EP1104491A4 - Sequen age d'adn par differenciation des periodes de decroissance d'emission de sondes fluorescentes et systemes a cet effet - Google Patents

Sequen age d'adn par differenciation des periodes de decroissance d'emission de sondes fluorescentes et systemes a cet effet

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Publication number
EP1104491A4
EP1104491A4 EP99941073A EP99941073A EP1104491A4 EP 1104491 A4 EP1104491 A4 EP 1104491A4 EP 99941073 A EP99941073 A EP 99941073A EP 99941073 A EP99941073 A EP 99941073A EP 1104491 A4 EP1104491 A4 EP 1104491A4
Authority
EP
European Patent Office
Prior art keywords
fluorescent
nucleic acid
detection region
radiation
fluorescence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP99941073A
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German (de)
English (en)
Other versions
EP1104491A1 (fr
Inventor
Morten J Jensen
J Wallace Parce
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Caliper Life Sciences Inc
Original Assignee
Caliper Technologies Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/213,297 external-priority patent/US6447724B1/en
Application filed by Caliper Technologies Corp filed Critical Caliper Technologies Corp
Publication of EP1104491A1 publication Critical patent/EP1104491A1/fr
Publication of EP1104491A4 publication Critical patent/EP1104491A4/fr
Ceased legal-status Critical Current

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

Definitions

  • the present invention provides for the analysis of mixtures of compounds. More particularly, the present invention involves tagging individual compounds with unique fluorescent markers having different fluorescence lifetimes. The analysis of the mixture is then accomplished by distinguishing individual compounds by their unique fluorescence lifetime, using a new apparatus as set forth below.
  • the radioactive nucleic acids are separated by a method such as gel electrophoresis and the positions of the nucleic acids are visualized by autoradiography.
  • a method such as gel electrophoresis and the positions of the nucleic acids are visualized by autoradiography.
  • Fluorescent-labeled oligonucleotide primers have been used in place of radiolabeled primers for sensitive detection of DNA fragments (see, e.g., U.S. Pat. No. 4,855,225 to Smith et al). Additionally, DNA sequencing products can be labeled with fluorescent dideoxynucleotides (U.S. Pat. No. 5,047,519 to Prober et al.) or by the direct incorporation of a fluorescent labeled deoxynucleotide (Voss et al. Nucl Acids Res. 17:2517 (1989)). As currently practiced, fluorescent sequencing reactions circumvent many of the problems associated with the use of radionuclides.
  • the devices are generally suitable for assays utilizing fluorophores which relate to the interaction of biological and chemical species, including enzymes and substrates, ligands and ligand binders, receptors and ligands, antibodies and antibody ligands, as well as many other assays. Because the devices provide the ability to mix fluidic reagents and assay mixing results in a single continuous process, and because minute amounts of reagents can be assayed, these microscale devices represent a fundamental advance for laboratory science.
  • fluorogenic and non- fluorogenic assays utilizing fluorescent labels in flowing microfluidic systems are provided, e.g., in Kopf-Sill et al, WO98/56956 "APPARATUS AND METHODS FOR CORRECTING FOR VARIABLE VELOCITY IN MICROFLUIDIC SYSTEMS," filed June 8, 1998.
  • a fluorogenic assay is an assay in which a product of the assay emits a label distinct from those of the reactants of the assay.
  • a non- fluorogenic assay is an assay in which the mobility of a product differs from those of labeled reactants (e.g., in a flowing electrokinetic system), but the emitted label is still essentially the same as the label found on a reactant.
  • Detection of non-fluorogenic assay products is possible e.g., in an electroosmotically driven microfluidic device using periodic injections of reaction mixture into a separation channel, in which reactants and products are separated by electrophoresis due to changes in the electrophoretic mobility resulting from the reaction (see also, A. R. Kopf-Sill, T. Nikiforov, L. Bousse, R. Nagel, & J. W. Parce, "Complexity and performance of on-chip biochemical assays," in Proceedings of Micro- and Nano fabricated Electro-Optical
  • Closed-loop biochemical microfluidic devices especially adapted to sequencing nucleic acids, as well as for high-throughput screening are described in WO98/45481 , entitled "CLOSED-LOOP BIOCHEMICAL ANALYZERS" by Knapp et al.
  • the results of a first sequencing reaction can be used to select primers, templates, or the like, for additional sequencing, or to select related families of compounds for screening in high- throughput assay methods. These primers or templates are then accessed by the system and the process continues.
  • fluorescent dyes are commonly detected/distinguished by their emission spectra.
  • appropriately selected fluorescent dyes can be distinguished by measuring the corresponding wavelengths (or frequencies).
  • This distinguishing characteristic has led to methodologies and techniques for sequencing nucleic acids by detecting the emission spectra of labeled nucleic acid samples as noted above.
  • a fluorescent labeled oligonucleotide is used to synthesize nucleic acid samples having a sequence complementary to the sequence under analysis.
  • the fluorescent labeled nucleic acid samples are separated by a method such as gel electrophoresis and the positions of the nucleic acids are determined by spectrally identifying the order of the fluorescent labels.
  • a method such as gel electrophoresis and the positions of the nucleic acids are determined by spectrally identifying the order of the fluorescent labels.
  • Such multicolor spectral detection techniques and devices tend to be quite costly and cumbersome, as they typically require a separate filter and detector for each color to be detected.
  • four filters and four detectors are required.
  • many different labels may need to be used for each technique.
  • each nucleotide in order to identify individual nucleotides, each nucleotide must bear a fluorescent marker that has by a unique absorbance and/or emission spectrum with a different absorbance or emission maximum.
  • the absorbance or emission maxima of each tag must be clearly resolved from those of every other tag.
  • fluorescence must be monitored at a number of different wavelengths in order to detect each of the maxima and a filtering system must be employed. This is cumbersome and increases the expense of the instrumentation. This situation is additionally complicated by the dependence of the abso ⁇ tion or emission maxima for a compound upon the environment surrounding that compound.
  • a method of detecting individual fluorescently labeled compounds within a mixture of compounds which relied on a characteristic of the fluorescent moiety other than its absorption and/or emission spectrum would represent a significant advance in the art.
  • the present invention provides such a method and apparatus for practicing the methods.
  • the present invention provides a method of distinguishing between a plurality of fluorescent species.
  • the fluorescent species are first electrokinetically transported through a microfluidic channel.
  • the fluorescent species are then excited by irradiating them with electomagnetic energy.
  • the excitation can occur either during the transporting or at the completion of the transporting.
  • the fluorescent molecules are allowed to return to their ground state. This process is accompanied by a fluorescence emission which is characteristic for each fluorescent species and which is characterized by a temporal duration referred to as the fluorescence lifetime.
  • the lifetimes for each of the fluorescent labels is detected at a detecting station and the labeled species are identified by measuring the characteristic fluorescence lifetime of the label to which they are conjugated.
  • the detection station can include, for example, a laser or pulse lamp to excite the fluorescent species.
  • any useful configuration of lenses, prisms, mirrors, diffraction gratings, monochromators and the like can be used to practice the present invention.
  • Useful detectors include fast, high sensitivity optical detectors like PMT, Avalanche Photo Diodes and Photo Diodes.
  • the detector can be coupled to a digital computer that receives incoming data from the detector and processes it into a form useful for distinguishing between the lifetimes of the labels.
  • the different fluorescent species present in the mixture can be detected and identified.
  • Single or overlapping emissions that are composed of species with different lifetimes can be mathematically resolved into individual lifetimes, allowing the identification of the individual fluorescent constituents contributing to the emission.
  • the method is generally useful for the detection and identification of a broad range of compounds. It can be used to identify individual molecules which range in size and functionality from small organic, inorganic or organometallic molecules to proteins, including enzymes, antibodies and the like.
  • the method of the invention can also be used to characterize and identify synthetic polymers and oligomers. These polymers and oligomers find utility in diverse fields of endeavor, including industrial applications, mechanical applications, drugs, foodstuffs and textiles. Synthetic, natural and modified polymers and oligomers of biomolecules such as amino acids, nucleic acids and saccharides can also be identified using the method of the invention.
  • the present invention provides a method of sequencing a nucleic acid polymer of interest.
  • the method comprises performing a sequencing reaction on the nucleic acid polymer. Any of the sequencing reactions known in the art is appropriate for use in this aspect.
  • methods which chemically or enzymatically degrade or synthesize nucleic acids are of use in practicing the present invention.
  • one or more fluorescent labels is inco ⁇ orated into either the nucleic acid being sequenced or a sequence complementary to the nucleic acid being sequenced.
  • Several methods for performing this inco ⁇ orating are known in the art. A non-limiting list includes the Sanger, Sanger dideoxy and Maxam-Gilbert sequencing methodologies.
  • Sequencing reaction mixtures that are useful in practicing the present invention include those that contain the nucleic acid to be sequenced and a fluorescent label.
  • the fluorescent label is attached to a first labeled nucleic acid selected from the group consisting of labeled nucleic acids, labeled nucleic acid polymers and combinations thereof.
  • the fluorescent species are electrokinetically transported through a microfluidic channel to resolve or partially resolve the mixture into separate components.
  • the fluorescent label will, following excitation, emit electromagnetic energy that is characterized by a distinct and detectable lifetime.
  • each of the labels will have a fluorescent lifetime that is distinct from other labels and thereby detectable.
  • the fluorescence emission is detected at a detecting station.
  • the present invention also provides apparatus that are useful, e.g., in practicing the methods of this invention.
  • the apparatus is capable of distinguishing between a plurality of fluorescent species, wherein each of the fluorescent species has a fluorescence emission, the emission having a characteristic fluorescence lifetime.
  • the apparatus of the invention comprises a microfluidic device that includes at least one microchannel fabricated within the body structure of the device.
  • the fluorescent species flows through the microchannel by means of, for example, pressure, electroosmosis, electrokinesis, capillarity and the like.
  • the microchannel is linked to a detecting station that is capable of detecting the fluorescent species in the microchannel.
  • the signal from the detector is sent to a digital computer that is operably linked to the detector.
  • the digital computer is appropriately configured or programmed to determine the fluorescence lifetimes of the fluorescent species.
  • the techniques of the present invention use modulated radiation to irradiate fluorophores in a detection region.
  • a beamsplitter element is optionally provided to selectively pass excitation signals to the detection region and to redirect fluorescence emissions toward a fluorescence detector.
  • the fluorescence detector outputs a signal proportional to the fluorescence emissions detected, and a processor analyzes the proportional signal to determine fluorescence lifetimes.
  • the processor measures the decay time directly; if the excitation source emits, or is modulated to emit, an oscillating excitation signal, the processor determines the fluorescence lifetimes by measuring the phase difference relative to an excitation modulation reference signal.
  • a phase-locked loop (PLL) is preferably used to determine phase differences.
  • an apparatus for use in measuring the fluorescent lifetimes of a plurality of fluorescent labels, where the labels are provided to a detection region of the apparatus.
  • the apparatus typically includes a radiation source.
  • the radiation source emits, or is modulated to emit, radiation for irradiating the detection region, where the radiation excites the fluorescent labels and causes the labels to fluoresce.
  • the apparatus also typically includes a fluorescence detector for detecting the fluorescent emissions from the fluorescent labels in the detection region, where each one of the plurality of fluorescent labels has a different fluorescent lifetime.
  • the apparatus normally has a processor, coupled to the fluorescence detector, for analyzing the fluorescent emissions to determine the fluorescent lifetimes of the labels.
  • an apparatus for use in determining the sequence of nucleotides in a nucleic acid sample having a plurality of overlapping nucleic acid fragments, where each fragment includes one of four different fluorescent labels. Each label has a different fluorescent lifetime, with each of the labels binding to a specific nucleotide.
  • the nucleic acid fragments of the nucleic acid sample are provided to a detection region.
  • the apparatus typically includes a radiation source. The radiation source emits, or is modulated to emit, radiation for irradiating the detection region, where the radiation excites the fluorescent labels and causes the labels to fluoresce.
  • the apparatus also typically includes a fluorescence detector for detecting the fluorescent emissions from the fluorescent labels in the detection region.
  • the apparatus ordinarily has a processor, coupled to the fluorescence detector, for analyzing the fluorescent emission to determine the fluorescent lifetimes of the labels, thereby determining the sequence of nucleotides in the nucleic acid.
  • a system for measuring the fluorescent lifetimes of a plurality of fluorescent labels.
  • the system typically includes a detection region, where the labels are provided to the detection region.
  • the system also typically includes a radiation source.
  • the radiation source emits, or is modulated to emit, radiation for irradiating the detection region, where the radiation excites the fluorescent labels and causes the labels to fluoresce.
  • the system also typically includes detection means for detecting the fluorescent emissions from the fluorescent labels in the detection region; and means, coupled to the fluorescence detector, for analyzing the fluorescent emissions to determine the fluorescent lifetimes of the labels.
  • a method of measuring the fluorescent lifetimes of a plurality of fluorescent labels is provided.
  • the method typically includes the steps of providing the labels to a detection region and of irradiating the detection region with radiation emitted from a radiation source, where the radiation excites the fluorescent labels and causes the labels to fluoresce.
  • the method also typically includes the steps of detecting the fluorescent emissions of the fluorescent labels using a fluorescence detector, where each one of the plurality of fluorescent labels has a different fluorescent lifetime, and of analyzing the fluorescent emissions using a processor coupled to the fluorescence detector to determine the fluorescent lifetimes of the labels.
  • Figure 1 depicts an example of a microfluidic device for use with certain aspects of the present invention
  • Figure 2 illustrates examples of fluorescence emission signals for long and short decay time fluorophores that have been excited by a pulsed excitation signal
  • Figure 3 illustrates examples of fluorescence emission signals for long and short decay time fluorophores that have been excited by a sinusoidally modulated excitation signal
  • Figure 4 is a block diagram of a fluorescence detection system according to one embodiment of the present invention
  • FIG. 5 is a block diagram of a fluorescence detection system according to an alternate embodiment of the present invention.
  • Figure 6 is a block diagram of a fluorescence detection system according to another alternate embodiment of the present invention.
  • the present invention provides a method of distinguishing between a plurality of fluorescent species.
  • the fluorescent species are first electrokinetically transported through a microfluidic channel.
  • the fluorescent molecules are then excited by irradiating them with electomagnetic energy.
  • the excitation can occur either during the transporting or at the completion of the transporting.
  • the fluorescent molecules are allowed to return to their ground state. This process is accompanied by a fluorescence emission which is characteristic for each fluorescent species and which is characterized by a temporal duration referred to as the fluorescence lifetime of that species.
  • the lifetimes for each of the fluorescent labels is detected at a detecting station and the labeled species are identified by measuring the characteristic fluorescence lifetime of the label to which they are conjugated.
  • the detection station can include, for example, a laser or pulse lamp to excite the fluorescent species.
  • any useful configuration of lenses, prisms, mirrors, diffraction gratings, monochromators and the like can be used to practice the present invention.
  • Useful detectors include fast, high sensitivity optical detectors like PMT, Avalanche Photo Diodes and Photo Diodes.
  • the detector can be coupled to a digital computer that receives incoming data from the detector and processes it into a form useful for distinguishing between the lifetimes of the fluorescent labels.
  • the methods and apparatus of the instant invention are used in the detection of fluorescence emission signals from analytical systems employing fluorescence detection in microscale fluidic channels. Examples include, e.g., fused silica capillary systems, i.e., CE, as well as microfluidic devices and systems that inco ⁇ orate microscale elements such as microfluidic channels. Such systems are generally described in U.S. Patent Application Nos. 08/845,754, filed April 25, 1997, PCT Application Publication No. 98/00231, filed June 24, 1997 and WO98/56956.
  • the methods of the present invention are generally carried out in "microfluidic devices” or “microlaboratory systems,” which allow for integration of the elements required for performing the assay, automation, and minimal environmental effects on the assay system, e.g., evaporation, contamination, human error, and the like.
  • a number of devices for carrying out the assay methods of the invention are described in substantial detail herein. However, it will be recognized that the specific configuration of these devices will generally vary depending upon the type of assay and/or assay orientation desired.
  • the screening methods of the invention can be carried out using a microfluidic device having two intersecting channels. For more complex assays or assay orientations, multichannel/intersection devices are optionally employed.
  • microfluidic generally refers to one or more fluid passages, channels, chambers or conduits which have at least one internal cross- sectional dimension, e.g., depth, width, length, diameter, etc., that is less than 500 ⁇ m, and typically between about 0.1 ⁇ m and about 500 ⁇ m.
  • the microscale channels or chambers preferably have at least one cross-sectional dimension between about 0.1 ⁇ m and 200 ⁇ m, more preferably between about 0.1 ⁇ m and 100 ⁇ m, and often between about 0.1 ⁇ m and 50 ⁇ m.
  • the microfluidic devices or systems prepared in accordance with the present invention typically include at least one microscale channel, usually at least two intersecting microscale channels, and often, three or more intersecting channels disposed within a single body structure. Channel intersections may exist in a number of formats, including cross intersections, "T" intersections, or any number of other structures whereby two channels are in fluid communication.
  • a “microfluidic” channel is a channel (enclosed groove, depression, tube, capillary, etc.) which is adapted to handle small volumes of fluid.
  • the channel is a tube, channel or conduit having at least one subsection with at least one cross-sectional dimension of between about 0.1 ⁇ m and 500 ⁇ m, and typically less than lOO ⁇ m; ordinarily, the channel is closed over a significant portion of its length, having top, bottom and side surfaces.
  • materials that are being analyzed e.g., subjected to optical analysis for fluorescence emission signals, in these microscale fluidic systems, are transported along the microscale fluid channels, past a detection point, where a detectable fluorescence emission signal is measured.
  • the signals within these channels typically result from the presence of fluorescent substances therein, e.g., fluorophores that inherently fluoresce, or are made to fluoresce, that are used as indicators of the presence or absence of some material or condition.
  • the body structure of the microfluidic devices described herein typically comprises an aggregation of two or more layers (which can be fused or bonded) which, when appropriately mated or joined together, form the microfluidic device of the invention, e.g., containing the channels and/or chambers described herein.
  • the microfluidic devices described herein will comprise a body structure having a top portion, a bottom portion, and an interior portion, wherein the interior portion, or microscale cavity, substantially defines the channels and chambers of the device.
  • Suitable substrate materials for the body structure are generally selected based upon their compatibility with the conditions present in the particular operation to be performed by the device. Such conditions can include extremes of pH, temperature, salt concentration, and application of electrical fields. Additionally, substrate materials are also selected for their inertness to critical components of an analysis or synthesis to be carried out by the device. Examples of useful substrate materials include, e.g., glass, quartz and silicon as well as polymeric substrates, e.g. plastics, particularly polyacrylates. In the case of conductive or semi-conductive substrates, it is occasionally desirable to include an insulating layer on the substrate. This is particularly important where the device inco ⁇ orates electrical elements, e.g., electrical fluid direction systems, sensors and the like.
  • the substrate materials may be rigid, semi-rigid, or non-rigid, opaque, semi-opaque or transparent, depending upon the use for which they are intended.
  • devices which include an optical, spectrographic, photographic or visual detection element will generally be fabricated, at least in part, from transparent materials to allow, or at least, facilitate that detection.
  • transparent windows of, e.g., glass or quartz are optionally inco ⁇ orated into the device for these types of detection elements.
  • the polymeric materials optionally have linear or branched backbones, and may be crosslinked or non-crosslinked.
  • polymeric materials include, e.g., polydimethylsiloxanes (PDMS), polyurethane, polyvinylchloride (PVC) polystyrene, polysulfone, polycarbonate and the like.
  • PDMS polydimethylsiloxanes
  • PVC polyvinylchloride
  • the methods and apparatus of the present invention are used for determining the sequence of nucleotides in a nucleic acid sample wherein the nucleotides are tagged or labeled with fluorescent labels as is well known in the art.
  • a fluorescent-labeled oligonucleotide is used to synthesize a nucleic acid having a sequence complementary to the sequence under analysis.
  • nucleic acids can be labeled with fluorescent dideoxynucleotides or by the direct inco ⁇ oration of a fluorescent labeled dideoxynucleotide.
  • the fluorescent-labeled nucleic acids are separated by a method such as gel electrophoresis and the positions of the nucleic acids are determined by identifying the order of the fluorescent markers or labels using the techniques of the present invention.
  • the present invention is useful for detecting and distinguishing any fluorophore labeled substances (for example, any fluorophore labeled substance in an electrophoretic separation medium or the like) and any fluorescing substances.
  • a fluorescent material or a fluorescent-labeled material is transported along the microscale channel and past a detection point.
  • transporting materials within these systems may be carried out by any of a variety of methods.
  • such material transport is optionally carried out through the application of pressures to the materials within the channels, through the inco ⁇ oration of microscale mechanical pumps, or through the application of electric fields, to move materials through the channels.
  • electrokinetic transport systems for moving material within the microfluidic channels.
  • electrokinetic material transport systems include systems which transport and direct materials within an interconnected channel and/or chamber containing structure, through the application of electrical fields to the materials, thereby causing material movement through and among the channel and/or chambers (i.e., cations will move toward the negative electrode, while anions will move toward the positive electrode).
  • electrokinetic material transport and direction systems include those systems that rely upon the electrophoretic mobility of charged species within the electric field applied to the structure. Such systems are more particularly referred to as electrophoretic material transport systems.
  • pressure-driven flow is used to move components in microfluidic channels or channel regions. Additional details on the movement of materials in microfluidic systems are found below.
  • Figure 1 depicts an example of a microfluidic device for use with certain aspects of the present invention.
  • the device 100 includes a body structure 102 which has an integrated channel network 104 disposed therein.
  • the body structure 102 includes a plurality of reservoirs 106-128, disposed therein, for holding reagents, sample materials, and the like.
  • buffer reservoir 130 Also included is buffer reservoir 130, as well as waste reservoirs 132, 134 and 136.
  • the reagents, samples, etc. are transported from their respective reservoirs, either separately or together with other reagents from other reservoirs into a main channel 138, and along main channel 138 toward waste reservoir 136, past detection zone or window 140.
  • Detection window 140 is typically transparent, and maybe comprised of a transparent region of the body structure, or a separate transparent window fabricated into the body structure.
  • the body structure is itself fabricated from a transparent material, e.g., glass or transparent polymers, thereby obviating the need for a separate transparent region to define the detection window.
  • Microfluidic devices of the sort described above are useful in performing a variety of analyses, such as electrophoretic separation of macromolecules, e.g., nucleic acids, proteins, etc. (see U.S. Application No.
  • the individual members of virtually any complex mixture can be distinguished and identified using the method of the invention.
  • Exemplary species include, for example, individual members of compound libraries (e.g., small organic molecules, peptides, nucleic acids) and the products of sequencing reactions.
  • the method is used to distinguish between a plurality of fluorescent products derived from a sequencing reaction performed on a nucleic acid, a peptide or an oligosaccharide.
  • the fluorescent products are derived from a dideoxy nucleotide chain termination method sequencing reaction mixture derived from one or more nucleic acids. See, for example, Sanger et al, Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977); U.S. Pat. No. 5,171,534, to Smith et al.
  • the means of practicing various embodiments of the present invention will be apparent from the theoretical and practical discussion that follows. Fluorescence Lifetime
  • ⁇ is the fluorescence lifetime
  • V is the emissive rate constant of the fluorophore
  • k is the rate constant of radiationless deca .
  • the lifetime of a particular fluorophore, in the absence of nonradiative processes, is called the intrinsic lifetime:
  • Both the quantum yield Q and the fluorescence lifetime ⁇ can be modified by factors which affect either of the rate constants T and k.
  • some molecules can be substantially non-fluorescent with a large rate of internal, radiationless conversion. Such molecules typically have low quantum yields and short fluorescence lifetimes.
  • scintillation agents which have high quantum yields as a result of high T values, typically have very short fluorescence lifetimes.
  • a fluorophore is a substance which itself fluoresces, or is made to fluoresce, or is a fluorescent analogue of an analyte.
  • any fluorophore now known, or later discovered can be used with the present invention.
  • Fluorescent species having lifetimes that fall within a broad range of measurable lifetimes are useful in the present invention.
  • the fluorescence lifetimes of fluorophores preferably range from about 0.1 nanoseconds to about 4000 nanoseconds, more preferably from about 0.1 nanoseconds to about 1000 nanoseconds, and even more preferably from about 0.1 nanoseconds to about 100 nanoseconds.
  • particularly preferred fluorophores have the following characteristics: a.
  • Excitation wavelengths greater than 350 nanometers reduce the background interference because most fluorescent substances responsible for background fluorescence in biological samples are excited below 350 nanometers. A greater Stoke's shift also allows for less background interference.
  • each compound in a set of fluorescent compounds used to analyze a mixture is selected to have a fluorescence lifetime, under relevant experimental conditions, which is distinguishable from the fluorescence lifetimes of some or all of the other compounds in the set.
  • fluorescence lifetimes are distinguishable from the fluorescence lifetimes of some or all of the other compounds in the set.
  • fluorophores and combinations of fluorophores will be apparent to those of skill in the art.
  • the fluorophore is derivatized with a reactive functionality through which the fluorophore is tethered to a component of the mixture that is being analyzed.
  • a reactive functionality through which the fluorophore is tethered to a component of the mixture that is being analyzed.
  • Many reactive fluorescent molecules are known by and readily available to those of skill in the art. Appropriate reactive fluorescent derivatives are commercially available (e.g., Molecular Probes Inc., Eugene, Oregon) or they can be synthesized by means well known in the art.
  • Fluorescent agents which are reactive towards amines e.g., isothiocyanates, carboxylic acids, succinimidyl esters, sulfonyl halides, dialdehydes), thiols (e.g., iodoacetamides, maleimides, alkyl halides, aziridines, epoxides, disulfides), alcohols (isocyanates, acylnitriles, acid chlorides), aldehydes, ketones, vicinal diols (hydrazine derivatives, amines) and carboxylic acids (amines, alkyl halides, trifluoromethansulfonates) are preferred for use in the present invention.
  • amines e.g., isothiocyanates, carboxylic acids, succinimidyl esters, sulfonyl halides, dialdehydes
  • thiols e.g., iodoacetamide
  • models of the conjugates are preferably characterized as to spectral characteristics including optimal excitation and emission wavelengths and fluorescence lifetimes. All of these properties of the conjugates can be determined using standard techniques.
  • the fluorescence lifetime of the conjugate may be dependent on the fluorophore to analyte ratio in the sample. The optimal ratio between the fluorophore and the analyte can be determined experimentally.
  • the following criteria generally pertain: a. Ideally, the fluorophores should have substantial overlap of abso ⁇ tion wavelengths so that they can all be efficiently excited at a single wavelength; b.
  • the emission wavelengths should have substantial overlap of emission bands so that the fluorescence contribution of each label can be monitored at a single wavelength; and c.
  • the differences in the fluorescence lifetimes between fluorophores is typically at least about 5 nanoseconds.
  • a set of fluorescent compounds with overlapping emission bands allows the excitation of all of the compounds of the set to occur in a substantially simultaneous manner.
  • each member of the set of compounds must have a unique abso ⁇ tion band and each compound must be excited at a different wavelength.
  • a set of compounds which have substantially similar emission maxima are preferred.
  • the use of a set of compounds having this characteristic allows the compounds to be detected and identified by monitoring their emission at one wavelength, or within a narrow range of wavelengths. In order to detect those compounds which are excited by, or which emit, electromagnetic energy at similar wavelengths, the compounds will preferably have lifetimes which are sufficiently different to allow them to be clearly distinguished.
  • a useful set of compounds will include a group of compounds whose lifetimes differ from each other by at least 5 nanoseconds.
  • a reactive fluorescent molecule or set of molecules that is appropriate to the practice of the present invention.
  • a broad range of appropriate fluorophores are commercially available from sources such as Molecular Probes Inc. (Eugene, Oregon). Measurement of Fluorescence Lifetimes
  • the pulse decay method the sample is excited with a pulse of light, or a series of pulses, and the time-dependent decay of the fluorescent emission is measured.
  • the phase-modulation method the sample is excited with light having a time-dependent intensity, e.g., sinusoidally modulated light, and the time-dependent fluorescence emission is detected. The phase shift of the fluorescence emission relative to the exciting light is used to calculate the fluorescence lifetime.
  • the detecting is provided by a pulse method or a phase-modulation method.
  • Figure 2 depicts an example of excitation pulses and of the resulting fluorescence emissions from fluorescent dyes having different fluorescence lifetimes.
  • the excitation pulses are represented as square waves or pulses.
  • Each excitation pulse is absorbed by the fluorescent dye and the fluorescent dye subsequently fluoresces with emission characteristics as shown depending on whether the particular dye has a long or short fluorescence lifetime.
  • the fluorescent dye having a long fluorescence lifetime exhibits a slower rate of attenuation relative to the fluorescent dye having a short fluorescent lifetime.
  • To determine the fluorescence lifetime a measurement is taken of the time it takes for the decaying fluorescent pulse to decay to 1/e, or approximately 37%, of the maximum amplitude.
  • Figure 3 depicts an example of a sinusoidally modulated excitation signal and of the resulting fluorescent emission signals from fluorescent dyes having different fluorescent lifetimes.
  • the excitation light is represented as a sinusoidal wave.
  • the excitation light is continually absorbed by the fluorescent dye, and the fluorescent dye subsequently fluoresces with emission characteristics as shown depending on whether the particular dye has a short or a long fluorescence lifetime.
  • the fluorescent dye having a longer fluorescence lifetime exhibits a greater phase shift relative to the excitation signal than does the fluorescent dye having a shorter fluorescence lifetime, as shown by the arrows in Figure 3.
  • ⁇ is the angular modulation frequency
  • fluorescence emission signals that reach the detector are measured as a function of time.
  • the overall detected signal is a supe ⁇ osition of several signals (e.g., a background signal and one analyte-specific signal; or signals from different analytes in the case of a multiple analyte assay, etc.).
  • the individual contributions to the overall fluorescence signal detected are distinguishable by the different fluorescence lifetimes (decay rates) exhibited by each signal.
  • the amplitude of each component of the overall fluorescence signal is proportional to the species responsible for that component.
  • the individual fluorescence lifetimes (fluorescent decay times) should be significantly different, which is often the case with the background signal compared to a fluorescent probe.
  • a first fluorescent label and a second fluorescent label have emission maxima that occur at substantially the same wavelength.
  • additional resolution and/or complexity of analysis can be accomplished by using labels which have distinguishable excitation and/or emission maxima.
  • the first fluorescent label and the second fluorescent label have an emission maximum that occurs at a substantially different wavelength.
  • individual compounds are identified at a detection station by stimulating and detecting their fluorescence and measuring the lifetime of the detected fluorescence.
  • Useful detection stations will typically include three components: an excitation source, an optical system and a detector.
  • an excitation source an optical system
  • a detector a detector
  • the detection station will use an excitation source which is a laser or a nanosecond flash lamp.
  • Useful lasers include, but are not limited to, argon ion pumped and mode-locked Ti: sapphire lasers which provide tunable femto- or picosecond pulses. Suitable argon and mode-locked Ti:sapphire lasers are available as models LNNOVA 420 and MIRA 900, respectively from the Laser Products Division of Coherent, Inc. (Palo Alto, CA). Other suitable lasers include Nd:YAG lasers such as models ANT ARES 76-S, 468-ASE, 7950, 701 and 7049 from the Laser Products Division of Coherent, Inc. (Palo Alto, CA).
  • Nanosecond flash lamps that generate pulses on the nanosecond time-scale are commercially available.
  • One suitable lamp is available from Photon Technology International (Monmouth Junction, NJ.) and generates pulses of 1.6 nanoseconds.
  • the optical system can be constructed to have any useful configuration known in the art and can comprise any number of lenses, mirrors, prisms, beam splitters and dispersive elements (e.g., monochronomators and diffraction gratings) and the like.
  • the detector can be any device that is capable of detecting photons including, but not limited to, photodiodes, photocathodes, photomultiplier tubes and the like.
  • a presently preferred detector utilizes a stroboscopic detection system such as that described in James et al, Rev. Sci. Instrum. 63:1710 (1991).
  • the reactants or components to be detected after labeling with fluorescent labels distinguishable by their decay times can be elements of essentially any assay or reaction which is adaptable to a flowing or electrophoretic format; thus, while often described in terms of sequencing reactions, it will be understood that the reactants or assay components herein can comprise a moiety derived from any of a wide variety of components, including, antibodies, antigens, ligands, receptors, enzymes, enzyme substrates, amino acids, peptides, proteins, nucleosides, nucleotides, nucleic acids, organic molecules, monomers, polymers, drugs, polysaccharides, lipids, liposomes, micelles, toxins, biopolymers, therapeutically active compounds, molecules from biological sources, blood constituents, cells or the like.
  • FIG. 4 is a block diagram of a fluorescence detection system 200 according to one embodiment of the present invention.
  • Detection system 200 includes an excitation source 210 for exciting fluorophores in a detection region 240 with one or more excitation pulses, and a sensor, or detector, 220 for detecting fluorescence emission signals from detection region 240.
  • Fluorescence detector 220 is coupled to a processor 230 which analyzes signals from fluorescence detector 220 to determine fluorescence lifetimes.
  • excitation source 210 is a radiation source that can be turned on and off very rapidly or which can be modulated rapidly at a rate up to many million times per second, either directly or by using an opto-mechanical device.
  • excitation source 210 emits radiation having a wavelength in the range of about 350 nm to about 1000 nm, and which is modulated with a reference signal having a frequency in the range of about 1MHz to about 100MHz. More preferably, excitation source 210 is a laser diode that emits visible radiation having a wavelength of approximately 635nm, and which is modulated at approximately 10MHz with pulses having a width of approximately 10ns.
  • excitation source 210 is a laser diode that emits visible radiation having a wavelength of approximately 635nm, and which is modulated at approximately 10MHz with pulses having a width of approximately 10ns.
  • One suitable laser diode is Hitachi's Laser Diode Model HL6320G.
  • Pulse generator 260 is provided according to this embodiment to pulse the excitation source at the desired frequency to obtain the desired modulation and pulse width characteristics.
  • the time between pulses is equal to or greater than about five times the decay time of each fluorophore.
  • One of skill in the art will, of course, be able to determine other suitable modulation frequencies and characteristics without undue experimentation depending on the particular characteristics of the fluorophores being analyzed.
  • Excitation source 210 includes other suitable excitation sources such as a laser, a flash-lamp, a light emitting diode (LED), or any other controllable radiation source that emits radiation at the desired wavelength(s).
  • suitable lasers suitable for use with the present invention include, but are not limited to, argon ion pumped lasers and mode- locked Ti: sapphire lasers that provide tunable mili- second, nano-second, pico-second or femto-second pulses.
  • Suitable mode-locked Ti:sapphire lasers and ND:YAG lasers are available as models MLRA 900 and INFINITY, respectively from the Laser Products Division of Coherent, Inc. (Palo Alto, CA).
  • Nd:YAG lasers such as models ANT ARES 76-S, 468- ASE, 7950, 701 and 7049 from the Laser Products Division of Coherent, Inc. (Palo Alto, CA). Flash-lamps that generate nanosecond pulses are commercially available.
  • One suitable lamp is available from Photon Technology International (Monmouth Junction, N. J.) and generates 1.6 nanoseconds pulses.
  • Pulse generator 260 is optionally provided for use with an excitation source requiring modulation, e.g., for pulsing a laser diode, flash-lamp or arclamp, for turning a laser, such as an excimer or HeNe laser, on and off, or for pulsing a flash-lamp- pumped laser.
  • An electro-optical chopping device (not shown), located between excitation source 210 and detection region 240, can also be used to physically chop a continuous excitation signal into a series of pulses as is well known.
  • Other techniques for generating excitation pulses such as, for example, mode locking and Q-switching, will be readily apparent to those of skill in the art.
  • beamsplitter element 250 is optionally provided in one embodiment to pass excitation light to detection region 240 and to reflect light, including fluorescent emissions from detection region 240 toward fluorescence detector 220. More specifically, beamsplitter element 250 is preferably selected so as to allow a substantial portion of the radiation emitted from the excitation source having a wavelength below a threshold wavelength to pass to detection region 240, and to reflect toward fluorescence detector 220 a substantial portion of radiation incident from detection region 240 having a wavelength above the threshold wavelength.
  • beamsplitter element 250 includes a planar wavelength-sensitive beamsplitter that allows substantially all incident light having a wavelength below about 650nm to pass, and reflects substantially all incident light having a wavelength above about 650nm. As shown in Figure 4, in one embodiment the planar beamsplitter is situated at a 45 angle relative to the incident light, but the beamsplitter may be situated at any desirable angle depending on the beamsplitter's specific properties and the relative position of detector 220.
  • beamsplitter element 250 can include a prism beamsplitter or a diffraction grating selected and appropriately positioned to reflect and transmit appropriate wavelengths depending on the selected excitation source. Additionally, beamsplitter element 250 can include a beamsplitter that is polarization-dependent.
  • fluorescence detector 220 includes a photo multiplier tube (PMT) that measures fast light signals with low intensity and outputs a corresponding proportional signal to processor 230.
  • PMT photo multiplier tube
  • Fluorescence detector 220 must be fast enough to convert the fluorescence emission signal into a proportional electrical signal. Therefore, fluorescence detector 220 must operate at a rate faster than the decay times of the particular fluorophores being distinguished.
  • fluorescence detector 220 can include an avalanche photo diode or a photodiode, or any other light detection device that measures fast light signals at low intensity and outputs a proportional signal to processor 230.
  • PMTs tend to decrease in efficiency as the wavelength of detected light increases, in some embodiments where fluorescence emissions in the red to infrared wavelengths are to be detected the use of an avalanche photodiode is preferred.
  • excitation pulses from excitation source 210 irradiate detection region 240 and excite fluorophores therein, thereby causing the fluorophores to fluoresce.
  • Fluorescence detector 220 detects the resulting fluorescence emissions, either directly or by reflection from beamsplitter element 250, and generates a proportional signal.
  • Processor 230 receives and analyzes the signal from fluorescence detector 220, which is proportional to the overall fluorescence emissions signal received by fluorescence detector 220 from detection region 240.
  • processor 230 is coupled to pulse generator 260.
  • processor 230 receives a reference signal from generator 260, which is proportional to the reference signal used to modulate excitation source 210.
  • Processor 230 uses this reference signal as a reference for determining the fluorescence lifetimes from the signal received from fluorescence detector 220. According to one embodiment, it is not necessary that processor 230 be able to quantify each fluorophore, but, rather that it is able to categorize and distinguish each fluorophore effectively. For example, when used to sequence a nucleic acid in a prepared nucleic acid sample that is separated in an electrophoretic gel transported across detection region 240, processor 230 determines the nucleotide sequence by the relative characteristics of the fluorescent decay times of the different fluorescent labels or probes used.
  • FIG. 5 is a block diagram of a fluorescence detection system 300 according to an alternate embodiment of the present invention.
  • Detection system 300 includes an excitation source 310 for exciting fluorophores in a detection region 340 with an excitation signal having a time-dependent intensity, e.g., sinusoidally modulated light, and a detector 320 for detecting fluorescence emission signals from detection region 340.
  • Fluorescence detector 320 is coupled to a processor 330 which analyzes signals from fluorescence detector 320 to determine fluorescence lifetimes.
  • a beamsplitter element 350 positioned between excitation source 310 and detection region 340, is optionally provided as discussed above to allow a substantial portion of the excitation signal incident from excitation source 310 to pass through to detection region 340, and to redirect a substantial portion of the radiation incident from detection region 340, including fluorescence emissions, toward fluorescence detector 320.
  • excitation source 310 is a radiation source that emits sinusoidally modulated radiation or which can be continuously modulated to emit sinusoidally varying radiation. According to preferred aspects, excitation source 310 emits radiation having a wavelength in the range of about 350 nm to about 1000 nm, and which is modulated with a reference signal having a frequency in the range of about 1MHz to about 100MHz. More preferably, excitation source 310 is a laser diode that emits visible radiation having a wavelength of approximately 635nm, and which is modulated at approximately 10MHz.
  • One suitable laser diode includes Hitachi's Model HL6320G.
  • Oscillator 360 is provided according to this embodiment to modulate the excitation source at the desired frequency and amplitude to obtain the desired excitation signal characteristics.
  • One of skill in the art will, of course, be able to determine other suitable modulation frequencies without undue experimentation depending on the particular characteristics of the fluorophores being analyzed.
  • excitation sources include any radiation source that emits, or which can be controlled to emit, radiation having a time-dependent intensity, such as a laser, a flash-lamp, a light emitting diode (LED), or the like.
  • Fluorescence detector 320 in one embodiment, includes a photomultiplier tube (PMT) that measures fast light signals with low intensity and outputs a corresponding proportional signal to processor 330.
  • fluorescence detector 320 can include an avalanche photodiode or a photodiode, or any other light detection device that measures fast light signals at low intensity and outputs a proportional signal to processor 330.
  • processor 320 includes, or is coupled to, a phase-locked loop (not shown).
  • a phase-locked loop PLL
  • the PLL compares a reference signal received from oscillator 360 with the signals received from the fluorescence detector 320 to determine the differences in phase between the signals.
  • Processor 330 determines the fluorescence lifetimes of the corresponding fluorophores based on the phase difference or demodulation factor determined by the PLL.
  • oscillator 360 generates a reference signal at the desired frequency.
  • Excitation source emits modulated excitation signals in response to the reference signal received from oscillator 360.
  • excitation signals from excitation source 310 irradiate detection region 340 and excite fluorophores therein, thereby causing the fluorophores to fluoresce.
  • Fluorescence detector 320 detects the resulting fluorescence emissions, either directly or by reflection from beamsplitter element 350, and generates a proportional signal.
  • Processor 330 receives and analyzes the signal from fluorescence detector 320, which is proportional to the overall fluorescence emissions signal received by fluorescence detector 320 from detection region 340. In one embodiment, processor 330 receives a reference signal from oscillator 360, which is proportional to the reference signal used to modulate excitation source 310. Alternatively, processor 330 receives a reference signal from excitation source 310.
  • Processor 330 uses the received reference signal as a reference for determining the fluorescence lifetimes from the signal received from fluorescence detector 320. For example, in one embodiment, a PLL compares the reference signal with the signals from fluorescence detector 320 to determine the phase differences. Processor 330 then determines the lifetimes of the fluorophores being studied. However, it is not necessary that processor 330 be able to quantify each fluorophore, but, rather that it is able to categorize and distinguish each fluorophore effectively.
  • FIG. 6 is a block diagram of a fluorescence detection system 400 according to another alternate embodiment of the present invention.
  • Detection system 400 includes an excitation source 410 for exciting fluorophores in a detection region 440 with an excitation signal, and a detector 420 for detecting fluorescence emission signals from detection region 440.
  • Fluorescence detector 420 is coupled to a processor 430 which analyzes signals from fluorescence detector 420 to determine fluorescence lifetimes.
  • a beamsplitter element 450 positioned between excitation source 410 and detection region 440, is optionally provided as discussed above to allow a substantial portion of the excitation signal incident from excitation source 410 to pass through to detection region 440, and to redirect a substantial portion of the radiation incident from detection region 440, including fluorescence emissions, toward fluorescence detector 420.
  • Modulator 460 is provided to modulate excitation source 410 to obtain the desired excitation signal characteristics.
  • modulator 460 includes an oscillator that generates a reference signal having a desired frequency and amplitude.
  • excitation source emits radiation having a time-dependent intensity, e.g., sinusoidally modulated light, in response to the reference signal.
  • modulator 460 includes a pulse generator that pulses excitation source 410.
  • Fluorescence detection system 400 also includes additional optical elements for enhancing the excitation and detection capabilities of system 400.
  • Optical elements 470 and 472, positioned between excitation source 410 and detection region 440, are optionally provided according to one embodiment to assist in directing and focusing the excitation signal onto detection region 440.
  • Optical elements 470 and 472 can include focusing lenses, mirrors, or any other optical elements as are well known and which are useful for collimating, directing and focusing radiation depending on the desired system layout and characteristics.
  • Optical element 474 is optionally provided in one embodiment to assist in directing and focusing the fluorescence emissions signals from detection region 440 onto fluorescence detector 420.
  • Optical element 474 in one embodiment includes a focusing lens selected accordingly depending on whether fluorescence emission signals are received by fluorescence detector 420 directly from detection region 440 or via reflection from beamsplitter element 450.
  • Filter elements 480 and 482 are optionally provided to avoid an overlap of the excitation source spectra and the detectable fluorescence emission spectra.
  • filter element 480 can be used to prevent undesirable wavelengths, other than the desired excitation wavelength, that may also be emitted by excitation source 410 from irradiating detection region 440.
  • Filter element 482 can be used to filter unwanted background noise (light) and fluorescence emissions from certain solid support materials in the detection region, e.g., microchannel capillary tubes and the like.
  • An electronic filter can also be used to filter out background noise and unwanted fluorescence emissions from the fluorescence signal received by fluorescence detector 420.
  • the signal from detector 420 is electronically filtered so that only the emitting frequency (e.g., 10MHz) is detected. The resulting signal can then be compared to the modulation reference signal to determine the fluorescence lifetimes or to determine which is the dominating fluorescence lifetime.
  • DNA or "deoxyribonucleic acid” shall be construed as collectively including DNA containing classical nucleotides, DNA containing one or more modified nucleotides (i.e., fluorescently tagged nucleotides containing a chemically modified base, sugar and or phosphate), DNA containing one or more nucleotide analogs, and combinations of the above).
  • modified nucleotides i.e., fluorescently tagged nucleotides containing a chemically modified base, sugar and or phosphate
  • nucleotide shall be construed as collectively including all of the forms of nucleotides described supra in addition to RNA and derivatives of RNA analogous to those of DNA discussed above.
  • polymer refers to molecules having two or more subunits (e.g., dinucleotides).
  • nucleic acid is used interchangeably with RNA and DNA and this term can refer to monomeric, oligomeric or polymeric species of these molecules.
  • the methods and devices of the invention can also be utilized to sequence polymeric and oligomeric molecules including, but not limited to, DNA, RNA, peptides, polysaccharides and the like.
  • sequence polymeric and oligomeric molecules including, but not limited to, DNA, RNA, peptides, polysaccharides and the like.
  • the discussion that follows focuses on techniques for sequencing nucleic acids.
  • the methods and apparatus of the invention can be utilized to sequence other polymeric molecules such as peptides, proteins, polysaccharides and the like.
  • the present invention provides a method of sequencing a nucleic acid polymer of interest.
  • the method comprises performing a sequencing reaction on the nucleic acid polymer to produce a nested set of sequence fragments. Any of the sequencing reactions known in the art is appropriate for use in this aspect.
  • methods which chemically or enzymatically degrade or synthesize nucleic acids are of use in practicing the present invention. See, for example Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 74: 560 (1977).
  • synthesize nucleic acid polymers an embodiment, involves producing a plurality of nucleic acid polymers complementary to a region of the nucleic acid polymer of interest. See, for example, Sanger et al, Proc. Natl. Acad. Sci. USA 74: 5463 (1977).
  • one or more fluorescent labels is inco ⁇ orated into either the nucleic acid being sequenced or a sequence complementary to the nucleic acid being sequenced.
  • Sequencing reaction mixtures that are useful in practicing the present invention include those that contain the nucleic acid to be sequenced and a fluorescent label.
  • the fluorescent label is attached to a first labeled nucleic acid selected from the group consisting of labeled nucleic acids, labeled nucleic acid polymers and combinations thereof.
  • the fluorescent label will, following excitation, emit electromagnetic energy that is characterized by a distinct and detectable lifetime. W en more than one fluorescent label is utilized in the sequencing reaction mixture, each of the labels will have distinct and detectable fluorescence lifetimes.
  • the sequencing reaction mixture further comprises a second labeled nucleic acid which is a member selected from the group consisting of labeled nucleic monomers and labeled nucleic acid polymers, wherein said nucleic acid bears a second fluorescent label.
  • the second fluorescent label has a fluorescence emission that has a characteristic fluorescence lifetime.
  • the second labeled nucleic acid can be a polymeric species such as an oligonucleotide (e.g., a primer or a dimer, trimer, etc.).
  • an oligonucleotide e.g., a primer or a dimer, trimer, etc.
  • the component bases of the polymer can be identical or they can be different over the length of the strand.
  • Useful monomers include, for example, nucleotides, deoxynucleotides, dideoxynucleotides and modified derivatives thereof.
  • the second labeled nucleic acid can be an oligonucleotide primer that is used to start nucleic acid synthesis at a second region of the nucleic acid being sequenced.
  • the second nucleic acid can also be a dideoxynucleotide such that chain elongation is terminated upon the dideoxynucleotide 's inco ⁇ oration into a growing nucleic acid.
  • the sequencing reaction mixture further comprises additional labeled nucleic acids.
  • the labels on these additional labeled nucleic acids will also have a fluorescence emission that has a characteristic fluorescence lifetime. It will be clear to one of skill in the art that any number of labeled nucleic acids can be used in a sequencing reaction mixture.
  • the first labeled nucleic acid bearing a first fluorescent label is a member of a plurality of unique labeled nucleic acid species. Similar to the above-described embodiments, the fluorescent label has a fluorescent emission that has a characteristic fluorescence lifetime.
  • the set of labeled nucleic acids can include a mixture of nucleic acid species.
  • a sequencing reaction mixture can include a primer or a primer and one or more labeled dideoxynucleotides.
  • another exemplary sequencing reaction mixture can include one or more labeled dideoxynucleotides and one or more deoxynucleotides with or without a primer present in the mixture.
  • Other useful sequencing reaction mixture compositions will be apparent and readily accessible to those of skill in the art.
  • the method of the invention can be carried out by combining all of the labeled species in a "one pot” reaction or, alternatively, one or more of the labeled species can be segregated into one or more reaction vessels.
  • the sequencing reaction is carried out with all of the fluorescently labeled species together as a mixture in a "one pot" reaction.
  • the sequencing reaction is performed following the Sanger procedure. See, for example, Sanger et al, Proc. Natl. Acad. Sci. USA 74: 5463 (1977).
  • each of the labeled nucleic acids bearing a different fluorescent tag is inco ⁇ orated into a polymeric nucleic acid.
  • This embodiment can utilize a "one pot" reaction or, alternatively, one or more labeled species can be segregated and reacted in a separate reaction vessel.
  • the labeled nucleic acids can be labeled primers, labeled deoxynucleotides, labeled dideoxynucleotides or combinations thereof.
  • the method further comprises a second sequencing reaction mixture comprising the nucleic acid polymer of interest and a second labeled nucleic acid bearing a second fluorescent label, wherein the second fluorescent label has a fluorescence emission, the emission having a characteristic fluorescence lifetime.
  • the method of the invention further comprises a third sequencing reaction mixture. Similar to the other sequencing reaction mixtures, the third sequencing mixture comprises the nucleic acid polymer of interest. The third sequencing reaction mixture also comprises a third labeled nucleic acid which is labeled with a third fluorescent label, wherein the third fluorescent label has a fluorescence emission which has a characteristic fluorescence lifetime.
  • the method of the invention further comprises a fourth sequencing reaction mixture that, similar to the mixtures discussed above, comprises a fourth labeled nucleic acid.
  • the present invention utilizes as many sequencing reaction mixtures as there are unique nucleic acid bases.
  • a particular sequencing reaction mixture is a member of a plurality of unique sequencing reaction mixtures.
  • Each reaction mixture comprises the nucleic acid polymer of interest and a unique labeled nucleic acid bearing a unique fluorescent label, wherein the unique fluorescent label has a fluorescence emission.
  • the emission has a characteristic fluorescence lifetime. The fluorescence lifetime is different for each unique fluorescent label.
  • nucleic acids e.g., labeled nucleic acids, labeled nucleic acid analogs, fluorescent labeled nucleic acids, oligonucleotides, etc.
  • solvents e.g., buffers, catalysts, acids, bases, surfactants, chelating agents, metal ions and the like.
  • the sequencing reaction mixture further comprises one or more members selected from the group consisting of polymerases, exonucleases, endonucleases, deoxynucleotides, deoxynucleotide diphosphates, deoxynucleotide triphosphates, dideoxynucleotides, dideoxynucleotide diphosphates, dideoxynucleotide triphosphates, nucleotide analogs and nucleoside analogs and combinations thereof.
  • the nucleic acids labeled with the fluorescent labels and that the nucleic acids are members selected from the group consisting of nucleotides, nucleosides, nucleoside diphosphates, nucleoside triphosphates, dideoxynucleosides, deoxynucleotides, deoxynucleoside diphosphates, deoxynucleoside triphosphates, dideoxynucleosides, dideoxynucleoside diphosphates, dideoxynucleoside triphosphates, nucleotide analogs and nucleoside analogs and combinations thereof.
  • nucleotide bearing a fluorescent label is a non-natural nucleotide.
  • the invention provides a sequencing reaction mixture as described above. In a still further preferred embodiment, the invention provides a kit comprising one or more sequencing mixtures as described above.
  • nucleic acid sequencing techniques can be used in conjunction with the present invention.
  • suitable sequencing techniques include those that use a chemical or enzymatic degradation process and those that use enzymatic synthesis of nucleic acids.
  • a DNA polymerase is an enzyme that has the ability to catalytically synthesize new strands of DNA in vitro.
  • the DNA polymerase carries out this synthesis by moving along a preexisting single DNA strand ("the template") and creating a new strand complementary to the existing strand by inco ⁇ orating single nucleotides one at a time into the new strand following the base-pairing rule.
  • exonuclease activity refers to the ability of an enzyme (an exonuclease) to cleave off a nucleotide at the end of a DNA strand.
  • Enzymes are known which can cleave successive nucleotides off a single DNA strand, working from the 5' end of the strand to the 3' end; such enzymes are termed single- stranded 5' to 3' exonucleases.
  • Other enzymes are known which perform this operation in the opposite direction (single-stranded 3' to 5' exonucleases).
  • Thermostable polymerases are also useful in performing the polymerase chain reaction in conjunction with the sequencing method of the invention. See, for example, U.S. Patent No. 4,683,202; Arnheim and Levinson, C&EN 36-47 (October 1, 1990), Kwoh et al, Proc. Nat l Acad. Sci. USA 86:1173 (1989).
  • the method of the invention further comprises the use of the polymerase chain reaction to amplify the DNA being sequenced.
  • Techniques for sequencing DNA generate fragments of labeled DNA, the lengths of which are sequence dependent, and separate the fragments according to their lengths, for example, by electric field induced migration in a gel or capillary. Such a pattern of sequence-dependent fragment lengths is known as a sequencing ladder.
  • the sequencing mixture will generally be submitted to a separation protocol that separates different populations of oligonucleotides on the basis of their size, charge, hydrophobicity and combinations of these properties.
  • the method of the invention further comprises separating the complementary nucleic acid polymers into distinct populations, each of the populations consisting of nucleic acid polymers of about the same size.
  • the separating is provided by a method selected from the group consisting of electrophoresis, electroosmosis, electrokinesis, chromatography and combinations thereof.
  • the fragments of a sequencing ladder can be generated by either: (a) cleaving the DNA in a base-specific manner, or (b) synthesizing a copy of the DNA wherein the synthesized strand terminates in a base-specific manner.
  • the Maxam-Gilbert technique for sequencing involves the specific chemical cleavage of DNA.
  • four samples of the same DNA are each subjected to a different chemical reaction to effect preferential cleavage of the DNA molecule at one or two nucleotides of a specific base identity.
  • DNA fragments are thus generated in each sample whose lengths are dependent upon the position within the DNA base sequence.
  • each sample contains DNA fragments of different lengths each of which ends in the same one or two of the four nucleotides. See, Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 74:560 (1977)
  • the plus/minus DNA sequencing method involves: (a) use of polymerases to generate complementary labeled DNA oligonucleotides of different lengths; (b) (the "minus” system) in four separate reaction vessels, reaction of one half of the generated DNA with DNA polymerase and three out of the four nucleotide precursors; (c) (the "plus system”) in four separate reaction vessels, reaction of the remaining half of the generated DNA with DNA polymerase and only one of each of the four nucleotide precursors. Each reaction mixture generated in steps (b) and (c) is subjected to a separation procedure and the generated fragments are separated from each other by migration. See, Sanger and Coulson, J. Mol. Biol. 94:441-448 (1975).
  • the dideoxy method relies on the enzymatic activity of a DNA polymerase to synthesize DNA fragments with lengths that are sequence dependent. See, Sanger et al, Proc. Natl. Acad. Sci. USA 74:5463 (1977).
  • the Sanger dideoxy method utilizes an enzymatically active fragment of the DNA polymerase termed E. coli DNA polymerase I, to carry out the enzymatic synthesis of new DNA strands.
  • the newly synthesized DNA strands include fragments of sequence-dependent length, generated through the use of inhibitors of DNA polymerase which cause the base-specific termination of synthesis.
  • Such inhibitors are dideoxynucleotides that, upon their inco ⁇ oration by the DNA polymerase, destroy the ability of the enzyme to further elongate the DNA chain due to the dideoxynucleotides' lack of a suitable 3'-OH necessary in the elongation reaction.
  • a dideoxynucleotide whose base can appropriately hydrogen bond with the template DNA is thus inco ⁇ orated into the DNA, synthesis of the growing polymer chain stops.
  • DNA fragments are generated by the DNA polymerase, the lengths of which are dependent upon the position within the DNA base sequence of the nucleotide whose base identity is the same as the inco ⁇ orated dideoxynucleotide. The fragments are then submitted to a separation procedure.
  • the boronated nucleotide is stocastically inco ⁇ orated into PCR products at varying positions along the PCR amplicon in a nested set of PCR fragments of the template.
  • An exonuclease which is blocked by inco ⁇ orated boronated nucleotides is used to cleave the PCR amplicons.
  • the cleaved amplicons are then separated by size using polyacrylamide gel electrophoresis, providing the sequence of the amplicon.
  • Sequencing methods which reduce the number of steps necessary for template preparation and primer selection have been developed and can be applied to the present invention.
  • One proposed variation on sequencing technology involves the use of modular primers for use in PCR and DNA sequencing.
  • Ulanovsky and co- workers have described the mechanism of the modular primer effect (Beskin et al. ,
  • Nucleic acids to serve as sequencing templates are optionally derived from a natural source or they can be synthetic or recombinant.
  • DNAs can be recombinant DNAs, e.g., plasmids, viruses or the like.
  • a wide variety of molecular and biochemical methods are available for making coding DNAs. Examples of appropriate molecular techniques for generating recombinant nucleic acids, and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimrnel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); as well as in Sambrook, and Ausubel (both supra).
  • oligonucleotides are used as sequencing primers, or as amplification primers. Most commonly, these DNA or RNA oligonucleotides are made synthetically. Synthetic oligonucleotides are typically synthesized chemically according to common solid phase phosphoramidite triester methods described, e.g., by Beaucage & Caruthers (1981) Tetrahedron Letts. 22(20):1859-1862, e.g., using an automated synthesizer, as described in Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168. Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to persons of skill. In other embodiments, oligonucleotides are made recombinantly according to standard techniques, described, e.g., in Berger, Sambrook and Ausubel, all supra.
  • Oligonucleotides are typically selected to have particular hybridization characteristics with a template DNA to form a duplex with the DNA.
  • the oligonucleotide is typically used as a primer for a processive DNA polymerase in either a sequencing or amplification reaction.
  • oligonucleotides are selected to be fully complementary to the selected template DNA, although a portion of the oligonucleotide can be non-complementary (e.g., a portion may act as a labeling or cloning element instead of participating in hybridization, or a single oligonucleotide can be used as a primer for multiple closely related templates in separate assays to reduce individual assay costs).
  • the oligonucleotides are preferably selected to have melting temperatures near the temperature of the reaction, to reduce background hybridization interactions. It is expected that one of skill is thoroughly familiar with the theory and practice of nucleic acid hybridization and selection of complementary oligonucleotides. See, e.g., Gait (ed.), OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, LRL Press, Oxford (1984); Kuijpers, Nucleic Acids Research 18(17):5197 (1994); Dueholm (1994) J. Org. Chem.
  • Fluorescent tags useful in practicing the present invention can be tethered to any location on a nucleic acid, including sites on the base segment and sites on the sugar segment.
  • the fluorescent label is covalently attached to a segment of a nucleic acid which is a member selected from the group consisting of the base segment, the sugar segment and both the base segment and the sugar segment.
  • the modified nucleic acid bears at least one fluorescent label and it serves as a primer for nucleic acid synthesis and the method of the invention further comprises annealing the nucleic acid polymer of interest with a primer nucleic acid polymer.
  • the fluorescent label is covalently attached to a labeled nucleic acid which is a member selected from the group consisting of the 3'-terminus, the 5'-terminus, an internal position and combinations thereof.
  • Chemical methods are available to introduce fluorescence into specific nucleic acid bases by the reaction of chloracetaldehyde with adenosine and cytidine to give fluorescent products.
  • the reaction can be controlled with respect to which of the two bases is derivatized by manipulating the pH of the reaction mixture; the reaction at 37 °C proceeds rapidly at the optimum pH of 4.5 for adenosine and 3.5 for cytidine. See, Barrio et al, Biochem. Biophys. Res. Commun. 46:597-604 (1972). This reaction is also useful for rendering fluorescent the deoxyribosyl derivatives of these bases. See, Kochetkov et al, Dokl Akad. Nauk. SSSR C 213:1327-1330 (1973).
  • DNA and RNA can be modified by reacting their cytidine residues with sodium bisulfite to form sulfonate intermediates that are then coupled to reactive nitrogen compounds such as hydrazides or amines. See, Viscidi et al. J. Clin. Microbiol. 23:311 (1986) and Draper and Gold, Biochemistry 19:1774 (1980).
  • RNA can also be labeled at the 3 ' terminus by selective oxidation.
  • the selective oxidation of the 3' ribose of RNA by periodate yields a dialdehyde which can then be coupled with an amine or hydrazide reagent.
  • Fluorescent G derivatives have also been prepared from the natural base upon its reaction with variously substituted malondialdehydes. See, Leonard and Tolman, in “Chemistry, Biology and Clinical Uses of Nucleoside Analogs," A. Bloch, ed., Ann. N. Y. Acad. Sci. 255:43-58 (1975).
  • At least three methods are available for fluorescently tagging a synthetic oligonucleotide. These methods utilize fluorescently tagged supports, fluorescently tagged 5' modification reagents and fluorescently tagged monomers. The first of these methods utilizes a fluorescently tagged linker that tethers the oligonucleotide strand to the solid support. When the oligonucleotide strand is cleaved from the solid support, the fluorescent tether remains attached to the oligonucleotide. This method affords an oligonucleotide that is fluorescently labeled at its 3 '-end.
  • the 3 '-end of the nucleic acid is labeled with a linker that bears an amine, or other reactive or masked reactive group, which can be coupled to a reactive fluorophore following cleavage of the oligonucleotide from the solid support.
  • a linker that bears an amine, or other reactive or masked reactive group, which can be coupled to a reactive fluorophore following cleavage of the oligonucleotide from the solid support.
  • This method is particularly useful when the fluorophore is not stable to the cleavage or deprotection conditions.
  • An exemplary derivatized solid support is shown below in Formula I:
  • n is an integer between 1 and 10 and X is a fluorophore or a reactive group such as, for example, NH 2 , SH, OH, COOH, or a protected derivative of a reactive group.
  • a fluorophore or a reactive group such as, for example, NH 2 , SH, OH, COOH, or a protected derivative of a reactive group.
  • Methods for protecting these and other reactive groups are known in the art. See, for example, Greene and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 2 nd Ed., John Wiley & Sons, N.Y., 1991.
  • a second method relies on the selective labeling of the 5' terminus of the oligonucleotide chain.
  • n is an integer between 1 and 10 and X is a fluorophore or a reactive group such as, for example, NH , SH, OH, COOH, or a protected derivative of a reactive group.
  • nucleotides can be derivatized with fluorescent moieties on the base or sugar components. Modification to the base can occur at exocyclic amines or at the carbons of the ring. See, for example, Levina et al, Bioconjug. Chem. 4:319-325 (1993). Modification of the sugar moiety can take place at the oxygens of the hydroxyl groups or the carbon atoms of the ribose ring. See, for example, Augustyns et al, Nucleic
  • the modified labeled nucleic acids can also be 2 '-deoxyribonucleic acids which are labeled at the 3 '-hydroxyl via, for example, alkylation or acylation. These labeled nucleic acids will function like dideoxynucleic acids, terminating synthesis, when used in the Sanger method.
  • fluorescent tags useful in practicing the present invention can be tethered to any location on a nucleic acid, including sites on the base segment and sites on the sugar segment.
  • the fluorescent label is covalently attached to a segment which is a member selected from the group consisting of the base segment, the sugar segment and both the base segment and the sugar segment.
  • nucleic acid lengths are within the art established ranges, preferably a size of from about 2 bases to about 100,000 bases, more preferably from about 100 bases to about 10,000 and still more preferably from about 300 bases to about 5000 bases.
  • the following exemplary embodiment illustrates a method of sequencing a nucleic acid using fluorescent labels and a chemical degradation pathway.
  • Fully protected oligodeoxyribonucleotides can be prepared on an Applied Biosystems DNA synthesizer using standard ⁇ -cyanoethyl phosphoramidite chemistry. See, Sinha et al, Nucleic Acids Res. 12:4539-4557 (1984). A portion of the material can be retained for a further synthetic cycle employing (S-trityl-3-mercaptopropyloxy), 2- cyanoethoxy N, N-diisopropylaminophosphine in the condensation step. This phosphoramidite has been synthesized and is known in the art. See, Ansorge et al, Nucleic Acids Res. 16:2203-6 (1988).
  • the S-trityl oligonucleotide can be purified by reverse-phase HPLC.
  • Detritylation with silver nitrate and subsequent reaction of the liberated thiol with 5- iodoacetamidofluorescein can be performed as described in Ansorge et al, Nucleic Acids Res. 15:4593-4602 (1987).
  • the excess dye can be removed by ethanol precipitations of the oligodeoxyribonucleotide.
  • the fluorescein labeled oligodeoxyribonucleotide can be purified by reverse-phase HPLC, prior to sequencing by chemical degradation.
  • oligonucleotides Chemical degradation of oligonucleotides can be performed essentially as described in Rosenthal et al, Methods Enzymol. 155:301-331 (1987) using Hybond M & G paper (Amersham). Approximately 5 pmol of fluorescein labeled oligomer can be applied to the carrier in 1 ⁇ l aliquots.
  • the following reagents can be used: G: with 1% DMS in 50 mM ammonium formate buffer, pH 3.5 for 10 min.;
  • the samples can be dissolved in 30%) aqueous formamide.
  • a lanthanide chelate serves as the fluorescent label.
  • the chelate is diethylenetriaminepentaacetic acid (DTPA) and it is tethered to the nucleic acid using the corresponding DTPA dianhydride (DTPAA).
  • DTPA diethylenetriaminepentaacetic acid
  • DTPAA DTPA dianhydride
  • the method and device of the invention is used simply to separate and identify, not sequence, different oligonucleotides.
  • a plasmid such as plasmid pBR322 is purified and digested according to art-recognized procedures. See, Mamatis et al. MOLECULAR CLONING: A LABORATORY MANUAL, Cold Springs Harbor Laboratory, Cold Springs Harbor, N.Y., pp. 100-106.
  • the digestion of pBR322 generates 10 fragments with staggered ends ranging from 75 base pairs to 1631 base pairs; the sequence of single-stranded bases at each end is ANT, where N denotes any nucleotide. It is assumed that the exocyclic amines on the exposed bases provide sites for attachment of the DTPA moiety via amide linkages formed between these amines and a carboxylate group of the DTPA.
  • the DTPAA is added to the plasmid digest and stirred at room temperature for at least 60 minutes. After storage overnight at 4 °C a lanthanide salt (e.g., terbium chloride) is added to the reaction mixture. The resulting mixture is shaken and allowed to stand for at least 30 minutes. Excess hydrolyzed chelate and lanthanide salt can be separated from the plasmid digest-chelate conjugate by passing the mixture through a column packed with Sephadex, such as Sephadex G 25-150. Suitable elution buffers include, for example, 10 mM 3-[N-mo ⁇ holino]propane sulfonic acid at pH 7. The DNA fractions can then be pooled and evaporated to dryness.
  • a lanthanide salt e.g., terbium chloride
  • the DNA fractions can then be loaded into a microfluidic device and sequenced.
  • the plasmid digest-chelate conjugate can be characterized by determining the DNA concentration by measuring the absorbance at 260 nm.
  • Label concentration can be determined by comparing the fluorescence of the purified labeled nucleotide conjugate with the fluorescence of the free chelate complexed with terbium. Suitable instrumentation for these measurements includes a Perkin-Elmer Lambda Array UV-Vis spectrometer and a Perkin-Elmer LS-5 spectrofluorimeter.
  • microscale systems and/or components which can be adapted to the present invention by inco ⁇ orating assay components and appropriate additional elements, e.g., related to signal detection as noted herein, are available.
  • Microfluidic devices which can be adapted to the present invention by the addition of signal detection elements or addition of fluorescent components as set forth herein, are described in various PCT applications and issued U.S. Patents by the inventors and their coworkers, including U.S. Patent Nos. 5,699,157 (J. Wallace Parce) issued 12/16/97; 5,779,868 (J. Wallace Parce et al.) issued 07/14/98; 5,800,690 (Calvin Y.H.
  • MICROSCALE FLUIDIC DEVICES WO 98/00231 provide pioneering technology for the integration of micro fluidics and sample selection and manipulation.
  • the microfluidic systems of the invention provide an integration of several elements, including a microfluidic device with interior microfluidic channels and reservoirs, optics for viewing labeled components, computer systems and software for recording and analyzing components and the like.
  • WO 98/00231 entitled “HIGH THROUGHPUT SCREENING ASSAY SYSTEMS IN MICROSCALE FLUIDIC DEVICES" by Parce et al. provides pioneering technology related to microscale fluidic devices, including electrokinetic devices.
  • the devices are generally suitable for assays relating to the interaction of biological and chemical species, including enzymes and substrates, ligands and ligand binders, receptors and ligands, antibodies and antibody ligands, as well as many other assays.
  • microscale devices provide the ability to mix fluidic reagents and assay mixing results in a single continuous process, and because minute amounts of reagents can be assayed, these microscale devices represent a fundamental advance for laboratory science.
  • Pioneering integrated systems for nucleic acid sequencing and other iterative fluid manipulation processes utilizing microfluidic fluid manipulation are described in, e.g., in "CLOSED LOOP BIOCHEMICAL ANALYZERS" by Knapp et al, WO98/45481.
  • a template nucleic acid is selected and introduced into a reaction channel in a microfluidic device of the invention.
  • the template is optionally amplified, e.g., by introducing PCR or LCR reagents into the channel and performing cycles of heating and cooling on the template.
  • Thermocycling in microscale devices is described in USSN 60/083,532, attorney docket number 100/01320 entitled "ELECTRICAL CURRENT FOR CONTROLLING FLUID TEMPERATURES IN MICROCHANNELS" filed April 29, 1998 by Calvin Chow, Anne R. Kopf-Sill and J. Wallace Parce and in related application 08/977,528, filed November 25, 1997.
  • thermocycling in microfluidic systems
  • WO 98/17910 examples of non-thermocyclic polymerase mediated reactions
  • WO 98/45481 examples of non-thermocyclic polymerase mediated reactions
  • the present invention optionally uses power sources that pass electrical current through the fluid in a channel for heating pu ⁇ oses, as well as for material transport (in alternate embodiments, heating is produced by application of external heating or cooling sources, or the like).
  • the fluid passes through a channel of a desired cross-section (e.g., diameter) to enhance thermal transfer of energy from the current to the fluid.
  • the channels can be formed on almost any type of substrate material such as, for example, amo ⁇ hous materials (e.g., glass, plastic, silicon), composites, multi-layered materials, combinations thereof, and the like.
  • substrate material such as, for example, amo ⁇ hous materials (e.g., glass, plastic, silicon), composites, multi-layered materials, combinations thereof, and the like.
  • the source of template is from an abundant sequence such as a cloned nucleic acid
  • further amplification can be unnecessary.
  • PCR-based sequencing methods available, such as a PCR nuclease chain termination procedure that can also be used for direct sequencing in the methods of the invention, by inco ⁇ orating fluorescent nucleotides which are distinguishable by their decay time into templates for sequencing.
  • Porter et al. (1997) Nucleic Acids Research 25(8):1611-1617 describe the biochemistry of exemplar PCR chain termination methods.
  • Sequencing reagents are added to the template nucleic acid, e.g., by electrokinetic or pressure-based flow of reagents into contact with the template nucleic acid, e.g., in a reaction channel, and a sequencing reaction is performed appropriate to the particular reaction in use. Many appropriate reactions are known, with the Sanger dideoxy chain termination method being the most common.
  • the primer used to prime synthesis is optionally selected from a pre-synthesized set of nucleic acid primers, preferably a set including many or all of the primers for a particular primer length.
  • modular primers are used. See, Beskin et al.
  • these primers can inco ⁇ orate fluorescent labels distinguishable by their decay times, or nucleotides inco ⁇ orated by primer extension can inco ⁇ orate such labels.
  • products are separated by size and/or charge in an analysis region of the microfluidic device.
  • Devices of the invention can be used to electrophoretically separate macromolecules by size and/or charge.
  • the separated products are detected as they pass a fluorescent detector (nucleic acids and other molecules are typically labeled with fluorophores that are distinguishable by their decay times in the present invention; accordingly, appropriate detectors include spectrophotometers, fluorescent detectors, microscopes (e.g., for fluorescent microscopy), etc.
  • the detection systems are adapted to measure fluorescence decay times.
  • a computer is optionally used to select a second primer from the pre-synthesized primer set which hybridizes to the sequenced region, and the process is iteratively repeated with the second primer, leading to sequencing of a second region, selection of a third primer hybridizing to the second region, etc.
  • a variety of commercially available hardware and software is available for digitizing, storing, and analyzing a signal or image such as that generated by the microfluidic device described herein.
  • a computer commonly used to transform signals from the detection device into reaction rates will be a PCTM-compatible computer (e.g., having a central processing unit (CPU) compatible with x86 CPUs, and running an operating system such as DOSTM, OS/2 Wa ⁇ TM, WINDOWS/NTTM, or WINDOWS 95TM), a MacintoshTM (running MacOSTM), or a UNIX workstation (e.g., a SUNTM workstation running a version of the SolarisTM operating system, or PowerPCTM workstation) are all commercially common, and known to one of skill in the art.
  • Data analysis software on the computer is then employed to determine the rate of formation and or mobility of any component which is labeled with a fluorescent label distinguishable by its decay time.
  • WO98/56956 "APPARATUS AND METHODS FOR CORRECTING FOR VARIABLE VELOCITY IN MICROFLUIDIC SYSTEMS" provides a variety of fluorogenic and non-fluorogenic assay formats for microfluidic systems which can be adapted to the present invention by the inco ⁇ oration of decay-time distinguishable labels. See also, A. R. Kopf-Sill, T. Nikiforov, L. Bousse, R. Nagel, & J. W. Parce, "Complexity and performance of on-chip biochemical assays," in Proceedings of Micro- and Nano fabricated Electro-Optical Mechanical Systems for Biomedical and Environmental Applications, SPLE, Vol. 2978, San Jose, California, February 1997, p. 172-179).
  • ligands and ligand binders such as an antibody and an antibody ligand, receptors and receptor ligands, biotin and avidin, proteins and complementary binding proteins, carbohydrates and carbohydrate binding moieties, nucleic acids, etc.
  • Reactants or, e.g., molecules which hybridize are contacted by flowing the components together in a microfluidic system. At least one of the components is typically labeled with a label distinguishable by its decay time. Products and reactants are detected and quantitated by observing, e.g., the movement of labels in the system.
  • Data correction for the effects of velocity of components can be applied, e.g., by considering conservation of flux in the flowing systems, by generating and applying data masking files, by using self-co ⁇ ecting fluid sampling techniques and the like. See, Kopf- Sill et al, supra. FLUID MOVEMENT IN MICROSCALE SYSTEMS
  • Fluid flow (and flow of materials suspended or solubilized within the fluid, including sequencing reagents, enzymes, enzyme substrates, catalysts, cells or other particles, etc.) is optionally regulated by pressure based mechanisms such as those based upon fluid displacement, e.g., using a piston, pressure diaphragm, vacuum pump, probe or the like to displace liquid and raise or lower the pressure at a site in the microfluidic system.
  • the pressure is optionally pneumatic, e.g., a pressurized gas, or uses hydraulic forces, e.g., pressurized liquid, or alternatively, uses a positive or negative displacement mechanism, i.e., a plunger fitted into a material reservoir, for forcing material through a channel or other conduit, or is a combination of such forces.
  • pneumatic e.g., a pressurized gas
  • hydraulic forces e.g., pressurized liquid
  • a positive or negative displacement mechanism i.e., a plunger fitted into a material reservoir, for forcing material through a channel or other conduit, or is a combination of such forces.
  • a vacuum source is applied to a reservoir or well at one end of a channel to draw the relevant materials through the channel (e.g., fluidic compositions comprising enzymes, buffers, substrates, reaction modulators, sequencing reagents or the like).
  • Pressure or vacuum sources are optionally supplied external to the device or system, e.g., external vacuum or pressure pumps sealably fitted to the inlet or outlet of the channel, or they are internal to the device, e.g., microfabricated pumps integrated into the device and operably linked to the channel. Examples of microfabricated pumps have been widely described in the art. See, e.g., published International Application No. WO 97/02357.
  • Hydrostatic, wicking and capillary forces can also be used to provide pressure for fluid flow of materials such as cells, biological molecules, particles and chemicals. See, e.g., "METHOD AND APPARATUS FOR CONTINUOUS LIQUID FLOW IN MICROSCALE CHANNELS USING PRESSURE INJECTION, WICKING AND ELECTROKINETIC INJECTION," by Alajoki et al. USSN 09/245,627, filed February 5, 1999.
  • an adsorbent material or branched capillary structure is placed in fluidic contact with a region where pressure is applied, thereby causing fluid to move towards the adsorbent material or branched capillary structure.
  • microfluidic systems can be inco ⁇ orated into centrifuge rotor devices, which are spun in a centrifuge. Fluids and particles travel through the device due to gravitational and centripetal centrifugal pressure forces.
  • Electrokinetic material transport systems include systems that transport and direct materials within a microchannel and/or chamber containing structure, through the application of electrical fields to the materials, thereby causing material movement through and among the channel and/or chambers, i.e., cations will move toward a negative electrode, while anions will move toward a positive electrode.
  • movement of fluids toward or away from a cathode or anode can cause movement of sequencing reagents or products or other biological or relevant molecules suspended within the fluid.
  • the fluid can be immobile or flowing and can comprise a matrix as in electrophoresis.
  • electrokinetic material transport and direction systems also include those systems that rely upon the electrophoretic mobility of charged species within the electric field applied to the structure. Such systems are more particularly referred to as electrophoretic material transport systems.
  • electrophoretic material transport systems For electrophoretic applications, the walls of interior channels of the electrokinetic transport system are optionally charged or uncharged. Typical electrokinetic transport systems are made of glass, charged polymers, and uncharged polymers. The interior channels are optionally coated with a material which alters the surface charge of the channel.
  • Modulating voltages are concomitantly applied to the various reservoirs to affect a desired fluid flow characteristic, e.g., continuous or discontinuous (e.g., a regularly pulsed field causing the sample to oscillate a direction of travel) flow of labeled components toward a waste reservoir.
  • a desired fluid flow characteristic e.g., continuous or discontinuous (e.g., a regularly pulsed field causing the sample to oscillate a direction of travel) flow of labeled components toward a waste reservoir.
  • modulation of the voltages applied at the various reservoirs can move and direct fluid flow through the interconnected channel structure of the device.
  • any of a number of methods and devices are suitable for use in the present invention for separating the components of a mixture; however, typically, the methods of the invention are practiced in the context of a microfluidic system.
  • Fluorescently labeled components (typically multiple components comprising one or more labels distinguishable by their decay time) are transported through a microfluidic channel.
  • Material transport and direction in the microfluidic channel is typically accomplished through electrokinesis, e.g. , electroosmosis or electrophoresis or by pressure-based fluid movement, or a combination thereof.
  • a preferred microfluidic apparatus has a body structure with at least two intersecting channels fabricated inside the body structure, as taught in the numerous references regarding microfluidic technology noted above.
  • the channels preferably have at least one cross-sectional dimension that is in the range of from about
  • the sequencing reaction is performed using the microfluidic device.
  • the microfluidic device also comprises a detecting station and the device is used for both separating and detecting the components of a mixture.
  • the microfluidic device is also used for identifying the compounds of the mixture by the differences in their fluorescence lifetimes.
  • the invention provides a microfluidic device with at least one microchannel, a detector for detecting fluorescence species in the channel and a digital computer which is operatively linked to the detector. The digital computer is used to determine the lifetimes of the fluorescent species.
  • a variety of controlling instrumentation is optionally utilized in conjunction with the microfluidic devices described above, for controlling the transport and direction of fluids and/or materials within the devices of the present invention, e.g., by pressure-based and/or electrokinetic control.
  • fluid transport and direction are controlled in whole or in part, using pressure based flow systems that inco ⁇ orate external or internal pressure sources to drive fluid flow.
  • Internal sources include microfabricated pumps, e.g., diaphragm pumps, thermal pumps, lamb wave pumps and the like that have been described in the art. See, e.g., U.S. Patent Nos. 5,271,724, 5,277,556, and 5,375,979 and Published PCT Application Nos. WO 94/05414 and WO 97/02357.
  • the systems described herein can also utilize electrokinetic material direction and transport systems.
  • differential flow rates on volumes are optionally accomplished by applying different pressures or vacuums at multiple ports, or preferably, by applying a single vacuum at a common waste port and configuring the various channels with appropriate resistance to yield desired flow rates.
  • Example systems are described in USSN 09/238,467, filed 1/28/99.
  • electrokinetic transport to control material movement in interconnected channel structures is an alternate preferred method of achieving material transport (and controllers can include both pressure-based and electrokinetic control elements).
  • This method of material control is particularly useful in electrophonetic separation of labeled components.
  • electrokinetic material transport is set forth, e.g., in WO 96/04547 and US 5,858,195 to Ramsey, as well as in a variety of other references noted herein.
  • An exemplary electrokinetic controller is described in U.S. 5,800,690.
  • modulating voltages are concomitantly applied to the various reservoirs to affect a desired fluid or material flow characteristic, e.g., continuous or discontinuous (e.g., a regularly pulsed field causing the sample to oscillate direction of travel) flow of labeled components toward a waste reservoir.
  • a desired fluid or material flow characteristic e.g., continuous or discontinuous (e.g., a regularly pulsed field causing the sample to oscillate direction of travel) flow of labeled components toward a waste reservoir.
  • modulation of the voltages applied at the various reservoirs can move and direct fluid flow through the interconnected channel structure of the device.
  • controller systems are appropriately configured to receive or interface with a microfluidic device or system element as described herein.
  • the controller and/or detector optionally includes a stage upon which the device of the invention is mounted to facilitate appropriate interfacing between the controller and or detector and the device.
  • the stage typically includes an appropriate mounting/alignment structural element, such as a nesting well, alignment pins and/or holes, asymmetric edge structures (to facilitate proper device alignment), and the like. Many such configurations are described in the references cited herein.
  • the controlling instrumentation discussed above is also used to provide for pressure-based or electrokinetic injection or withdrawal of material downstream of the region of interest to control an upstream flow rate.
  • the same instrumentation and techniques described above are also utilized to inject a fluid into a downstream port to function as a flow control element.
  • the present invention provides for the use of any of the apparatus elements described herein, e.g., for practicing any of the methods or assays set forth herein.
  • the invention provides for the use of a fluorescent molecule in any of the assays set forth herein, and for the use of the apparatus herein to detect such molecules.
  • Kits are provided which inco ⁇ orate, e.g., any of the apparatus elements herein, e.g., in conjunction with instructions for practicing the methods herein, packaging, reagents, fluorescent molecules such as control molecules, containers for holding apparatus or reagent elements, or the like.

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Abstract

La présente invention porte sur des procédés, des appareils et des systèmes permettant de différencier divers fluorophores sur la base des durées de vies des fluorescences associées grâce à une modulation du rayonnement d'excitation des fluorophores d'une région étudiée. Le principe est d'utiliser un détecteur de fluorescence produisant un signal qui est fonction des émissions de fluorescence détectées et qu'un processeur analyse de façon à mesurer le temps de décroissance de fluorescence. Si la source d'excitation produit des impulsions, ou se fait moduler de façon à en émettre, le processeur est capable de mesurer directement le temps de décroissance de fluorescence. Par contre, si source d'excitation émet un signal d'excitation à variation sinusoïdale, ou se fait moduler de façon à en émettre un, pour calculer le temps de décroissance de fluorescence, le processeur utilise une mesure du déphasage ou de la démodulation par rapport à un signal de référence de la modulation d'excitation. L'invention concerne également un procédé d'identification de composants d'un mélange par marquage de chacun des composants au moyen d'agents fluorescents se différenciant par les temps de décroissance de fluorescence. Ce procédé consiste à filtrer les composants, à isoler les marqueurs fluorescents et à en mesurer les temps de décroissance de fluorescence. Ces mesures permettent d'identifier les composants de mélanges réunissant des petites molécules organiques, des polymères, des peptides, des saccharides et des acides nucléiques.
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US09/213,297 US6447724B1 (en) 1998-08-11 1998-12-15 DNA sequencing using multiple fluorescent labels being distinguishable by their decay times
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