitted light or energy for a nanoparticle or nanoparticle population using any suitable method. In some embodiments, the intensity of emitted light or energy from the disclosed nanoparticle or population thereof does not decrease by more than about 20% (and in some embodiments, not more than about 10%) upon prolonged or continuous irradiation (under moderate to high excitation rate). In some

embodiments, the intensity of emitted light from the disclosed nanoparticle or population thereof does not decrease by more than about 20%, about 15%, about 10%, about 5% or less upon irradiation from about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours to about 4 hours, under moderate to high excitation energy.

[00304] In some embodiments, the photostable nanoparticles provided herein further demonstrate enhanced stability in which they exhibit a reduction in or absence of spectral shifting during prolonged excitation. In the conventional nanoparticles previously described in the art, increased exposure to an excitation source - whether via increase time or power - results in a spectral shift of the wavelength emission wavelength profile of a nanoparticle and populations thereof from a longer wavelength to an increasingly shorter wavelength. Such spectral shifting of emission wavelength represents a significant limitation as precise resolution of emission spectra is required for applications which require rapid detection, multi-color analysis, and the like. Shifting of any significance then requires that the wavelength emissions used in an assay be sufficiently separated to permit resolution, thus reducing the number of colors available as well as increasing signal to noise ratio to an unacceptable level as the initial spectral profile cannot be relied upon once spectral shifting begins. Such shifting may require shortened observation times or use of fluorophores with widely separated emission spectra. The nanoparticles provided herein have little to no spectral shift, particularly over extended periods of excitation.

[00305] Wavelength emission spectra can be assessed by any suitable method. For example, spectral characteristics of nanoparticles can generally be monitored using any suitable light- measuring or light- accumulating instrumentation. Examples of such instrumentation are CCD (charge-coupled device) cameras, video devices, CIT imaging, digital cameras mounted on a fluorescent microscope, photomultipliers, fluorometers and luminometers, microscopes of various configurations, and even the human eye. The emission can be monitored continuously or at one or more discrete time points. The photostability and sensitivity of nanoparticles allow recording of changes in electrical potential over extended periods of time.

[00306] Thus, in some embodiments, the photostable nanoparticle and population thereof has a wavelength spectrum of emitted light which does not change more than about 10% upon prolonged or continuous exposure to an appropriate energy source (e.g. irradiation) over about 4 minutes to about 10 minutes, under moderate to high excitation energy. In some embodiments, the wavelength emission spectra does not change more than about 5%, more than about 10% , more than about 20% over 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours to about 4 hours.

[00307] It should be appreciated that there can be various other objective indicia of nanoparticle photostability. For example, a nanoparticle can be classified as photostable when the nanoparticle, under moderate to high excitation, emits about 1,000,000 to about 100,000,000 photons or more preferably about 100,000,001 to about 100,000,000,000 photons or even more preferably more than about 100,000,000,000 photons before becoming non-emissive (i.e., bleached).

[00308] A nanoparticle with modulated, reduced or no fluorescent intermittency (e.g., continuous, non-blinking, etc.); reduced or absent spectral shifting; low to no photobleaching; high quantum yield; and sufficient FRET efficiency can be of any suitable size. Typically, it is sized to provide fluorescence in the UV-visible portion of the electromagnetic spectrum as this range is convenient for use in monitoring biological and biochemical events in relevant media. The disclosed nanoparticle and population thereof can have any combination of the properties described herein.

[00309] Thus, in some embodiments the nanoparticle or population thereof has modulated or no blinking, are photostable (e.g., limited or no photobleaching, limited or no spectral shift), has high quantum yield, have high FRET efficiency, has a diameter of less than about 15 nm, is spherical or substantially spherical shape, or any combination of all these properties as described herein.

[00310] Likewise, in some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more of the individual nanoparticles in a population of nanoparticles have modulated or no blinking, are photostable (e.g., limited or no photobleaching, limited or no spectral shift), have high quantum yield, have high FRET efficiency, have diameters of less than about 15 nm, are spherical or substantially spherical shape, or any combination of or all of these properties as described herein.

[00311] In one aspect, the FRET capable, non-blinking and/or photostable nanoparticle or population thereof provided herein has a maximum diameter of less than about 20 nm. In some embodiments, the nanoparticle(s) can be less than about 15 nm, less than about 10 nm, less than about 8 nm, less than about 6 nm, less than about 5 nm, less than about 4 nm, less than about 3 nm or less in its largest diameter when measuring the core/shell structure. Any suitable method may be used to determine the diameter of the nanoparticle(s). The nanoparticle(s) provided herein can be grown to the desired size using any of the methods disclosed herein. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more of the individual members of a population of nanoparticles have maximum diameters (when measuring the core, core/shell or core/shell/ligand structure) which are less than about 20 nm, less than about 15 nm, less than about 10 nm, less than about 8 nm, less than about 6 nm, less than about 5 nm, less than about 4 nm, less than about 3 nm or less.

[00312] The FRET capable, non-blinking and/or photostable nanoparticle(s) provided herein and populations thereof can be spherical or substantially spherical. In some embodiments, a substantially spherical nanoparticle can be one where any two radius measurements do not differ by more than about 10%, about 8%, about 5%, about 3% or less. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more of the individual members of a population of nanoparticles are spherical or substantially spherical.

[00313] Nanoparticles can be synthesized in shapes of different complexity such as spheres, rods, discs, triangles, nanorings, nanoshells, tetrapods, nanowires and so on. Each of these geometries can have distinctive properties: spatial distribution of the surface charge, orientation dependence of polarization of the incident light wave, and spatial extent of the electric field. In some embodiments, the nanoparticles are substantially spherical or spheroidal.

[00314] For embodiments where the nanoparticle is not spherical or spheroidal, e.g. rod-shaped, it may be from about 1 to about 15 nm, from about 1 nm to about 10 nm, or 1 nm to about 5 nm in its smallest dimension. In some such embodiments, the nanoparticles may have a smallest dimension of about 0.5 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm and ranges between any two of these values.

[00315] The single-color preparation of the nanoparticles disclosed herein can have individual nanoparticles which are of substantially identical size and shape. Thus, in some embodiments, the size and shape between the individual nanoparticles in a population of nanoparticles vary by no more than about 20%, no more than about 15%, no more than about 10%, no more than about 8%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3% or less in at least one measured dimension. In some embodiments, disclosed herein is a population of nanoparticles, where at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, and ideally about 100% of the particles are of the same size. Size deviation can be measured as root mean square ("rms") of the diameter, with the population having less than about 30% rms, preferably less than about 20% rms, more preferably less than about 10% rms. Size deviation can be less than about 10% rms, less than about 9% rms, less than about 8% rms, less than about 7% rms, less than about 6% rms, less than about 5% rms, less than about 3% rms, or ranges between any two of these values. Such a collection of particles is sometimes referred to as being a "monodisperse" population.

[00316] The color (emitted light) of a nanoparticle can be "tuned" by varying the size and composition of the particle. Nanoparticles as disclosed herein can absorb a wide spectrum of wavelengths, and emit a relatively narrow wavelength of light. The excitation and emission wavelengths are typically different, and non-overlapping. The nanoparticles of a monodisperse population may be characterized in that they produce a fluorescence emission having a relatively narrow wavelength band. Examples of emission widths include less than about 200 nm, less than about 175 nm, less than about 150 nm, less than about 125 nm, less than about 100 nm, less than about 75 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, and less than about 10 nm. In some embodiments, the width of emission is less than about 60 nm full width at half maximum (FWHM), or less than about 50 nm FWHM, and sometimes less than about 40 nm FWHM, less than about 30 nm FWHM or less than about 20 nm FWHM. In some embodiments, the emitted light preferably has a symmetrical emission of wavelengths.

[00317] The emission maxima of the disclosed nanoparticle and population thereof can generally be at any wavelength from about 200 nm to about 2,000 nm. Examples of emission maxima include about 200 nm, about 400 nm, about 600 nm, about 800 nm, about 1,000 nm, about 1,200 nm, about 1,400 nm, about 1,600 nm, about 1,800 nm, about 2,000 nm, and ranges between any two of these values.

[00318] As discussed previously, the disclosed nanoparticle or populations thereof can comprise a core and a layered shell, wherein the shell includes at least one inner (intermediate) shell layer comprising a first shell material and at least one outer (external) shell layer comprising a second shell material, and wherein the layered shell is substantially uniform in coverage around the core and is substantially free of defects.

[00319] Thus, in one aspect, the nanoparticle or population thereof comprises a core (M^XY) and a layered shell, wherein the shell comprises m inner shell monolayers comprising a first shell material (M 1 X)_m and n outer shell monolayers comprising a second shell material (M 2 X)_n, wherein M can be a metal atom and X can be a non-metal atom, each of m and n is independently an integer from 1 to 10, and the layered shell is substantially uniform in coverage around the core and is substantially free of defects. In specific embodiments, the sum of m + n is 3-20, or 5-14, or 6- 12, or 7-10.

[00320] In certain embodiments, the disclosed nanoparticles can further comprise one or more additional shell layers between the at least one inner shell layer and the at least one outer shell layer. [00321] In some embodiments, the nanoparticle core and population thereof can have a first bandgap energy and the first shell material can have a second bandgap energy, wherein the second bandgap energy can be greater than the first bandgap energy.

[00322] In a further aspect, provided herein is a nanoparticle or population thereof comprising a core and a layered shell, wherein the shell comprises sequential monolayers comprising an alloyed multi-component shell material of the form M 1 _XM2 _yX, where M 1 and M2 can be metal atoms and X can be a non metal atom, where the composition becomes successively enriched in M as the monolayers of shell material are deposited, where x and y represent the ratio of M 1 and M 2 in the shell material, and wherein the monolayered shell is substantially uniform in coverage around the core and is substantially free of defects. In some embodiments, the layered shell sometimes has about 3-20 monolayers of shell material, sometimes about 5-14 monolayers of shell material, sometimes about 6-12 monolayers of shell material, or sometimes about 7-10 monolayers of shell material.

[00323] In one aspect, provided herein is a nanoparticle or population thereof comprising a core and a layered shell having a gradient potential, wherein the shell comprises at least one inner shell layer and at least one outer shell layer, and wherein the layered shell is substantially uniform in coverage around the core and is substantially free of defects.

[00324] The layered shell may be engineered such that the sequential monolayers are selected to provide a gradient potential from the nanoparticle core to the outer surface of the nanoparticle shell. The steepness of the potential gradient may vary depending on the nature of the shell materials selected for each monolayer or group of monolayers. For example, a nanoparticle comprising several sequential monolayers of the same shell material may reduce the potential through a series of steps, while a more continuous gradient may be achievable through the use of sequential monolayers of a multi-component alloyed shell material. In some embodiments, both single component and multi-component shell materials may be applied as different monolayers of a multi-layer shell on a nanoparticle.

[00325] The nanoparticles can be synthesized as disclosed to the desired size by sequential, controlled addition of materials to build and/or apply monolayers of shell material to the core. This is in contrast to conventional methods of adding shells where materials (e.g., diethylzinc and bis(trimethylsilyl)sulfide) are added together. Sequential addition permits the formation of thick (e.g., >2 nm) relatively uniform individual shells (e.g., uniform size and depth) on a core. The layer additions generally require the addition of an appropriate amount of the shell precursors to form a single monolayer, based on the starting size of the underlying core. This means that as each monolayer of shell material is added, a new "core" size must be determined by taking the previous "core" size and adding to it the thickness of just-added shell monolayer. This leads to a slightly larger volume of the following shell material needing to be added for each subsequent monolayer of shell material being added.

[00326] Each monolayer of shell material can be independently selected, and may be made up of a single component, or may comprise a multi-component (e.g., alloyed, etc.) shell material. In some embodiments, it is suitable to apply one or more sequential monolayers of a first shell material, followed by one or more sequential monolayers of a second shell material. This approach allows the deposition of at least one inner shell layer of a material having a bandgap and lattice size compatible with the core, followed by the deposition of at least one outer shell layer of a material having a bandgap and lattice size compatible with the inner shell layer. In some embodiments, multiple sequential monolayers of a single shell material can be applied to provide a uniform shell of a desired number of monolayers of a single shell material; in these embodiments, the first and second shell materials are the same. In other embodiments, sequential monolayers of an alloyed shell material are applied, where the ratio of the components varies such that the composition becomes successively enriched in one component of the multi-component mixture as the successive monolayers of shell material are deposited.

[00327] In some embodiments, the layered shell can be about 3-20 monolayers of shell material thick, sometimes about 5-14 monolayers of shell material thick, sometimes about 6-12 monolayers of shell material thick or sometimes about 7-10 monolayers of shell material thick. In some embodiments, at least one inner shell layer can be comprised of about 3-5 monolayers, sometimes about 3-7 monolayers, of the first shell material. In other embodiments, at least one outer shell layer can be comprised of about 3-5 monolayers, sometimes about 3-7 monolayers, of the second shell material. In some embodiments, the inner shell layer can be at least 3 monolayers thick; in other embodiments, the outer shell layer can be at least 3 monolayers thick. The individual monolayers can be formed by the controlled, sequential addition of the layer materials methods described herein. The monolayers may not always be completely distinct as they may, in some embodiments, be a latticing between the surfaces of contacting monolayers.

[00328] In certain embodiments, provided herein are nanoparticles having a thick, uniform, layered shell, as described herein, wherein the core comprises CdSe, the at least one inner shell layer comprises CdS, and the at least one outer shell layer comprises ZnS. In a particular embodiment, provided herein is a nanoparticle or population thereof having a CdSe core and a layered shell comprising 4CdS + 3.5ZnS layers. In some embodiments, provided herein is a nanoparticle which consists essentially of CdSe/4CdS-3.5ZnS.

[00329] Also disclosed herein are methods of making a nanoparticle and population thereof with modulated, reduced or no fluorescence intermittency or "blinking". These nanoparticles can be small, photostable, bright, highly FRET efficient or some combination thereof. These

nanoparticles can have a multi- shell layered core achieved by a sequential shell material deposition process, whereby one shell material is added at a time, to provide a nanoparticle having a substantially uniform shell of desired thickness which is substantially free of defects.

[00330] In one aspect, provided herein is a method for making a nanoparticle or population thereof with modulated, reduced or no fluorescence intermittency, comprising: providing a mixture comprising a core and at least one coordinating solvent; adding a first inner shell precursor alternately with a second inner shell precursor in layer additions, to form an inner shell layer which is a desired number of layers thick; and adding a first outer shell precursor alternately with a second outer shell precursor in layer additions, to form an outer shell layer which is a desired number of layers thick. If the coordinating solvent of is not amine, the method further comprises an amine in.

[00331] In some embodiments, the mixture can be heated to a temperature which is suitable for shell formation before and/or after every sequential addition of a shell precursor. In some embodiments, the shell is substantially uniform in coverage around the core and is substantially free of defects. In some embodiments, the resulting nanoparticles have a diameter of less than about 15 nm. In other embodiments, the nanoparticles have a diameter of between about 6 nm to about 10 nm. The nanoparticles made by this method can have quantum yields greater than about 80%. The nanoparticle made by this method can have on-time fractions (i.e., ratio of the time which nanoparticle emission is turned "on" when the nanoparticle is excited) of greater than about 0.80 (under moderate to high excitation energy).

[00332] In another aspect, provided herein is a method for making a FRET capable nanoparticle and populations thereof with modulated, reduced or no fluorescence intermittency, comprising: (a) providing a mixture comprising a plurality of nanocrystal cores and at least one coordinating solvent; (b) adding a first intermediate shell precursor alternately with a second intermediate shell precursor in layer additions to form an intermediate shell layer on each of the plurality of nanocrystal cores, wherein the intermediate shell layer is comprised of more than one monolayer; (c) adding a first external shell precursor alternately with a second external shell precursor in layer additions to form an external shell layer on each of the plurality of nanocrystal cores, wherein the external shell layer is disposed on top of the intermediate shell layer and is comprised of more than one monolayer; (d) adding an aqueous solution comprising a hydrophilic ligand; and (e) maintaining the mixture under conditions which cause the plurality of nanocrystals to migrate into an aqueous phase. If the coordinating solvent is not an amine, at least one amine can be included in step (a). In some embodiments, the resulting population of FRET capable non- blinking nanoparticles has a a_on value which is less than about 1.4. In other embodiments, the resulting population of FRET capable non-blinking nanoparticles has an on-time fraction of least about 0.8 (under moderate to high excitation energy). In some embodiments, the resulting population of FRET capable non-blinking nanoparticles has diameters which are less than about 15nm. In some embodiments, the resulting population of FRET capable non-blinking

nanoparticles has a FRET efficiency of at least 20%. In some embodiments, the resulting population of FRET capable non-blinking nanoparticles has a quantum yield of at least about 40%.

[00333] In some embodiments, the methods disclosed above utilize a one step or a two step ligand exchange process to replace the hydrophobic ligands on the nanoparticles with hydrophilic ligands to cause the plurality of nanocrystals to migrate into the aqueous phase. See PCT Application Serial No. PCT/US09/053018 and PCT/US09/059456 which are expressly incorporated herein by reference as if set forth in full.

[00334] In another aspect, provided herein is a method for making a FRET capable nanoparticle and populations thereof with modulated, reduced or no fluorescence intermittency, comprising: providing a mixture comprising a plurality of nanocrystal cores, functionalized

organophosphorous-based hydrophilic ligands and at least one coordinating solvent; adding a first intermediate shell precursor alternately with a second intermediate shell precursor in layer additions to form an intermediate shell layer on each of the plurality of nanocrystal cores; and adding a first external shell precursor alternately with a second external shell precursor in layer additions to form an external shell layer on each of the plurality of nanocrystal cores. In some embodiments, the resulting population of FRET capable non-blinking nanoparticles has an a_on value which is less than about 1.4. In other embodiments, the resulting population of FRET capable non-blinking nanoparticles has an on-time fraction of least about 0.8. In some

embodiments, the resulting population of FRET capable non-blinking nanoparticles has diameters which are less than about 15nm. In some embodiments, the resulting population of FRET capable non-blinking nanoparticles has a FRET efficiency of at least 20%. In some embodiments, the resulting population of FRET capable non-blinking nanoparticles has a quantum yield of at least about 40%.

[00335] In some embodiments, the functionalized organophosphorous-based hydrophilic ligands are multi-functional surface ligands which include a phosphonate/phosphinate nanocrystal binding center, a linker, and a functional group, which imparts functionality on the nanocrystal. As used herein the term "functional group" may refer to a group which affects reactivity, solubility, or both reactivity and solubility when present on a multi-functional surface ligand. Embodiments can include a wide variety of functional groups which can impart various types of functionality on the nanocrystal including hydrophilicity, water- solubility, or dispersibility and/or reactivity, and the functionality may generally not include only hydrophobicity or only solubility in organic solvents without increasing reactivity. For example, a functional group which is generally hydrophobic but which increases reactivity such as an alkene or alkyne and certain esters and ethers can be encompassed by embodiments, whereas alkyl groups, which do not generally impart reactivity but increase hydrophobicity may be excluded.

[00336] In certain embodiments, the FRET capable and non-blinking nanoparticles produced by the disclosed methods may be coated with ligands which impart water solubility and/or reactivity on the nanoparticle obviating the need for ligand replacement. Without wishing to be bound by theory, eliminating ligand replacement may provide more consistent thermodynamic properties, which may lead to reduction in variability of coating and less loss of quantum yield, among other improvements in the properties of nanoparticles produced by the methods embodied herein.

Eliminating ligand replacement may also allow for the production of nanoparticles having a wide variety of functional groups associated with the coating. In particular, while ligand replacement is generally limited to production of nanoparticles having amine and/or carboxylic acid functional groups, in various embodiments, the skilled artisan may choose among numerous functional groups when preparing the multi-functional ligands and may, therefore, generate nanoparticles which provide improved water- solubility or water-dispersity and/or support improved crosslinking and/or improved reactivity with cargo molecules. See PCT Application Serial No.

PCT/US09/059117 which is expressly incorporated herein by reference as if set forth in full.

[00337] In another aspect, provided herein is a method of making a nanoparticle or population thereof comprising a core and a layered gradient shell, wherein the shell comprises an multi-

1 2

component (e.g. , alloy, etc.) shell material of the form M _XM _yX, where x and y represent the ratio of M 1 and M 2 in the shell material. The method comprising: (a) providing a mixture comprising a core, at least one coordinating solvent; (b) heating said mixture to a temperature suitable for formation of the shell layer; and (c) adding a first inner shell precursor comprising

d M 2y alternately with a second inner shell precursor comprising X in layer additions, wherein the ratio of y to x gradually increases in sequential layer additions, such that the shell layers becomes successively enriched in M , to form a layered gradient shell which is a desired number of monolayers thick. If the coordinating solvent is not an amine, at least one amine can be included in step (a).

[00338] In one embodiment, the method described above provides a nanoparticle having a layered gradient shell, wherein the core comprises CdSe and the shell comprises sequential layers of Cd_xZn_yS, where the ratio of y to x increases gradually from the innermost shell layer to the outermost shell layer, to provide a layered gradient shell with a finely graded potential. In some such embodiments, the outermost shell layer is essentially pure ZnS. In some embodiments, the percent of Zn in the gradient shell varies from less than about 10% at the innermost shell layer to greater than about 80% at the outermost shell layer.

[00339] Typically, the heating steps in the disclosed methods are conducted at a temperature within the range of about 150-350°C, more preferably within the range of about 200-300°C. In some embodiments, the temperature suitable for formation of at least one inner shell layer is about 215°C. In some embodiments, the temperature suitable for formation of at least one outer shell layer is about 245 °C. It is understood that the above ranges are merely exemplary and are not intended to be limiting in any manner as the actual temperature ranges may vary, dependent upon the relative stability of the precursors, ligands, and solvents. Higher or lower temperatures may be appropriate for a particular reaction. The determination of suitable time and temperature conditions for providing nanoparticles is within the level of skill in the art using routine experimentation .

[00340] It can be advantageous to conduct the nanoparticle-forming reactions described herein with the exclusion of oxygen and moisture. In some embodiments the reactions are conducted in an inert atmosphere, such as in a dry box. The solvents and reagents are also typically rigorously purified to remove moisture and oxygen and other impurities, and are generally handled and transferred using methods and apparatus designed to minimize exposure to moisture and/or oxygen. In addition, the mixing and heating steps can be conducted in a vessel which is evacuated and filled and/or flushed with an inert gas such as nitrogen. The filling can be periodic or the filling can occur, followed by continuous flushing for a set period of time.

[00341] In some embodiments, the at least one coordinating solvent comprises a

trialkylphosphine, a trialkylphosphine oxide, a phosphonic acid, or a mixture of these.

Sometimes, the at least one coordinating solvent comprises TOP, TOPO, TDPA, OPA or a mixture of these. The solvent for these reactions often comprises a primary or secondary amine, for example, decylamine, hexadecylamine, or dioctylamine. In some embodiments, the amine is decylamine. In some embodiments, the first inner shell precursor is Cd(OAc)₂ and the second inner shell precursor is bis(trimethylsilyl)sulfide (TMS₂S). Sometimes, the first and second inner shell precursors are added as a solution in TOP. In some embodiments, the first outer shell precursor is Et₂Zn and the second inner shell precursor is TMS₂S. Sometimes, the first and second outer shell precursors are added as a solution in TOP.

[00342] In certain embodiments, the disclosed nanoparticles may be prepared using the method described herein to build a layered CdS-ZnS shell on a CdSe quantum size core. The shells for these materials can have varying numbers of layers of CdS and ZnS. Prototypical materials containing a CdSe core and approximately 4 monolayers CdS and 3.5 monolayers of ZnS (the final 0.5 monolayer is essentially pure Zn), or a CdSe core and 9 monolayers CdS and 3.5 monolayers of ZnS were prepared as described in the examples.

[00343] In some embodiments, for either the inner or outer layer, or both, less than a full layer of the appropriate first shell precursor can be added alternately with less than a full layer of the appropriate second shell precursor, so the total amount of the first and second shell precursor required is added in two or more portions. Sometimes, the portion is about 0.25 monolayers of shell material, so that the 4 portions of 0.25 monolayer of first shell precursor are added alternately with 4 portions of 0.25 monolayer of second shell precursor; sometimes the portion is about 0.5 monolayers of shell material, and sometimes about 0.75 monolayers of shell material.

[00344] Examples of compounds useful as the first precursor can include, but are not limited to: organometallic compounds such as alkyl metal species, salts such as metal halides, metal acetates, metal carboxylates, metal phosphonates, metal phosphinates, metal oxides, or other salts. In some embodiments, the first precursor provides a neutral species in solution. For example, alkyl metal species such as diethylzinc (Et₂Zn) or dimethyl cadmium are typically considered to be a source of neutral zinc atoms (Zn°) in solution. In other embodiments, the first precursor provides an ionic species (i.e., a metal cation) in solution. For example, zinc chloride (ZnCl₂) and other zinc halides, zinc acetate (Zn(OAc)₂) and zinc carboxylates are typically considered to be sources of Zn²⁺ cations in solution.

[00345] By way of example only, suitable first precursors providing neutral metal species include dialkyl metal sources, such as dimethyl cadmium (Me₂Cd), diethyl zinc (Et₂Zn), and the like. Suitable first precursors providing metal cations in solution include, e.g. , cadmium salts, such as cadmium acetate (Cd(OAc)₂), cadmium nitrate (Cd(N0₃)₂), cadmium oxide (CdO), and other cadmium salts; and zinc salts such as zinc chloride (ZnCl₂), zinc acetate (Zn(OAc)₂), zinc oleate (Zn(oleate)₂), zinc chloro(oleate), zinc undecylenate, zinc salicylate, and other zinc salts. In some embodiments, the first precursor is salt of Cd or Zn. In some embodiments, it is a halide, acetate, carboxylate, or oxide salt of Cd or Zn. In other embodiments, the first precursor is a salt of the form M(0₂CR)X, wherein M is Cd or Zn; X is a halide or 0₂CR; and R is a C4-C24 alkyl group which is optionally unsaturated. Other suitable forms of Groups 2, 12, 13 and 14 elements useful as first precursors are known in the art.

[00346] Precursors useful as the "second" precursor in the disclosed methods include compounds containing elements from Group 16 of the Periodic Table of the Elements (e.g., S, Se, Te, and the like), compounds containing elements from Group 15 of the Periodic Table of the Elements (N, P, As, Sb, and the like), and compounds containing elements from Group 14 of the Periodic Table of the Elements (Ge, Si, and the like). Many forms of the precursors can be used in the disclosed methods. It will be understood that in some embodiments, the second precursor will provide a neutral species in solution, while in other embodiments the second precursor will provide an ionic species in solution.

[00347] When the first precursor comprises a metal cation, the second precursor can provide an uncharged (i.e. , neutral) non-metal atom in solution. In frequent embodiments, when the first precursor comprises a metal cation, the second precursor contributes a neutral chalcogen atom, most commonly S°, Se° or Te°.

[00348] Suitable second precursors for providing a neutral chalcogen atom include, for example, elemental sulfur (often as a solution in an amine, e.g. , decylamine, oleylamine, or dioctylamine, or an alkene, such as octadecene), and tri-alkylphosphine adducts of S, Se and Te. Such

trialkylphosphine adducts are sometimes described herein as R3P=X, wherein X is S, Se or Te, and each R is independently H, or a C1-C24 hydrocarbon group which can be straight-chain, branched, cyclic, or a combination of these, and which can be unsaturated. Exemplary second precursors of this type include tri-n (butylphosphine)selenide (TBP=Se), tri-n- (octylphosphine)selenide (TOP=Se), and the corresponding sulfur and tellurium reagents, TBP=S, TOP=S, TBP=Te and TOP=Te. These reagents are frequently formed by combining a desired element, such as Se, S, or Te with an appropriate coordinating solvent, e.g. , TOP or TBP.

Precursors which provide anionic species under the reaction conditions are typically used with a first precursor which provides a neutral metal atom, such as alkylmetal compounds and others described above or known in the art.

[00349] In some embodiments, the second precursor provides a negatively charged non-metal ion in solution (e.g. , S-2, Se-2 or Te-2). Examples of suitable second precursors providing an ionic species include silyl compounds such as bis(trimethylsilyl)selenide ((TMS)₂Se),

bis(trimethylsilyl)sulfide ((TMS)₂S) and bis(trimethylsilyl)telluride ((TMS)₂Te). Also included are hydrogenated compounds such as H2Se, H2S, H2Te; and metal salts such as NaHSe, NaSH or NaHTe. In this situation, an oxidant can be used to oxidize a neutral metal species to a cationic species which can react with the anionic precursor in a 'matched' reaction, or an oxidant can be used increase the oxidation state of the anionic precursor to provide a neutral species which can undergo a 'matched' reaction with a neutral metal species.

[00350] Other exemplary organic precursors are described in U.S. Pat. Nos. 6,207,229 and 6,322,901 to Bawendi et al., and synthesis methods using weak acids as precursor materials are disclosed by Qu et al., (2001), Nano Lett., l(6):333-337, the disclosures of each of which are incorporated herein by reference in their entirety.

[00351] Both the first and the second precursors can be combined with an appropriate solvent to form a solution for use in the disclosed methods. The solvent or solvent mixture used to form a first precursor solution may be the same or different from that used to form a second precursor solution. Typical coordinating solvents include alkyl phosphines, alkyl phosphine oxides, alkyl phosphonic acids, alkyl phosphinic acids, or carboxylic acid containing solvents, or mixtures of these.

[00352] Suitable reaction solvents include, by way of illustration and not limitation,

hydrocarbons, amines, alkyl phosphines, alkyl phosphine oxides, carboxylic acids, ethers, furans, phosphoacids, pyridines and mixtures thereof. The solvent may actually comprise a mixture of solvents, often referred to in the art as a "solvent system". In some embodiments, the solvent comprises at least one coordinating solvent. In some embodiments, the solvent system comprises a secondary amine and a trialkyl phosphine (e.g., TBP or TOP) or a trialkylphosphine oxide (e.g. , TOPO). If the coordinating solvent is not an amine, an amine can be included. [00353] A coordinating solvent might be a mixture of an essentially non-coordinating solvent such as an alkane and a ligand as defined below.

[00354] Suitable hydrocarbons include alkanes, alkenes and aromatic hydrocarbons from 10 to about 30 carbon atoms; examples include octadecene and squalane. The hydrocarbon may comprise a mixture of alkane, alkene and aromatic moieties, such as alkylbenzenes (e.g., mesitylene).

[00355] Suitable amines include, but are not limited to, monoalkylamines, dialkylamines, and trialkylamines, for example dioctylamine, oleylamine, decylamine, dodecylamine,

hexyldecylamine, and so forth. Alkyl groups for these amines typically contain about 6-24 carbon atoms per alkyl, and can include an unsaturated carbon-carbon bond, and each amine typically has a total number of carbon atoms in all of its alkyl groups combined of about 10-30 carbon atoms.

[00356] Exemplary alkyl phosphines include, but are not limited to, the trialkyl phosphines, tri-n- butylphosphine (TBP), tri-n-octylphosphine (TOP), and so forth. Alkyl groups for these phosphines contain about 6-24 carbon atoms per alkyl, and can contain an unsaturated carbon- carbon bond, and each phosphine has a total number of carbon atoms in all of its alkyl groups combined of about 10-30 carbon atoms.

[00357] Suitable alkyl phosphine oxides include, but are not limited to, the trialkyl phosphine oxide, tri-n-octylphosphine oxide (TOPO), and so forth. Alkyl groups for these phosphine oxides contain about 6-24 carbon atoms per alkyl, and can contain an unsaturated carbon-carbon bond, and each phosphine oxide has a total number of carbon atoms in all of its alkyl groups combined of about 10-30 carbon atoms.

[00358] Exemplary fatty acids include, but are not limited to, stearic, oleic, palmitic, myristic and lauric acids, as well as other carboxylic acids of the formula R-COOH, wherein R is a C6-C24 hydrocarbon group and can contain an unsaturated carbon-carbon bond. It will be appreciated that the rate of nanocrystal growth generally increases as the length of the fatty acid chain decreases.

[00359] Exemplary ethers and furans include, but are not limited to, tetrahydrofuran and its methylated forms, glymes, and so forth.

[00360] Suitable phosphonic and phosphinic acids include, but are not limited to hexylphosphonic acid (HPA), tetradecylphosphonic acid (TDPA), and octylphosphinic acid (OPA), and are frequently used in combination with an alkyl phosphine oxide such as TOPO. Suitable phosphonic and phosphinic acids are of the formula RPO₃H₂ or R₂P02H, wherein each R is independently a C6-C24 hydrocarbon group and can contain an unsaturated carbon-carbon bond. [00361] Exemplary pyridines include, but are not limited to, pyridine, alkylated pyridines, nicotinic acid, and so forth.

[00362] Suitable alkenes include, e.g. , octadecene and other C4-C24 hydrocarbons which are unsaturated.

[00363] Nanoparticle core or shell precursors can be represented as a M-source and an X-donor. The M-source can be an M-containing salt, such as a halide, carboxylate, phosphonate, carbonate, hydroxide, or diketonate, or a mixed salt thereof (e.g. , a halo carboxylate salt, such as

Cd(halo)(oleate)), of a metal, M, in which M can be, e.g. , Cd, Zn, Mg, Hg, Al, Ga, In, or Tl. In the X-donor, X can be, e.g. , O, S, Se, Te, N, P, As, or Sb. The mixture can include an amine, such as a primary amine (e.g., a C8-C20 alkyl amine). The X donor can include, for example, a phosphine chalcogenide, a bis(trialkylsilyl)chalcogenide, a dioxygen species, an ammonium salt, or a tris(trialkylsilyl)phosphine, or the like.

[00364] The M-source and the X donor can be combined by contacting a metal, M, or an M- containing salt, and a reducing agent to form an M-containing precursor. The reducing agent can include an alkyl phosphine, a 1,2-diol or an aldehyde, such as a C₆-C₂₀ alkyl diol or a C₆-C₂₀ aldehyde.

[00365] Suitable M-containing salts include, for example, cadmium acetylacetonate, cadmium iodide, cadmium bromide, cadmium chloride, cadmium hydroxide, cadmium carbonate, cadmium acetate, cadmium oxide, zinc acetylacetonate, zinc iodide, zinc bromide, zinc chloride, zinc hydroxide, zinc carbonate, zinc acetate, zinc oxide, magnesium acetylacetonate, magnesium iodide, magnesium bromide, magnesium chloride, magnesium hydroxide, magnesium carbonate, magnesium acetate, magnesium oxide, mercury acetylacetonate, mercury iodide, mercury bromide, mercury chloride, mercury hydroxide, mercury carbonate, mercury acetate, aluminum acetylacetonate, aluminum iodide, aluminum bromide, aluminum chloride, aluminum hydroxide, aluminum carbonate, aluminum acetate, gallium acetylacetonate, gallium iodide, gallium bromide, gallium chloride, gallium hydroxide, gallium carbonate, gallium acetate, indium acetylacetonate, indium iodide, indium bromide, indium chloride, indium hydroxide, indium carbonate, indium acetate, thallium acetylacetonate, thallium iodide, thallium bromide, thallium chloride, thallium hydroxide, thallium carbonate, or thallium acetate. Suitable M-containing salts also include, for example, carboxylate salts, such as oleate, stearate, myristate, and palmitate salts, mixed halo carboxylate salts, such as M(halo)(oleate) salts, as well as phosphonate salts. [00366] Alkyl is a branched or unbranched saturated hydrocarbon group of 1 to 100 carbon atoms, preferably 1 to 30 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Optionally, an alkyl can contain 1 to 6 linkages selected from the group consisting of -0-, -S-, -M- and -NR- where R is hydrogen, or C1-C8 alkyl or lower alkenyl.

[00367] The X donor is a compound capable of reacting with the M-containing salt to form a material with the general formula MX. The X donor is generally a chalcogenide donor or a phosphine donor, such as a phosphine chalcogenide, a bis(silyl) chalcogenide, dioxygen, an ammonium salt, or a tris(trialkylsilyl) phosphine. Suitable X donors include dioxygen, elemental sulfur, bis(trimethylsilyl) selenide ((TMS)₂Se), trialkyl phosphine selenides such as (tri-n- octylphosphine) selenide (TOPSe) or (tri-n-butylphosphine) selenide (TBPSe), trialkyl phosphine tellurides such as (tri-n-octylphosphine) telluride (TOPTe) or hexapropylphosphorustriamide telluride (HPPTTe), bis(trimethylsilyl)telluride ((TMS)₂Te), sulfur, bis(trimethylsilyl)sulfide ((TMS)₂S), a trialkyl phosphine sulfide such as (tri-n-octylphosphine) sulfide (TOPS),

tris(dimethylamino) arsine, an ammonium salt such as an ammonium halide (e.g. , NH₄C1), tris(trimethylsilyl) phosphide ((TMS)₃P), tris(trimethylsilyl) arsenide ((TMS)₃As), or

tris(trimethylsilyl) antimonide ((TMS)₃Sb). In certain embodiments, the M donor and the X donor can be moieties within the same molecule.

Ligand Exchange Processes for Coating Nanoparticles

[00368] Provided herein are ligand exchange processes that permit efficient conversion of a conventional hydrophobic nanoparticle or population thereof into a water-dispersible and functionalized nanoparticle or population of nanoparticles. It also permits preparation of small nanoparticles which are highly stable and bright enough to be useful in biochemical and biological assays. The resulting nanoparticles can also be linked to a target molecule or cell or enzyme (e.g. , polymerase) of interest.

[00369] Typically, the nanoparticle used for this process is a core/shell nanocrystal which is coated with a hydrophobic ligand such as tetradecylphosphonic acid (TDPA), trioctylphosphine oxide (TOPO), trioctyl phosphine (TOP), octylphosphonic acid (OPA), and the like, or a mixture of such ligands; these hydrophobic ligands typically have at least one long-chain alkyl group, i.e. an alkyl group having at least 8 carbons, or for the phosphine / phosphine oxide ligands, this hydrophobic character may be provided by two or three alkyl chains on a single ligand molecule having a total of at least 10 carbon atoms. Therefore, in some embodiments, the surface of the core/shell nanocrystal or population thereof can be coated with varying quantities of TDPA hydrophobic ligands prior to replacement with hydrophilic ligand(s). For example, TDPA can represent at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, at least about 95%, at least about 98%, at least about 99% or more of the total surface ligands coating the core/shell nanoparticles. Moreover, certain hydrophobic ligands show an unexpected and apparent ease of replacement with the hydrophilic ligand. For example, nanoparticles with OPA on the surface have been observed to transfer into aqueous buffer more readily and more completely than the same type of core- shell with TDPA on the surface. Therefore, in some embodiments, the surface of the core/shell nanocrystal or populations thereof can be coated with varying quantities of OPA hydrophobic ligands prior to replacement with hydrophilic ligand(s). For example, OPA can represent at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, at least about 95%, at least about 98%, at least about 99% or more of the total surface ligands coating the core/shell nanocrystal.

[00370] In one aspect, provided herein is a "one-step" ligand exchange process to apply various types of ligands to the surface of a nanoparticle, by substituting a desired hydrophilic ligand for a conventional hydrophobic ligand like TOPO, TOP, TDPA, OPA, and the like. The process steps, comprising: providing a nanocrystal coated with a surface layer comprising a hydrophobic ligand, and dissolved or dispersed in a non-aqueous solvent, contacting the nanocrystal dispersion with a phase transfer agent and an aqueous solution comprising a hydrophilic ligand, to form a biphasic mixture having an aqueous phase and a non-aqueous phase and maintaining the mixture under conditions that cause the nanocrystal to migrate from the non-aqueous solvent into the aqueous phase. See PCT Application Serial No. PCT/US09/053018which is expressly incorporated herein by reference as if set forth in full.

[00371] The One-step' ligand exchange process described herein utilizes phase transfer catalysts which are particularly effective, and provide faster exchange reactions. Butanol has been utilized as a phase transfer catalyst for this type of exchange reaction; however, the reaction takes several days typically, and requires heating to about 70°C. The time for this reaction exposes the nanoparticles to these reaction conditions for a long period of time, which may contribute to some reduction in its ultimate stability. The embodiments disclosed herein provide more efficient conditions which achieve ligand exchange more rapidly, thus better protecting the nanoparticles. As a result of accelerating the exchange reaction and allowing use of milder conditions, these phase transfer catalysts produce higher quality nanoparticles.

[00372] The phase transfer agent for this process can be a crown ether, a PEG, a

trialkylsulfonium, a tetralkylphosphonium, and an alkylammonium salt, or a mixture of these. In some embodiments, the phase transfer agent is 18-crown-6, 15-crown-5, or 12-crown-4. In some embodiments, the phase transfer agent is a PEG, which can have a molecular weight from about 500 to about 5000. In some embodiments, the phase transfer agent is a trialkylsulfonium, tetralkylphosphonium, or alkylammonium (including monoalkylammonium, dialkylammonium, trialkylammonium and tetralkylammonium) salt.

[00373] Tetralkylammonium salts are sometimes preferred as phase transfer agents. Examples of suitable tetralkylammonium salts include triethylbenzyl ammonium, tetrabutylammonium, tetraoctylammonium, and other such quaternary salts. Other tetralkylammonium salts, where each alkyl group is a C1-C12 alkyl or arylalkyl group, can also be used. Typically, counting all of the carbons on the alkyl groups of a trialkylsulfonium, tetralkylphosphonium, and alkylammonium salt, the phase transfer agent will contain a total of at least 2 carbons, at least 10 carbons and preferably at least 12 carbon atoms. Each of the trialkylsulfonium, tetralkylphosphonium, and alkylammonium salts has a counterion associated with it; suitable counterions include halides, preferably chloride or fluoride; sulfate, nitrate, perchlorate, and sulfonates such as mesylate, tosylate, or triflate; mixtures of such counterions can also be used. The counterion can also be a buffer or base, such as borate, hydroxide or carbonate; thus, for example, tetrabutylammonium hydroxide can be used to provide the phase transfer catalyst and a base. Specific phase transfer salts for use in these methods include tetrabutylammonium chloride (or bromide) and

tetraoctylammonium bromide (or chloride).

[00374] Suitable hydrophilic ligands are organic molecules which provide at least one binding group to associate tightly with the surface of a nanocrystal. The hydrophilic ligand typically is an organic moiety having a molecular weight between about 100 and 1500, and contains enough polar functional groups to be water soluble. Some examples of suitable hydrophilic ligands include small peptide having 2-10 amino acid residues (preferably including at least one histidine or cysteine residue), mono- or polydentate thiol containing compounds.

[00375] Following ligand exchange, the surface layer can optionally be crosslinked. [00376] In another aspect, provided herein is a "two-step" ligand exchange process to apply various types of ligands to the surface of a nanoparticle, by substituting a desired hydrophilic ligand for a conventional hydrophobic ligand like TOPO, TOP, TDPA, OPA, and the like. The process involves the removal of phosphonate or phosphinate ligands from the surface of a nanoparticle or nanocrystal by treatment with sulfonate reagents, particularly silylsulfonate derivatives of weak bases or other poorly coordinating groups.

[00377] The process steps, comprising: providing a nanocrystal whose surface comprises a phosphonate ligand, contacting the nanocrystal with a sulfonate reagent in an organic solvent, contacting the sulfonate ligand coated nanocrystal with a functionalized organic molecule (i.e., hydrophilic ligand) comprising at least one nanocrystal surface attachment group, contacting the nanocrystal dispersion with an aqueous solution to form a biphasic mixture having an aqueous phase and a non-aqueous phase, and maintaining the biphasic mixture under conditions which cause the nanocrystal to migrate from the non-aqueous phase into the aqueous phase. See PCT Application Serial No. PCT/US09/59456 which is expressly incorporated herein by reference as if set forth in full.

[00378] The result of this removal of phosphonate ligands is replacement of the phosphonates with the weakly coordinating groups. One example is the use of silyl sulfonates, such as trimethylsilyl triflate, to form a sulfonate-coated nanoparticle. Triflate is a conventional / common name for a trifluoromethanesulfonyloxy group, CF₃SO₂O-.

[00379] The same type of replacement process can also occur on nanoparticles having phosphinic acid ligands of the formula R₂P(=0)-OH or on nanoparticles having carboxylic acid ligands of the formula RC(=0)-OH, which could be incorporated on the surface of a nanocrystal by known methods; R can be a C₁-C₂₄ hydrocarbon group in these phosphinates, and the two R groups can be the same or different. Thus, it is understood that when phosphonate-containing nanocrystals are described herein, phosphinate-containing nanocrystals can be used instead, with similar results.

[00380] This process provides a mild and selective method for removing phosphonate, phosphinate, and carboxylate ligands from the surface of a nanocrystal. As a result, it provides a way for a user to remove these groups and replace them, without removing other ligands which are not displaced or affected by the silylsulfonate.

[00381] The sulfonate ligands can comprise an alkyl or aryl moiety linked to -SO₃X, where X can represent whatever the sulfonate group is attached to. For example, where the sulfonate ligand is a sulfonate anion (i.e., triflate), X would represent a nanocrystal, or the surface of a nanocrystal. Some of the sulfonate embodiments disclosed herein can also be described with reference to feature 'A' of Formula I, as set forth below.

I

[00382] wherein R¹, R², R³ and A are each, independently, CI -CIO alkyl or C5-C10 aryl; and each alkyl and aryl is optionally substituted.

[00383] The alkyl groups for Formula I compounds are independently selected, and can be straight chain, branched, cyclic, or combinations of these, and optionally can include a C1-C4 alkoxy group as a substituent. Typically, the alkyl groups are lower alkyls, e.g., C1-C4 alkyl groups which are linear or branched. Methyl is one suitable example.

[00384] The aryl group for the compounds of Formula I can be phenyl, naphthyl or a heteroaryl having up to 10 ring members, and can be monocyclic or bicyclic, and optionally contain up to two heteroatoms selected from N, O and S as ring members in each ring. (It will be understood by those skilled in the art that the 5-membered aryl is a heteroaryl ring.) Phenyl is a preferred aryl group; and an aryl group is typically only present if the other organic groups on the silicon other than the sulfonate are lower alkyls, and preferably they are each Me.

[00385] Examples of silylsulfonate ligands can include, but are not limited to:

(trimethylsilyl)triflate, (triethylsilyl)triflate, (t-butyldimethylsilyl)triflate,

(phenyldimethylsily)triflate, trimethylsilyl fluoromethanesulfonate, trimethylsilyl

methanesulfonate, trimethylsilyl nitrophenylsulfonate, trimethylsilyl trifluoroethylsulfonate, trimethylsilyl phenylsulfonate, trimethylsilyl toluenesulfonate, diisopropylsilyl

bis(trifluoromethanesulfonate), tertbutyldimethylsilyl trifluoromethanesulfonate, triisopropylsilyl trifluoromethanesulfonate and trimethylsilyl chlorosulfonate.

[00386] Examples of other sulfonate ligands can include, but are not limited to:

trifluoromethanesulfonate (triflate), fluoromethanesulfonate, methanesulfonate (mesylate), nitrophenylsulfonate (nosylate), trifluorethylsulfonate, phenylsulfonate (besylate) and

toluenesulfonate (tosylate).

[00387] Some suitable examples of the hydrophilic ligand are disclosed, for example, in Naasani, U.S. Patents No. 6,955,855; 7,198,847; 7,205,048; 7,214,428; and 7,368,086. Suitable hydrophilic ligands also include imidazole containing compounds such as peptides, particularly dipeptides, having at least one histidine residue, and peptides, particularly dipeptides, having at least one cysteine residue. Specific ligands of interest for this purpose can include carnosine (which contains beta-alanine and histidine); His-Leu; Gly-His; His-Lys; His-Glu; His-Ala; His-His; His- Cys; Cys-His; His-Ile; His-Val; and other dipeptides where His or Cys is paired with any of the common alpha-amino acids; and tripeptides, such as Gly-His-Gly, His-Gly-His, and the like. The chiral centers in these amino acids can be the natural L-configuration, or they can be of the D- configuration or a mixture of L and D. Thus a dipeptide having two chiral centers such as His-Leu can be of the L,L-configuration, or it can be L,D- or D,L; or it can be a mixture of diastereomers.

[00388] Furthermore, suitable hydrophilic ligands can also include mono- or polydentate thiol containing compounds, for example: monodentate thiols such as mercaptoacetic acid, bidentate thiols such as dihydrolipoic acid (DHLA), tridentate thiols such as compounds of Formula II - VII as shown below, and the like.

II

III

[00389] In compounds of Formula II - VII, R¹, R², R³ can independently be H, halo, hydroxyl, (- (C=0)-Ci-C₂₂, -(C=0)CF₃, ) alkanoyl, Ci-C₂₂ alkyl, Ci-C₂₂ heteroalkyl, ((CO)OCi-C₂₂) alkylcarbonato, alkylthio (Ci-C₂₂) or (-(CO)NH(Ci-C₂₀) or -(CO)N(Ci-C₂o)₂) alkylcarbamoyl. In some embodiments, R¹, R², and R³ are different. In other embodiments, R¹, R², and R³ are the same. [00390] In compounds of Formula II - VII, R⁴, and R⁵ can independently be H, C C₂o alkyl, C₆- C₁₈ aryl, CrC₂₂ heteroalkyl or CrC₂₂ heteroaryl. In some embodiments, R⁴ and R⁵ are different. In other embodiments, R⁴ and R⁵ are the same.

[00391] In compounds of Formula II - VII, R⁶ can be H or a polyethylene glycol based moiety of Formula VIII:

VIII

[00392] In certain embodiments of Formula VIII, R⁷ can be -NH₂, -N₃, -NHBoc, -NHFmoc, - NHCbz, -COOH, -COOt-Bu, -COOMe, iodoaryl, hydroxyl, alkyne, boronic acid, allylic alcohol carbonate, -NHBiotin, -(CO)NHNHBoc, -(CO)NHNHFmoc or -OMe. In some embodiments, n can be an integer from 1 to 100.

[00393] In still further embodiments, the tridentate thiol ligands can be a compound of Formula IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII or XXIV:

X

XIV 101

XIX

XXI

OH O

XXIII

XXIV

Functionalized TDPA Ligands on Nanoparticles

[00394] Provided herein are methods for preparing water-soluble semi-conducting, insulating, or metallic nanoparticles including the steps of admixing one or more nanocrystal precursors and one or more multi-functional surface ligands with a solvent to form a solution and heating the solution to a suitable temperature, and in certain embodiments, methods may include the steps of admixing nanocrystal cores, one or more nanocrystal precursors, and one or more multi-functional surface ligands with a solvent to form a solution and heating the solution to a suitable temperature. In such embodiments, the one or more multi-functional surface ligands may at least include a nanocrystal binding center, a linker, and a functional group, which imparts functionality on the nanocrystal. As used herein the term "functional group" may refer to a group which affects reactivity, solubility, or both reactivity and solubility when present on a multi-functional surface ligand. Embodiments can include a wide variety of functional groups which can impart various types of functionality on the nanocrystal including hydrophilicity, water- solubility, or

dispersibility and/or reactivity, and the functionality may generally not include only

hydrophobicity or only solubility in organic solvents without increasing reactivity. For example, a functional group which is generally hydrophobic but which increases reactivity such as an alkene or alkyne and certain esters and ethers can be encompassed by embodiments, whereas alkyl groups, which do not generally impart reactivity but increase hydrophobicity may be excluded.

[00395] In certain embodiments, the nanoparticles produced by the methods of such embodiments may be coated with ligands which impart water solubility and/or reactivity on the nanoparticle obviating the need for ligand replacement. Without wishing to be bound by theory, eliminating ligand replacement may provide more consistent thermodynamic properties, which may lead to reduction in variability of coating and less loss of quantum yield, among other improvements in the properties of nanoparticles produced by the methods embodied herein. Eliminating ligand replacement may also allow for the production of nanoparticles having a wide variety of functional groups associated with the coating. In particular, while ligand replacement is generally limited to production of nanoparticles having amine and/or carboxylic acid functional groups, in various embodiments, the skilled artisan may choose among numerous functional groups when preparing the multi-functional ligands and may, therefore, generate nanoparticles which provide improved water- solubility or water-dispersity and/or support improved crosslinking and/or improved reactivity with cargo molecules. See for example PCT Application Serial No. PCT/US09/59117 filed September 30, 2009 which are expressly incorporated herein by reference as if set forth in full.

[00396] The methods, compositions, systems and kits disclosed herein can involve the use of surfaces (e.g., solid surfaces) which can be attached covalently or non-covalently with the nanoparticles and/or the biomolecules (polymerases, nucleotides, target nucleic acid molecules, primers, and/or oligonucleotides) described herein. The attachment can be reversible or irreversible. The immobilized biomolecules include the: polymerases, nucleotides, target nucleic acid molecules, primer molecules and/or oligonucleotides which are components in the nucleotide binding and/or nucleotide incorporation reactions. The immobilized nanoparticles and/or biomolecules may be attached to the surface in a manner that they are accessible to components of the nucleotide incorporation reaction and/or in a manner which does not interfere with nucleotide binding or nucleotide incorporation. The immobilized nanoparticles and/or biomolecules may be attached to the surface in a manner which renders them resistant to removal or degradation during the incorporation reactions, including procedures which involve washing, flowing, temperatures or pH changes, and reagent changes. In another aspect, the immobilized nanoparticles and/or biomolecules may be reversibly attached to the surface.

[00397] The surface may be a solid surface, and includes planar surfaces, as well as concave, convex, or any combination thereof. The surface may comprise texture (e.g., etched, cavitated or bumps). The surface includes the inner walls of a capillary, a channel, a well, groove, channel, reservoir, bead, particle, sphere, filter, gel or a nanoscale device. The surface can be optically transparent, minimally reflective, minimally absorptive, or exhibit low fluorescence. The surface may be non-porous. The surface may be made from materials such as glass, borosilicate glass, silica, quartz, fused quartz, mica, polyacrylamide, plastic polystyrene, polycarbonate,

polymethacrylate (PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramics, silicon, semiconductor, high refractive index dielectrics, crystals, gels, polymers, or films (e.g., films of gold, silver, aluminum, or diamond). The surface can include a solid substrate having a metal film or metal coat.

[00398] The immobilized nanoparticles and/or biomolecules may be arranged in a random or ordered array on a surface. The ordered array includes rectilinear and hexagonal patterns. The distance and organization of the immobilized molecules may permit distinction of the signals generated by the different immobilized molecules. The surface can be coated with an adhesive and/or resist layer which can be applied to the surface to create the patterned array and can be applied to the surface in any order. The adhesive layer can bind/link the nanoparticle or biomolecules (e.g., polymerases, nucleotides, target nucleic acid molecules, primers, and/or oligonucleotides). The resist layer does not bind/link, or exhibits decreased binding/linking, to the nanoparticle or biomolecules (e.g., polymerases, nucleotides, target nucleic acid molecules, primers, and/or oligonucleotides).

[00399] The immobilized nucleic acid molecules (e.g., target and/or primer molecules) may be attached to the surface at their 5' ends or 3' ends, along their length, or along their length with a 5' or 3' portion exposed. The immobilized proteins (e.g., polymerases) can be attached to the surface in a manner which orients them to mediate their activities (nucleotide binding or nucleotide incorporation).

[00400] The surface can be coated to facilitate attachment of nucleic acid molecules (target and/or primers). For example, a glass surface can be coated with a polyelectrolyte multilayer (PEM) via light-directed attachment (U.S. Patent Nos. 5,599,695, 5,831,070, and 5,959,837) or via chemical attachment. The PEM chemical attachment can occur by sequential addition of polycations and polyanions (Decher, et al., 1992 Thin Solid Films 210:831-835). In one embodiment, the glass surface can be coated with a polyelectrolyte multilayer which terminated with polyanions or polycations. The polyelectrolyte multilayer can be coated with biotin and an avidin-like compound. Biotinylated molecules (nucleic acid molecules or polymerases or nanoparticles) can be attached to the PEM/biotin/avidin coated surface (Quake, U.S. Patent Nos: 6,818,395;

6,911,345; and 7,501,245).

[00401] In one embodiment, the at least one type of nucleotide can include 3-10 phosphate groups or substituted phosphate groups, or a combination of phosphate groups and substituted phosphate groups. The nucleotide can include a terminal phosphate group or terminal substituted phosphate group which can be linked to the energy transfer acceptor moiety. The nucleotide can include the energy transfer acceptor moiety which is linked the base, sugar, or any phosphate group or substituted phosphate group. The nucleotide can be adenosine, guanosine, cytosine, thymidine, uridine, or any other type of nucleotide.

[00402] In one embodiment, more than one type of nucleotide can be contacted with the polymerase. Each of the different types of nucleotides can be linked to the same or to different types of energy transfer acceptor moieties, or any combination of the same or different types of acceptor moieties. [00403] The methods, compositions, systems and kits disclosed herein can include nucleotides. The nucleotides can be linked with at least one energy transfer moiety (Figure 1). The energy transfer moiety can be an energy transfer acceptor or donor moiety. The different types of nucleotides (e.g., adenosine, thymidine, cytidine, guanosine, and uridine) can be labeled with a different type energy transfer acceptor or donor moiety so that the detectable signals (e.g., energy transfer signals) from each of the different types nucleotides can be distinguishable to permit base identity. In one embodiment, the different types of nucleotides (e.g., adenosine, thymidine, cytidine, guanosine, and uridine) can be labeled with a different type of energy transfer acceptor moiety so that the detectable signals (e.g., energy transfer signals) from each of the different types nucleotides can be distinguishable to permit base identity. The nucleotides can be labeled in a way that does not interfere with the events of nucleotide polymerization. For example the attached energy transfer acceptor moiety does not interfere with: nucleotide binding; nucleotide

incorporation; cleavage of the nucleotide; or release of the cleavage product. See for example, U.S. Serial No. 61/164,091, Ronald Graham, concurrently filed March 27, 2009. See for example U.S. Patent Nos. 7,041,812, 7,052,839, 7,125,671, and 7,223,541; U.S. Pub. Nos. 2007/0072196 and 2008/0091005; Sood et al., 2005, J. Am. Chem. Soc. 127:2394-2395; Arzumanov et al., 1996, J. Biol. Chem. 271:24389-24394; and Kumar et al., 2005, Nucleosides, Nucleotides & Nucleic Acids, 24(5):401-408.

[00404] In one aspect, the energy transfer acceptor moiety may be linked to any position of the nucleotide. For example, the energy transfer acceptor moiety can be linked to any phosphate group (or substituted phosphate group), the sugar or the base. In another example, the energy transfer moiety can be linked to any phosphate group (or substituted phosphate group) which is released as part of a phosphate cleavage product upon incorporation. In yet another example, the energy transfer acceptor moiety can be linked to the terminal phosphate group (or substituted phosphate group). In another aspect, the nucleotide may be linked with an additional energy transfer acceptor moiety, so that the nucleotide is attached with two or more energy transfer acceptor moieties. The additional energy transfer acceptor moiety can be the same or different as the first energy transfer acceptor moiety. In one embodiment, the energy transfer acceptor moiety can be a FRET acceptor moiety.

[00405] In one aspect, the nucleotide may be linked with a label which is not an energy transfer moiety. For example, the label can be a fluorophore. [00406] In one aspect, the one or more labels (e.g., energy transfer moiety) can be attached to the nucleotide via a linear or branched linker moiety. An intervening linker moiety can connect the one or more labels, e.g., energy transfer acceptor moieties, with each other and/or to the nucleotide, in any combination of linking arrangements.

[00407] In another aspect, the nucleotides comprise a sugar moiety, base moiety, and at least three, four, five, six, seven, eight, nine, ten, or more phosphate groups (or substituted phosphate groups) linked to the sugar moiety by an ester or phosphoramide linkage. The phosphates can be linked to the 3' or 5' C of the sugar moiety.

[00408] In some embodiments, different linkers can be used to operably link the different nucleotides (e.g., A, G, C, T or U) to the labels, e.g., to the energy transfer moieties. For example, adenosine nucleotide can be attached to one type of energy transfer moiety using one type of linker, and guanosine nucleotide can be linked to a different type of energy transfer moiety using a different type of linker. In another example, adenosine nucleotide can be attached to one type of energy transfer moiety using one type of linker, and the other types of nucleotides can be attached to different types of energy transfer moieties using the same type of linker. One skilled in the art will appreciate that many different combinations of nucleotides, energy transfer moieties, and linkers are possible.

[00409] In one aspect, the distance between the nucleotide and the energy transfer moiety can be altered. For example, the linker length and/or number of phosphate groups (or substitute phosphate groups) can lengthen or shorten the distance from the sugar moiety to the energy transfer moiety. In another example, the distance between the nucleotide and the energy transfer moiety can differ for each type of nucleotide (e.g., A, G, C , T or U).

[00410] In another aspect, the number of energy transfer moieties which are linked to the different types of nucleotides (e.g., A, G, C, T or U) can be the same or different. For example: A can have one dye, and G, C, and T have two; A can have one dye, C has two, G has three, and T has four; A can have one dye, C and G have two, and T has four. One skilled in the art will recognize that many different combinations are possible.

[00411] In another aspect, the concentration of the labeled nucleotides used to conduct the nucleotide binding or nucleotide incorporation reactions, or the concentration included in the systems or kits, can be about 0.0001 nM-1 μΜ, or about 0.0001 nM-0.001 nM, or about 0.001 nM- 0.01 nM, or about 0.01 nM-0.1 nM, or about 0.1 nM-1.0 nM, or about 1 nM-25 nM, or about 25 nM-50 nM, or about 50 nM-75 nM, or about 75 nM-100 nM, or about 100 nM-200 nM, or about 200 nM-500 nM, or about 500 nM-750 nM, or about 750 nM-1000 nM, or about 0.1 μΜ-20 μΜ, or about 20 μΜ-50 μΜ, or about 50 μΜ-75 μΜ, or about 75 μΜ-100 μΜ, or about 100 μΜ-200 μΜ, or about 200 μΜ-500 μΜ, or about 500 μΜ-750 μΜ, or about 750 μΜ-1000 μΜ.

[00412] In some embodiments, the concentration of the different types of labeled nucleotides, which are used to conduct the nucleotide binding or incorporation reaction, can be the same or different from each other.

[00413] In some embodiments, more than one type of nucleotide can be contacted with the polymerase in the same sequencing reaction. Each of the different types of nucleotides can be linked to the same or to different types of energy transfer acceptor moieties, or any combination of the same or different types of acceptor moieties.

[00414] In another embodiment, a plurality of one or more different types of nucleotides can be included in the nucleotide incorporation reaction to permit successive nucleotide incorporation.

[00415] The nucleotides typically comprise a hetero cyclic base which includes substituted or unsubstituted nitrogen-containing parent heteroaromatic ring which is commonly found in nucleic acids, including naturally-occurring, substituted, modified, or engineered variants. The base is capable of forming Watson-Crick and/or Hoogstein hydrogen bonds with an appropriate complementary base. Exemplary bases include, but are not limited to, purines and pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, Ν⁶-Δ²- isopentenyladenine (6iA), N⁶-A²-isopentenyl-2-methylthioadenine (2ms6iA), N⁶-methyladenine, guanine (G), isoguanine, N -dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and 0⁶-methylguanine; 7-deaza-purines such as 7- deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C), 5- propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O⁴- methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines;

hydroxymethylcytosines; 5-methycytosines; base (Y); as well as methylated, glycosylated, and acylated base moieties; and the like. Additional exemplary bases can be found in Fasman, 1989, in: Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Florida, and the references cited therein.

[00416] The nucleotides typically comprise phosphate groups which can be linked to the 2', 3' and/or 5' position of the sugar moiety. The phosphate groups include analogs, such as

phosphoramidate, phosphorothioate, phosphorodithioate, and O-methylphosphoroamidite groups. In one embodiment, at least one of the phosphate groups can be substituted with a fluoro and/or chloro group. The phosphate groups can be linked to the sugar moiety by an ester or

phosphoramide linkage. Typically, the nucleotide comprises three, four, five, six, seven, eight, nine, ten, or more phosphate groups linked to the 5' position of the sugar moiety.

[00417] The methods, compositions, systems and kits disclosed herein can include non- hydrolyzable nucleotides. The nucleotide binding and nucleotide incorporation methods can be practiced using incorporatable nucleotides and non-hydrolyzable nucleotides. In the presence of the incorporatable nucleotides (e.g., labeled), the non-hydrolyzable nucleotides (e.g., non-labeled) can compete for the polymerase binding site to permit distinction between the complementary and non-complementary nucleotides, or for distinguishing between productive and non-productive binding events. In the nucleotide incorporation reaction, the presence of the non-hydrolyzable nucleotides can alter the length of time, frequency, and/or duration of the binding of the labeled incorporatable nucleotides to the polymerase.

[00418] The non-hydrolyzable nucleotides can be non-labeled or can be linked to a label (e.g., energy transfer moiety). The labeled non-hydrolyzable nucleotides can be linked to a label at any position, such as the sugar, base, or any phosphate (or substituted phosphate group). For example, the non-hydrolyzable nucleotides can have the general structure:

[00419] Where B can be a base moiety, such as a hetero cyclic base which includes substituted or unsubstituted nitrogen-containing heteroaromatic ring. Where S can be a sugar moiety, such as a ribosyl, riboxyl, or glucosyl group. Where n can be 1-10, or more. Where P can be one or more substituted or unsubstituted phosphate or phosphonate groups. Where R_{ll 5} if included, can be a label (e.g., a fluorescent dye). In one embodiment, the non-hydrolyzable nucleotide having multiple phosphate or phosphonate groups, the linkage between the phosphate or phosphonate groups can be non-hydrolyzable by the polymerase. The non-hydrolyzable linkages include, but are not limited to, amino, alkyl, methyl, and thio groups. The phosphate or phosphonate portion of the non-hydrolyzable nucleotide can have the general structure:

R₄ R₃ R₂

R₇— P— R₆— P— R₅— P— Ri— S— B

Rio R9 Re [00420] Where B can be a base moiety and S can be a sugar moiety. Where any one of the R - R₇ groups can render the nucleotide non-hydrolyzable by a polymerase. Where the sugar C5 position can be CH₂, CH₂0, CH=, CHR, or CH₂ CH₂. Where the R_x group can be O, S, CH=, CH(CN), or NH. Where the R₂, R₃, and R₄, groups can independently be O, BH₃, or SH. Where the R5 and R₆ groups can independently be an amino, alkyl, methyl, thio group, or CHF, CF₂, CHBr, CC1₂, O-O, or -C≡C- . Where the R₇ group can be oxygen, or one or more additional phosphate or phosphonate groups, or can be a label. Where Rg can be SH, BH₃, CH₃, NH₂, or a phenyl group or phenyl ring. Where R9 can be SH. Where can be CH₃, N₃CH₂CH₂, NH₂, ANS, N₃, MeO, SH, Ph, F, PhNH, PhO, or RS (where Ph can be a phenyl group or phenyl ring, and F can be a fluorine atom or group). The substituted groups can be in the S or R configuration.

[00421] The non-hydrolyzable nucleotides can be alpha-phosphate modified nucleotides, alpha- beta nucleotides, beta-phosphate modified nucleotides, beta-gamma nucleotides, gamma- phosphate modified nucleotides, caged nucleotides, or di-nucleotides.

[00422] Many examples of non-hydrolyzable nucleotides are known (Rienitz 1985 Nucleic Acids Research 13:5685-5695), including commercially-available ones from Jena Bioscience (Jena, Germany).

[00423] In some embodiments, the exchange-based sequencing methods can be performed using nucleotides that do not include a blocking group. A blocking group can include any group or moiety that inhibits, impedes, prevents or delays further incorporation of nucleotides by a polymerase once the nucleotide including the blocking group is incorporated by the polymerase into a nucleic acid molecule. Typically, such inhibition, impedance, prevention or delay is measured relative to the rate of incorporation by the same polymerase of a reference nucleotide that lacks the blocking group but possesses an otherwise identical structure under identical reaction conditions. In some embodiments, the blocking effect of the blocking group is reversible, and ceases upon suitable treatment or a specified change in reaction conditions. For example, some blocking groups can be removed upon exposure to suitable light or other radiation, via enzymatic cleavage, or via chemical cleavage. Sequencing methods using nucleotides that lack blocking groups typically involve the continuous incorporation of multiple nucleotides in succession, whereas sequencing methods that use nucleotides including blocking groups typically proceed in step-wise fashion, where the blocking group of each nucleotide must be neutralized following incorporation of that nucleotide and prior to incorporation of the next nucleotide. Use of nucleotides including blocking groups typically stops or pauses the synthesis of the nascent nucleic acid molecule following each nucleotide incorporation; detection and identification of the incorporated nucleotide can be performed during such pause. The incorporated label is typically removed before, or simultaneously with, neutralization of the blocking group. Following such neutralization, the next nucleotide including a blocking group is incorporated, and the process repeats. However, the use of blocking nucleotides typically requires additional steps to remove or otherwise neutralize the blocking group, as well as wash steps to remove unincorporated nucleotide prior to detection of the sequence-specific signal emitted by the incorporated nucleotide. In methods using nucleotides that lack blocking groups, it may not be necessary to include wash steps, and the detecting can be performed while synthesis of the nascent nucleic acid molecule is ongoing.

[00424] The compositions, methods, systems and kits disclosed herein involve the use of one or more polymerases. In some embodiments, the polymerase incorporates one or more nucleotides into a nascent nucleic acid molecule. In some embodiments, the polymerase has an active site. The nucleotide can bind the active site.

[00425] In some embodiments, the polymerase provided herein can offer unexpected advantages over polymerases that are traditionally used for nucleotide polymerization reactions. In some embodiments, the polymerases can be enzymatically active when conjugated to an energy transfer moiety (e.g., donor moiety). In some embodiments, the polymerases have altered kinetics for nucleotide binding and/or nucleotide incorporation which improve distinction between productive and non-productive nucleotide binding events. In some embodiments, the polymerases having altered kinetics for nucleotide binding and/or nucleotide incorporation can be used in combination with labeled nucleotides having six or more phosphate groups (or substituted phosphate groups), which improves distinction between productive and non-productive binding events. In some embodiments, the polymerases have improved photo- stability compared to polymerases traditionally used for nucleotide polymerization. Examples of polymerases having altered kinetics for nucleotide binding and/or nucleotide incorporation include B103 polymerases disclosed in U.S. Serial Nos. 61/242,771, 61/293,618, and any one of SEQ ID NOS: l-5.

[00426] In some embodiments, the polymerase can be unlabeled. Alternatively, the polymerase can be linked to one or more labels. In some embodiments, the label comprises at least one energy transfer moiety.

[00427] The polymerase may be linked with at least one energy transfer donor or acceptor moiety. One or more energy transfer donor or acceptor moiety can be linked to the polymerase at the amino end or carboxyl end or may be inserted at any site therebetween. Optionally, the energy transfer donor or acceptor moiety can be attached to the polymerase in a manner which does not significantly interfere with the nucleotide binding activity, or with the nucleotide incorporation activity of the polymerase. In such embodiments, the energy transfer donor or acceptor moiety is attached to the polymerase in a manner that does not significantly interfere with polymerase activity.

[00428] In one aspect, a single energy transfer donor or acceptor moiety can be linked to more than one polymerase and the attachment can be at the amino end or carboxyl end or may be inserted within the polymerase.

[00429] In another aspect, a single energy transfer donor or acceptor moiety can be linked to one polymerase.

[00430] In one aspect, the energy transfer donor moiety can be a nanoparticle (e.g., a fluorescent nanoparticle) or a fluorescent dye. The polymerase, which can be linked to the nanoparticle or fluorescent dye, typically retains one or more activities that are characteristic of the polymerase, e.g., polymerase activity, exonuclease activity, nucleotide binding, and the like.

[00431] The polymerase can be a DNA-dependent or RNA-dependent polymerase, or a reverse transcriptase. In some embodiments, the polymerases can be replicases, DNA-dependent polymerases, primases, RNA-dependent polymerases (including RNA-dependent DNA polymerases such as, for example, reverse transcriptases), strand-displacement polymerases, or thermo-stable polymerases. In another aspect, the polymerase can be any Family A or B type polymerase. Many types of Family A (e.g., E. coli Pol I), B (e.g., E. coli Pol II), C (e.g., E. coli Pol III), D (e.g., Euryarchaeotic Pol II), X (e.g., human Pol beta), and Y (e.g., E. coli UmuC/DinB and eukaryotic RAD30/xeroderma pigmentosum variants) polymerases are described in Rothwell and Watsman 2005 Advances in Protein Chemistry 71:401-440.

[00432] In yet another aspect, the polymerases can be isolated from a cell, or generated using recombinant DNA technology or chemical synthesis methods. In another aspect, the polymerases can be expressed in prokaryote, eukaryote, viral, or phage organisms. In another aspect, the polymerases can be post-translationally modified proteins or fragments thereof.

[00433] In one aspect, the polymerase can be a recombinant protein which is produced by a suitable expression vector/host cell system. The polymerases can be encoded by suitable recombinant expression vectors carrying inserted nucleotide sequences of the polymerases. The polymerase sequence can be linked to a suitable expression vector. The polymerase sequence can be inserted in-frame into the suitable expression vector. The suitable expression vector can replicate in a phage host, or a prokaryotic or eukaryotic host cell. The suitable expression vector can replicate autonomously in the host cell, or can be inserted into the host cell's genome and be replicated as part of the host genome. The suitable expression vector can carry a selectable marker which confers resistance to drugs (e.g., kanamycin, ampicillin, tetracycline, chloramphenicol, or the like), or confers a nutrient requirement. The suitable expression vector can have one or more restriction sites for inserting the nucleic acid molecule of interest. The suitable expression vector can include expression control sequences for regulating transcription and/or translation of the encoded sequence. The expression control sequences can include: promoters (e.g., inducible or constitutive), enhancers, transcription terminators, and secretion signals. The expression vector can be a plasmid, cosmid, or phage vector. The expression vector can enter a host cell which can replicate the vector, produce an RNA transcript of the inserted sequence, and/or produce protein encoded by the inserted sequence. The recombinant polymerase can include an affinity tag for enrichment or purification, including a poly-amino acid tag (e.g., poly His tag), GST, and/or HA sequence tag. Methods for preparing suitable recombinant expression vectors and expressing the RNA and/or protein encoded by the inserted sequences are well known (Sambrook et al,

Molecular Cloning (1989)).

[00434] The polymerases may be DNA polymerases and include without limitation bacterial DNA polymerases, prokaryotic DNA polymerase, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases and phage DNA polymerases. The polymerase can be a commercially available polymerase.

[00435] In some embodiments, the polymerase can be a DNA polymerase and include without limitation bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases and phage DNA polymerases.

[00436] Suitable bacterial DNA polymerase include without limitation E. coli DNA polymerases I, II and III, IV and V, the Klenow fragment of E. coli DNA polymerase, Clostridium

stercorarium (Cst) DNA polymerase, Clostridium thermocellum (Cth) DNA polymerase and Sulfolobus solfataricus (Sso) DNA polymerase.

[00437] Suitable eukaryotic DNA polymerases include without limitation the DNA polymerases α, δ, ε, η, ζ, γ, β, σ, λ, μ, ι, and κ, as well as the Revl polymerase (terminal deoxycytidyl transferase) and terminal deoxynucleotidyl transferase (TdT). [00438] Suitable viral and/or phage DNA polymerases include without limitation T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Phi- 15 DNA polymerase, Phi-29 DNA polymerase (see, e.g., U.S. Patent No. 5,198,543; also referred to variously as Φ29 polymerase, phi29 polymerase, phi 29 polymerase, Phi 29 polymerase, and Phi29 polymerase); Φ15 polymerase (also referred to herein as Phi-15 polymerase); Φ21 polymerase (Phi-21 polymerase); PZA polymerase; PZE polymerase, PRD1 polymerase; Nf polymerase; M2Y polymerase; SF5 polymerase; fl DNA polymerase, Cp-1 polymerase; Cp-5 polymerase; Cp-7 polymerase; PR4 polymerase; PR5 polymerase; PR722 polymerase; L17 polymerase; M13 DNA polymerase, RB69 DNA polymerase, Gl polymerase; GA-1 polymerase, BS32 polymerase; B103 polymerase;

BA103 polymerase, a polymerase obtained from any phi-29 like phage or derivatives thereof, etc. See, e.g., U.S. Patent No. 5,576,204, filed Feb. 11, 1993; U.S. Pat. Appl. No. 2007/0196846, published Aug. 23, 2007.

[00439] Suitable archaeal DNA polymerases include without limitation the thermostable and/or thermophilic DNA polymerases such as, for example, DNA polymerases isolated from Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavus (Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase as well as Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase or Vent DNA polymerase, Pyrococcus sp. GB-D polymerase, "Deep Vent" DNA polymerase, New England Biolabs), Thermotoga maritima (Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase; Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp. 9° N-7 DNA polymerase; Thermococcus sp. NA1; Pyrodictium occultum DNA polymerase; Methanococcus voltae DNA polymerase;

Methanococcus thermoautotrophicum DNA polymerase; Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNA polymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcus horikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase; Thermococcus fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase; the heterodimeric DNA polymerase DP1/DP2, etc.

[00440] Suitable RNA polymerases include, without limitation, T3, T5, T7, and SP6 RNA polymerases. [00441] Suitable reverse transcriptases include without limitation reverse transcriptases from HIV, HTLV-I, HTLV-II, FeLV, FIV, SIV, AMV, MMTV and MoMuLV, as well as the

commercially available "Superscript" reverse transcriptases, (Life Technologies Corp., Carlsbad, CA) and telomerases.

[00442] In some embodiments, the polymerase is selected from the group consisting of: Phi-29 DNA polymerase, a variant of Phi-29 DNA polymerase, B103 DNA polymerase and a variant of B 103 DNA polymerase.

[00443] In another aspect, the polymerases can include one or more mutations that improve the performance of the polymerase in the particular biological assay of interest. The mutations can include amino acid substitutions, insertions, or deletions.

[00444] The selection of the polymerase for use in the disclosed methods can be based on the desired polymerase behavior in the particular biological assay of interest. For example, the polymerase can be selected to exhibit enhanced or reduced activity in a particular assay, or enhanced or reduced interaction with one or more particular substrates.

[00445] For example, in some embodiments the polymerase is selected based on the

polymerization kinetics of the polymerase either in unconjugated form or when linked to a label (labeled polymerase conjugate). For example, the polymerase can be a polymerase having altered nucleotide binding and/or altered nucleotide incorporation kinetics which are selected on the basis of kinetic behavior relating to nucleotide binding (e.g., association), nucleotide dissociation (intact nucleotide), nucleotide fidelity, nucleotide incorporation (e.g., catalysis), and/or release of the cleavage product. The selected polymerase can be wild-type or mutant.

[00446] In one embodiment, polymerases may be selected that retain the ability to selectively bind complementary nucleotides. In another embodiment, the polymerases may be selected which exhibit a modulated rate (faster or slower) of nucleotide association or dissociation. In another embodiment, the polymerases may be selected which exhibit a reduced rate of nucleotide incorporation activity (e.g., catalysis) and/or a reduced rate of dissociation of the cleavage product and/or a reduced rate of polymerase translocation (after nucleotide incorporation). Some modified polymerases which exhibit nucleotide binding and a reduced rate of nucleotide incorporation have been described (Rank, U.S. published patent application No. 2008/0108082; Hanzel, U.S.

published patent application No. 2007/0196846).

[00447] In polymerases from different classes (including DNA-dependent polymerases), an active-site lysine can interact with the phosphate groups of a nucleoside triphosphate molecule bound to the active site. The lysine residue has been shown to protonate the pyrophosphate leaving-group upon nucleotidyl transfer. Mutant polymerases having this lysine substituted with leucine, arginine, histidine or other amino acids, exhibit greatly reduced nucleotide incorporation rates (Castro, et al., 2009 Nature Structural and Molecular Biology 16:212-218). One skilled in the art can use amino acid alignment and/or comparison of crystal structures of polymerases as a guide to determine which lysine residue to replace with alternative amino acids. The sequences of Phi29 (SEQ ID NOS:6-12), RB69 (SEQ ID NO: 13), B103 (SEQ ID NOS: l-5), and Klenow fragment can be used as the basis for selecting the amino acid residues to be modified (for B103 polymerase, see Hendricks, et al., U.S. Serial No. 61/242,771, filed on September 15, 2009, or U.S. Serial No. 61/293,618, filed on January 8, 2010). In one embodiment, a modified phi29 polymerase can include lysine at position 379 and/or 383 substituted with leucine, arginine or histidine.

[00448] In other embodiments, the polymerase can be selected based on the combination of the polymerase and nucleotides, and the reaction conditions, to be used for the nucleotide binding and/or nucleotide incorporation reactions. For example, certain polymerases in combination with nucleotides which comprise 3, 4, 5, 6, 7, 8, 9, 10 or more phosphate groups can be selected for performing the disclosed methods. In another example, certain polymerases in combination with nucleotides which are linked to an energy transfer moiety can be selected for performing the nucleotide incorporation methods.

[00449] The polymerases, nucleotides, and reaction conditions, can be screened for their suitability for use in the nucleotide binding and/or nucleotide incorporation methods, using well known screening techniques. For example, the suitable polymerase may be capable of binding nucleotides and/or incorporating nucleotides. For example, the reaction kinetics for nucleotide binding, association, incorporation, and/or dissociation rates, can be determined using rapid kinetics techniques (e.g., stopped- flow or quench flow techniques). Using stopped-flow or quench flow techniques, the binding kinetics of a nucleotide can be estimated by calculating the 1/k_d value. Stopped-flow techniques which analyze absorption and/or fluorescence spectroscopy properties of the nucleotide binding, incorporation, or dissociation rates to a polymerase are well known in the art (Kumar and Patel 1997 Biochemistry 36: 13954-13962; Tsai and Johnson 2006 Biochemistry 45:9675-9687; Hanzel, U.S. published patent application No. 2007/0196846). Other methods include quench flow (Johnson 1986 Methods Enzymology 134:677-705), time-gated fluorescence decay time measurements (Korlach, U.S. Patent No. 7,485,424), plate-based assays (Clark, U.S. published patent application No. 2009/0176233), and X-ray crystal structure analysis (Berman 2007 EMBO Journal 26:3494). Nucleotide incorporation by a polymerase can also be analyzed by gel separation of the primer extension products. In one embodiment, stopped-flow techniques can be used to screen and select combinations of nucleotides with polymerases having a t_poi value (e.g., l k_pol ) which is less than a (e.g., l/k-i ) value. Stopped-flow techniques for measuring t_poi (MP Roettger 2008 Biochemistry 47:9718-9727; M Bakhtina 2009 Biochemistry 48:3197-320) and (M Bakhtina 2009 Biochemistry 48:3197-3208) are known in the art.

[00450] For example, some phi29 or B103 (SEQ ID NOS: l, 2, or 3) polymerases (wild-type or mutant) exhibit t_poi values which are less than Li values, when reacted with tetraphosphate, pentaphosphate or hexaphosphate nucleotides. These polymerases can offer improvements in distinguishing between productive and non-productive nucleotide binding events compared to other polymerases. In another embodiment, polymerases can be modified by binding it to a chemical compound or an antibody, in order to inhibit nucleotide incorporation.

[00451] In some embodiments, the selection of the polymerase may be determined by the level of processivity desired for conducting nucleotide incorporation or polymerization reactions. The polymerase processivity can be gauged by the number of nucleotides incorporated for a single binding event between the polymerase and the target molecule base-paired with the

polymerization initiation site. For example, the processivity level of the polymerase may be about 1, 5, 10, 20, 25, 50, 100, 250, 500, 750, 1000, 2000, 5000, or 10,000 or more nucleotides incorporated with a single binding event. Processivity levels typically correlate with read lengths of a polymerase. Optionally, the polymerase can be selected to retain the desired level of processivity when conjugated to a label.

[00452] The selection of the polymerase may be determined by the level of fidelity desired, such as the error rate per nucleotide incorporation. The fidelity of a polymerase may be partly determined by the 3'→ 5' exonuclease activity associated with a DNA polymerase. The fidelity of a DNA polymerase may be measured using assays well known in the art (Lundburg et al., 1991 Gene, 108: 1-6). The error rate of the polymerase can be one error per about 100, or about 250, or about 500, or about 1000, or about 1500 incorporated nucleotides. In some embodiments, the polymerase is selected to exhibit high fidelity. Such high-fidelity polymerases include those exhibiting error rates typically of about 5xl0^~6 per base pair or lower.

[00453] In some embodiments, the selection of the polymerase may be determined by the rate of nucleotide incorporation such as about one nucleotide per 2-5 seconds, or about one nucleotide per second, or about 5 nucleotides per second, or about 10 nucleotides per second, or about 20 nucleotides per second, or about 30 nucleotides per second, or more than 40 nucleotides per second, or more than 50-100 per second, or more than 100 per second. In one embodiment, polymerases exhibiting reduced nucleotide incorporation rates include mutant phi29 polymerase having lysine substituted with leucine, arginine, histidine or other amino acids (Castro 2009

Nature Structural and Molecular Biology 16:212-218).

[00454] In some embodiments, the polymerase can be selected to exhibit either reduced or enhanced rates of nucleotide incorporation when reacted with nucleotides linked at the terminal phosphate group with an energy transfer acceptor.

[00455] In some embodiments, the polymerase can be selected to exhibit either reduced or enhanced nucleotide binding times for a particular nucleotide of interest. In some embodiments, the nucleotide binding time of the selected polymerase for the particular labeled nucleotide of interest can be between about 20 msec and about 300 msec, typically between about 55 msec and about 100 msec. In some embodiments, the nucleotide binding time of the selected polymerase for the particular labeled nucleotide of interest can be between about 1.5 and about 4 times the nucleotide binding time of the corresponding wild-type polymerase for the labeled nucleotide. These polymerases can offer improvements in distinguishing between productive and non-productive nucleotide binding events compared to other polymerases.

[00456] In some embodiments, the polymerase can be selected, mutated, modified, evolved or otherwise engineered to exhibit either reduced or enhanced entry of nucleotides, particularly labeled nucleotides, into the polymerase active site. These polymerases can offer improvements in distinguishing between productive and non-productive nucleotide binding events compared to other polymerases.

[00457] In some embodiments, the polymerase can be selected to exhibit a reduced K_sub for a substrate, particularly a labeled nucleotide. In some embodiments, the polymerase can comprise one or more mutations resulting in altered K_cat/K_sub and/or V_max/K_sub for a particular labeled nucleotide. In some embodiments, the K_cat/K_sub, the V_max/K_sub, or both, are increased compared to the wild type polymerase.

[00458] In one embodiment, mutant polymerases having altered nucleotide binding kinetics and/or altered nucleotide incorporation kinetics can be selected for use in the nucleotide

incorporation methods. The altered kinetics for nucleotide binding and/or for nucleotide

incorporation include: polymerase binding to the target molecule; polymerase binding to the nucleotide; polymerase catalyzing nucleotide incorporation; the polymerase cleaving the phosphate group or substituted phosphate group; and/or the polymerase releasing the cleavage product. These polymerases can offer improvements in distinguishing between productive and non-productive nucleotide binding events compared to other polymerases.

[00459] In one embodiment, the selected polymerases can have improved photo-stability compared to polymerases traditionally used in nucleotide polymerization reactions. The desirable polymerases can remain enzymatically active during and/or after exposure to electromagnetic energy (e.g., light). For example, the desirable polymerase can retain a level of enzymatic activity, and/or be enzymatically active for a greater length of time, compared to polymerases traditionally used in nucleotide polymerization reactions after exposure to electromagnetic energy. Methods for measuring enzymatic activity are well known in the art.

[00460] In one embodiment, the selected polymerase can be enzymatically active when conjugated to an energy transfer moiety (e.g., nanoparticle or fluorescent dye). The selected polymerase, as part of a polymerase-energy transfer moiety conjugate, can polymerize nucleotides. For example, various forms of B103 polymerase (SEQ ID NOS: 1, 2, and 3) retain enzymatic activity when linked to a nanoparticle or fluorescent dye. Conjugates having these types of selected polymerases offer advantages over other polymerases which may lose most or all enzymatic activity when linked to an energy transfer moiety. In some embodiments, the polymerase can comprise any of the polymerases, including the B103 polymerase variants, disclosed in U.S. Patent Application Ser. No. 12/748,359, filed March 26, 2010, incorporated by reference in its entirety.

[00461] In some embodiments, the polymerase can be a deletion mutant which retains nucleotide polymerization activity but lacks the 3'→ 5' or 5'→ 3' exonuclease activity (SEQ ID NOS: l- 12). For example, mutant phi29 polymerases having exonuclease-minus activity, or reduced exonuclease activity, can optionally comprise the amino acid sequence of SEQ ID NO:7-12 and further comprise one or more amino acid substitutions at positions selected from the group consisting of: 12, 14, 15, 62, 66, 165 and 169 (wherein the numbering is relative to the amino acid sequence of wild type phi29 according to SEQ ID NO:6). In some embodiments, the polymerase is a phi29 polymerase comprising the amino acid sequence of SEQ ID NO:6 and one or more of the following amino acid substitutions: D12A, E14I, E14A, T15I, N62D, D66A, Y165F, Y165C, and D169A, wherein the numbering is relative to SEQ ID NO:6. [00462] In one embodiment, the mutant phi29 polymerases include one or more amino acid mutations at positions selected from the group consisting of: 132, 135, 250, 266, 332, 342, 368, 371, 375, 379, 380, 383, 387, 390, 458, 478, 480, 484, 486 and 512, wherein the numbering is relative to the amino acid sequence of SEQ ID NO:6. In some embodiments, the phi29

polymerase can comprise an amino acid deletion, wherein the deletion includes some of all of the amino acids spanning positions 306 to 311 (relative to the numbering in SEQ ID NO:6).

[00463] In one embodiment, the mutant phi29 polymerase includes one or more amino acid mutations selected from the group consisting of: K132A, K135A, K135D, K135E, V250A,

V250C, Y266F, D332Y, L342G, T368D, T368E, T368F, K370A, K371E, T372D, T372E, T372R, T372K, E375A, E375F, E375H, E375K, E375Q, E375R, E375S, E375W, E375Y, K379A,

Q380A, K383E, K383H, K383L, K383R, N387Y, Y390F, D458N, K478D, K478E, K478R,

L480K, L480R, A484E, E486A, E486D, K512A K512D, K512E, K512R, K512Y,

K371E/K383E/N387Y/D458N, Y266F/Y390F, Y266F/Y390F/K379A/Q380A, K379A/Q380A, E375Y/Q380A/K383R, E375Y/Q380A/K383H, E375Y/Q380A/K383L, E375Y/Q380A/V250A, E375Y/Q380A/V250C, E375Y/K512Y/T368F, E375Y/K512Y/T368F/A484E, K379A/E375Y, K379A/K383R, K379A/K383H, K379A/K383L, K379A/Q380A, V250A/K379A,

V250A/K379A/Q380A, V250C/K379A/Q380A, K132A/K379A and deletion of some or all of the amino acid residues spanning R306 to K311, wherein the numbering is relative to the amino acid sequence of SEQ ID NO:6.

[00464] Without being bound to any particular theory, it is thought that the domain comprising amino acid residues 304-314 of the amino acid sequence of SEQ ID NO: 6 (Phi-29 polymerase), or homologs thereof, can reduce or otherwise interfere with DNA initiation and/or elongation by inhibiting access to the Phi-29 polymerase active site, and that this region must be displaced in order to allow access to the active site. See, e.g., Kamtekar et al., "The Φ29 DNA polymerase:protein primer structure suggests a model for the initiation to elongation transition", EMBO J., 25: 1335-1343 (2005).

[00465] In another embodiment, the polymerase can be a B 103 polymerase comprising the amino acid sequence of SEQ ID NOS: l-5. The B103 polymerase can optionally include one or more mutations that reduce the exonuclease activity of the polymerase. Optionally, such mutations can include any one or a combination of mutations at the following amino acid positions: 2, 9, 11, 12, 14, 15, 58, 59, 63, 162, 166, 377 and 385, wherein the numbering is relative to SEQ ID NOS: l or 2. In some embodiments, the B103 polymerase can optionally comprise the amino acid sequence of SEQ ID NOS: l or 2, and further comprise one or more amino acid substitutions selected from the group consisting of: D9A, El l A, El II, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relative to SEQ ID NOS: l or 2.

[00466] In some embodiments, the B103 polymerase can optionally the amino acid sequence of SEQ ID NOS: l or 2, and further comprise one or more amino acid substitutions selected from the group consisting of (in single letter amino acid code): H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, D507H, D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y and K509F, wherein the numbering is relative to the sequence shown in SEQ ID NOS: l or 2. The B103 polymerase can optionally further comprise the amino acid sequence of any of the polymerases disclosed by Hendricks, in U.S. Serial No. 61/242,771, filed on September 15, 2009, or U.S. Serial No. 61/293,618, filed on January 8, 2010.

[00467] Polymerases having desirable properties, including those having altered nucleotide binding and/or nucleotide incorporation kinetics, having improved photo-stability, and/or having improved enzymatic activity when conjugated to an energy transfer moiety, include polymerases according to SEQ ID NOS: l-5.

[00468] SEQ ID NO: 1

MPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFH

NLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIY

DSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNAIEIIARALDIQFKQ

GLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIG

EGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQ

IKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEF

IDKWTYVKTHEKGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEY

KDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYW

AHES TFKR AKYLRQKTYIQDIY AKEVDGKLIECS PDE ATTTKFS VKC AGMTDTIKKKVTF

DNFR VGFS S TGKPKP VQ VNGG V VLVDS VFTIK

[00469] SEQ ID NO:2

MPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFH

NLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIY

DSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNDIEIIARALDIQFKQ

GLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIG

EGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQ IKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEF IDKWTYVKTHEKGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEY KDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYW AHES TFKR AKYLRQKTYIQDIY AKEVDGKLIECS PDE ATTTKFS VKC AGMTDTIKKKVTF DNFR VGFS S TGKPKP VQ VNGG V VLVDS VFTIK

[00470] SEQ ID NO:3

MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGSKHMPRKMFSCDFETTTKLDDCRVW

AYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSN

EGLPNTYNTIIS KMGQWYMIDICFGYKGKRKLHTVIYDS LKKLPFPVKKIAKDFQLPLLKG

DIDYHAERPVGHEITPEEYEYIKNAIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKK

FNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPY

GAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELY

LTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTREKGAKKQLAKLML

NS LYGKF AS NPD VTGKVP YLKEDGS LGFRVGDEE YKDP V YTPMG VFIT A WARFTTIT A A

QACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAK

EVDGKLIECS PDE ATTTKFS VKC AGMTDTIKKKVTFDNFRVGFS S TGKPKP VQ VNGG VVL

VDSVFTIK

[00471] SEQ ID NO:4

MNHLVHHHHHHIEGRHMELGTLEGSMKHMPRKMFSCDFETTTKLDDCRVWAYGYMEI

GNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTY

NTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAE

RPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPK

LSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQG

KYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELYLTNVDLEL

IQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTHEKGAKKQLAKLMLNSLYGKFA

SNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIY

CDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIE

CSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVLVDSVFTIK

[00472] SEQ ID NO:5

MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGSKHMPRKMFSCDFETTTKLDDCRVW

AYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSN

EGLPNTYNTIIS KMGQWYMIDICFGYKGKRKLHTVIYDS LKKLPFPVKKIAKDFQLPLLKG

DIDYHAERPVGHEITPEEYEYIKNAIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKK

FNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPY

GAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELY

LTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTHEKGAKKQLAKLML

NS LYGKF AS NPD VTGKVP YLKEDGS LGFRVGDEE YKDP V YTPMG VFIT A WARFTTIT A A

QACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAK

EVDGKLIECS PDE ATTTKFS VKC AGMTDTIKKKVTFDNFRVGFS S TGKPKP VQ VNGG VVL

VDSVFTIK

[00473] SEQ ID NO:6

MKHMPRKM YS CDFETTTKVEDCRVW A YG YMNIEDHS EYKIGNS LDEFM A WVLKVQ A DLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKI HTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEAL LIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRF

KEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEG

YIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATT

GLFKDFID KWT YIKTTS EG AIKQLAKLMLNS LYGKF AS NPD VTGKVP YLKENG ALGFRL

GEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPK

KLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDK

IKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK

[00474] SEQ ID NO:7

MGLRRAS LHHLLGGGGS GGGGS A A AGS A ARKM YS CDFETTTKVEDCRVW A YG YMNIE

DHS EYKIGNS LDEFM A WVLKVQ ADLYFHNLKFDG AFIINWLERNGFKWS ADGLPNT YNT

IISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERP

VGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSL

GLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKY

VWDED YPLHIQHIRCEFELKEG YIPTIQIKRS RF YKGNE YLKS S GGEIADLWLS N VDLELM

KEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASN

PDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCD

TDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEG

SPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK

[00475] SEQ ID NO:8

MHHHHHHLLGGGGS GGGGS A A AGS A ARKM YS CDFETTTKVEDCRVW A YGYMNIEDHS

EYKIGNS LDEFM A WVLKVQ ADLYFHNLKFDG AFIINWLERNGFKWS ADGLPNT YNTIIS R

MGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGY

KITPEEYAYIKNAIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD

KEVR Y A YRGGFTWLNDRFKEKEIGEGM VFD VNS LYP AQM YS RLLP YGEPF FEGKY VW

DEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEH

YDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDV

TGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDS

IHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPD

DYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK

[00476] SEQ ID NO:9

MNHLVHHHHHHIEGRHMELGTLEGS MKHMPRKM YS C AFETTTKVEDCR VWA YG YMNI

EDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAGAFIINWLERNGFKWSADGLPNTYN

TIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKER

PVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLS

LGLD KEVR Y A YRGGFTWLNDRFKEKEIGEGM VFD VNS LYP AQM YS RLLP YGEPIVFEGK

Y VWDED YPLHIQHIRCEFELKEG YIPTIQIKRS RF YKGNEYLKS S GGEIADLWLS N VDLEL

MKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFAS

NPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTrrAAQACYDRIIYC

DTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVE

GSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK

[00477] SEQ ID NO: 10

MNHLVHHHHHHIEGRHMELGTLEGS MKHMPRKM YS C AFETTTKVEDCR VWA YG YMNI EDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAGAFIINWLERNGFKWSADGLPNTYN TIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKER PVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLS LGLD KEVR Y A YRGGFTWLNDRFKEKEIGEGM VFD VNS LYP AQM YS RLLP YGEPIVFEGK

Y VWDED YPLHIQHIRCEFELKEG YIPTIQIKRS RF YKGNEYLKS S GGEIADLWLS N VDLEL MKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKALAKLMLNSLYGKFAS NPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTrrAAQACYDRIIYC DTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVE GSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK

[00478] SEQ ID NO: ! !

Y VWDED YPLHIQHIRCEFELKEG YIPTIQIKRS RF YKGNEYLKS S GGEIADLWLS N VDLEL MKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNGLYGKFAS NPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTrrAAQACYDRIIYC DTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVE GSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK

[00479] SEQ ID NO: 12

MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGSKHMPRKMYSCAFETTTKVEDCRVW

AYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAGAFIINWLERNGFKWSA

DGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKG

DIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKF

KKVFPTLS LGLD KEVRY A YRGGFTWLNDRFKEKEIGEGM VFD VNS LYP AQM YS RLLP YG

EPP FEGKYVWDED YPLHIQHIRCEFELKEG YIPTIQIKRSRFYKGNEYLKSSGGEIADLW

LS N VDLELMKEH YDLYN VE YIS GLKFKATTGLFKDFID KWT YIKTTS EG AIKQLAKLMLN

SLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQ

ACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMK

EVDGKLVEGS PDD YTDIKFS VKC AGMTDKIKKEVTFENFKVGFS RKMKPKP VQVPGG V V

LVDDTFTIK

[00480] SEQ ID NO: 13

MKEFYLTVEQIGDSIFERYIDSNGRERTREVEYKPSLFAHCPESQATKYFDIYGKPCTRK LFANMRDASQWIKRMEDIGLEALGMDDFKLAYLSDTYNYEIKYDHTKIRVANFDIEVTSP DGFPEPS QAKHPID AITHYDS IDDRFYVFDLLNSPYGNVEEWS IEIA AKLQEQGGDEVPS EIIDKIIYMPFDNEKELLMEYLNFWQQKTPVILTGWNVESFDIPYVYNRIKNIFGESTAK RLS PHRKTR VKVIENM YGS REIITLFGIS VLD YIDLYKKFS FTNQPS YS LD YIS EFELN V GKLKYDGPIS KLRES NHQRYIS YNIID V YR VLQID AKRQFINLS LDMG Y Y AKIQIQS VFS PIKTWD AIIFNS LKEQNKVIPQGRS HP VQP YPG AFVKEPIPNR YKY VMS FDLTS LYPS II RQVNIS PETIAGTFKV APLHD YIN A V AERPS D V YS CS PNGMM Y YKDRDG V VPTEITKVFN QRKEHKG YMLA AQRNGEIIKE ALHNPNLS VDEPLD VD YRFDFS DEIKEKIKKLS AKS LNE MLFRAQRTEVAGMTAQINRKLLINSLYGALGNVWFRYYDLRNATAITTFGQMALQWIER KVNEYLNEVCGTEGEAFVLYGDTDSIYVSADKIIDKVGESKFRDTNHWVDFLDKFARER MEPAIDRGFREMCEYMNNKQHLMFMDREAIAGPPLGSKGIGGFWTGKKRYALNVWDM EGTRY AEPKLKIMGLETQKS S TPKA VQKALKECIRRMLQEGEES LQE YFKEFEKEFRQLN YIS IAS VS S ANNIAKYD VGGFPGPKCPFHIRGILT YNRAIKGNID APQ V VEGEKV Y VLPLRE GNPFGDKCIAWPSGTEITDLIKDDVLHWMDYTVLLEKTFIKPLEGFTSAAKLDYEKKASL FDMFDF

[00481] In one aspect, the polymerase can be a fusion protein comprising the amino acid sequence of a nucleic acid-dependent polymerase (the polymerase portion) linked to the amino acid sequence of a second enzyme or a biologically active fragment thereof (the second enzyme portion). The second enzyme portion of the fusion protein may be linked to the amino or carboxyl end of the polymerase portion, or may be inserted within the polymerase portion. The polymerase portion of the fusion protein may be linked to the amino or carboxyl end of the second enzyme portion, or may be inserted within the second enzyme portion. In some embodiments, the polymerase and second enzyme portions can be linked to each other in a manner which does not significantly interfere with polymerase activity of the fusion or with the ability of the fusion to bind nucleotides, or does not significantly interfere with the activity of the second enzyme portion. In the fusion protein, the polymerase portion or the second enzyme portions can be linked with at least one energy transfer donor moiety. The fusion protein can be a recombinant protein having a polymerase portion and a second enzyme portion. In some embodiments, the fusion protein can include a polymerase portion chemically linked to the second enzyme portion.

[00482] The polymerase can be a modified polymerase having certain desired characteristics, such as an evolved polymerase selected from a directed or non-directed molecular evolution procedure. The evolved polymerase can exhibit modulated characteristics or functions, such as changes in: affinity, specificity, or binding rates for substrates (e.g., target molecules,

polymerization initiation sites, or nucleotides); binding stability to the substrates (e.g., target molecules, polymerization initiation sites, or nucleotides); nucleotide incorporation rate;

nucleotide permissiveness; exonuclease activity (e.g., 3'→5' or 5'→3'); rate of extension;

processivity; fidelity; stability; or sensitivity and/or requirement for temperature, chemicals (e.g., DTT), salts, metals, pH, or electromagnetic energy (e.g., excitation or emitted energy). Many examples of evolved polymerases having altered functions or activities can be found in U.S.

provisional patent application No. 61/020,995, filed January 14, 2008.

[00483] Methods for creating and selecting proteins and enzymes having the desired

characteristics are known in the art, and include: oligonucleotide-directed mutagenesis in which a short sequence is replaced with a mutagenized oligonucleotide; error-prone polymerase chain reaction in which low-fidelity polymerization conditions are used to introduce point mutations randomly across a sequence up to about 1 kb in length (R. C. Caldwell, et al., 1992 PCR Methods and Applications 2:28-33; H. Gramm, et al, 1992 Proc. Natl. Acad. Sci. USA 89:3576-3580); and cassette mutagenesis in which a portion of a sequence is replaced with a partially randomized sequence (A. R. Oliphant, et al., 1986 Gene 44: 177-183; J. D. Hermes, et al., 1990 Proc. Natl.

Acad. Sci. USA 87:696-700; A. Arkin and D. C. Youvan 1992 Proc. Natl. Acad. Sci. USA

89:7811-7815; E. R. Goldman and D. C. Youvan 1992 Bio/Technology 10: 1557-1561; Delagrave et al., 1993 Protein Engineering 6: 327-331; Delagrave et al., 1993 Bio/Technology 11: 1548- 155); and domain shuffling.

[00484] Methods for creating evolved antibody and antibody-like polypeptides can be adapted for creating evolved polymerases, and include applied molecular evolution formats in which an evolutionary design algorithm is applied to achieve specific mutant characteristics. Many library formats can be used for evolving polymerases including: phage libraries (J. K. Scott and G. P.

Smith 1990 Science 249:386-390; S. E. Cwirla, et al. 1990 Proc. Natl. Acad. Sci. USA 87:6378- 6382; J. McCafferty, et al. 1990 Nature 348:552-554) and lad (M. G. Cull, et al., 1992 Proc. Natl. Acad. Sci. USA 89: 1865-1869).

[00485] Another adaptable method for evolving polymerases employs recombination (crossing- over) to create the mutagenized polypeptides, such as recombination between two different plasmid libraries (Caren et al. 1994 Bio/Technology 12: 517-520), or homologous recombination to create a hybrid gene sequence (Calogero, et al., 1992 FEMS Microbiology Lett. 97: 41-44;

Galizzi et al., WO91/01087). Another recombination method utilizes host cells with defective mismatch repair enzymes (Radman et al., WO90/07576). Other methods for evolving

polymerases include random fragmentation, shuffling, and re-assembly to create mutagenized polypeptides (published application No. U.S. 2008/0261833, Stemmer). Adapting these

mutagenesis procedures to generate evolved polymerases is well within the skill of the art.

[00486] In some embodiments, the polymerase can be fused with, or otherwise engineered to include, DNA-binding or other domains from other proteins that are capable of modulating DNA polymerase activity. For example, fusion of suitable portions of the Single-Stranded DNA Binding Protein (SSBP), thioredoxin and/or T7 DNA polymerase to bacterial or viral DNA polymerases has been shown to enhance both the processivity and fidelity of the DNA polymerase. Similarly, other groups have described efforts to engineer polymerases so as to broaden their substrate range. See, e.g., Ghadessy et al, Nat. Biotech., 22 (6):755-759 (2004). Similarly, the conjugates of the disclosure can optionally comprise any polymerase engineered to provide suitable performance characteristics, including for example a polymerase fused to intact SSBP or fragments thereof, or to domains from other DNA-binding proteins (such as the herpes simplex virus UL42 protein.)

[00487] In some embodiments, a blend of different conjugates, each of which comprises a polymerase of unique sequence and characteristics, can be used according to the methods described herein. Use of such conjugate blends can additionally increase the fidelity and processivity of DNA synthesis. For example, use of a blend of processive and non-processive polymerases has been shown to result in increased overall read length during DNA synthesis, as described in U.S. Published App. No. 2004/0197800. Alternatively, conjugates comprising polymerases of different affinities for specific acceptor-labeled nucleotides can be used so as to achieve efficient incorporation of all four nucleotides.

[00488] In one embodiment, the polymerase can be a mutant which retains nucleotide

polymerization activity but lacks the 3'→5' or 5'→ 3' exonuclease activity (SEQ ID NOS: l-12). In another embodiment, the polymerase can be an exonuclease minus mutant which is based on wild type phi29 polymerase (SEQ ID NO:6) (Blanco, U.S. Patent No. 5,001,050, 5,198,543, and 5,576,204; and Hardin PCT/US2009/31027 with an International filing date of January 14, 2009) and comprising one or more substitution mutations, including: D12A, D66A, D169A, H61R, N62D, Q380A, and/or S388G, and any combination thereof.

[00489] In some embodiments, the polymerase can comprise the amino acid sequence of any polymerase disclosed in U.S. Provisional Application Nos. 61/242,771, filed on September 15, 2009; 61/263,974, filed on November 24, 2009 and 61/299,919, filed on January 29, 2010, or any variant thereof.

[00490] In some embodiments, the polymerase (or polymerase fusion protein) may be linked with at least one label, for example an energy transfer moiety. In the polymerase fusion protein, the label can be attached to the polymerase portion or to the second enzyme portion. In some

embodiments, one or more energy transfer moieties are linked to the amino end or the carboxyl end of the polymerase (or polymerase fusion protein); alternatively, the one or more energy transfer moieties can be inserted in the interior of the polymerase (or fusion protein sequence).

The energy transfer moiety can be attached to the fusion protein in a manner which does not interfere with the nucleotide binding activity, or with the nucleotide incorporation activity, or with the activity of the second enzyme.

[00491] In some embodiments, the polymerase can be labeled with one or more fluorescent nanoparticles, for example one or more quantum dots. Examples of labeled polymerases, including quantum-dot labeled polymerases are disclosed, for example, in U.S. Patent Application Ser. No. 12/748,314, filed March 26, 2010; and in U.S. Patent Application Ser. No. 12/748,355, filed March 26, 2010, both of which disclosures are incorporated by reference in their entireties.

[00492] In one embodiment, a single label is operably attached to more than one polymerase (or more than one polymerase fusion protein) and the attachment can be at the amino end or carboxyl end or may be inserted within the polymerase (or fusion protein sequence). In another

embodiment, a single label can be linked to one polymerase or polymerase fusion protein. In yet another embodiment, a plurality of labels are linked to a single polymerase (or polymerase fusion protein).

[00493] The components of the sequencing reaction that can be exchanged include any component of a nucleotide binding or nucleotide incorporation reaction, including but not limited to any type of: target nucleic acid molecule; template strand of a target nucleic acid molecule; primer; polymerization initiation site; polymerase; nucleotides (e.g., hydrolyzable, non- hydrolyzable, chain-terminating, or labeled or non-labeled nucleotides); the synthesized nascent nucleic acid molecule; compounds which reduce photo-damage; buffers; salts; co-factors; divalent cations; and chelating agents. The fresh reagents can be the same or different types of reagents compared to the old reagents.

[00494] The reagent exchange methods can be practiced using any type of nucleotide binding or nucleotide incorporation reactions, including but not limited to: the energy transfer methods disclosed herein; any type of discontinuous reactions (e.g., synchronous nucleotide incorporation methods described in: U.S. Serial No. 61/184,774, filed on June 5, 2009; U.S. Serial No.

61/242,762, filed on September 15, 2009; and U.S. Serial No. 61/180,811, filed on May 22, 2009; U.S. Serial No. 61/295,533, filed on January 15, 2010); and any type of continuous reactions (e.g., asynchronous nucleotide incorporation methods as described in: (U.S. Serial No. 61/077,090, filed on June 30, 2008; U.S. Serial No. 61/089,497, filed on August 15, 2008; U.S. Serial No.

61/090,346, filed on August 20, 2008; PCT application No. PCT/US 09/049324, filed on June 30, 2009; U.S. Serial No. 61/164,324, filed on March 27, 2009; and U.S. Serial No. 61/263,974, filed on November 24, 2009; U.S. Serial Nos. 61/289,388; 61/293,616; 61/299,917; 61/307,356).

[00495] The reagent exchange methods can be practiced using any type of format using an immobilized: primer; target molecule; synthesized strand; and/or polymerase. The reagent exchange methods can be practiced on a single target nucleic acid molecule, or on random or organized arrays of single nucleic acid molecules, and using any type of solid surface (U.S. Serial No. 61/220174, filed on June 24, 2009; and U.S. Serial No. 61/245248, filed on September 23, 2009; U.S. Serial No. 61/302,475). The target molecules and synthesized strands can be genomic, recombinant, DNA, RNA, double- stranded, or single-stranded nucleic acid molecules. The target nucleic acid molecules can be linear or circular. The target nucleic acid molecules can be self- priming molecules or can be associated with primer molecules. The target nucleic acid molecules can be immobilized using any method, including the methods depicted in any of Figures 1-7.

[00496] In practicing the reagent exchange methods and related compositions, systems, kits and apparatuses of the disclosure, any combination of capture molecule, primer, target molecule, and/or synthesized strand, can be removed from the sequencing reaction using suitable physical, chemical, and/or enzymatic methods, in any combination and in any order.

[00497] In some embodiments, the target molecule, synthesized strand, polymerase, primer, capture molecule, or any other reagent in the sequencing reaction can be removed using fluid flow, washing, and/or aspiration. In one exemplary embodiment, the target molecule, primer molecule or synthesized strand is linked, either directly or indirectly, to the solid surface in a manner which withstands flowing, washing, aspirating, and changes in salt, temperature, chemical, enzymatic, and/or pH conditions. A fresh supply of polymerase, nucleotides, reagents, primer molecules, splinter molecules, and/or adaptor molecules, can be added to the immobilized nucleic acid molecules following such washing. The polymerase (e.g., donor-labeled) and nucleotides (e.g., and acceptor-labeled) can be added to the immobilized nucleic acid molecules under conditions which are suitable for nucleotide binding and/or nucleotide incorporation to occur. The fresh polymerase, nucleotides, and reagents, can be the same or different from the old polymerase, nucleotides, and/or reagents.

[00498] In one exemplary embodiment, the target nucleic acid molecule is linked to a surface, the polymerase and the nucleotides are flowed into the flow cell and contacted with the target under polymerization conditions. One or more components of the sequencing reaction is then removed from the mixture, and a replacement component is then flowed into the cell.

[00499] In some embodiments, the synthesis of the same nascent nucleic acid molecule is continued after the exchange of a particular sequencing reaction component is complete. In such embodiments, the portions of the template strand that are sequenced prior to and following such exchange are non- overlapping and can optionally be contiguous with each other. Hence, the sequence information obtained prior to the exchange is not redundant to the sequence information obtained following the exchange; in effect, different portions of the template are sequenced before and after the exchange.

[00500] In other embodiments, the synthesis of the same nascent nucleic acid molecule is not continued following the exchange; instead, synthesis of a new nascent nucleic acid molecule is reinitated afresh. In such embodiments, the portions of the template strand that are sequenced prior to and following such exchange typically overlapping and can in some instances be the same. Hence, the sequence information obtained prior to the exchange is at least partially redundant to the sequence information obtained following the exchange; in effect, the same or overlapping portions of the template are sequenced before and after the exchange.

[00501] It should be understood, however, that the process steps outlined for the methods disclosed herein are not limited to any particular order or sequence of operation. That is, the sequence of operation of the process steps in the disclosed methods can change depending on the requirements of the particular application.

[00502] In some embodiments, the disclosure relates generally to apparatuses or devices for sequencing a target nucleic acid molecule according to any of the methods disclosed herein. In some embodiments, the apparatus can include one or more flow cells in with the target nucleic acid molecule or template strand (or several target nucleic acid molecules or template strands to be sequenced in parallel) are immobilized. The target nucleic acid molecule can be attached to the surface through at least one covalent bond, or through at least one non-covalent bond (e.g., biotin- avidin interaction, etc.).

[00503] The flow cell can include one or more inlets and/or one or more outlets. Sequence reaction reagents can be introduced through the one or more inlets, and "spent" sequencing reaction mixture can be removed through the one or more outlets. Exchange of one or more sequencing reagents can be performed by introducing the one or more flow cell volumes of sequencing reaction mixture including the new reagent through an inlet, and removing one or more flow cell volumes of sequencing reaction mixture through an outlet.

[00504] In designing an effective flow cell for sequencing including reagent exchange, several considerations should be addressed, namely channel geometry, fluid handling architecture, and temperature control.

[00505] In some embodiments, the channel geometry within the flow cell can be constructed to maximize the effectiveness of reagent exchange by minimizing the carryover of reagents and minimizing the shear drag forces on template molecules during fluid transfers. Such design will reduce cross contamination of molecules that may harm sequencing reaction chemistry and will reduce the amount of template loss that may occur after each cycle. Depending on the method of fluid transfer, channel geometries can be constructed for optimal reagent exchange performance. For example, for efficient exchange of reagents in a pressure driven flow system, the width of the channel can be minimized. The minimum width can be determined by the field of view of the imaging objective, which can be approximated by the field number divided by the magnification. The following examples can be used for standard, i.e., "off-the-shelf objectives: For a standard 20x objective, 1.5 mm channel width; for a standard 60x objective, 0.5 mm channel width; for a standard lOOx objective, 0.3 mm channel width.

[00506] In some embodiments, especially embodiments including non-covalently bound target nucleic acid molecules or template strands, and depending on target/template length, it can be desirable to keep shear rates as low as possible, for example, at less than 2000 Is. Shear rates will be proportional to flow rate, but will be inversely proportional to the square of the channel height and inversely proportional to the width. Therefore, increasing flow rates or decreasing flow cell geometries will have a negative impact on shear rates. Another factor to consider in the flow cell geometry is the pressure drop across the channel. Excessive pressure with high numerical aperture systems can cause movement of the observation plane due to the thin substrates that the objectives are designed for. Figure 16 shows an example of one configuration for a disposable flow cell with multiple fields of view. Figure 17 depicts a plot showing the relationships between the pressure drop, flow rate, and channel height for a cartridge having the configuration depicted in Figure 16 with a 16mm x 2mm channel. The predicted "safe design" regions are under the magenta curve (i.e., at shear rates of 2000 Is or below).

[00507] Some exemplary flow cell geometries are depicted in Figure 18. A so-called "push system" includes a small diameter to minimize dead volume, and a large diameter outlet to minimize back pressure. A so-called "push-pull" system includes a small diameter inlet to minimize dead volume, and an outlet having an impedance that is matched to the inlet geometry for load balancing.

[00508] In embodiments involving a non-multiplexing configuration, it can be desirable to have each field of view being independently fluidically addressable in order to minimize exposure to unnecessary shear stresses and reagents. One option is to create independent fluidic paths in the flow cell. In some embodiments, however, such an approach can be problematic because the higher the number of independent field of views in a cartridge, the great the number of fluidic interface ports that are required, making it more difficult to make reliable connections and maintain flatness. In some embodiments, the flatness can optionally be improved by supporting the interfaces as shown in the exemplary embodiment depicted in Figure 19.

[00509] In some embodiments, the flow cell is an electro wetting flow cell, which affords flexibility in field of views, minimizes shear forces, provides for optimal reagent exchange efficiency, and minimizes the number of fluidic ports is an electro wetting flow cell. Figure 20A depicts a cross section an exemplary electro wetting flow cell; Figure 20B depicts the top view of this exemplary electro wetting flow cell.

[00510] In some embodiments, the flow cell is used in conjunction with a fluid handling system. The type of devices and plumbing used in the fluid handling system can influence the efficiency of retention of the molecules of interest. For example, syringe type pumps can have effects from the stepper motors and sticktion of the syringe plunger that causes instability of flow (Figure 21 A). This instability can cause problems with robust attachment of molecules. Pressure driven flow can be more suitable for producing stable flow rates that causes less disturbance to the attached molecules. An exemplary system producing pressure-driven flow is depicted in Figure 21B.

[00511] In some embodiments, the flow cell is used in conjunction with a temperature control system. Temperature control can be useful in creating an environment that is optimized for the sequencing enzyme and/or to facilitate efficient binding of primers. The architecture of the underlying system can influence the thermal control approach. In the case of systems including prism-TIRF based detection, where excitation launch is performed on one side and detection on the other, both the objective and the prism can be heated.

[00512] In some embodiments, the heater element can be included between the optical layers without interfering with detection or excitation. In one exemplary embodiment, optically transparent, electrically conductive thin film heaters are deposited into the assembly (Figure 22A), allowing for thermal coupling to the sample and minimized interference with detection. In other embodiments, where it is desirable to localize the heat to a particular field of view, but only one optical side is required for detection, one or more laminated heater circuits can be integrated with a lower cost consumable (Figure 22B).

EXAMPLES

[00513] In the following embodiments (e.g., Figures 1-7), the "N" can be any nucleotide base, and the "I" can be a universal base such as inosine. [00514] Example 1

[00515] In one exemplary embodiment, a template strand can be immobilized through ligation of the template strand to an immobilized capture molecule using a splinter oligonucleotide that hybridizes both to the target molecule and capture oligonucleotide. The immobilized template strand can then be contacted with a sequencing enzyme, for example with T4 DNA polymerase, (see Figure 1). A primer can be annealed to the immobilized target molecule, and a first nascent nucleic acid molecule can be synthesized using a polymerase and nucleotides. Physical, chemical, and/or enzymatic conditions can be used to remove the first nascent nucleic acid molecule, polymerase, and/or nucleotides, while leaving behind the target nucleic acid molecule (or at least the template strand) capable of supporting a fresh round of template-dependent replication. The remaining target nucleic acid molecule (or remaining template strand) can be contacted with fresh reagents to permit re-sequencing the same template strand of the target nucleic acid molecule.

[00516] Example 2

[00517] Figure 2 depicts another example of a method involving re-sequencing the same target molecule, in a direction away from the solid surface. A "two-pass" method for re-sequencing the same nucleic acid molecule has been described (Harris, et al., 2008 Science 320: 106-109, and supporting online material).

[00518] In another embodiment, a polynucleotide tail (e.g., poly-A, -G, -C, or -T) can be added to a target nucleic acid molecule (or to a template strand), for example using a terminal transferase enzyme (TdT in Figure 2). The tailed target nucleic acid molecule (or template strand) can be ligated to an immobilized capture molecule using a splinter oligonucleotide (which can hybridize to the target molecule and capture oligonucleotide) and enzymes for ligation and/or nucleotide polymerization (e.g., T4 ligase and T4 DNA polymerase, respectively). A primer can be annealed to the immobilized target nucleic acid molecule (or immobilized template strand), and a synthesized strand can be produced using a polymerase and nucleotides. Physical, chemical, and/or enzymatic conditions can be used to remove the synthesized strand, polymerase, and nucleotides. The remaining target nucleic acid molecule (or template strand) can be contacted with fresh reagents to permit re-sequencing the same target molecule. Figure 2 depicts re- sequencing the same target molecule, in a direction away from the solid surface.

[00519] Example 3

[00520] In yet another embodiment, a target nucleic acid molecule (or template strand thereof) can be ligated to an immobilized hairpin capture molecule, where a portion of the capture molecule can hybridize to the target molecule (see Figure 3). The target nucleic acid molecule (or template strand) can be ligated to the hairpin capture molecule using enzymes for ligation and/or nucleotide polymerization (e.g., T4 ligase and T4 DNA polymerase, respectively). The hairpin adaptor molecule can include a recognition sequence for cleavage (scission) by an endonuclease enzyme. For example, the recognition sequence can be an RNA portion which can be 3-6 nt in length, to form a DNA/RNA hybrid. The RNA portion can be 4 nt in length. The RNA portion can include purines (A and G) in any order. The RNA portion of the RNA/DNA duplex can be a substrate for cleavage by an endoribonuclease (e.g., RNase H). In another example, the recognition sequence can be an AP site (apurinic/apyrimidinic) having a THF substrate

(tetrahydrofuran) which can be cleaved by an AP endonuclease. In another example, the recognition sequence can include nucleotide analogs (e.g., 8-oxo-7,8-dihydroguanine, 8- oxoguanine, or 8-hydroxyguanine) which can be cleaved by DNA glycosylase OGG1. In yet another example, the recognition sequence can include any sequence which can be cleaved by a nicking enzyme. After scission, a primer can be annealed to the target nucleic acid molecule (or template strand), and a synthesized strand can be produced using a polymerase and nucleotides. Physical, chemical, and/or enzymatic conditions can be used to remove the synthesized strand, polymerase, and nucleotides. The remaining target nucleic acid molecule (or template strand) can be contacted with fresh reagents to permit re-sequencing the same target nucleic acid molecule (or template strand). Figure 3 depicts re-sequencing the same target molecule, in a direction away from the solid surface.

[00521] Example 4

[00522] In yet another embodiment, the 5' end of the template strand of a target molecule can be ligated to an adaptor molecule using T4 ligase (Figure 4A). The adaptor molecule can be annealed with a primer having a blocked 3' end (Figure 4A). The template strand can be reacted with terminal transferase to add a poly-nucleotide tail (e.g., poly-A, -G, -C, or -T) (TdT in Figure 4A). The tailed target molecule can be captured by an immobilized oligonucleotide (Figure 4B). The immobilized oligonucleotide can be used to produce a synthesized strand, using a polymerase and nucleotides (Figure 4B). Physical, chemical, and/or enzymatic conditions can be used to remove the target nucleic acid molecule (or template strand), polymerase, and nucleotides. A primer can be annealed to the remaining synthesized strand. A newly synthesized strand can be produced using a polymerase and nucleotides. Physical, chemical, and/or enzymatic conditions can be used to remove the newly synthesized strand, polymerase, and nucleotides. The remaining synthesized strand can be contacted with fresh reagents to permit re-sequencing the same synthesized strand. Figures 4A and 4B depict re-sequencing the same synthesized strand, in a direction towards the solid surface.

[00523] Example 5

[00524] In yet another embodiment, the target molecule can be reacted with terminal transferase to add a poly-nucleotide tail (e.g., poly-A, -G, -C, or -T) (TdT in Figure 5A). The tailed target molecule can be captured by an immobilized capture oligonucleotide (Figure 5A). The immobilized capture oligonucleotide can be used to generate a synthesized strand, using a polymerase and nucleotides (Figure 5B). The 3' end of the synthesized strand can be ligated to an adaptor molecule. Physical, chemical, and/or enzymatic conditions can be used to remove the target molecule, polymerase, and nucleotides. The 3' end of the remaining synthesized strand can be annealed to a primer. A newly synthesized strand can be generated with a polymerase and nucleotides. Physical, chemical, and/or enzymatic conditions can be used to remove the newly synthesized strand, polymerase, and nucleotides. The remaining synthesized strand can be contacted with fresh reagents to permit re-sequencing the same synthesized strand. Figures 5A and 5B depict re-sequencing the same synthesized strand, in a direction towards the solid surface.

[00525] Example 6

[00526] In yet another embodiment, the target molecule can be reacted with terminal transferase to add a poly-nucleotide tail (e.g., poly-A, -G, -C, or -T) (TdT in Figure 6). The tailed target molecule can be circularized. The circularized target molecule can be captured by an immobilized oligonucleotide. The 3' end of the capture oligonucleotide can be used to generate a synthesized strand using a polymerase and nucleotides, in a rolling circle replication mode. A strand- displacement DNA polymerase can be used for the rolling circle replication.

[00527] Example 7

[00528] In another embodiment, stem-loop adaptor molecules can be ligated to both ends of a double-stranded target molecule using T4 ligase (Figure 7) to produce a closed-ended molecule. The resulting molecule can be captured by an immobilized oligonucleotide via complementary sequences in one of the stem-loop adaptor molecules. The immobilized capture oligonucleotide can be used as a primer to generate the synthesized strand, using a polymerase and nucleotides. [00529] Example 8

[00530] In this Example, different portions of a nucleic acid template strand were sequencing successively using different labeled polymerases. An overview of the sequencing system of this Example is depicted in Figure 8.

[00531] The buffers used in this example were as follows:

[00532] TBST buffer pH 7.5: 50mM Tris-HCl (pH 7.5), 50mM NaCl, 0.3% BSA, 0.05% Tween- 20

[00533] MOPS-OSS pH 6.5: MOPS (pH 6.8), lOOmM, Potassium Acetate 50mM, Trolox (in EtOH)10mM, Tween-20 0.1%, BSA 0.3%, Glucose Oxidase 125U/ml, Katalase lOOOOunits/ml

[00534] The sequencing system used in this example was as follows:

[00535] Biotinylated single-stranded nucleic acid template strands were immobilized to a PEG- Streptavidin functionalized surface. Contiguous portions of the immobilized template strands were successively sequenced using different polymerases, each labeled with a quantum dot (FRET donor, referred to herein as the "sDot" label), and using four different types of nucleotide polyphosphates (A, C, G and T), each type of nucleotide being labeled with a different FRET acceptor on the terminal phosphate group. The quantum dot-labeled polymerases are referred to herein as "conjugates". Incorporation of an acceptor-labeled nucleotide polyphosphate by the sDot-labeled polymerase results in the emission of a sequence specific signal due to FRET between the sDot and the acceptor of the incorporating nucleotide, and these sequence- specificsignals are recorded in real-time to determine the sequence of the template strand. After a first portion of the template strand was sequencing using a first sDot-labeled polymerase, the first sDot-labeled polymerase was removed and replaced with a second sDot-labeled polymerase and sequencing was continued from the position where the first sDot-labeled polymerase left off; further cycles of polymerase exchanged were performed as described in further detail below. In theory, such "relay mode" sequencing can be continued for an infinite number of further cycles, with the sequence being extended further with each exchange. The principle underlying such polymerase exchange is is that each new labeled polymerase binds to the template strand and continues nucleic acid synthesis from the point where the previous polymerase had stopped prior to the exchange, thus enabling removal of "damaged" or sick enzymes and replenishment of the sequencing reaction with a robust enzyme to continue nucleic acid synthesis.

[00536] Functionalized cover-glasses were assembled onto a 9-channel cartridge to facilitate injection and aspiration of reagents through the chambers. The cartridge consists of the top plastic over, a middle layer including a double-sided pressure sensitive adhesive which is 170um thick and in which channels are defined by die-cutting, laser cutting or plotter cutting. The

functionalized glass cover slips were adhered to the adhesive forming reaction chambers in the cut slits space with the functionalized surface facing the inside of the reaction chamber. The top of the cartridge also contained 9 entry ports to inject reagents. Biotinylated template (at ΙΟρΜ) was immobilized for 30 minutes on the streptavidin coated PEG-biotin surface of the glass slides. Excess unbound template was washed away using 1ml TBST buffer. Each chamber was then rinsed with a lOOul of 50mM NaOH, immediately followed by a rinse with TBST.

[00537] Multiple assays were performed; some assays utilized a synthetic biotinylated template comprising the following sequence: (80 bases template,

[00538] 5' CAGGATTAGAGAGTACCTTTAATTGCTCCTTTTGATAAGAG

GTCATTTTTGCGGATGGCTTAGAGCTTAATTTTTACCCCGACACGGAGGTT CTATCA

3' ) (Template LC27)

[00539] In some assays, the template comprised an Ml 3 amplicon (108 bases template, of the following nucleic acid sequence:

[00540] 5 ' TTC ATGTTTC AG A AT A ATAGGTTCCG A A AT AGGC AGGGGGC ATT A ACTGTT

TATACGGGCACTGTTACTCAAGGCACTGACCCCGTTAAAACTTATTACCTTTACCCCG

ACACGGAGGTTCTATCA 3' (Template T008). .Sequencing using this template was continued over 3 cycles of polymerase exchange (each cycle consisting of a 3 minute movie).

[00541] In some experiments, data was also collected using a PCR amplicon (258 base template) having the following sequence:

5 ' GGTGGCGGTACTA A ACCTCCTG AGTACGGTG AT AC ACCT ATTCCGGGCT AT ACTTAT ATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTA ATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAGGTTC CGAAATAGGCAGGGGGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCACTGAC CCCGTTAAAACTTATTACCTTTACCCCGACACGGAGGTTCTATCA-3 ' (template GB6902- 76). Sequencing using this template was perfomed for 6 reagent exchange runs (each consisting of 3 minute movies).

[00542] Part (A): Four-color sequencing using acceptor-labeled nucleotides and polymerase exchange

[00543] In four-color sequencing assays, an oligonucleotide primer comprising the following nucleic acid sequence was used: [00544] 5' TG ATA GAA CCT CCG TGT C 3' (Oligonucleotide #315)

[00545] This oligonucleotide primer was added at 200 nM and hybridized to the immobilized DNA templates for 30 minutes at room temperature. Excess oligonucleotide primer was washed away with 1 ml of TBST buffer. Subsequently, the chip was mounted on the microscope, and all solutions on the microscope were aspirated through the chambers using a custom designed pump.

[00546] The sequencing apparatus used for this example comprised a fluorescence microscope equipped with a 405nm laser to excite the sDot label of the polymerase, and a 2 camera detection system with 5 spectrally separate channels; in addition to these the donor channel included a notch filter, and a neutral density filter of 0.9OD. The data were collected at a defined laser power density at defined integration time.

[00547] lOOul of E.coli single strand binding protein (SSB) in MOPS OSS buffer, pH 6.5 was injected into the channel, followed immediately by injection of the sDot-polymerase conjugate (4- 6nM in MOPS OSS buffer, pH 6.5). After a few seconds, the excess conjugate was rinsed out by injection of MOPS OSS buffer, pH 6.5 including 500nM of a short 3'dd-DNA hairpin. The laser was then turned on, the plane of focus was located by manual focusing of the donor channel, the laser was then shut and the stage moved to a new FOV (without illumination). Data collection was started, the laser was turned on and a reaction mixture including all 4 acceptor-labeled nucleotides and catalytic metal ions was injected almost simultaneously. This reaction mixture included all four types of nucleotide polyphosphates, each type of nucleotide polyphosphate conjugated to different a Alexa Fluor dye, and further included 0.5mM MnC12, 2mM (ASP)4 in MOPS-OSS pH 6.5. The incorporation of labeled nucleotide polyphosphates was allowed to occur, and data was collected for three minutes. For each experiment, data were collected from one field of view (FOV) for 3 min. at a frame rate of 16ms.

[00548] While the data collection was ongoing, a pipette tip containing the removing agent Guanidine Hydrochloride was loaded in to the inlet port of the chamber of interest. As soon as the first movie collection was completed, lOOul of 8M Guanidine Hydrochloride was injected in to the chamber to denature and remove polymerase conjugates from the extended DNA molecules. Immediately, the channel was then rinsed with 1ml TBST buffer, and lOOul of MOPS OSS buffer, pH 6.5, before repeating the ssb-conjugate-DNA block wash-reaction mix injection-detection cycle. This sequence of injections and data collection was done three times from the same field of view. The resulting FRET-based sequence-specific signals were detected and analyzed using software to determine the sequence of the templates (data not shown). [00549] Example 8(B): Duplex binding and mapping assay

[00550] In the following assay, a primer labeled with the fluorescent dye Alexa Fluor 647

(Af647) comprising the following nucleic acid sequence:

[00551] 5' TG ATA GAA CCT CCG T-AF647GT C 3' (Primer INV37)

[00552] was used to form a duplex with the template strand on the surface as described above, and then the duplex was bound to the sDot-labeled polymerase.

[00553] After binding and washing off of excess (unbound) labeled polymerase conjugates, data were collected at 100ms integration time for 10 frames using both the 633nm laser (to excite the Alexa Fluor 647-labeled duplexes) and the 405nm laser (to excite the quantum dot label of the polymerase conjugate). The polymerase conjugates were removed and exchanged with fresh conjugates as described in Part (A) above, and the data was collected again. This binding-data collection-conjugate removal cycle is repeated 6 times. The data were then analyzed using software and the labeled polymerase conjugates (detected through the donor channel) were mapped to the Alexa-Fluor-647-labeled duplexes. The percentage of mapping between the labeled primer-template duplexs and the labeled polymerases was calculated and plotted for each binding cycle according to the following equation:

% mapping= Number of mapped conjugates and Af647 duplexes÷Total number of Af647 duplexesxlOO

[00554] Representative results are depicted in Figure 9.

[00555] Example 8(C) Determination of conjugate removal efficiency

[00556] To measure the polymerase exchange efficiency, primer-template duplex was bound to labeled polymerase conjugates as described above. The template-bound labeled polymerases were then removed from the primer-template duplex by washing in with buffers comprising different removing agents, and the efficiency of polymerase removal from the template was determined. The various buffers tested included:

1) MOPS-SodP04-NaCl: 50mM MOPS (pH7.2)-50mM Potassium Acetate, 0.3% BSA, 0.1% Tween-20, 0.5mg/ml glucose oxidase, lOkU/ml Katalase, 200mM Sodium Phosphate (pH8.0), 1M NaCl, (10 minute incubation)

2) MOPS-SodP04-NaCl-NaOH: 50mM MOPS (pH7.2)-50mM Potassium Acetate, 0.3% BSA, 0.1% Tween-20, 0.5mg/ml glucose oxidase, lOkU/ml Katalase, 200mM Sodium Phosphate (pH8.0), 1M NaCl, (10 minute incubation), followed by 50mM NaOH, 30 seconds wash.

3) Urea (4M or 8M) 30 seconds wash.

4) 8M Guanidine hydrochloride 30 seconds wash.

[00557] Data was collected for 1 minute after injection of reaction mix, and once again after conjugate removal using the above mentioned buffers. The percentage of conjugates washed off was determined according to the following equation:

Number of conjugates detected after wash÷Total number of conjugates detected before removal x 100

[00558] Representative results are depicted in Figure 10A.

[00559] Example 8(D) Mapping of unremoved polymerase conjugates

[00560] In these experiments, an Alexa Fluor 647-labeled primer (INV37) comprising the following nucleic acid sequence was used:

[00561] 5' TG ATA GAA CCT CCG T-Af647GT C 3'

[00562] A primer-template duplex was formed on the surface following the conditions described above. After binding and aspiration of excess conjugates, data were collected at 100ms integration time for 10 frames. Then the labeled polymerase conjugates were removed by a GndHCl wash, and data were then again collected using the 633nm and 405nm lasers at 100ms integration time for 10 frames, then fresh conjugates bound as described above, and the data are collected again. This binding-data collection-polymerase conjugate removal cycle was repeated 6 times. The data were then analyzed using software and the number of conjugates (detected through the 405nm laser) mapped to the Alexa Fluor-647 labeled duplexes (detected through the 633nm laser) were tabulated and the percentage of mapping between the duplex and the conjugates were calculated and plotted for each binding cycle according to the following equations:

% mapping= Number of mapped conjugates and Alexa Fluor 647 duplexes ÷ Total number of Alexa Fluor-647 duplexesxlOO

% unwashed conjugates mapped to duplexes= unwashed number of conjugates mapped to Alexa Fluor-647 duplex ÷ Total number of Alexa Fluor-647 duplexesxlOO. % mapping between unwashed dots in Run 1 and freshly bound dots in run2 = # of mapped dots in runl wash and run to freshly bound dots ÷ Total number of dots freshly bound in run2xl00

[00563] Representative results are depicted in Figures 10B and IOC.

[00564] The number of duplexes detected after each wash were also counted to monitor loss of template from each cycle. Representative results are depicted in Figure 11.

[00565] Experiment 8(E): Unremoved conjugates in an extension assay

[00566] The primer-template duplex was immobilized and conjugates were bound as described above, then a nucleotide mix in a buffer containing 5mM CaCl₂ (non-catalytic ions) was injected, data was collected for 3 minutes, then the conjugates were removed using 8M guanidine

Hydrochloride. After a 1ml TBST wash, reaction mix containing labeled nucleotides and MnCl₂ (catalytic ions) were injected and data was collected at 16ms integration time with the 405nm laser. The data were subjected to software analysis, and conjugates detected in the first round were counted, then numbers of conjugates that remain unremoved following the GndHCl wash were counted. The detected fluorescence traces were examined for the presence of the leader sequence (i.e., the first 11 bases GGGGTTAAAAA). Using such methods, it was determined that the residual unremoved (i.e., unwashed) dots are unable to extend DNA without addition of fresh conjugates (data not shown).

[00567] Example 8(E): Reagent exchange efficiency calculation

[00568] The reagent exchange efficiency evaluation data from reagent exchange runs was calculated. The total number of labeled polymerases detected, the number of labeled polymerases mapped in 3 runs as % of total labeled polymerases detected (i.e. detected at the same spot in 3 runs÷total number of labeled polymerasesxlOO).

[00569] Of the labeled polymerases that mapped in 3 runs, the percentage that provides signals indicating the presence of a leader sequence (i.e., the first 10 bases, GGGGTAAAAA), and then the % of mapped polymerases yielding proper leader sequence in more then one run, can be expressed as a % of the mapped donors in a Venn diagram. Representative results are depicted in Figure 13. [00570] Example 8(F) Measurement of read length

[00571] The reagent exchange data were analyzed using software and mapped conjugates were identified, then the list of tuples were examined by eye and the sequences obtained were mapped to the known sequence to determine sequence length obtained from each run.

[00572] Sequence obtained from a single run were compared to the sequences obtained from tuples from more than one run to tabulate average read length from one run vs. average read length from 3 runs. Representative results are depicted in Figures 12 and 14.

[00573] Results

[00574] Mapping experiments to determine the efficiency of conjugate binding to the duplex showed 70% binding to a Al^'647 labeled duplex in any given reagent exchange run (Figure 9).

[00575] After the dissociating agent wash -75-85% of the polymerase is washed away in each reagent exchange run (Figure 10A, 10B, IOC).

[00576] Furthermore, the dissociating agent (GndHCl) is not affecting the functionalized surface significantly, as observed by monitoring its effect on the distribution of a labeled duplex after the

GndHCl washes (Figures 1 1 A & 1 IB). Roughly ~ 18% duplex loss after 6 cycles of 8M GndHCl washes was observed. Control experiments show that the reaction efficiency in the subsequent run, i.e., after the dissociating agent wash, is not affected adversely (Figure 12).

[00577] The efficiency of reagent exchange is shown in Figure 13. In the 6, 3 runs experiments shown here, 35-47% mapping between donors in 3 runs has been observed.

[00578] Of the mapped donors, 10-18% of donors yield decipherable sequence in at least 2 of the

3 reagent exchange runs (Figure 13).

[00579] The mean read length is proportional to the number of sDot-pol exchange cycles, and an improvement in the average read length with the additional cycles is observed (Figures 12 and 14).

[00580] Example 9: Nicking a long nucleic acid template and binding of labeled

polymerases

[00581] In this example, genomic DNA from the bacteriophage lambda was labeled with biotin and digoxigenin (bound at the ends of the same strand) and randomly nicked using DNase I.

Labeled polymerase conjugates were bound to the nicked DNA and visualized using a single molecule detection system.

[00582] The structure of the ends of the lambda genomic DNA included the structure depicted in Figure 23. This was converted to a DNA product having ends including the following structure: 5'(DIG)ttctgccccgggttcctcattctctGGGCGGCGACCTCGC//ACGgggcggcgacct(Bio)3' gacggggcccaaggagtaagagacccgccgctggaGCG//TGCCCCGCCGCTGGA

[00583] To make this product: a biotinylated oligonucleotide having the following structure was first ligated to one end of the lambda genomic DNA (this was destined to be the proximal end to be attached to the surface):

5 ' -pGGGCGGCG ACCT-TEG-Biotin-3 ' (Oligo "Lambda L-end")

[00584] A digoxigenin-containing double-stranded adaptor was ligated to the other end of the same strand of the lambda genomic DNA, where the adaptor had the following structure:

5'-(phos)AGGTCGCCGCCCAGAGAATGAGGAACCCGGGGCAG

TCTCTTACTCCTTGGGCCCCGTCTT(DIG)

[00585] The lambda genomic DNA was then phosphorylated and the biotinylated oligonucleotide was then ligated to the lambda DNA according to the following procedure:

[00586] Molarity of ends Lambda stock 0.5 ug/ul

[00587] 0.5 ug/ul÷[48500 x 650] x 2 = 32xl0^"9 = 32 nM ends

[00588] Molarity of ends if I put 40 ug in 200ul reaction:

[00589] 40,000.0 ng ÷ 200 ul = 200 ng/ul = 0.2 ug/ul

[00590] [(0.2 ug/ul)÷(48500 x 650)] x 2 = 13 xlO⁹ = 13 nM

[00591] Phosphorylation:

[00592] V=100 ul

[00593] 40μ1 Lambda DNA (SOOng/μΙ or 16 nM stock) - total 20μg (0.63 pmole) and final concentration 6.4 nM

[00594] Heat to 65°C 5 min, then cool on ice, add:

[00595] 10 ul lOx T4 ligase buffer (NEB)

[00596] 2 ul lOU/ul T4 PNK (Ensimatics)

[00597] 48 ul H₂0

[00598] Incubate 30 min at 37°C, then put on ice,

[00599] Annealing of biotinylated oligonucleotide and ligation:

[00600] Add 6.4 ul #oligo Lambda-bio L-end (10 uM, or 10 pmole/ul)

[00601] 1 ul of 5M NaCl (to make final concentration -50 mM)

[00602] Heat at 80°C for 5 min, then cool slowly to RT

[00603] Bring volume of ligation mix to 200 ul by adding the following: [00604] 10 ul lOx T4 Ligase Buffer (NEB)

[00605] 5 ul T4 Ligase (400U ) (NEB)

[00606] 77.6 ul H₂0

[00607] Incubate at 15^°C overnight

[00608] Precipitate once with EtOH:

[00609] Add 40ul NaOAc pH5.3 and 880 ul EtOH, gently mix (DNA "ball" formed),

[00610] Spin at RT at 13K rpm 1 min, wash with cold 70% EtOH (spin 1 min)

[00611] Re-dissolve in 200ul T10E0.1, then add 155 ul H₂0,

[00612] Ligation to Dig-adaptor

[00613] The biotinlyated lambda genomic DNA was then labeled with digoxigenin via ligation to the digoxigenin-labeled adaptor as described below:

[00614] V=400 ul

[00615] 354 ul re-dissolved DNA

[00616] 1.25 ul of 50 uM Dig-adaptor

[00617] 40 ul lOx T4 Ligase Buffer

[00618] 5 ul T4 Ligase (400U ) (NEB)

[00619] Incubate at 15^°C overnight

[00620] Limited DNase I digestion of Bio-Dig-Lambda DNA (titration) to generate random nicks

[00621] The biotin- and digoxigenin-labeled lambda genomic DNA was then subjected to random nicking using DNasel according to the following procedure:

[00622] DNasel (NEB, 2U/ul)

[00623] Lambda Bio-Dig as template

[00624] Vreaction =20ul

[00625] Prepare master mix for 10 reactions:

[00626] 2 ul Lambda bio-Dig DNA x 10 = 20ul

[00627] 2 ul lOx DNasel buffer x 10 = 20 ul

[00628] 14 ul H₂0 x 10 =140 ul

[00629] Prepare dilutions of DNasel stock in lx DNasel Buffer (NEB) as follows:

[00630] 10^"2 4 ul 10^_1+ 36 ul

[00631] 5xl0^"3 lOul 10^"2+10ul

[00632] 10^"3 4 ul 10^"2+ 36 ul -4

[00633] 5x10 lOul 10^"3+ lOul

[00634] 10 ,-4

4 ul 10^"3+ 36 ul

[00635] 5x10-5 lOul 10^"4+ lOul

[00636] 10 ,-5 4 ul 10^"4+ 36 ul

[00637] 5x10-6 lOul 10^"5+ lOul

[00638] 10 ,-6 4 ul 10^"5+ 36 ul

[00639] Distribute 2 ul of serially diluted DNasel (or 2 ul H₂0 for Rxn 1 - control minus DNase) in 0.2ml PCR tubes in cold block, then add 18 ul of cold Master mix; transfer onto pre-heated to 30°C PCR cycler; run 15 min at 30°C, 10 min at 75°C (inactivation), +4°C hold.

[00640] Products were resolved on an agarose gel (data not shown).

[00641] Flow cell preparation and on-chip manipulation

[00642] The nicked and biotin- and digoxigenin-labeled DNA was then immobilized onto the surface of a flow cell as described below:

[00643] Wash PEG-biotin flow-cell (170 urn deep flow cell) with 1 ml TBST

[00644] Inject 200 ul TBST+5nM streptavidin (SA), hold 30 min

[00645] Wash with 1 ml TBST

[00646] Dilute each DNasel reaction 7 and 8 50-fold: lul Rxn + 49 ul TBST,

[00647] Deposit Rxn7 into Lnl-4 and Rxn8 into Ln5-9 for 6 min to bind DNase I treated DNA to the surface.

[00648] Wash with 200ul TBST.

[00649] On- Chip Treatment with T7 exonuclease (to generate gaps from nicks introduced by DNase I)

[00650] The nicks within the immobilized nicked labeled DNA within the flow cell were expanded into gaps via limited digestion with T7 exonuclease, and then bound to polymerases labeled with quantum dots (polymerase-dot conjugates, prepared according to the disclosure of U.S. Patent Application Ser. No. 12/748,355, filed March 26, 2010) as described below:

[00651] Prepare T7exo (lOU/ul NEB) serial 5-fold dilutions in NEB4 lx buffer on ice.

[00652] Prepare 9 rxn tubes with serial dilutions of T7 exonuclease:

[00653] V=50 ul

[00654] 0.5ul BSA

[00655] 5ul lOx NEB4

[00656] 42.5ul H₂0 [00657] 2 ul T7exo (diluted or not: undiluted lOU/ul; 5-fold - 2U/ul; 25-fold - 0.4U ; 125-fold - 0.08U ; 625-fold - 0.016U )

[00658] Inject 45 ul per lane of each reaction at 2ul/sec and hold at RT for 30 min; wash 1^st with

45 ul TBST, then with 200 ul TBST

[00659] Lnl Dnasel Rxn7 + 0.4 U/ul final T7exo

[00660] Ln2 Dnasel Rxn7 + 0.08 U/ul final T7exo

[00661] Ln3 Dnasel Rxn7 + 0.016 U/ul final T7exo

[00662] Ln4 Dnasel Rxn7 + 0.0032 U/ul final T7exo

[00663] Ln5 Dnasel Rxn8 + 0.4 U/ul final T7exo

[00664] Ln6 Dnasel Rxn8 + 0.08 U/ul final T7exo

[00665] Ln7 Dnasel Rxn8 + 0.016 U/ul final T7exo

[00666] Ln8 Dnasel Rxn8 + 0.0032 U/ul final T7exo

[00667] Ln9 Dnasel Rxn8 + 0.00064 U/ul final T7exo

[00668] Inject polymerase labeled with quantum dots at 41 nM in TBST for 5 min, wash with 200ul TBST; perform imaging at +/- flow in MOPS+OSS (0.1M trolox stock in H20/DMSO was used) with 405 nm laser to visualize Dot-Pols, -/+ flow- stretching (flow-rate Q=10ul/sec)

[00669] Using the above procedure, an average of 2-6 discrete polymerase-dot conjugates were observed per molecule of lambda genomic DNaseI/T7exo-treated DNA subjected to flow stretching as described above (data not shown).

Claims

CLAIMS What is claimed:

1. A method for obtaining sequence information from a target nucleic acid molecule, comprising:

providing a target nucleic acid molecule including a template strand;

sequencing a first portion of the template strand using a first polymerase, wherein sequencing the first portion includes:

synthesizing a first nascent nucleic acid molecule by contacting the template strand with the first polymerase in the presence of nucleotides under conditions where the first polymerase binds to the template strand and catalyzes one or more template- dependent nucleotide incorporations; and

detecting, during the synthesizing, a sequence- specific signal indicating a template- dependent nucleotide incorporation by the first polymerase; and

sequencing a second portion of the template strand using a second polymerase, wherein sequencing the second portion includes:

synthesizing a second nascent nucleic acid molecule by contacting the template strand with the second polymerase in the presence of nucleotides under conditions where the second polymerase binds to the template strand and catalyzes one or more template-dependent nucleotide incorporations; and

detecting, during the synthesizing, a sequence- specific signal indicating a template- dependent nucleotide incorporation by the second polymerase;

wherein the first and second portions of the template strand are contiguous with each other.

2. The method of claim 1, wherein the target nucleic acid molecule is linked to a solid or semi-solid substrate.

3. The method of claim 1, wherein the second polymerase synthesizes the second nascent nucleic acid molecule by extending the first nascent nucleic acid molecule synthesized by the first polymerase.

4. The method of claim 1, further including exchanging the first polymerase is exchanged for the second polymerase following sequencing the first portion and before sequencing the second portion.

5. The method of claim 4, wherein the exchanging further includes removing the first polymerase from the template strand after sequencing the first portion and prior to sequencing the second portion using the second polymerase.

6. The method of claim 5, wherein the removing is performed in a manner such that where the first nascent nucleic acid molecule can be extended by the second polymerase after the removing.

7. The method of claim 1, wherein the first nascent nucleic acid molecule hybridizes to the template strand to form a synthesized nucleic acid duplex.

8. The method of claim 7, further comprising removing the first polymerase from the template strand after sequencing the first portion without completely denaturing the synthesized nucleic acid duplex.

9. The method of claim 1, wherein none of the labeled nucleotides includes a blocking group.

10. The method of claim 1, wherein at least one of the first and second polymerases includes a polymerase label, at least one nucleotide includes a nucleotide label, and the sequence specific signal includes a FRET signal between a polymerase label and a nucleotide label.

11. A method for obtaining sequence information from two or more target nucleic acid molecules in parallel, comprising: simultaneously sequencing a plurality of target nucleic acid molecules in parallel according to the method of claim 1.

12. A method for obtaining sequence information from a target nucleic acid molecule, comprising:

providing a target nucleic acid molecule including a template strand; sequencing a first portion of the template strand using a first polymerase, wherein sequencing the first portion includes:

detecting, during the synthesizing, a sequence- specific signal indicating a template- dependent nucleotide incorporation catalyzed by the first polymerase; sequencing a second portion of the template strand using a second polymerase, wherein sequencing the second portion includes:

detecting, during the synthesizing, a sequence- specific signal indicating a template- dependent nucleotide incorporation catalyzed by the second polymerase;

wherein the first and second portions of the template strand overlap with each other.

13. The method of claim 12, wherein the target nucleic acid molecule is linked to a solid or semi-solid substrate.

14. The method of claim 12, wherein the first nascent nucleic acid molecule hybridizes to the template strand to form a synthesized nucleic acid duplex.

15. The method of claim 14, further comprising denaturing the synthesized nucleic acid duplex after sequencing the first portion and prior to sequencing the second portion.

16. The method of claim 15, wherein the first portion includes at least a third portion and a fourth portion, the third portion and fourth portion being contiguous with each other, and sequencing the first portion further includes:

sequencing the third portion using a third polymerase,

removing the third polymerase from the template strand, and sequencing the fourth portion using a fourth polymerase.

17. The method of claim 16, wherein sequencing the third portion further includes synthesizing a third nascent nucleic acid molecule by contacting the template strand with the third polymerase in the presence of nucleotides under conditions where the third polymerase binds to the template strand and catalyzes one or more template-dependent nucleotide incorporations; and detecting, during the synthesizing, a sequence- specific signal indicating a template-dependent nucleotide incorporation catalyzed by the third polymerase.

18. The method of claim 17, wherein sequencing the fourth portion further includes synthesizing a fourth nascent nucleic acid molecule by contacting the template strand with the fourth polymerase in the presence of nucleotides under conditions where the fourth polymerase binds to the template strand and catalyzes one or more template-dependent nucleotide

incorporations; and detecting, during the synthesizing, a sequence- specific signal indicating a template-dependent nucleotide incorporation catalyzed by the fourth polymerase.

19. The method of claim 18, wherein the removing includes exchanging the third polymerase for the fourth polymerase.

20. The method of claim 18, wherein the fourth polymerase synthesizes the fourth nascent nucleic acid molecule by extending the third nascent nucleic acid molecule.

21. The method of claim 20, wherein the first, second, third and fourth polymerases are different.

22. The method of claim 12, wherein at least one polymerase includes a polymerase label, at least one nucleotide includes a nucleotide label, and the sequence specific signal includes a FRET signal between a polymerase label and a nucleotide label.

23. A method for obtaining sequence information from two or more target nucleic acid molecules in parallel, comprising: simultaneously sequencing a plurality of target nucleic acid molecules in parallel according to the method of claim 12.

24. The method of claim 12, further including introducing a first polymerization initiation site into the target nucleic acid molecule prior to sequencing the first portion, and introducing a second polymerization initiation site into the target nucleic acid molecule prior to sequencing the second portion, wherein the introducing includes nicking one or more strands of the target nucleic acid molecule.

25. An apparatus for obtaining sequencing information from a target nucleic acid molecule according to the method of claim 1 or claim 12.

26. A method of obtaining sequence information from a target nucleic acid molecule, comprising:

providing a target nucleic acid molecule including a template strand;

sequencing a portion of the template strand using a polymerase, wherein sequencing the portion includes synthesizing a nascent nucleic acid molecule via template-dependent nucleotide incorporation and forming a synthesized nucleic acid duplex through hybridization of the nascent nucleic acid molecule and the template strand;

denaturing the synthesized nucleic acid duplex, and

resequencing at least some of the same portion of the template strand using another polymerase.

27. The method of claim 26, further comprising repeating the denaturing and resequencing at least once.

28. The method of claim 26, wherein the portion of the template strand includes at least a first subportion and a second subportion that are contiguous with each other, and sequencing the portion includes:

sequencing the first subportion using a first polymerase,

removing the first polymerase from the template strand, and

sequencing the second subportion of the nucleic acid template using a second polymerase.

29. The method of claim 28, wherein sequencing the first subportion includes contacting the template strand with the first polymerase and a plurality of labeled nucleotides under conditions where the polymerase binds to the template strand and polymerizes one or more labeled nucleotides in a template-dependent fashion to form a synthesized nucleic acid duplex, and identifying, during the polymerizing, at least one labeled nucleotide polymerized by the first polymerase.

30. The method of claim 28, wherein sequencing the second subportion includes contacting the template strand with the first polymerase and a plurality of labeled nucleotides under conditions where the polymerase binds to the template strand and polymerizes one or more labeled nucleotides in a template-dependent fashion to form a synthesized nucleic acid duplex, and identifying, during the polymerizing, at least one labeled nucleotide polymerized by the first polymerase.

31. The method of claim 30, wherein the method further includes removing the first polymerase from the template strand after sequencing the first subportion and prior to sequencing the second subportion. .

32. The method of claim 31, wherein the removing includes contacting the first polymerase with a removing agent.

33. The method of claim 31, wherein the nascent nucleic acid molecule synthesized by the first polymerase hybridizes to the template strand to form a synthesized nucleic acid duplex, which is further extended by the second polymerase after the removing.

34. The method of claim 26, wherein at least one polymerase includes a polymerase label, at least one nucleotide includes a nucleotide label, and the sequence specific signal includes a FRET signal between a polymerase label and a nucleotide label.

35. A method for obtaining sequence information from a target nucleic acid molecule, comprising:

providing a target nucleic acid molecule including a template strand;

sequencing a first portion of the template strand, wherein sequencing the first portion includes synthesizing a first nascent nucleic acid molecule by contacting the template strand with the a polymerase in the presence of a first set of nucleotides under conditions where the first polymerase binds to the template strand and catalyzes one or more template-dependent nucleotide incorporations; and

sequencing a second portion of the template strand, wherein sequencing the second portion includes: synthesizing a second nascent nucleic acid molecule by contacting the template strand with a polymerase in the presence of a second set of nucleotides under conditions where the second polymerase binds to the template strand and catalyzes one or more template-dependent nucleotide incorporations.

36. The method of claim 35, wherein the first and second portions of the template strand are contiguous with each other.

37. The method of claim 35, wherein the first and second portions of the template strand overlap with each other partly or completely.

38. The method of claim 35, wherein the the first set of nucleotides includes four types of nucleotides, each type associated with a first label, and the second set of nucleotides includes the same four types of nucleotides, each type being associated with a second label, and wherein the first and second labels associated with any given type of nucleotide are different from each other.

39. The method of claim 35, wherein sequencing the first portion, sequencing the second portion, or both sequencing the first portion and sequencing the second portion further includes detecting, during the synthesizing, a sequence- specific signal indicating a template-dependent nucleotide incorporation catalyzed by the polymerase.