WO2010091046A2

WO2010091046A2 - Systems and methods for high throughput, high fidelity, single molecule nucleic acid sequencing using time multiplexed excitation

Info

Publication number: WO2010091046A2
Application number: PCT/US2010/022988
Authority: WO
Inventors: Xioliang Sunney Xie; Peter A. Sims; William J. Greenleaf
Original assignee: President & Fellows Of Harvard College
Priority date: 2009-02-03
Filing date: 2010-02-03
Publication date: 2010-08-12
Also published as: WO2010091046A3

Abstract

A method is disclosed of using microscopy in high throughput sequencing of a target nucleic acid. The method includes the steps of disposing in a microreactor a mixture in solution phase comprising a copy of a target nucleic acid, providing a first illumination beam having a center frequency of ωi that is selected to effect fluorescence of a first label associated with a first nucleotide, providing a second illumination beam having a center frequency of ct^ that is selected to effect fluorescence of a second label associated with a second nucleotide, and modulating the first illumination beam and the second illumination beam such that a multiplexed excitation beam is provided that includes illumination from the first illumination beam and the second illumination beam. The method also includes the steps of directing and focusing the multiplexed excitation beam toward a common focal volume, and detecting radiation at an array detector from within the common focal volume. The radiation is associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid to achieve high throughput sequencing of the target nucleic acid.

Description

SYSTEMS AND METHODS FOR

HIGH THROUGHPUT, HIGH FIDELITY, SINGLE MOLECULE NUCLEIC ACID SEQUENCING USING TIME MULTIPLEXED EXCITATION

PRIORITY

This application claims priority to U.S. Ser. No. 61/149,503 filed February 3, 2009,

the disclosure of which is hereby incorporated in its entirety.

BACKGROUND The invention generally relates to systems and methods for nucleic acid

sequencing, and relates in particular to systems and methods for high throughput single molecule nucleic acid sequencing.

Conventional techniques for the direct sequencing of nucleic acid, including ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), often involve sequencing ~

by - synthesis approaches that use a polymerase to generate a new polynuceotide

strand that is complementary to the strand being sequenced. The incoming,

sequentially incorporated nucleic acid bases are identified using fluorescent tags that

are attached to the nucleotides. Most methods involve attaching a different fluorescent

tag to each species of nucleotide, and identifying by emission spectroscopy the

nucleotide that has been incorporated by the color of the fluorescence generated by its tag. Because each tag has a different emission spectrum, the identity of the

incorporated nucleotide may be uniquely determined.

Single molecule sequencing sensitivity requires the ability to detect a single fluorophore. Many efforts for achieving single molecule detection are limited by the presence of background fields. For example, the use of fluorescently labeled nucleotides for single-molecule sequencing - by - synthesis has been explored (see U.S. Patents Nos. 6,911,345 and 7,033,764) with limited success because the required high concentrations of fluorescently labeled nucleotides in the reaction mixture overwhelm the signal from incorporation on a single template.

In one approach to avoid this overwhelming background signal in DNA sequencing, the four deoxynucleotide triphosphates (dNTPs), which are the basic building blocks of DNA, are repeatedly flowed in and out of the sample cell, one at a time with stringent wash steps (See U.S. Patent No. 6,911,345). This approach however, does not allow continuous enzymatic turnovers by a single enzyme on a single template and hence reduces the speed of detection and increases costs. Another shortcoming of this approach is that it necessitates the immobilization of either the DNA or the polymerase, both of which can further perturb the system. In addition, this method faces serious difficulty when attempting to sequence homopolymer templates, as the incorporation of many identical bases becomes difficult to detect and quantify. Moreover, the base moiety of the nucleotides is labeled with a fluorophore, which hinders subsequent polymerase reactions and must be chemically removed after each incorporation. Despite the removal of these dye labels, the synthesized DNA is still non-natural, reducing the read length of the sequencing reaction. Only short reads averaging 25 bases have been demonstrated with this approach, which is a serious limitation to de novo sequencing. Sanger sequencing provides the highest demonstrated, continuous read lengths for sequencing at approximately 800 bases.

Another approach circumvents the problem of short reads by the use of γ-labeled nucleotides (see U, S. Patent No. 7,033,764). This approach allows for the release of the fluorophore upon formation of a phosphodiester bond, leaving a natural DNA. Production of natural DNA allows for the possibility of long read lengths. In order to circumvent the overwhelming background signal from the fluorescent label attached to the γ-phosphate of nucleotides, a zero-order-wave guide is used to reduce significantly the optical probe volume. The enzyme (and hence the DNA) is immobilized at a nanometric metal structure of the zero order wave guide. The small volume of the metallic structure however, may hinder enzymatic activity and require stringent surface chemistry treatment. Furthermore, the binding of γ-labeled nucleotides onto the DNA template gives rise to a signal, even if nucleotide incorporation does not occur. It is difficult therefore, to distinguish between nucleotide binding to the complementary strand without incorporation and binding with actual incorporation, potentially leading to spurious signals, and therefore incorrect sequence identification.

Gamma-labeled fluorogenic nucleotide triphosphates (NTPs) have been developed for bulk measurement applications (see U.S. Patent No. 7,041,812). These fluorogenic NTPs are not fluorescent until hydrolysis of the label from the phosphate, providing for a background-free detection of the incorporation of the NTP into a nucleic acid. These reagents however, have not been employed in single-molecule detection because of technical difficulties.

A further approach for performing single molecule detection of DNA involves placing an enzyme on a surface in proximity to a metal particle to thereby provide metal enhanced fluorescence of the product and/or increased plasmon resonance of the particle (see U.S. Patent Application Publication No. 2008/0241866). This approach however, requires fluorescent elements to be present one at a time and for a sufficient period of time that they may be accurately detected.

Conventional techniques of achieving nucleic acid sequencing have also employed excitation spectroscopy as a means of distinguishing fluorescently labeled nucleotides (see U.S. Patent No. 6,995,841). Such systems, however, do not include the necessary components for high-throughput, single molecule sequencing. Achieving high-throughput, cost-effective DNA sequencing of human genomes would open many applications in medicine and scientific research. A significant reduction in cost and increase in speed are needed however, for large scale marketing of genetic analysis systems.

There remains a need, therefore, for more economical and efficient methods for continuous single-molecule nucleic acid sequencing, including, for example, methods with long read lengths free from the complications of enzyme or nucleic acid immobilization and from the inability to distinguish nucleotide binding and incorporation events, as well as efficient and economical methods of detecting multiple distinct events such as the sequencing of the four dNTPs in DNA. SUMMARY

The invention provides a method and system of using microscopy in high throughput sequencing of a target nucleic acid. In accordance with an embodiment, the method includes the steps of disposing in a microreactor a mixture in solution phase comprising a copy of a target nucleic acid, providing a first illumination beam having a center frequency of ωj that is selected to effect fluorescence of a first label associated with a first nucleotide, providing a second illumination beam having a center frequency of ω^ that is selected to effect fluorescence of a second label associated with a second nucleotide, and modulating the first illumination beam and the second illumination beam such that a multiplexed excitation beam is provided that includes illumination from the first illumination beam and the second illumination beam. The method also includes the steps of directing and focusing the multiplexed excitation beam toward a common focal volume, and detecting radiation at an array detector from within the common focal volume. The radiation is associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid to achieve high throughput sequencing of the target nucleic acid.

In accordance with another embodiment, the method includes the steps of optically modulating a first illumination beam such that a multiplexed excitation beam is provided that includes illumination from a first optical modulation of the first illumination beam and a second optical modulation from the first illumination beam to provide a time multiplexed excitation beam, and detecting radiation at an array detector from within the common focal volume associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid to achieve high throughput sequencing of the target nucleic acid.

In accordance with a further embodiment, the invention provides a microscopy system for use in sequencing a target nucleic acid. The microscopy system includes a microreactor, an illumination source, a modulation unit, optics and an array detector. The microreactor is for receiving a mixture in solution phase comprising a copy of the target nucleic acid, and the microreactor includes a microreactor array of a plurality of nanoreators. The illumination source is for providing a first illumination beam having a center frequency of ωj that is selected to effect fluorescence of a first label associated with a first nucleotide, and is for providing a second illumination beam having a center frequency of a>₂ that is selected to effect fluorescence of a second label associated with a second nucleotide. The modulation unit is for modulating the first illumination beam and the second illumination beam such that a multiplexed excitation beam is provided that includes illumination from the first illumination beam and the second illumination beam. The optics are for directing and focusing the multiplexed excitation beam toward a common focal volume within a portion of the microreactor. The array detector is for detecting radiation from within the common focal volume associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid to achieve single molecule sequencing of the target nucleic acid. In accordance with further embodiments, the array detector includes an array of a plurality of sensors, each of which is associated with a different sequence reactor and is associated with a nanoreactor in the microreactor array.

BRIEF DESCRIPTION OF THE DRAWINGS The following description may be further understood with reference to the accompanying drawings in which:

Figures IA - IF show illustrative diagrammatic views of single-molecule sequencing using a coupled enzyme assay in a system in accordance with an embodiment of the invention; Figures 2A - 2F show illustrative diagrammatic views of a procedure for fabricating microreactors for use in a system in accordance with an embodiment of the invention;

Figures 3A and 3B show illustrative bright field and fluorescence images respectively of microreactors filled with a fluorogenic dye; Figure 4 shows an illustrative diagrammatic view of the excitation and emission spectrums in an emission spectroscopy system for detecting two different species using two detectors;

Figure 5 shows an illustrative view of a detection combinations table for the system of Figure 4; Figure 6 shows an illustrative diagrammatic view of the excitation and emission spectrums in an excitation spectroscopy system for detecting two different species using two lasers and one detector; Figure 7 shows an illustrative view of a detection combinations table for the system of Figure 6;

Figure 8 shows an illustrative diagrammatic view of a microscopy system for use in time multiplexed excitation spectroscopy in accordance with another embodiment of the invention;

Figure 9 shows an illustrative micro-photographic image of a microreactor including an array of nanoreactors for use in a system in accordance with an embodiment of the invention;

Figure 10 shows an illustrative diagrammatic view of the excitation and emission spectrums in an excitation spectroscopy system for detecting two different species using one laser and one detector;

Figure 11 shows an illustrative view of a detection combinations table for the system of Figure 10;

Figure 12 shows an illustrative diagrammatic view of the excitation and emission spectrums in an excitation spectroscopy system for detecting three different species using two lasers and one detector;

Figure 13 shows an illustrative view of a detection combinations table for the system of Figure 12;

Figure 14 shows an illustrative diagrammatic view of a microscopy system for use in time multiplexed excitation spectroscopy in accordance with another embodiment of the invention employing more two detectors;

Figure 15 shows an illustrative diagrammatic view of the excitation and emission spectrunis in an excitation spectroscopy system for detecting four different species using two lasers and two detectors;

Figure 16 shows an illustrative view of a detection combinations table for the system of Figure 15; Figure 17 shows an illustrative diagrammatic view of the excitation and emission spectrums in an excitation spectroscopy system for detecting four different species using four lasers and two detectors; and

Figure 18 shows an illustrative view of a detection combinations table for the system of Figure 17; The drawings are shown for illustrative purposes only.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

The invention provides a system and method for performing high throughput single molecule nucleic acid sequencing using time multiplexed excitation. In accordance with an embodiment, first and second illumination beams are modulated such that a time multiplexed excitation beam is provided that includes illumination from the first illumination beam and illumination from the second illumination beam.

An array detector is used to detect radiation from within the common focal volume, wherein the radiation is associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid to achieve high throughput sequencing of the target nucleic acid.

Previous work in the field of single molecule fluorescence detection has taken advantage of differentially exciting a sample containing multiple fluorophores (see for example, U.S. Patent Application Publication No. 2007/0109536 Al). This methodology however, does not take advantage of differential excitation as disclosed herein to distinguish at least two fluorophores with fluorescence emission spectra that are not sufficiently separable for emission spectroscopy to be effective. Instead, a system for excitation spectroscopy is combined with a system of fluorophores with well-separated emission spectra such that as disclosed therein, the emission can be examined in terms of wavelength, polarization, and excitation-emission time-interval and conventional emission-based ratiometric analysis of emission onto two spatially distinct detectors or Forster resonance energy transfer (FRET) can be realized. In accordance with the present invention, the use of pure excitation spectroscopy without regard to differences in emission spectra for distinguishing two or more fluorophores, reduces the detector area per unit sample area, which increases signal-to-noise and throughput.

In accordance with an embodiment, the invention employs continuous single-molecule sequencing of nucleic acids based on the continuous measurement of the incorporation of fluorogenic nucleotides in microreactors. The methods and systems permit the unambiguous determination of sequence, continuous sequencing, and long read lengths.

In particular and in one aspect, the invention provides a method for sequencing a nucleic acid by providing a mixture in solution phase and including a single copy of a target nucleic acid, a nucleic acid replicating catalyst (e.g., DNA polymerase, RNA polymerase, ligase, RNA-dependent RNA polymerase, or reverse transcriptase), and a mixture of nucleotides that includes a first nucleotide having a first label that is substantially non-fluorescent until after incorporation of the first nucleotide into a nucleic acid based on complementarity to the target nucleic acid. The mixture in solution phase, e.g., having a volume of 0.0001 fL - 1000 fL, is disposed in a microreactor, and continuous template-dependent replication of the target nucleic acid is allowed to occur. The target nucleic acid is then sequenced by detecting in real time the individual incorporation of the first nucleotide during template-dependent replication by monitoring fluorescence emission resulting from the first label. The detection step may be repeated as desired to continue sequencing the target nucleic acid by detecting incorporation of the next nucleotide, e.g., for 10, 25, 100, 300, 1000, or 10,000 base pairs.

In certain embodiments, the mixture in solution phase further includes an activating enzyme that renders the first label fluorescent. Examples of activating enzymes include an alkaline phosphatase, acid phosphatase, galactosidase, horseradish peroxidase, phosphodiesterase, phosphotriesterase, pyruvate kinase, lactic dehydrogenase, maltose phosphorylase, glucose oxidase, lipase, or combination thereof.

In other embodiments, the first label is photobleached after fluorescence detection. The first label may also be a phosphate label that is cleaved from the first nucleotide during incorporation. The mixture of nucleotides may further include a second, third, and/or fourth nucleotide having a second, third, and/or fourth label that is substantially non-fluorescent until incorporation of the corresponding nucleotide into a nucleic acid based on complementarity to the target nucleic acid.

DNA or RNA may be sequenced in accordance with various embodiments of the present invention. For DNA or RNA, a primer may be employed. Preferably, the method sequences the target nucleic acid continuously. The methods of the invention may also be multiplexed to determine the sequence of more than one target nucleotide at the same time or sequentially.

The invention further features a system for sequencing a nucleic acid that includes a plurality of microreactors each of which is capable of holding a mixture in solution phase of a single copy of a target nucleic acid, a nucleic acid replicating catalyst, and a mixture of nucleotides, at least one of which has a label that is substantially non-fluorescent until after incorporation of that nucleotide into a nucleic acid based on complementarity to the target nucleic acid; and a fluorescent microscope for imaging the plurality of microreactors to sequence target nucleic acids in the microreactors by the methods described herein. The system may further include a fluidic delivery system capable of delivering liquids to each of said plurality of microreactors or a light source capable of photobleaching said label after detection. In certain embodiments, the excitation source of the fluorescent microscope is capable of photobleaching the label. The term "microreactor" as used herein means a vessel having a volume such that a light microscope can detect a freely diffusing fluorophore using a sensitive photon detector, e.g., capable of detecting a single molecule, In accordance with an embodiment, the microreactor includes an array of nanoreators.

The terms "fluorogenic" and "substantially non-fluorescent" are used herein to mean not emitting a significant amount of fluorescence at a given wavelength until after a chemical reaction has occurred.

The term "sequencing a nucleic acid" as used herein means identifying one or more nucleotides in, or complementary to, a target nucleic acid. This sequencing may include the determination of the individual bases in sequence, the determination of the presence of an oligonucleotide sequence, or the determination of the class of nucleotide present, e.g., member of A-T, A-U, or G-C pair, or purine base or pyrimidine base.

The term "continuous sequencing" as used herein means employing a sequencing by synthesis that results in the generation of a single complementary nucleic acid, e.g., of 10, 25, 100, 300, 1000, or 10,000 base pairs. Continuous sequencing is contrasted with shotgun sequencing in which many smaller, overlapping sequences are generated, and the overall sequence is determined by aligning the various fragments. Continuous sequencing is advantageous for determination of the number of repeats of a particular sequence. The phrase does not imply that the sequencing occurs at a constant rate. In addition, replication may occur as a result of catalysis activity by different copies of a catalyst, i.e., a single enzyme molecule need not catalyze synthesis of the entire complementary nucleic acid. The term "detecting in real time" as used herein means detecting light emitted from a label after incorporation of a labeled nucleotide into a nucleic acid but prior to incorporation of a subsequent labeled nucleotide.

The term "incorporation of a nucleotide into a nucleic acid" as used herein means the formation of a chemical bond, e.g., a phosphodiester bond, between the nucleotide and another nucleotide in the nucleic acid. For example, a nucleotide may be incorporated into a replicating strand of DNA via formation of a phosphodiester bond. Other types of bonds may be formed if non-natural Iy occurring nucleotides are employed. The term "nucleotide" as used herein means a natural or synthetic ribonucleosidyl, 2'-deoxyribonucleosidyl radical, Locked Nucleic Acid, peptide nucleic acid, glycerol nucleic acid, morpholino nucleic acid, or threose nucleic acid connected, e.g., via the 5', 3' or 2' carbon of the radical, to a phosphate group and a base. The nucleotide may include a purine or pyrimidine base, e.g., cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thioinosine, thiouracil, pseudouracil, 5,6-dihydrouracil, and 5-bromouracil. The purine or pyrimidine may be substituted as is known in the art, e.g., with halogen (i.e., fluoro, bromo, chloro, or iodo), alkyl (e.g., methyl, ethyl, or propyl), acyl (e.g., acetyl), or amine or hydroxyl protecting groups. In certain embodiments when DNA is being sequenced, the nucleotides employed are dATP, dCTP, dGTP, and dTTP. In other embodiments when RNA is being sequenced, the nucleotides employed are ATP, CTP, GTP, and UTP. A target DNA sequence can also be sequenced with riboside bases using RNA polymerase, and a target RNA sequence can also be sequenced with deoxyriboside bases using reverse transcriptase. The term includes moieties having a single base, e.g., ATP, and moieties having multiple bases, e.g., oligonucleotides.

The term "nucleotide replicating catalyst" as used herein means any catalyst, e.g., an enzyme, that is capable of producing a nucleic acid that is complementary to a target nucleic acid. Examples include DNA polymerases, RNA polymerases, reverse transcriptases, ligases, and RNA-dependent RNA polymerases.

Fluorogenic single molecule sequencing In accordance with an embodiment, the invention employs a method for detecting the synthesis of a single nucleic acid using fluorogenic nucleotides that are substrates for nucleic acid replicating catalysts and that become able to emit light as a result of incorporation of the nucleotide into a nucleic acid. The methods employ microreactors to contain the sequencing reaction. Advantages of such a process include: 1) Confinement of an isolated single nucleic acid, the reaction of which can be followed continuously allowing unambiguous determination of sequence. 2) Restriction of the diffusion of the fluorescent label generated to a volume that is sufficiently small such that a single molecule can be detected above the background, e.g., Raman or autofluorescence. 3) Reduction of fluorescence signal from autohydrolysis of the fluorogenic substrate in the absence of incorporation of the labeled nucleotide. 4) Allows for a regular, dense array of microreactors enabling high-throughput, parallel nucleic acid sequencing. 5) Reduction in the amount and the cost of reagents (enzyme, labeled nucleotide, DNA, etc.) required for high -throughput sequencing. 6) Confinement makes surface immobilization of the single nucleic acid or replicating catalyst unnecessary, avoiding perturbation of enzyme activities. 7) No change in the reactants is necessary during sequencing. 8) Use of fluorogenic substrates eliminates background from unincorporated labeled nucleotides. 9) Continuous sequencing of thousands of nucleotides is possible, in principle.

As shown in Figure IA, a process for replicating a strand of nucleic acid 10 (e.g., DNA) using fluorogenic single-molecule sequencing employing a coupled enzyme assay includes a primer strand 20 having combinations of four bases 12, 14, 16 and 18. As shown in Figure IB, a polymerase 30 selectively adds the next appropriate nucleotide 22, 24, 26 or 28 (e.g., 26 as shown) that is complementary to the next base (e.g., 16 as shown).

Once added and bound to the primer strand, the nucleotide (e.g., 26) releases a dark fluorophore (e.g., 36 as shown in Figure 1C) conjugated to two phosphates 40. The remaining nucleotides (e.g., 22, 24 and 28) each also include a dark fluorophore 32, 34 and 38, each of which is also conjugated to two phosphates 40 as shown.

A phosphatase 50 as shown in Figure ID receives the released dark fluorophore

(e.g., 36) together with two phosphates 40, and then cleaves one of these two phosphates 40 of from the fluorophore 36 as shown in Figure IE. The phosphatase 50 then cleaves the other phosphate 40, generating a fluorescent molecule (since fluorphore 36 now fluoresces) that can be detected as shown in Figure IF.

The above method is employed in connection with sequencing by synthesis, in which the incorporation of an individual nucleotide, e.g., including a single base or multiple bases, into a nucleic acid during replication is detected. As nucleotides (22, 24, 26, 28) are incorporated into a nucleic acid that is complementary to the target nucleic acid, the label is rendered able to emit light, e.g., by cleavage from the incorporated nucleotide (e.g., when bound to the beta or gamma phosphate of an NTP). Because nucleotides are incorporated sequentially during replication, the incorporation of an individual nucleotide can be measured in real time as a result of the emitted light. Preferably, the fluorescent label (32, 34, 36, 38) is substantially non-emitting when diffusing free in solution to reduce background that could interfere with real time detection of incorporation. Tens of thousands of bases on a single nucleic acid can be read continuously with high speeds up to 10-100 bp/sec. The technique may easily distinguish incorporation from false binding (i.e., temporary hybridization not resulting in bond formation), and no zero-order waveguide is required.

In accordance with certain embodiments, the label may not be immediately fluorescent upon cleavage from the nucleotide. In these embodiments, chemical modification of the label or groups pendant on the label must first occur. For example, certain dyes are non-fluorescent when conjugated to a phosphate group; removal of the phosphate group, e.g., via a phosphatase, then renders the label fluorescent. Other chemical mechanisms that may be involved include acid and base catalyzed reactions and other catalytic processes described herein. Labels may alternatively become able to emit merely as a result of cleavage from the growing nucleic acid. For example, a label may be quenched or otherwise rendered non-emitting by proximity to the nitrogenous base of a nucleotide or a moiety associated with the base.

When each nucleotide is added to the synthesized strand, the nucleotide added is preferably identified. By detecting which of the labels is added at a given point in synthesis, the corresponding nucleotide added may be identified, and, when present, the sequence of a target nucleic acid may be determined, by virtue of its complementary nature. Methods for detecting four or more optically distinguishable labels are well known in the art.

Alternatively, fewer labels may be employed. Two labels may be employed when a target double stranded nucleic acid or a single stranded nucleic acid and its complement are sequenced. Another example of two-label detection is to label one nucleotide with a first label and the other three nucleotides with another label. Binary sequencing may also be employed in which two nucleotides are labeled with one label, while the other two nucleotides are labeled with a second label. A subsequent sequencing in which the label in one of each pair is changed, may be employed to obtain nucleotide specificity. One label may be employed, where the other three nucleotides are not labeled but are kept at lower concentrations, where the time, and therefore position, between the detection of each label is determined. Subsequent experiments using the same label with a different nucleotide can be used to provide the remaining sequence information. When more than one labeled nucleotide is employed, each label may become light emitting as a result of different mechanisms. For example, one label may require additional chemical reaction after cleavage from the synthesized nucleic acid, while a second label may become light emitting upon cleavage without additional reaction.

Sequencing may also be performed using ligase, in which oligonucleotides hybridized adjacent to one another on a template strand are ligated together. Each oligonucleotide employed may be uniquely labeled. Oligonucleotides having the sequence complementary to a region of repeated sequence may be added sequentially using the methods of the invention, and the number of repeats determined by the number of oligonucleotides ligated.

An individual sequencing reaction may be controlled by controlling the introduction of Mg or Mn ions, nucleotides, and other co-factors necessary to effect replication. Other methods for controlling replication include introducing or removing substances that promote or discourage complex formation between the target and catalyst. The catalyst or target may also be rendered inoperative to end sequencing, e.g., through denaturation or cleavage. Multiplexing, i.e., detection of more than one replication at a time, may be employed to increase throughput as discussed further below.

Microreactor

The reagents for synthesis of nucleic acids are disposed in a microreactor. Exemplary microreactors hold volumes of 0.0001 fL to 1000 fL, although larger volumes are possible. Conducting single molecule sequencing in a microreactor imparts several advantages as described herein. A target nucleic acid or replicating catalyst may be immobilized within the microreactor, although the methods of the invention do not require immobilization. Methods for immobilizing nucleic acids or catalysts are well known in the art. To use the coupled-enzyme assay of the present invention however, the biochemical reaction must be conducted in a confined volume such that the freed, phosphate-attached fiuorophores do not diffuse away from the site of the DNA incorporation. Materials that are useful in forming the microreactors include glass, glass with surface modifications, silicon, metals, semiconductors, high refractive index dielectrics, crystals, gels, lipids, and polymers Single molecules of target nucleic acid (or replicating catalyst) can be delivered to a microreactor using methods known in the art. One method for delivery is to provide a dilute solution of nucleic acid so that each microreactor, on average, holds less than one molecule. Using this approach some microreactors will have no target nucleic acid, some will have a single target nucleic acid, and a very small number will have more than one.

For example, a method to achieve this confinement is the use of sub-micron lipid vesicles to entrap DNA, substrate, DNA polymerase, and phosphatase. These nanoreaclors of the microreactor were then immobilized on the coverslide of a fluorescence microscope (See Okumus et al. Biophys. J. 2004, 87(4), 2798-2806). Further approaches may include processes that utilize standard nanofabrication techniques to generate femtoliter-sized indentations in polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA), or quartz, which may seal against the surface of a coverslide (See Rondelez et al. Nature Biotechnology. 2005, 23, 361-5).

Microreactors for use in a system in accordance with an embodiment of the invention may be generated through a variant of so-called nanosphere lithography (See Hulteen et al. J. Vac. Sci. Technol. A 1995 13(3), 1553-1558). In particular, as shown in Figure 2A, polystyrene beads 60 are close-packed onto a flat glass surface 62. In particular, 500 nm to 2000 nm polystyrene or glass beads are we evaporated onto glass slides to create a close-packed monolayer of beads. Polydimethylsiloxane (PDMS) 64 is poured and cured onto these beads as shown in Figure 2B in a 60 ⁰C oven overnight, and the PDMS is then removed as shown in Figure 2C leaving a pattern of nanoscale indentations 78 that may be, for example, be in the form of a honeycomb. Any beads 60 on the cured PDMS 64 are removed mechanically, and the coupled -enzyme reaction mixture 74 is placed between the patterned PDMS 64 and a coverslip 790 that is spin-coated with PDMS 74 as shown in Figures 2D and 2E. Upon application of pressure by plate 76, the mixture 74 that is not entrapped within the cavities 78 becomes entrapped as the excess mixture 74 is squeezed out the sides as shown in Figure 2F to generate a regular array of nanoreactors 80 that contain on the order of 5 to 0.1 fL. Sealed microreactors are thereby formed and may be imaged from below with a light microscope. Figure 3A shows at 90 a bright-field image of an array of nanoreactors 80 filled with a fluorescent DDAO using 1.5 micron polystyrene beads, and Figure 3B shows at 92 a fluorescent image of the array of nanoreactors imaged in Figure 3A. Other materials for microreactor fabrication include polytetrafluoroethylene, perfluoropolyethers, and parylene, Additionally, lipid vesicles can be generated using standard lipid extrusion techniques (Okumus et al. Biophys. J. 2004, 87(4), 2798-2806) and used to confine the reaction. Another method of generating microreactors is the creation of an emulsion of the reaction mixture in an immiscible solvent such as mineral oil or silicon oil. Further methods for manufacturing microreactors are known in the art, for example, see U.S. Patent No. 7,081,269. Fluorogenic Labels

Any label that becomes able to emit light as a result of incorporation of a nucleotide to a synthesized nucleic acid may be employed in the methods of the invention. Exemplary labels include resorufin and

9H-(l,3-dichloro-9,9-dimethylacridin— 2-one (DDAO). Additional labels are known in the art, for example, as disclosed in U.S. Patents Nos. 7,041,812, 7,052,839, 7,125,671, 7,223,541, and 7,244,566. Labels may be attached to nucleotides at a variety of locations. Attachment may be made either with or without a bridging linker to the nucleotide. The label may be attached to the base, sugar, or phosphate of the nucleotide. Preferably the label is attached to the beta or gamma phosphate so that it is cleaved from the nucleotide during replication. Labels may also be attached to non-naturally occurring portions of a nucleotide, e.g., to the delta or epsilon phosphate in a tetra- or pentaphosphate containing nucleotide. Alternatively, labels may be attached to the alpha phosphate and displaced during incorporation of a nucleotide in a synthesized strand. In certain embodiments, the label is destroyed (or rendered non detectable) once detected. One method to destroy the label is photob leaching. Another method is to employ a catalyst that chemically alters the label after detection. In other embodiments, the label is not destroyed after detection, and the incorporation of nucleotides having the same label is monitored via the incremental increase of signal.

Activating Catalyst

Any catalyst that is capable of acting on a label to render it fluorescent after a nucleotide incorporation event may be used in systems and methods of the present invention. Preferably, the activating catalyst does not act on the label prior to incorporation. Preferred catalysts include enzymes such as alkaline phosphatases (e.g., bacterial alkaline phosphatase, shrimp alkaline phosphatase, calf intestinal phosphatase, and antarctic phosphatase), acid phosphatases, galactosidases, horseradish peroxidase, phosphodiesterase, phosphotriesterase, pyruvate kinase, lactic dehydrogenase, lipase, or combinations of enzymes and substrates in a coupled enzyme system such as maltose, maltose phosphorylase, glucose oxidase, horseradish peroxidase, and amplex red (PIPER™ phosphate detection kit, Invitrogen). The activating catalyst may also be an ion in solution, e.g., iodide, hydroxide, or hydronium, a zeolite or other porous catalytic surface, or a metal surface, e.g., platinum, palladium, or molybdenate. Other biological and synthetic catalysts may also be employed. Multiple copies of a particular catalyst may be present to reduce the time required for interaction with the label. Nucleic Acids and Nucleotides

The invention may be employed with any nucleic acid (e.g., DNA, RNA, and DNA/RNA) using any appropriate nucleic acid replicating catalyst. Nucleotides may be naturally occurring or synthetic, e.g., synthetic ribonucleosidyl, 2'-deoxyribonucleosidyl, Locked Nucleic Acid, peptide nucleic acid, glycerol nucleic acid, morpholino nucleic acid, or threose nucleic acid connected, e.g., via the 5', 3' or 2' carbon of the radical, to a phosphate group and a base. The nucleotide may include a purine or pyrimidine base, e.g., cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thioinosine, thiouracil, pseudouracil, 5,6-dihydrouracil, and 5-bromouracil. The purine or pyrimidine may be substituted as known in the art, e.g., with halogen (i.e., fluoro, bromo, chloro, or iodo), alkyl (e.g., methyl, ethyl, or propyl), acyl (e.g., acetyl), or amine or hydroxyl protecting groups.

In certain embodiments when DNA is being sequenced, the nucleotides employed are dATP, dCTP, dGTP, and dTTP. In other embodiments when RNA is being sequenced, the nucleotides employed are ATP, CTP, GTP, and UTP. Ribosides may also be employed for sequencing DNA, e.g., when or (DNA-dependent) RNA polymerase is employed. Ribosides may also be employed for sequencing RNA, e.g., when RNA-dependent RNA polymerase is employed. Deoxyribosides may also be employed for sequencing RNA, e.g., when reverse transcriptase is employed.

In preferred embodiments, the sequencing methods of the invention produce a nucleic acid that is complementary to the target nucleic acid and that includes only naturally occurring nucleotides, i.e., the label is removed during replication. Alternatively, nucleotides may include a moiety that is retained in the synthesized nucleic acid. Such moieties are preferably present on fewer than all of the labeled nucleotides employed, e.g., only one, two, or three, to minimize disruption of replicating catalyst activity.

Nucleic Acid Replicating or Synthesizing Catalysts

Exemplary replicating catalysts include DNA polymerases, RNA polymerases, reverse transcriptases, ligases, and RNA-dependent RNA polymerases. Exemplary DNA polymerases include E. coli DNA polymerase I, E. coli DNA polymerase I Large Fragment (Klenow fragment), Sequenase™ phage T7 DNA polymerase, T4 DNA polymerase, Phi-29 DNA polymerase, thermophilic polymerases (e.g., Thermus aquaticus (Taq) DNA polymerase, Thermus flavus (TfI) DNA polymerase, Thermus Thermophilus (Tth) DNA polymerase, Thermococcus litoralis (TIi) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, Vent™ DNA polymerase, or Bacillus stearothermophilus (B st) DNA polymerase, Theπninator™, Therminator II™), and a reverse transcriptase (e.g., AMV reverse transcriptase, MMLV reverse transcriptase, or HIV-I reverse transcriptase). Other suitable DNA polymerases are known in the art. Exemplary RNA polymerases include T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerases. Exemplary ligases are known in the art. Exemplary RNA-dependent RNA polymerases are known in the art. Catalysts may bind to a target at any appropriate site as is known in the art. Multiple copies of the replicating catalyst may be present. If a particular catalyst molecule disassociates from the template strand, another catalyst molecule may bind and continue replication without affecting the sequencing function.

Detection

The incorporation of an individual nucleotide may be detected by detecting the light emitted from its corresponding label by any appropriate method. For fluorescent labels, one or more excitation sources may be employed, depending on the nature and number of labels. Methods for single molecule detection are known in the art. Examples are conventional fluorescence microscopy, total internal reflection fluorescence microscopy, and confocal fluorescence microscopy. As described in more detail below, the methods of the invention may be employed in a multiplexed mode, where the sequences of multiple target nucleic acids are determined simultaneously, e.g., using a wide field of view array detector such as a charge-coupled device (CCD) or multiple detectors in accordance with various embodiments of the present invention.

A wide variety of chromophores may be employed as labels in accordance with various embodiments of the invention, and these may be divided into two main categories. The first category consists of fluorogenic dyes, Fluorogenic dyes are chromophores that are either substantially nonfluorescent or their fluorescence excitation and/or emission are shifted significantly in wavelength when covalently linked in a particular way to a second molecule. The second molecule may be a nucleotide substrate and the fluorogenic dye labels the terminal phosphate. When a fluorogenic dye becomes unconjugated from the second molecule, it becomes fluorescent or its excitation and/or emission wavelengths change significantly. There are many classes of fluorogenic dyes including fluorophores derived from coumarins, xanthenes, phenoxazines, benzophenoxazines, acridines, etc such as fluorescein, 6-carboxytetrachlorofluorescein, 6-carboxyhexachlorofluorescein, Oregon Green ^rM 488, Oregon Green™ 514, 3-O-alkylfluorescein, naphthofluorescein, carboxynaphthofluorescein, resorufin,

7-hydroxy-9H-(l,3-dichloro-9,9-dimethylacridin- 2-one) (DDAO), 7-hydroxy-9H-(9,9-dimethylacridin-2-one) (DAO), umbelliferone, 3-cyanocoumarin, 6,8-difluoro-4-methylumbelliferone, rhodamine, cresyl violet, and rhodol green. Additional specific examples are listed in U.S. Patent No. 7,125,671.

The second category consists of chromophores that are fluorescent and maintain their spectral properties when conjugated to a second molecule such as the terminal phosphate of a nucleotide substrate. This class of chromophores is very large and includes fluorophores derived from not only from coumarins, xanthenes, phenoxazines, benzophenoxazines, and acridines as in the case of fluorogenic dyes, but also includes cyanines, bodipies, anthracenes, and semiconductor nanocrystals.

Further examples in both categories may be employed given the numerous modifications to these dyes that are commonly used to alter their chemical stability, solubility, spectral properties, photostability, and quantum yields such as sulfonation, halogenation, alkylation, acetylation, etc. Chromophores in either or both categories may be used for labeled nucleotide substrates and combined in accordance with their spectral properties to form systems of labels that can distinguished from one another using excitation spectroscopy or the combination of excitation and emission spectroscopy for nucleic acid sequencing.

Example

In an example, deoxynucleotide triphosphates (dNTPs) derivatives that are linked through the γ-phosphate to different dyes, which are essentially non-fluorescent at relevant wavelengths in solution, are synthesized. High concentrations of substrate may thus be present in solution without fluorescence background, as these substrate molecules are dark. Once a DNA polymerase incorporates a labeled dNTP, cleaving between the α- and β-phosphates of the nucleotide, the liberated fluorophore becomes fluorescent, either directly upon cleavage from the dNTP, or after further enzymatic action of other enzymes (See Sood et al. J. Am. Chem. Soc, 2005, 127, 2394-2395; Kumar et al. Nucleotides, Nucleosides, and Nucleic Acids, 2005, 24, 401-408).

These newly fluorescent molecules are then detected using standard fluorescence detection techniques such as total internal reflection fluorescence, epifluorescence, or confocal microscopy (See English et al. Nat. Chem. Biol., 2006, 2, 87-946). The color of the fluorescence reports the identity of the incorporated nucleotide, and thus the underlying DNA sequence.

In an implementation of the invention, two different fluorogenic species are employed to extract sequence information from DNA, namely fluorescent DDAO

and fluorescent resorufin

Resorufin is not fluorescent when conjugated to dNTPs, while for DDAO the fluorescence and absorption spectra change significantly when it is conjugated to dNTPs to provide the γ-labeled dATP fluorogenic substrate

and the γ-labeled dTTP fluorogenic substrate

Upon cleavage from the dNTP through the action of DNA polymerase, these molecules still have phosphate groups covalently linked to the fluorophore, which must be removed before the molecule may become fluorescent. A large class of phosphatase enzymes, such as shrimp alkaline phosphatase, can quickly remove these phosphate groups from the fluorophore (but do not react with γ -labeled dNTP substrates), generating a single fluorescent dye molecule for every incorporation of the DNA polymerase as discussed above with reference to Figures IA - IF.

Excitation spectroscopy

In emission spectroscopy, the detection of two different species may be achieved using two different detectors where the two species respond to the same excitation as shown in Figure 4. In particular, responsive to an excitation spectrum 100 at a center wavelength (coi), a first species will produce an emission spectrum 102 that may be detected by a first detector having a wavelength range as shown at 104. Responsive to either the same excitation spectrum 100 or a later excitation spectrum 106 at the same center wavelength (ωj), a second species will produce a second emission spectrum 108 that may be detected by a second detector having a wavelength range as shown at 110. Figure 5 shows at 112 a table of the possible combinations for using the two detectors for identifying two species using one laser (ωj).

The present invention involves employing a different approach to determining the identity of the incorporated nucleotide. Rather than detecting the differences in emission spectra between the different dye tags, it is proposed herein to probe differences in the excitation spectra. If it is postulated that two different fluorescent species, species 1 and species 2, may have near-identical emission spectra, but significantly different excitation spectra, then the identity of an incorporated base may be determined by alternately exciting these molecules with two laser sources, one which strongly excites species 1 and another which strongly excites species 2. By measuring the relative strength of the signal generated from interrogations of the sample with these different excitation sources, the identity of the fluorescent label, and thus the incorporated nucleotide, can be uniquely determined. For example, as shown in Figure 6, responsive to a first excitation spectrum

120 at a center wavelength (ω_s) a first species will produce an emission spectrum 122 that may be detected by a detector having a wavelength range as shown at 124. Responsive to a second excitation spectrum 126 at a center wavelength (ω₂) a second species will produce an emission spectrum 128 that may be detected by the same detector having the wavelength range as shown at 124. Figure 7 shows at 130 a table of the possible combinations for using the two lasers (at ooi and ω₂) and one detector for identifying two species.

Figure 8 shows a microscopy system in accordance with an embodiment of the invention that includes a first laser source 140 for providing a first illumination beam 141 at a center frequency of coj, a second laser source 142 for providing a second illumination beam 143 at a center frequency of ω₂, an optional third laser source 144 for providing a third illumination beam 145 at a center frequency of 00₃, and an optional fourth laser source 146 for providing a fourth illumination beam 147 at a center frequency of ω₄. Each illumination beam is passed through a modulator (150, 152, 154 and 156 respectively as shown), and the modulated illumination beams are directed beam splitter mirrors 160, 162, 164 and 166 respectively to provide a combined time multiplexed illumination beam 170 that is focused by an objective 172 toward a nanoreactor 174 within a microreactor 176 that includes a plurality of further nanoreactors as shown.

A modulator 180 is employed to modulate the illumination beams 141, 143,

145 and 147 based on modulation signals 181, 183, 185 and 187 respectively. Associated modulation signals 191, 193, 195 and 197 are provided to detectors 200,

202, 204 and 206. Each detector 200, 202, 204 and 206 is arranged to detect emission spectrum 210, 212, 214 and 216 respectively from the sample via dichroic mirrors 220, 222, 224 and 226. The associated modulation signals 191, 193, 195 and 197 permit the detectors to select from the illumination emission spectrum associated with a specific excitation spectrum and time. In accordance with an embodiment, the array detectors each have an array of a plurality of picture elements, each of which is associated with a nanoreator in the microreator array.

In accordance with further embodiments, the laser sources 140, 142, 144 and

146 may not be separate lasers, but may all be created from a single device that provides synchronized multiple laser outputs, such as by using frequency conversion and/or using an optical parametric oscillator. In still further embodiments, the modulation may be provided as the multiple laser outputs are being combined to provide multiplexed excitation beam.

While excitation spectroscopy could well be applied to bulk fluorescence sequencing, it has especially clear application to single-molecule sequencing, where signal-to-noise and throughput considerations are of paramount importance.

High-throughput, multi-color fluorescence spectral imaging with emission spectroscopy requires one to disperse the emission spectrum of a given location in a sample onto spatially distinct regions of an array detector (e.g., a CCD camera) which reduces the signal per unit area of the detector and increases the detector area required to image a given sample region. As a result, both throughput and signal-to-noise are compromised. These effects can be avoided using excitation spectroscopy. For example two fluorophores, even with very similar emission spectra, may be distinguished without spectral dispersion onto a detector if they have sufficiently distinct excitation spectra and can be excited at two different wavelengths.

A pair of fluorophores may be designed whereby excitation at one wavelength results in a small signal for one fluorophore at a single point on the detector, but a large signal for the other at the same point, and vice-versa. In this scheme, all of the photons emitted by a given fluorophore are projected onto a single point on the detector thereby reducing the required detector area and increasing signal-to-noise. By extension of this example, a more complicated set of fluorophores can be distinguished using excitation spectroscopy, and a practical system can be realized using multiple laser lines and a series of fast shutters or modulators to toggle the excitation wavelength.

Figure 9 shows at a picture from a scanning electron microscope of a microreactor including an array of nanoreators for use with a system in accordance with an embodiment of the invention. Each picture elements of the array detector, detects radiation from within a common focal volume of a nanoreactor associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid. The m icroscopy imaging system integrates the array of nanoreactors with the array detector such that each picture element of the array detector is associated with a different sequence reactor and is associated with a nanoreactor in the microreactor array to achieve high throughput sequencing of the target nucleic acid. It has been discovered therefore, that excitation spectroscopy may be used to achieve extremely inexpensive, high-throughput nucleic acid sequencing without nucleic acid amplification when combined with multiplexed single molecule detection.

For single molecule, digital detection of fluorophore identity, a number of mechanisms relying on the absolute fluorescence signal intensity may be employed. In principle, assuming excellent signal-to-noise properties of the dyes to be distinguished and a priori knowledge that only one or zero molecules is present in the probe volume, one laser may be used to differentially excite two fluorophores, generating different absolute signals on a point detector depending on the species present. For example, as shown in Figure 10, responsive to a first excitation spectrum

240 at a center wavelength (α>i) a first species will produce an emission spectrum 242 that may be detected by a detector having a wavelength range as shown at 244. Responsive to a second excitation spectrum 246 at a center wavelength (ω₂) a second species will produce an emission spectrum 248 that may be detected by the same detector having the wavelength range as shown at 244. Unlike the detection scheme of Figure 7 above, however, the detection scheme associated with Figures 10 and 11 provide that the species 1 will be identified when the laser one is on, and the species 2 will be identified by laser one being off, which means that laser 2 must be on. Figure 7 shows this at 250, which is a table of the possible combinations for using the two lasers (at ωi and ω₂) and one detector for identifying two species wherein the state of laser 1 being on or all provides the needed information. This scheme may be extended to multiple laser lines, allowing, for example, identification of three fluorophores using relative fluorescence measurements under different illumination conditions. For example as shown in Figure 12, responsive to a first excitation spectrum 260 at a center wavelength (ωi) a first species will produce an emission spectrum 262 that may be detected by a detector having a wavelength range as shown at 264. Responsive to a second excitation spectrum 266 a second species will produce an emission spectrum 268 that may be detected by the same detector having the wavelength range as shown at 244. Responsive to a third excitation spectrum 270 a third species will produce an emission spectrum 272 that may be detected by the same detector having the wavelength range as shown at 244. The center wavelength of the third excitation spectrum 270 may be located between the frequencies (e.g., ω₂ - (ύ\). Given very optimal noise characteristics of different dyes, such schemes may allow for identification of 4 or more dyes. Figure 13 shows at 280 a table of the possible combinations for using the two lasers (at OD₁, ω₂, and ω₂ - α>i) and one detector for identifying two species. Figure 14 shows a microscopy system in accordance with a further embodiment of the invention that provides hybrid excitation / emission spectroscopy. The system includes Figure 8 shows a microscopy system in accordance with an embodiment of the invention that includes a first laser source 290 for providing a first illumination beam 291 at a center frequency of α>i, a second laser source 292 for providing a second illumination beam 293 at a center frequency of ω₂, an optional third laser source 294 for providing a third illumination beam 295 at a center frequency of 003, and an optional fourth laser source 296 for providing a fourth illumination beam 297 at a center frequency of (0₄. Modulation of the laser sources 290, 292, 294 and 296 is provided by a modulation controller 300 that provides modulation signals 301, 303, 305 and 307 to the laser sources. In accordance with further embodiments, the laser sources 290, 292, 294 and 296 may not be separate lasers, but may all be created from a single device that provides synchronized multiple laser outputs, such as by using frequency conversion and/or using an optical parametric oscillator. In still further embodiments, the modulation may be provided as the multiple laser outputs are being combined to provide multiplexed excitation beam. The modulated illumination beams 291, 293, 295 and 297 are combined to provide a multiplexed excitation beam 310 that is directed by optics including a dichroic beam splitter 312 toward and focused by an objective 314 toward a nanoreactor 316 within a microreactor 318 that includes a plurality of further nanoreactors as shown. Fluorescence from the sample is collected by the objective, passes through the dichroic beam splitter 312, and is imaged with a tube lens 320. The iris defines the relevant field of view, and a second dichroic beam splitter splits fluorescence generated by one species (e.g., resorufin) from fluorescence generated by another species (e.g., DDAO) into two separate channels that are then imaged using lenses 322 and 324 as shown. The images are offset laterally slightly, then recombined by a third dichroic beam splitter 326, and imaged on an electron multiplied charged coupled device (EMCCD) camera 328. A lock-in amplifier 330 is also coupled to the modulation controller 300 as well as the camera 328 to provide demodulation of the time multiplexed excitation illumination. The lock-in amplifier 330 as well as the use of the two-color fluorescence permits the detectors to select from the illumination emission spectrum associated with a specific excitation spectrum and time. In accordance with an embodiment, the array detectors within the camera 328 each have an array of a plurality of picture elements, each of which is associated with a nanoreator in the microreator array.

Figures 15 and 16, for example, show such the functionality of such a hybrid system. In particular, as shown in Figure 15, responsive to a first excitation spectrum 340 at a center wavelength (α>i) a first species will produce an emission spectrum 342 that may be detected by a first detector having a wavelength range as shown at 344. Responsive to a second excitation spectrum 346 at the center wavelength (coi) (that may be at the same or a later time) a second species will produce an emission spectrum 348 that may be detected by a second detector having the wavelength range as shown at 350. Responsive to a third excitation spectrum 352 at a center wavelength (ω₂) a third species will produce an emission spectrum 354 that may be detected by the first detector having the wavelength range as shown at 344. Responsive to a fourth excitation spectrum 356 at a center wavelength (ω₂) (that may be at the same or a later time as emission spectrum 352), a fourth species will produce an emission spectrum 358 that may be detected by the second detector having the wavelength range as shown at 350. Figure 16 shows at 360 a table of the possible combinations for using the two lasers (Co₁, ω₂) and two detectors for identifying four species.

Figures 17 and 18, for example, show such the functionality of such another hybrid system. In particular, as shown in Figure 17, responsive to a first excitation spectrum 370 at a center wavelength (ω_\) a first species will produce an emission spectrum 372 that may be detected by a first detector having a wavelength range as shown at 374. Responsive to a second excitation spectrum 376 at the center wavelength (ωj) (that may be at the same or a later time as emission spectrum 370) a second species will produce an emission spectrum 378 that may be detected by the first detector having the wavelength range as shown at 374. Responsive to a third excitation spectrum 380 at a center wavelength (0)₃) a third species will produce an emission spectrum 382 that may be detected by a second detector having the wavelength range as shown at 384. Responsive to a fourth excitation spectrum 386 at a center wavelength (ω₄) a fourth species will produce an emission spectrum 388 that may be detected by the second detector having the wavelength range as shown at 384. Figure 16 shows at 390 a table of the possible combinations for using the four lasers (ωι, 00₂, G0₃, and ω₄) and two detectors for identifying four species.

In accordance with further embodiments therefore, excitation spectroscopy may be combined with emission spectroscopy to identify four or more fluorescent species while always maximally exciting the species being detected. In principle, two lasers can achieve this. In such schemes for example, one set of two dyes may have similar excitation spectra, but different emission spectra. A second set of two dyes may have excitation spectra that are different from the first set of dyes, but emission spectra which are similar. By using two different detectors, and two different excitation lasers, all four dye species can be identified unambiguously. Alternately, the schemes of several of the above Figures (e.g., Figure 12) could be replicated over more different regions of the spectrum to unambiguously differentiate four different dye species using four lasers and two detectors. Those skilled in the art will appreciate that numerous modifications and variations may be made to the above disclosed embodiments without departing from the spirit and scope of the invention. What is claimed is:

Claims

L A method of using microscopy in high throughput sequencing of a target nucleic acid, said method comprising the steps of: disposing in a microreactor a mixture in solution phase including a copy of a target nucleic acid, said microreactor including a microreactor array of a plurality of nanoreactors; providing a first illumination beam having a center frequency of ωι that is selected to effect fluorescence of a first label associated with a first nucleotide; providing a second illumination beam having a center frequency of ω_∑ that is selected to effect fluorescence of a second label associated with a second nucleotide; modulating the first illumination beam and the second illumination beam such that a multiplexed excitation beam is provided that includes illumination from the first illumination beam and the second illumination beam; directing and focusing the multiplexed excitation beam toward a common focal volume; and detecting radiation at an array detector from within the common focal volume, said radiation being associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid to achieve high throughput sequencing of the target nucleic acid.

2. The method as claimed in claim 1 , wherein said array detector includes an array of a plurality of sensors, each of which is associated with a different sequence reactor and is associated with a nanoreactor in the microreactor array.

3. The method as claimed in claim 1, wherein the microreactor is a mixture in solution phase comprising a single copy of the target nucleic acid, a nucleic acid replicating catalyst, and a mixture of nucleotides, wherein said mixture of nucleotides comprises the first nucleotide comprising the first label that is substantially non-fluorescent until after incorporation of said first nucleotide into a nucleic acid based on complementarity to said target nucleic acid and the second nucleotide comprising the second label that is substantially non-fluorescent until after incorporation of said second nucleotide into a nucleic acid based on complementarity to said target nucleic acid.

4. The method as claimed in claim 1, wherein said step of detecting radiation from within the common focal volume associated with the incorporation of the individual nucleotide during template-dependent replication of the copy of the target nucleic acid involves detecting at a detector that includes a plurality of multiplexed detection areas.

5. The method as claimed in claim 1, wherein said method further includes the step of providing a third illumination beam having a center frequency of ωj that is selected to effect fluorescence of a third label associated with a third nucleotide.

6. The method as claimed in claim 4, wherein said method further includes the step of providing a fourth illumination beam having a center frequency of ω₄ that is selected to effect fluorescence of a fourth label associated with a fourth nucleotide

7. The method as claimed in claim 1, wherein said step of detecting radiation from within the common focal volume associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid involves detecting fluorescent radiation at a single location point detector.

8. The method as claimed in claim 1 , wherein said step of detecting radiation from within the common focal volume associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid involves detecting fluorescent radiation at an array detector.

9. The method as claimed in claim 1, wherein the time multiplexed excitation beam includes alternating illumination from the first illumination beam and the second illumination beam.

10. A method of using microscopy in high throughput sequencing a target nucleic acid, said method comprising the steps of: disposing in a microreactor a mixture in solution phase comprising a copy of a target nucleic acid, said microreactor including a microreactor array of a plurality of nanoreactors; providing a first illumination beam having a center frequency of coj that is selected to effect fluorescence of a first label associated with a first nucleotide; optically modulating the first illumination beam such that a multiplexed excitation beam is provided that includes illumination from a first optical modulation of the first illumination beam and a second optical modulation from the first illumination beam to provide a time multiplexed excitation beam; directing and focusing the time multiplexed excitation beam toward a common focal volume; and detecting radiation at an array detector from within the common focal volume associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid to achieve high throughput sequencing of the target nucleic acid.

11. The method as claimed in claim 10, wherein said array detector includes an array of a plurality of sensors, each of which is associated with a different sequence reactor and is associated with a nanoreactor in the microreactor array.

12. The method as claimed in claim 10, wherein the microreactor is a mixture in solution phase comprising a single copy of the target nucleic acid, a nucleic acid replicating catalyst, and a mixture of nucleotides, wherein said mixture of nucleotides comprises the first nucleotide comprising the first label that is substantially non-fluorescent until after incorporation of said first nucleotide into a nucleic acid based on complementarity to said target nucleic acid.

13. A microscopy system for use in sequencing a target nucleic acid, said microscopy system comprising: a microreactor for receiving a mixture in solution phase comprising a copy of the target nucleic acid, said microreactor including a microreactor array of a plurality of nanoreators; an illumination source for providing a first illumination beam having a center frequency of ω; that is selected to effect fluorescence of a first label associated with a first nucleotide, and for providing a second illumination beam having a center frequency of <x>₂ that is selected to effect fluorescence of a second label associated with a second nucleotide; a modulation unit for modulating the first illumination beam and the second illumination beam such that a multiplexed excitation beam is provided that includes illumination from the first illumination beam and the second illumination beam; optics for directing and focusing the multiplexed excitation beam toward a common focal volume within a portion of the microreactor; and an array detector for detecting radiation from within the common focal volume associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid to achieve single molecule sequencing of the target nucleic acid.

14. The microscopy system as claimed in claim 13, wherein said array detector includes an array of a plurality of sensors, each of which is associated with a different sequence reactor and is associated with a nanoreactor in the microreactor array.

15. The microscopy system as claimed in claim 13, wherein said multiplexed excitation beam is a time multiplexed excitation beam that includes alternating illumination from the first illumination beam and the second illumination beam.

16. The microscopy system as claimed in claim 13, wherein said detector includes a plurality of detector areas, each of which is associated with a different target nucleic acid.

17. The microscopy system as claimed in claim 13, wherein said system further includes a third illumination source for providing a third illumination beam having a center frequency of a>₃ that is selected to effect fluorescence of a third label associated with a third nucleotide.

18. The microscopy system as claimed in claim 17, wherein said system further includes a fourth illumination source for providing a fourth illumination beam having a center frequency of ω^ that is selected to effect fluorescence of a fourth label associated with a fourth nucleotide.

19. The microscopy system as claimed in claim 13, wherein at least one of the first illumination beam having a center frequency of a>_/ and the second illumination beam having a center frequency of a>₂ is selected to effect fluorescence of at least two labels associated with at least two nucleotides,

20. The microscopy system as claimed in claim 13, wherein said common focal volume includes a mixture in solution phase comprising a single copy of the target nucleic acid, a nucleic acid replicating catalyst, and a mixture of nucleotides, and wherein said mixture of nucleotides comprises the first nucleotide comprising the first label that is substantially non-fluorescent until after incorporation of said first nucleotide into a nucleic acid based on complementarity to said target nucleic acid and the second nucleotide comprising the second label that is substantially non-fluorescent until after incorporation of said second nucleotide into a nucleic acid based on complementarity to said target nucleic acid.

21. The microscopy system as claimed in claim 13, wherein said illumination source system includes a first illumination source for providing the first illumination beam having the center frequency of ωi that is selected to effect fluorescence of the first label associated with the first nucleotide, and a second illumination source for providing the second illumination beam having the center frequency of co^ that is selected to effect fluorescence of the second label associated with the second nucleotide.

22. The microscopy system as claimed in claim 13, wherein said illumination source system provides broadband illumination from which said first illumination source and said second illumination source are extracted.