WO2011011738A2

WO2011011738A2 - Illumination in biotech instruments

Info

Publication number: WO2011011738A2
Application number: PCT/US2010/043131
Authority: WO
Inventors: Liana Ilkova; Casey Mcfarland
Original assignee: Life Technologies Corporation
Priority date: 2009-07-24
Filing date: 2010-07-23
Publication date: 2011-01-27
Also published as: WO2011011738A3

Abstract

A system may include at least one reaction region including a biological sample; a temperature control mechanism configured to control the temperature of the at least one reaction region; an electrodeless lamp configured to illuminate the at least one reaction region; and a detector configured to detect at least one signal from the at least one reaction region. A method of detecting at least one target in a biological sample may comprise controlling the temperature of a biological sample comprising at least one target; exciting at least one marker to produce an emission signal in relation to the target present in the biological sample, the exciting including illuminating the biological sample with light from an electrodeless lamp; and detecting the emission signal.

Description

Illumination in Biotech Instruments

RELATED APPLICATION

[0001 ] The present application claims priority under 35 U. S. C §119(e) to provisional application No. 61/228,436, filed on July 24, 2009, the entire contents of which is hereby incorporated by reference in its entirety for all purposes as if fully set forth herein.

BACKGROUND OF THE INVENTION

[0002] Biotech instruments, such as RT-PCR instruments and

sequencers, often involve exciting samples with light and detecting emitted signals, e.g., fluorescence.

[0003] Light sources for fluorescence excitation in biotech instruments have tended to involve high brightness sources with short life or low brightness sources with long life. For example, LEDs offer long life but limited brightness. Traditional arc lamps have high brightness but limited lifetime. Incandescent lamps are generally not as bright as arc lamps and not as long living as LEDs.

SUMMARY OF THE INVENTION

[0004] In one or more aspects, embodiments of the present invention are directed to a system, comprising at least one reaction region comprising a biological sample; a temperature control mechanism configured to control the temperature of the at least one reaction region; an electrodeless lamp configured to illuminate the at least one reaction region; and a detector configured to detect at least one signal from the at least one reaction region.

[0005] In one or more aspects, embodiments of the present invention are directed to a method of detecting at least one target in a biological sample, comprising: controlling the temperature of a biological sample comprising at least one target; exciting at least one marker to produce an emission signal in relation to the target present in the biological sample, the exciting comprising illuminating the biological sample with light from an electrodeless lamp; and detecting the emission signal.

[0006] In one or more aspects, embodiments of the present invention are directed to a method of determining a sequence of at least one nucleic acid, comprising exciting at least one marker to produce an emission signal in relation to the at least one nucleic acid, the exciting comprising illuminating the at least one nucleic acid with light from an electrodeless lamp; detecting the emission signal; and determining a sequence of the at least one nucleic acid.

[0007] Additional embodiments are set forth in part in the description that follows, and in part will be apparent from the description, or may be learned by practice of the various embodiments described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Embodiments of the present invention are further described in the description of invention that follows, in reference to the noted plurality of non-limiting drawings, wherein:

[0009] Fig. 1 is a diagram showing a system according to an embodiment of the present invention.

[0010] Fig. 2 shows halogen lamp well uniformity in a system of the prior art.

[0011 ] Fig. 3 shows electrodeless lamp well uniformity in a system according to an embodiment of the present invention.

[0012] Fig. 4 shows dual reporter real-time PCR performance in a system according to an embodiment of the present invention.

[0013] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide a further explanation of the various embodiments of the present teachings.

DESCRIPTION OF THE INVENTION

[0014] Reference will now be made to various exemplary embodiments, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.

[0015] Unless otherwise stated, a reference to a compound or component includes the compound or component by itself, as well as in combination with other compounds or components, such as mixtures of compounds.

[0016] Before further discussion, a definition of the following terms will aid in the understanding of the present invention.

[0017] The term "biological sample" as used herein refers to any biological or chemical substance, typically in an aqueous solution with

luminescent dye that can produce emission light in relation to nucleic acid present in the solution. The biological sample can include one or more nucleic acid sequences to be incorporated as a reactant in polymerase chain reaction (PCR) and other reactions such as ligase chain reaction, antibody binding reaction, oligonucleotide ligations assay, hybridization assay and isothermal amplification. The biological sample can include one or more nucleic acid sequences to be identified for DNA sequencing.

[0018] The term "nucleic acid" as used herein refers to DNA, RNA, PNA, variations thereof, and other oligonucleotides or their analogs.

[0019] The term "protein" as used herein refers to oligopeptides, peptides, and proteins.

[0020] The term "luminescent dye" as used herein refers to fluorescent or phosphorescent dyes that can be excited by electromagnetic radiation (e.g., visible light or ultraviolet radiation) or the like. Luminescent dyes can be used to provide different colors depending on the dyes used. Several dyes will be apparent to one skilled in the art of dye chemistry. One or more colors can be collected for each dye to provide identification of the dye or dyes detected. The dye can be a dye-labeled fragment of nucleotides. The dye can be a marker triggered by a fragment of nucleotides. The dye can provide identification of nucleic acid sequence in the biological sample by association, for example, bonding to or reacting with a detectable marker, for example, a respective dye and quencher pair. The respective identifiable component can be positively identified by the luminescence of the dye. The dye can be normally quenched, then can become unquenched in the presence of a particular nucleic acid sequence in the biological sample or be quenched and become unquenched. The fluorescent dyes can be selected to exhibit respective and, for example, different, excitation and emission wavelength ranges. The luminescent dye can be measured to quantitate the amount of nucleic acid sequences in the biological sample. The luminescent dye can be detected in real-time to provide information about the identifiable nucleic acid sequences throughout the reaction. Examples of fluorescent dyes with desirable excitation and emission wavelengths can include 5-FAM™, TET™, and VIC™. The term

"luminescence" as used herein refers to low-temperature emission of light including fluorescence, phosphorescence, electroluminescence, and the like.

[0021 ] As an overview, in various embodiments, the present invention relates to a system comprising at least one reaction region configured to contain, hold, or support a biological sample; a temperature control mechanism configured to control the temperature of the at least one reaction region and/or a biological sample; an electrodeless lamp configured to illuminate the at least one reaction region; and a detector configured to detect at least one signal from the at least one reaction region.

[0022] Also as an overview, in various embodiments, the present invention is directed to a method of detecting at least one target in a biological sample, comprising: controlling the temperature of a biological sample comprising at least one target; exciting at least one marker to produce an emission signal in relation to the target present in the biological sample, the exciting comprising illuminating the biological sample with light from an electrodeless lamp; and detecting the emission signal.

[0023] Also as an overview, in various embodiments, the present invention is directed to a method of determining a sequence of at least one nucleic acid, comprising exciting at least one marker to produce an emission signal in relation to the at least one nucleic acid, the exciting comprising illuminating the at least one nucleic acid with light from an electrodeless lamp; detecting the emission signal; and determining a sequence of the at least one nucleic acid.

[0024] The systems may include at least one reaction region comprising a biological sample. The biological sample may comprise nucleic acids and/or proteins.

[0025] The at least one reaction region may contain components for promoting a nucleic acid sequence amplification reaction, such as a polymerase chain reaction, and/or for promoting a DNA sequencing reaction. For instance, the at least one reaction region may comprise a nucleic acid sequence amplification dye.

[0026] The at least one reaction region may comprise a plurality of reaction regions.

[0027] The at least one reaction region may take various forms known forms. For instance, the at least one reaction region may be within at least one container. The at least one reaction region may be within at least one sample well.

[0028] Thus, the reaction region may be contained within a sample chamber. Sample chambers may comprise any structure that provides containment to a sample. The sample chamber can be open or transparent to provide entry to excitation light and egress to fluorescent light. The

transparency can be provided by glass, plastic, fused silica, etc. The sample chamber can take any shape including a well, a tube, a vial, a cuvette, a tray, a multi-well tray, a microcard, a microslide, a capillary, an etched channel plate, a molded channel plate, an embossed channel plate, etc. The sample chamber can be part of a combination of multiple sample chambers grouped into a row, an array, an assembly, etc. Multi-chamber arrays can include 12, 24, 36, 48, 96, 192, 384, 1536, 3072, 6144, or more sample chambers. The sample chamber can be shaped to a multi-well tray under the SBS microtiter format.

[0029] Other examples of the at least one reaction region include, but are not limited to, an oil encapsulated slug, an emulsion, and at least one region defined by a hydrophobic coating.

[0030] The systems typically include a temperature control mechanism configured to control the temperature of the at least one reaction region. For instance, the temperature control mechanism may comprise at least one thermal cycler. The temperature control mechanism may comprise at least one thermoelectric temperature control device, such as Peltier devices and resistive heaters.

[0031 ] The systems typically include an electrodeless lamp configured to illuminate the at least one reaction region. In contrast with other electrical lamps that use electrical connections through the lamp envelope to transfer power to the lamp, the power needed to generate light in electrodeless lamps is transferred from the outside of the lamp envelope by means of electromagnetic fields.

[0032] In various embodiments, the electrodeless lamp comprises a sealed glass tube containing a spot of metal, e.g., pure metal, on the inner surface of the tube and a minute amount of a rare gas, such as neon or argon. This is subjected to a high frequency field to produce ionization of the residual gas in the tube and hence produce a discharge at the characteristic wavelength of the pure metal in the tube. The tube may be made from quartz.

[0033] In various embodiments, the tube of the electrodeless lamp is positioned at or near a point in the cavity where the electric field, e.g., created by microwave energy or radio frequency, is at a maximum. In certain embodiments, the support structure for the tube is of a size and composition that does not interfere with the electromagnetic field, e.g., resonating microwaves, as interference reduces the efficiency of the lamp.

[0034] In various electrodeless lamps, the tube contains a noble gas combined with a light emitter, i.e., a second element or compound which may comprise sulfur, selenium, a compound containing sulfur or selenium, or any one of a number of metal halides. Exposing the contents of the tube to an electromagnetic field, e.g., microwave energy, of high intensity causes the noble gas to become a plasma. The free electrons within the plasma excite the light emitter within the tube. When the light emitter returns to a lower electron state, radiation is emitted.

[0035] The spectrum of light emitted depends upon the characteristics of the light emitter within the tube. The light emitter may be chosen to cause emission of visible light and/or ultraviolet bands of the electromagnetic spectrum. In various embodiments, the electrodeless lamp generates light at a wavelength corresponding to at least one marker, such as a fluorescent dye. Thus, a large percentage of the light is at a useful wavelength. For instance, at least 70%, such as at least 75%, at least 80%, or at least 85%, of the

illumination energy from the electrodeless lamp consists of light having a wavelength ranging from 450 nm to 750 nm. Alternatively, at least 75%, such as at least 80%, at least 85%, or at least 90%, of the illumination energy from the electrodeless lamp consists of light having a wavelength ranging from 400 nm to 800 nm.

[0036] In various embodiments, the electrodeless lamp has a long life. For instance, electrodeless lamps may have an operational lifetime of at least 1000 hours, such as at least 5000 hours or at least 10,000 hours.

[0037] In various embodiments, the electrodeless lamp has low spectral variability. For instance, the electrodeless lamp may have a spectral variability of ± 20 %, such as ± 15% or ± 10 %, for any given range of 100 nm within the wavelength range of 450 nm to 750 nm. As an example, the spectral variability may be ± 20 %, such as ± 15% or ± 10 %, for the wavelength range of 500 nm to 600 nm.

[0038] In various embodiments, the electrodeless lamp has a numerical aperture ranging from 0.4 to 0.7, such as 0.5 to 0.6.

[0039] In various embodiments, the electrodeless lamp has a correlated color temperature (CCT) ranging from 4000 K to 6000 K, such as 4500 K to 5500 K.

[0040] Electrodeless lamps may provide beam shaping. In this regard, the shaping of electrodeless lamps tends to be more efficient than halogen lamps and less efficient than arc lamps. As a result, the present systems may include or omit parabolic reflectors.

[0041 ] Examples of electrodeless lamps include, but are not limited to a dielectric waveguide integrated plasma lamps, radio frequency plasma lamps, laser plasma lamps, and microwave plasma lamps.

[0042] Dielectric waveguide integrated plasma lamps are disclosed in U.S. Patent No. 6,737,809, which is incorporated herein by reference in its entirety.

[0043] In radio frequency plasma lamps, an electrodeless tube is filled with gas and irradiated with radio waves, causing the contents of the bulb to form a plasma and glow.

[0044] In various embodiments, the electrodeless lamp comprises a coil surrounding a krypton lamp driven by a radio frequency input source tapped across an end part of the coil and with a coil surrounding a mercury lamp tapped across the connection of the input central to the krypton-lamp coil and a point of the krypton-lamp coil electrically away from both input connections. Each coil is connected in parallel with separate capacitors which form resonant circuits at the input frequency.

[0045] In various embodiments, the electrodeless lamp comprises a radio frequency excited gas arc lamp for producing sufficient light to pump a laser comprising a transparent envelope containing an inert gas, namely krypton, xenon or argon, with a coil around or adjacent the envelope and a source of RF voltage to apply sufficient voltage to the coil to maintain a plasma in the gas. For pumping a laser rod, the lamp may be mounted adjacent to the laser rod and generally is cooled, as by mounting the lamp envelope and laser rod in a cooled chamber. A reflector may be arranged to direct back toward the rod that light from the lamp which is not initially absorbed in the rod, and a filter may be mounted to filter out at least a portion of any unwanted components of light from the lamp (e.g., the ultraviolet component) before it impinges on the laser rod. The lamp envelope may be an annular chamber with a laser rod mounted through the annulus.

[0046] Microwave plasma lamps are disclosed in U.S. Patent Nos.

6,922,021 and 7,525,253, which are incorporated herein by reference in their entireties. U.S. Patent Nos. 4,954,755; 4,975,625; 4,978,891 ; 5,021 ,704;

5,448,135; 5,594,303; 5,841 ,242; 5,910,710; and 6,031 ,333, each of which is incorporated herein by reference in their entireties, disclose electrodeless lamps in which the plasma lamps direct microwave energy into an air cavity, with the air cavity enclosing a bulb containing a mixture of substances that can ignite, form a plasma, and emit light.

[0047] Electrodeless lamp systems may further include a control device configured to activate or control the electrodeless lamp.

[0048] The system typically includes at least one detector configured to detect at least one signal from the at least one reaction region. Examples of detectors include, but are not limited to, at least one of a camera, a charge- coupled device (CCD), back-side-thinned, cooled CCD, front-side illuminated CCD, a CCD array, a photodiode, a photodiode array, a photo-multiplier tube (PMT), a PMT array, complimentary metal-oxide semiconductor (CMOS) sensors, CMOS arrays, a charge-injection device (CID), CID arrays, etc.

[0049] The detector can be adapted to relay information to a data collection device for storage, correlation, and/or manipulation of data, for example, a computer, or other signal processing system. In various embodiments, the detector can be a pixel filter array imaging detector as described in U.S. Patent No. 6,756,618, which is incorporated herein by reference in its entirety. In various embodiments, the pixel filter array can include a dielectric thin film coating providing sharper cut-offs and higher transmission than would be realized with filters made from dyed photoresists. In various embodiments, a color-imaging detector can be multi-color pixel imaging detector as described in U.S. Patent No. 5,965,875 and U.S. Published

Application No. 2006/0252070, which are incorporated herein by reference in their entireties.

[0050] The system may include further optics. In various embodiments, the system includes a mirror arranged between both the electrodeless lamp and the at least one reaction region, and the detector and the at least one reaction region.

[0051 ] As noted above, most of the light from the electrodeless lamp may be within a useful range. Thus, in various embodiments, the system does not include a hot mirror. In other various embodiments, the system includes a hot mirror disposed between the electrodeless lamp and the at least one reaction region.

[0052] In various embodiments, the system comprises a dichroic mirror disposed between the electrodeless lamp and the at least one reaction region.

[0053] In various embodiments, the system includes an excitation wavelength-excluding device disposed between the electrodeless lamp and the at least one reaction region. Examples of wavelength-excluding devices include, but are not limited to, longpass filters, multiple notch filters, and bandpass filters.

[0054] In various embodiments, an optical filter prevents excitation light from reaching the detector. The detector can be custom fabricated to provide three layers whose thickness can be optimized to balance the signal-to-noise ratio and condition number for three dyes such as FAM, VIC, and ROX. In various embodiments, lack of a filter can be combined with filters, for example, red, blue emission, all excitation light, red excitation light, blue, all red light, etc. In such an embodiment, not all the pixels have filters. This may be a better quantification for one color.

[0055] In various embodiments, the system includes a light pipe disposed between the electrodeless lamp and the at least one reaction region.

[0056] In various embodiments, the system includes a holographic beam shaper disposed between the electrodeless lamp and the at least one reaction region.

[0057] In various embodiments, the system includes a diffuser disposed between the electrodeless lamp and the at least one reaction region.

[0058] An electrodeless lamp may be incorporated into the system disclosed in U.S. Patent No. 6,518,068, which is incorporated herein by reference in its entirety. Thus, various embodiments involve a luminescence detecting apparatus and method for analyzing luminescent samples.

Luminescent samples are placed in a plurality of sample wells in a tray, and the tray is placed in a visible-light impervious chamber containing a charge coupled device camera. The samples may be injected in the wells, and the samples may be injected with buffers and reagents, by an injector. In the chamber, light from the luminescent samples may pass through a collimator, a Fresnel field lens, a filter, and a camera lens, whereupon a focused image may be created by the optics on a charge-coupled device (CCD) camera. The use of a Fresnel field lens, in combination with a collimator and filter, may reduce crosstalk between samples. Various embodiments of the luminescence detecting apparatus and method may include central processing control of all operations, multiple wavelength filter wheel, and robot handling of samples and reagents. Various embodiments may include processing software for mechanical alignment, outlier shaving, masking, manipulation of multiple integration times to expand the dynamic range, crosstalk correction, dark subtraction interpolation and drift correction, multi-component analysis applications specifically tailored for luminescence, and/or uniformity correction. [0059] An electrodeless lamp may be incorporated into the system disclosed in U.S. Patent No. 7,460,223, which is incorporated herein by reference in its entirety. In various embodiments, an electrodeless lamp may be used with an inverted microplate for conducting a thermocycled amplification reaction of polynucleotide. The microplate can comprise a main body having a first and second surfaces and a plurality of wells disposed in the first surface. Each of the plurality of wells can comprise a well opening and a well bottom and be sized to receive an assay. A sealing cover can be operably coupled to the first surface of the main body to seal the well openings of the plurality of wells when the main body is inverted so that the assay is in contact with the sealing covering cover.

[0060] In an exemplary embodiment shown in Fig. 1 , a detection system 100 can include reaction regions 102 adapted to receive a biological sample 104 containing one or more luminescent-light emitting dyes, for example, fluorescent dyes. The system 100 includes an electrodeless lamp 106 and a detector 112 for detecting luminescent light emitted from the biological sample 104. In the illustrated embodiment, system 100 further includes a large field lens 108 and well lenses 1 10.

[0061 ] The electrodeless lamp 106 can be configured to provide beams of light 1 16. As shown, the light beams have converging paths. In some

embodiments, the fluorescent detection system 100 can include one or more additional light sources (not shown). Each of the additional light sources can provide a beam of light whose optical spectrum differs from one another and/or from that of beams of light 1 16. For example, each additional beam of light can have a substantially different wavelength from one another and/or from that of the beams of light 116.

[0062] In various embodiments, the system 100 can include a hot mirror 120. The mirror 120 can be positioned in the path of the light beams 1 16. In various embodiments, the hot mirror 120 may be omitted. [0063] The system 100 may include an excitation wavelength-excluding device 122. Examples of wavelength-excluding devices include, but are not limited to, longpass filters, multiple notch filter, bandpass filters, and

combinations thereof.

[0064] The system 100 can include one or more optical blenders such as, for example, a beam splitter 130. Beam splitters, including dichroic, geometric, and 50-50, may be configured to combine beams of light by substantially reflecting a first light beam and substantially transmitting a second light beam. The reflective/transmissive properties of beam splitters can be based on wavelength differentiation or physical properties such as angle of incidence. Wavelength differentiation can be achieved by depositing thin-film interference coatings on either or both of the beam splitter surfaces. The beam splitter 130 can be positioned in the path of the light beams 1 16. The beam splitter 130 can be configured to output beams 132 toward the biological samples 104.

[0065] Thus, light leaving the wavelength-excluding device 122 is directed by the beam splitter 130 toward a luminescent dye that produces emission light in relation to nucleic acid present in the biological sample 104. The large field lens 108 and well lenses 1 10 can be between the beam splitter 130 and the biological sample 104.

[0066] In various embodiments, the detection assembly 1 12 can be configured to detect one or more particular wavelengths and, therefore, can detect luminescent light emitted by the biological sample having one of the detectable wavelengths. In various embodiments, the detection assembly 1 12 can include one or more detectors capable of detecting different emission light from different luminescent dyes in the biological sample 104. In embodiments having more than one detector, each detector can be set to receive a respective range of wavelengths of fluorescent light different than those of the other detector(s). In various embodiments, the detector assembly can include a plurality of detectors arranged as an array. In various embodiments, an array of transmission grating beam splitters can be provided to diffract the fluorescent light into a first contribution detectable by a first detector and a second contribution detectable by a second detector.

[0067] In various embodiments, the presence of various luminescent dyes in the biological sample 104 utilizing respective excitation wavelength ranges can be used to identify various nucleic acids in the sample 104. In various embodiments, the luminescent dyes can be fluorescent dyes chosen such that each dye possesses a discrete or substantially discrete optimum excitation wavelength range. In some embodiments, the emission wavelength ranges of various dyes can overlap. Each excitation light source can emit a respective wavelength range of light to cause fluorescence of a different fluorescent dye. The detection assembly 1 12 can be configured to detect the emission wavelengths of the various fluorescent dyes in the biological sample 104.

[0068] In various embodiments, the plurality of excitation light sources can provide excitation light selected respectively from the red, green, blue, violet, and/or ultra-violet spectra.

[0069] In various embodiments, the large field lens 108 and well lenses 1 10 can be configured to focus excitation light into the biological sample and/or collect and shape emission light emitted from the sample. The lenses can include any known lens, for example, a Fresnel lens.

[0070] The system 100 can include other devices known in the art arranged to direct, separate, filter, or focus the excitation light and/or emission light. For example, the system 100 can include a prism, a grating, a diffractive optical element, or a mask. In various embodiments, the system 100 can include a prism in combination with a lens (not shown).

[0071 ] In various embodiments, the sample chamber 102 can be one well of a multi-well tray including a plurality of wells. The field lens 108, well lenses 1 10, and detector 1 12 can form a unit that can be operatively aligned to direct excitation light toward the sample chamber 102 and/or direct emission light to the detector 1 12 from the sample chamber 102. In various embodiments, a control device (not shown) can be provided to activate the unit.

[0072] In various embodiments, the system 100 can include a light pipe (not shown) to facilitate the direction of excitation light to a desired sample chamber 102. In various embodiments, cross-talk between the excitation light and fluorescent emission light from each sample chamber can be substantially reduced by using a mask (not shown), as is known by practitioners in the art. The mask can include a mask, masking elements, or a masking layer. In various embodiments, each sample chamber 102 can include a cover (not shown) on the well capable of operating as a collimating lens.

[0073] In various embodiments, the system 100 may include an emission filter 140. The emission filter 140 may be disposed between the samples 104 and the detector 1 12. The emission filter filters emission light 142 from the biological sample 104.

[0074] In various embodiments, the system 100 may include an emission lens 150. The emission lens 150 may be disposed between the samples 104 and the detector 1 12.

[0075] As noted above, embodiments of the present invention also include methods of detecting at least one target in a biological sample. The method includes controlling the temperature of a biological sample comprising at least one target. The method further includes exciting at least one marker to produce an emission signal in relation to the target present in the biological sample, the exciting comprising illuminating the biological sample with light from an electrodeless lamp. Still further, the method includes detecting the emission signal.

[0076] It should be noted that the emission signal may increase or decrease with the amount of target present. In other words, the emission signal may be directly or inversely related to the amount of target.

[0077] Controlling the temperature may comprise heating the biological sample. For instance, the method may comprise thermally cycling the biological sample for amplification of at least one nucleic acid. As another example, the method may comprise real-time polymerase chain reaction.

[0078] To amplify DNA (Deoxyribose Nucliec Acid) using the PCR process, a specially constituted liquid reaction mixture may be cycled through several different temperature incubation periods. The reaction mixture may be comprised of various components including the DNA to be amplified and at least two primers sufficiently complementary to the sample DNA to be able to create extension products of the DNA being amplified. Thermal cycling involves alternating steps of melting DNA, annealing short primers to the resulting single strands, and extending those primers to make new copies of double-stranded DNA. In thermal cycling the PCR reaction mixture is repeatedly cycled from high temperatures of around 90 °C for melting the DNA, to lower temperatures of approximately 40 °C to 70 °C for primer annealing and extension.

[0079] In various embodiments, the electrodeless lamp is illuminated for a relatively short time, which facilitates increased throughput sensing. For example, the electrodeless lamp may be illuminated for less than 100 ms, such as less than 75 ms, less than 50 ms, less than 25 ms, less than 10 ms. For instance, the illumination time may range from 5 ms to 100 ms, such as 6 ms to 75 ms, 7 ms to 50 ms, or 8 ms to 25 ms.

[0080] The exciting the at least one marker may comprise simultaneous excitation of a plurality of reaction regions.

[0081 ] The at least one marker may comprise at least one luminescent dye. For instance, the at least one marker may comprise at least one

fluorescent dye.

[0082] In various embodiments, the method may further comprise separating nucleic acids in the biological sample by electrophoresis.

[0083] In various embodiments, the method may further comprise determining a sequence of the at least one nucleic acid.

[0084] For instance, an electrodeless lamp may be incorporated into the devices and methods disclosed in U.S. Published Application No. 2009/0062129, which is incorporated herein by reference in its entirety. In various embodiments, methods for determining a nucleic acid sequence involve performing successive cycles of duplex extension along a single stranded template. The cycles typically comprise steps of extension, ligation, and cleavage. In certain embodiments, the methods make use of extension probes containing phosphorothiolate linkages and agents capable of cleaving such linkages. Electrodeless lamps may be used in methods of determining information about a sequence using at least two distinguishably labeled probe families as well as methods of performing multiple sequencing reactions on a single template. Electrodeless lamps may be combined with automated sequencing systems, flow cells, image processing methods, and computer- readable media that store computer-executable instructions and/or sequence information that can be used in accordance with such methods.

[0085] As another example, an electrodeless lamp may be incorporated into the devices and methods disclosed in U.S. Published Application No.

2008/0003571 , which is incorporated herein by reference in its entirety. In various embodiments, an electrodeless lamp may be used in methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles may comprise steps of extension, ligation, and, generally, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. In certain

embodiments the methods make use of extension probes containing an abasic residue or a damaged base and employ agents appropriate to cleave linkages between a nucleoside and an abasic residue and/or agents appropriate to remove a damaged base from a nucleic acid. In various embodiments, an electrodeless lamp is used in methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to beads, which are immobilized in or on a semi-solid support.

[0086] Although the above description has focused on PCR and sequencing of nucleic acids, it should be noted that the devices, systems, and methods according to the present invention are also applicable to other uses. In various embodiments, the at least one target comprises at least one protein. For instance, the method may comprise determining a concentration and/or conformation of the at least one protein. Still other uses include assays involving ligation, polymerization, and ELISA.

[0087] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about."

Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by

embodiments of the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0088] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing

measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range of "less than 10" includes any and all subranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all subranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 5.

[0089] It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly and unequivocally limited to one referent. Thus, for example, reference to "an electrodeless lamp" includes two or more electrodeless lamps. As used herein, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[0090] Embodiments of the present invention will be further illustrated by way of the following Examples. These examples are non-limiting and do not restrict the scope of the invention. Unless stated otherwise, all percentages, parts, etc. presented in the examples are by weight.

Example

[0091 ] A LIFI 4000 lamp (available from Luxim Corporation, Sunnyvale, CA) was mounted onto a 7500 Fast Real-Time PCR system (available from Life Technologies Corporation, Carlsbad, CA), replacing the tungsten halogen lamp. The lamp was powered by an external power supply rather than running on the instrument power.

[0092] The following calibrations were performed using the default calibration modules in the SDS 1.4 software using material from the 7500 Fast Spectral Calibration Kit:

Region of Interest (ROI) calibration

Background calibration

Optical calibration - using the ROI plate

FAM pure spectra

VIC pure spectra

ROX pure spectra [0093] After performing calibrations, a solid state fluorescence target (Ultem resin, available from SABIC Innovative Plastics, Pittsfield, MA) was read on the system. The collection protocol was 10 cycles at 60 °C. The exposure times of the system were altered from the factory default to account for the increase in signal from the tungsten halogen lamp. In particular, while the exposure time with the halogen lamp was 50 ms, the exposure time with the Lifi 4000 electrodeless lamp was 10 ms. The resulting data was analyzed for image uniformity and overall signal intensity. Fig. 2 shows image uniformity and overall signal intensity for the halogen lamp, while Fig. 3 shows the same data for the electrodeless lamp. In comparison to the tungsten halogen lamp, the

electrodeless lamp system showed the potential of a 500% increase in signal without significantly sacrificing uniformity.

[0094] A TaqMan chemistry experiment was also performed to prove feasibility of the light source in the real-time PCR instrument. The reaction was prepared using the following materials:

TaqMan Fast Universal PCR Master Mix

(available from Life Technologies, Carlsbad, CA)

FAM-labeled TG Fβ TaqMan assay

(available from Life Technologies, Carlsbad, CA)

VIC-labeled 18s TaqMan assay

(available from Life Technologies, Carlsbad, CA)

Raji Human cDNA

[0095] A plate was prepared with all 96-wells containing the same reaction at 10 μL per well. The experimental protocol used the default fast thermal cycling conditions of the 7500 Fast system. The results were analyzed for Ct standard deviation as a way of measuring the system uniformity

performance. The results (see Fig. 4) show that electrodeless lamps may be used in real-time PCR systems in place of tungsten halogen lamps. In particular, with two outliers removed, the results were as follows:

[0096] The foregoing embodiments and advantages are merely

exemplary and are not to be construed as limiting the present invention. The description of the present invention is intended to be illustrative, and not to limit the scope of the claims. Many alternatives, modifications, and variations will be apparent to those skilled in the art.

[0097] Having now fully described this invention, it will be understood to those of ordinary skill in the art that the methods of the present invention can be carried out with a wide and equivalent range of conditions, formulations, and other parameters without departing from the scope of the invention or any embodiments thereof.

[0098] All patents and publications cited herein are hereby fully incorporated by reference in their entirety. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that such publication is prior art or that the present invention is not entitled to antedate such publication by virtue of prior invention.

Claims

WHAT IS CLAIMED IS:

1. A system, comprising:

at least one reaction region comprising a biological sample;

a temperature control mechanism configured to control the temperature of the at least one reaction region;

an electrodeless lamp configured to illuminate the at least one reaction region; and

a detector configured to detect at least one signal from the at least one reaction region.

2. The system of claim 1 , wherein the electrodeless lamp has an operational lifetime of at least 1000 hours.

3. The system of claim 1 , wherein the electrodeless lamp has an operational lifetime of at least 5000 hours.

4. The system of claim 1 , wherein at least 80% of the illumination energy from the electrodeless lamp consists of light having a wavelength ranging from 450 nm to 750 nm.

5. The system of claim 1 , wherein the electrodeless lamp is configured to have a spectral variability of ± 20 % in a wavelength range of 500 nm to 600 nm.

6. The system of claim 1 , wherein the electrodeless lamp comprises a dielectric waveguide integrated plasma lamp.

7. The system of claim 1 , wherein the electrodeless lamp comprises a radio frequency plasma lamp.

8. The system of claim 1 , wherein the electrodeless lamp comprises a laser plasma lamp.

9. The system of claim 1 , wherein the electrodeless lamp comprises a microwave plasma lamp.

10. The system of claim 1 , wherein the system does not include a parabolic reflector.

1 1. The system of claim 1 , wherein the temperature control mechanism comprises a thermal cycler.

12. The system of claim 1 , wherein the temperature control mechanism comprises a thermoelectric temperature control device.

13. The system of claim 1 , wherein the temperature control mechanism comprises a Peltier device.

14. The system of claim 1 , wherein the temperature control mechanism comprises a resistive heater.

15. The system of claim 1 , wherein the at least one reaction region contains components for promoting a nucleic acid sequence amplification reaction.

16. The system of claim 15, wherein the nucleic acid sequence amplification reaction is a polymerase chain reaction and the at least one reaction region comprises a nucleic acid sequence amplification dye.

17. The system of claim 1 , wherein the at least one reaction region comprises a plurality of reaction regions.

18. The system of claim 1 , wherein the at least one reaction region is within at least one container.

19. The system of claim 1 , wherein the at least one reaction region is within at least one sample well.

20. The system of claim 1 , wherein the at least one reaction region comprises at least one of an oil encapsulated slug, an emulsion, and at least one region defined by a hydrophobic coating.

21. The system of claim 1 , wherein the detector comprises at least one of a camera, a charge-coupled detector, a photodiode, a photomultiplier, and a complimentary metal-oxide semiconductor.

22. The system of claim 1 , further comprising a control device configured to activate the electrodeless lamp.

23. The system of claim 1 , further comprising a mirror arranged between both the electrodeless lamp and the at least one reaction region, and the detector and the at least one reaction region.

24. The system of claim 1 , wherein the system does not include a hot mirror.

25. The system of claim 1 , further comprising a dichroic mirror disposed between the electrodeless lamp and the at least one reaction region.

26. The system of claim 1 , further comprising an excitation wavelength-excluding device disposed between the electrodeless lamp and the at least one reaction region.

27. The system of claim 26, wherein the excitation wavelength- excluding device comprises at least one of a longpass filter, a multiple notch filter, and a bandpass filter.

28. The system of claim 1 , further comprising a light pipe disposed between the electrodeless lamp and the at least one reaction region.

29. The system of claim 1 , further comprising a holographic beam shaper disposed between the electrodeless lamp and the at least one reaction region.

30. The system of claim 1 , further comprising a diffuser disposed between the electrodeless lamp and the at least one reaction region.

31. A method of detecting at least one target in a biological sample, comprising:

controlling the temperature of a biological sample comprising at least one target;

exciting at least one marker to produce an emission signal in relation to the target present in the biological sample, the exciting comprising illuminating the biological sample with light from an electrodeless lamp; and

detecting the emission signal.

32. The method of claim 31 , wherein the electrodeless lamp comprises a dielectric waveguide integrated plasma lamp.

33. The method of claim 31 , wherein the electrodeless lamp comprises a radio frequency plasma lamp.

34. The method of claim 31 , wherein the electrodeless lamp comprises a laser plasma lamp.

35. The method of claim 31 , wherein the electrodeless lamp comprises a microwave plasma lamp.

36. The method of claim 31 , wherein the controlling the temperature comprises heating the biological sample.

37. The method of claim 31 , wherein the electrodeless lamp is illuminated for less than 100 ms.

38. The method of claim 31 , wherein the exciting comprises simultaneous excitation of a plurality of reaction regions.

39. The method of claim 31 , wherein the at least one marker comprises at least one luminescent dye.

40. The method of claim 31 , wherein the at least one marker comprises at least one fluorescent dye.

41. The method of claim 31 , wherein the at least one target comprises at least one nucleic acid.

42. The method of claim 41 , further comprising thermally cycling the biological sample for amplification of the at least one nucleic acid.

43. The method of claim 41 , further comprising separating the nucleic acids in the biological sample by electrophoresis.

44. The method of claim 41 , further comprising determining a sequence of the at least one nucleic acid.

45. The method of claim 41 , wherein the method comprises real-time polymerase chain reaction.

46. The method of claim 31 , wherein the at least one target comprises at least one protein.

47. The method of claim 46, further comprising determining a concentration of the at least one protein.

48. The system of claim 1 , wherein the biological sample is supported by the at least one reaction region.

49. The system of claim 1 , wherein the at least one reaction region contains components for promoting determination of a sequence of at least one nucleic acid.

50. A method of determining a sequence of at least one nucleic acid, comprising:

exciting at least one marker to produce an emission signal in relation to the at least one nucleic acid, the exciting comprising illuminating the at least one nucleic acid with light from an electrodeless lamp;

detecting the emission signal; and

determining a sequence of the at least one nucleic acid.

51. The method of claim 50, wherein the electrodeless lamp comprises a dielectric waveguide integrated plasma lamp.

52. The method of claim 50, wherein the electrodeless lamp comprises a radio frequency plasma lamp.

53. The method of claim 50, wherein the electrodeless lamp comprises a laser plasma lamp.

54. The method of claim 50, wherein the electrodeless lamp comprises a microwave plasma lamp.

55. The method of claim 50, wherein the electrodeless lamp is illuminated for less than 100 ms.

56. The method of claim 50, wherein the exciting comprises simultaneous excitation of a plurality of reaction regions.

57. The method of claim 50, wherein the at least one marker comprises at least one luminescent dye.

58. The method of claim 50, wherein the at least one marker comprises at least one fluorescent dye.