Patent Application
20050208539
This invention pertains to amplification and/or detection of target nucleic acid molecules. More particularly, this invention provides systems, devices and methods suitable for the amplification and detection of a small number of copies, or even a single copy, of a target nucleic acid molecule.
There is a need for methods and devices for detecting and amplifying extremely small amounts of nucleic acid target material while minimizing interference by other, non-target, nucleic acid material.
The present invention provides methods and devices for quantitative counting of a small number of target nucleic acid molecules. For example, the small number of target nucleic acid molecules may be hundreds of copies, or tens of copies, less than about ten copies, or less than about 5 copies, and may be only one copy. In some embodiments, methods herein comprise real-time PCR methods such as Taqman® methods and assays to detect the presence and amplification of target molecules.
Devices having pathways, substrates, barriers, or other elements configured to reduce the dispersion of amplified polynucleotides from a site of amplification may be used to provide small regions containing polynucleotides copied from a single or a small number of target polynucleotide(s). Such small regions of copies of target polynucleotides may be detected by optical or other methods. Polynucleotides comprising copies of a single or a small number of target polynucleotide(s) may be detected, and may be recovered from such regions for analysis, sequencing, or further amplification or processing.
In some embodiments, a device is provided for amplifying and/or detecting a small number of nucleic acid molecules in a sample, the device having a first well region configured to receive probe molecules and primer molecules, a second well region configured to receive target nucleic acid molecules, and a connecting region between the first well region and the second well region. The probe molecules and primer molecules may each be configured to hybridize with at least a portion of a target nucleic acid molecule. The connecting region provides a restricted pathway between the well regions, allowing contact between probe molecules, primer molecules and target nucleic acid molecules, so that a small number of target nucleic acid molecules may be amplified and/or detected. A restricted pathway may be, for example, a capillary with a small bore, a shallow groove with a small cross-section, a pathway containing material that slows or occludes fluid or solute flow, a long or tortuous pathway, or other pathway that restricts the passage of material such as a fluid or a material dissolved or suspended in the fluid. For example, the small number of copies of target polynucleotides may be less than about twenty copies, may be less than about ten copies, or may be less than about 5 copies, and may be only one copy of the target polynucleotide.
In some embodiments, a device for amplifying and/or detecting a small number of polynucleotides in a sample has a well with an interior region including a well base. The device is configured to receive probe molecules, primer molecules, and target nucleic acid molecules. The well base has more than one base location, and comprises a thin gel layer that is configured to accept probe, primer and target molecules so that at least some of the probe, primer and target molecules may diffuse into said thin gel layer. Target molecules that have diffused into the thin gel layer are dispersed to different locations so that at a given location within the thin gel layer as few as a small number of target nucleic acid molecules may be contacted by probe molecules and primer molecules. In this way, a small number of target molecules may be amplified and/or detected at a given location by action of the probe and primers. In some embodiments, target nucleic acid molecules may be dispersed within a thin gel layer at a density of less than about ten target nucleic acid molecules per square micron (as viewed from above), or less than about one target nucleic acid molecule per square micron, or less than about 10−1 target nucleic acid molecules per square micron, or less than about 10−2 target nucleic acid molecules per square micron, or less than about 10−3 target nucleic acid molecules per square micron, or less than about 10−4 target nucleic acid molecules per square micron, or less than about 10−5 target nucleic acid molecules per square micron, or less than about 10−6 target nucleic acid molecules per square micron.
In some embodiments, a device for amplifying and/or detecting a small number of nucleic acid molecules in a sample has a well with an interior volume including a gel. The well is configured to receive probe molecules, primer molecules, and target nucleic acid molecules, and the gel is configured so that at least some of the probe, primer and target molecules may diffuse into the gel and/or be dispersed in the gel. In some embodiments, probe and/or primer molecules are dispersed in the gel before application of target to gel. Target molecules diffused into the gel disperse to different locations within the interior volume so that at a given location as few as a small number of target nucleic acid molecules may be contacted by probe molecules and primer molecules. In this way, as few as a small number of target molecules may be amplified and/or detected at a given location by action of the probe and primers. In some embodiments, target nucleic acid molecules may be dispersed within a gel volume at a density of less than about ten target nucleic acid molecules per cubic micron, or less than about one target nucleic acid molecule per cubic micron, or less than about 10−1 target nucleic acid molecules per cubic micron, or less than about 10−2 target nucleic acid molecules per cubic micron, or less than about 10−3 target nucleic acid molecules per cubic micron, or less than about 10−4 target nucleic acid molecules per cubic micron, or less than about 10−5 target nucleic acid molecules per cubic micron, or less than about 10−6 target nucleic acid molecules per cubic micron.
In some embodiments, a device for amplifying and/or detecting a small number of nucleic acid molecules in a sample comprises multiple hydrophilic wells separated from each other by a hydrophobic surface. The wells have an interior volume configured to receive probe molecules, primer molecules, and target nucleic acid molecules, and are sized and spaced to receive as few as a small number of target nucleic acid molecules. Contact between the small number of target molecules and probe and primer molecules within a single well is such that as few as a small number of target molecules may be amplified and/or detected within a single well.
In some embodiments, a device for amplifying and/or detecting a small number of nucleic acid molecules in a sample has a substantially planar hydrophobic surface with a plurality of walls disposed substantially perpendicular to that surface. The walls define multiple wells, and define an opening opposite the substantially planar hydrophobic surface. The wells comprise one or more depressions in the planar hydrophobic surface, the depressions having a hydrophilic surface that defines a small volume configured to receive probe molecules, primer molecules, and target nucleic acid molecules. The probe and primer molecules are each configured to hybridize with at least a portion of a target nucleic acid molecule so that as few as a small number of the target molecules may be amplified and/or detected within a depression.
In some embodiments, systems including devices for amplification and/or detection of small numbers of target nucleic acid molecules are provided. Systems described herein comprise such devices and at least one other element, or assemblies such as an optical assembly, a reaction assembly, and an observation assembly, or an analytical assembly and a detection assembly. A system may comprise a detector for detecting copies of target nucleic acid molecules or for detecting the amplification (e.g., PCR amplification) of a single or a small number of target nucleic acid molecules. A system may comprise mechanical means for holding, transporting, and otherwise manipulating a device, or a detector, or other elements of the system. Detection may be by eye and/or by a detector. A detector may comprise an optical detector, such as a video camera, a charge-coupled device, or other instrument capable of an detecting an image or an optical signal such as fluorescence. Lenses, filters, mirrors, and other optical elements may also be comprised in systems. A system may also comprise a light source, or other illumination element. A system may comprise a controller, which may comprise a computer, for controlling the operation of a system, and for coordinating the operation of the various elements of the system. A system may comprise a fluid delivery system, such as a dispenser for delivering solutions to a device. A system may comprise a fluid collection system for removing liquids.
In some embodiments, devices and systems comprising component assemblies that are operably connected together to form operable assemblages are provided. Such devices may comprise, for example, optical assemblies having, e.g., a light source a lens, a filter, or other optical elements; thermal assemblies having, e.g., a heat source, a cooling element, or thermal element; and disk assemblies having, e.g., a reaction chamber configured to react target nucleic acid molecules with reagents. Such reagents may be prepositioned (e.g., pre-dried) in a portion or portions of a component assembly prior to addition of a sample comprising a target nucleic acid molecule. In embodiments, such devices may be dis-assembled into separate assemblies after use. In embodiments, a (one or more) component assembly may be replaced by a different assembly after use for re-use in a re-assembled device comprising the replacement assembly or assemblies. Such replacement assemblies are typically the same or a similar type of component assembly as the assembly that has been removed (e.g., a used reaction assembly may be replaced by a fresh, unused reaction assembly).
Also provided are methods for amplifying and/or detecting small numbers of nucleic acid molecules. In some embodiments, a method for amplifying and/or detecting small numbers of nucleic acid molecules comprises contacting a channel with a first solution containing a primer molecule and a probe molecule. The channel is configured to conduct a solution along the channel. The solution may be conducted along a channel by capillary action, by pressure, by suction, or by other means or combination of means. The first solution is then dried, so that the primer and probe molecules are retained within the channel. A second solution containing one or more target nucleic acid molecules is contacted with the channel, which conducts the second solution along the channel, effective to contact the target nucleic acid molecule, dried probe and primer molecules with the second solution to form a target-primer-probe mixture. In some embodiments, the channel is configured to enhance separation between target nucleic acid molecules in a solution. The mixture is then thermocycled to cause amplification of at least one target nucleic acid. In related methods, the order of application of the solutions is altered, for example, the order may be reversed, so that the first solution to be applied is a solution containing one or more target nucleic acid molecules, and the second solution to be applied is a solution containing a primer molecule and a probe molecule, and the mixture is then thermocycled to cause amplification of at least one target nucleic acid.
In some embodiments, methods are provided for amplifying and/or detecting small numbers of nucleic acid molecules, including steps of contacting a solution that comprises a primer molecule and a probe molecule with a gel within a well. The probe and the primer molecules are each configured to hybridize with at least a portion of a target nucleic acid molecule. At least some of the primer and probe molecules diffuse into the gel, or are allowed to diffuse into the gel. A solution including at least one target nucleic acid molecule is contacted with the gel, mixing the target nucleic acid molecule with probe and primer molecules within the gel. The mixture is thermocycled to cause amplification of at least one target nucleic acid. Thermocycles may be repeated a number of times to produce nucleic acid copies of said target nucleic acid molecule effective to amplify and/or detect said target nucleic acid molecule. In some embodiments, the number of times the thermal cycles may be repeated may be less than about fifty times, or less than about thirty times, or less than about twenty times, or less than about ten times. In some embodiments, the number of times the thermal cycles may be repeated may be between about thirty and about fifty times, or between about twenty and about thirty times, or between about ten and about twenty times.
Also provided are methods for amplifying and/or detecting small numbers of nucleic acid molecules comprises placing a hydrophilic well in contact with a solution, removing the hydrophilic well from contact with the solution, placing at least a portion of the device in contact with a hydrophobic liquid, making copies of said target nucleic acid molecule within said solution in contact with a well; and detecting the presence of said target nucleic acid molecule or copies thereof. The hydrophilic well forms at least a portion of a device that has a hydrophilic well surrounded by a hydrophobic surface. As before, the solution comprises a target nucleic acid molecule, a primer molecule and a probe molecule, the probe molecules and primer molecules each being configured to hybridize with at least a portion of the target nucleic acid molecule. When the well is removed from contact with the solution, a portion of the solution remains in contact with the hydrophilic well, although substantially no solution remains in contact with the hydrophobic surface.
Methods of making copies herein may comprise steps of applying heat, to raise the temperature of the mixed target nucleic acid, probe and primer molecules, and of allowing the temperature to become reduced, these steps comprising a thermal cycle. These thermal cycles may be repeated a number of times. In some embodiments, the number of times the thermal cycles may be repeated may be less than about fifty times, or less than about thirty times, or less than about twenty times, or less than about ten times. In some embodiments, the number of times the thermal cycles may be repeated may be between about thirty and about fifty times, or between about twenty and about thirty times, or between about ten and about twenty times.
Also provided are methods for amplifying and/or detecting small numbers of nucleic acid molecules comprising steps of placing a portion of a substrate comprising a hydrophilic well surrounded by a hydrophobic surface in contact with a first solution including a primer and a probe molecule. The probes and primers are each configured to hybridize with at least a portion of a target nucleic acid molecule. The substrate is then dried to dry the probe and primer molecules into at least a portion of the hydrophilic well. A second solution containing a target nucleic acid molecule is then placed in contact with the hydrophilic well, and then excess second solution is removed, so that a mixture of target nucleic acid molecules, probes and primers remains in contact with the well. At least a portion of the substrate is placed into contact with a hydrophobic liquid, so that the mixture remains in contact with the well; copies of the target nucleic acid molecule are made within or adjacent the mixture, so that the presence of the target nucleic acid molecules or its copies can be detected. In related methods for amplifying and/or detecting small numbers of nucleic acid molecules, the order of application may be altered. For example, the order of application may be reversed, so that a solution containing a target nucleic acid molecule is first placed in contact with a hydrophilic well, and then a solution including a primer and a probe molecule is placed in contact with a hydrophilic well, so that a mixture of target nucleic acid molecules, said probe and said primer remains in contact with the well.
Methods and devices herein are suitable for detecting and/or amplifying a small number of copies, or even only a single copy, of target nucleic acid molecules, and can be used to detect and/or amplify a few or a single target molecule where a solution contains only a single type of target nucleic acid molecule or where a solution contains mixtures of multiple target molecules, where the separation and dilution of the sample solution allows detection of a few or of an individual target nucleic acid molecule.
Devices, assemblies and systems may comprise probes and/or primer molecules. Thus, for example, probes and/or primer molecules may be deposited in a reaction chamber, or in an assay chamber, or may be deposited as part of the methods disclosed herein. Systems comprising optical assemblies, thermal assemblies and reaction assemblies (having reaction chambers for amplification of target nucleic acid molecules) are provided in which used reaction assemblies may be replaced to provide reusable devices. Systems comprising analytical assemblies and detection assemblies are provided in which an assay cartridge having assay chambers may engage a thermal assembly for amplification of target nucleic acid molecules.
Methods and devices herein are suitable for detecting and/or amplifying a small number of copies, or even only a single copy, of one target nucleic acid molecule while also detecting and/or amplifying a small number of copies, or even only a single copy, of one or more other target nucleic acid molecules, so that multiple targets may be detected and/or amplified. Thus, methods and devices disclosed herein are suitable for detecting and/or amplifying targets disposed in a solution containing mixtures of multiple target molecules.
These novel methods and devices are suitable for use with such assays as the Taqman® assay and other fluorescence assays to detect amplification products derived from a single target molecule. Identification is enhanced by spatial isolation (from other targets) of target molecules of interest. The methods and devices provide highly accurate quantification of target copy molecules resulting from the amplification steps, allowing highly accurate counting and comparison between assays. Embodiments of the methods and devices provide multiple assays in a single step, or only a few steps, which may include a sample preparation step. The novel methods and devices disclosed herein may provide results that are similar to colony growing methods (which take days), but instead take only one or a few tens of minutes.
Methods and devices are presented in the Figures and described in the following. The Figures show configurations where nucleic acid target molecules are distributed at such a low concentration that a small number of copies, or even only a single copy, of a nucleic acid target molecule can be amplified and/or detected without mixing substantially with other targets. A single target may be identified, amplified, and/or quantified with methods and devices disclosed herein. Multiple targets disposed in a sample, or disposed in or on a device, may also be identified, amplified, and/or quantified with methods and devices disclosed herein. Nucleic acid dispersion can be accomplished by allowing solute diffusion through small bores or channels, by allowing solute diffusion through a thin gel layer, by having barrier segments keep nucleic acid targets separate for independent amplification and detection, or by other means. Multiple target nucleic acid molecules may be individually detected in pooled samples containing multiple targets (e.g., about 10 targets, or about 30 targets, or about 100 targets, or more).
Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present teachings. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
As used herein, “target”, “target nucleic acid,” “target nucleic acid,” “target polynucleotide”, “target nucleic acid sequence,” “target sequence” and the like refer to a specific polynucleotide sequence that is the subject of hybridization with a complementary nucleic acid polymer (e.g., an oligomer). The nature of the target sequence is not limiting, and can be any nucleic acid polymer of any sequence, composed of, for example, DNA, RNA, substituted variants and analogs thereof, or combinations thereof. The target can be single-stranded or double-stranded. In primer extension processes, the target polynucleotide which forms a hybridization duplex with the primer may also be referred to as a “template.” A template serves as a pattern for the synthesis of a complementary polynucleotide. A target sequence may be derived from any living or once living organism, including but not limited to prokaryote, eukaryote, plant, animal, and virus, as well as non-natural, synthetic and/or recombinant target sequences.
As used herein, the term “probe” refers to a nucleic acid oligomer that is capable of forming a duplex structure by complementary base pairing with a sequence of a target polynucleotide, and further where the duplex so formed may be detected, visualized, measured and/or quantitated. In some embodiments, the probe is fixed to a solid support, such as in column, a chip or other array format.
As used herein, the term “primer” refers to a nucleic acid oligomer of defined sequence that hybridizes with a complementary portion of a target sequence and is capable of initiating the enzymatic polymerization of nucleotides (i.e., is capable of undergoing primer extension). A primer, by functional definition, is enzymatically extendable.
The term “primer extension” means the process of elongating an extendable primer that is annealed to a target in the 5′→3′ direction using a template-dependent polymerase. The extension reaction uses appropriate buffers, salts, pH, temperature, and nucleotide triphosphates, including analogs and derivatives thereof, and a template-dependent polymerase. Suitable conditions for primer extension reactions are well known in the art. The template-dependent polymerase incorporates nucleotides complementary to the template strand starting at the 3′-end of an annealed primer, to generate a complementary strand.
The terms “annealing” and “hybridization” are used interchangeably and mean the base-pairing interaction of one polynucleotide with another polynucleotide that results in formation of a duplex or other higher-ordered structure. The primary interaction is base specific, i.e., A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding.
The term “sample” as used herein is used in its broadest sense. A “sample” is typically, but not exclusively, of biological origin, and can refer to any type of material obtained from animals or plants (e.g., any fluid or tissue), cultured cells or tissues, cultures of microorganisms (prokaryotic or eukaryotic), any fraction or products produced from a living (or once living) culture or cells, or synthetically produced or in vitro sample. A sample can be unpurified (e.g., crude or minimally processed) or can be purified. A purified sample can contain principally one component, e.g., total cellular RNA, total cellular mRNA, cDNA or cRNA. In some embodiments, a sample can comprise material from a non-living source, such as synthetically produced nucleic acid polymers (e.g., oligomers).
As used herein, the term “polymerase extension” refers to any template-dependent polymerization of a polynucleotide by any polymerase enzyme. It is not intended that the present invention be limited to the use of any particular polymerase. A polymerase can be an RNA-dependent DNA polymerase (i.e., reverse transcriptase, e.g., Moloney murine leukemia virus [MMLV] reverse transcriptase), DNA-dependent RNA polymerase (e.g., T7 polymerase, SP6 polymerase, T3 polymerase), or a DNA-dependent DNA polymerase (e.g., Taq DNA polymerase, Bst DNA polymerase, Klenow fragment, SEQUENASE™). A polymerase may or may not be thermostable, and may or may not have 3′→5′ exonuclease activity. Polymerase extension is not limited to polymerase activity that requires a primer to initiate polymerization. For example, T7 RNA polymerase does not require the presence of a primer for polymerase initiation and extension.
As used herein, the term “amplification” refers generally to any process that results in an increase in the amount of a molecule. As it applies to polynucleotides, amplification means the production of multiple copies of a polynucleotide, or a part thereof, from one or few copies or small amounts of starting material. For example, amplification of polynucleotides can encompass a variety of chemical and enzymatic processes. The generation of multiple DNA copies from one or a few copies of a template DNA molecule during a polymerase chain reaction (PCR) is a form of amplification. Amplification is not limited to the strict duplication of the starting molecule. For example, the generation of multiple RNA molecules from a single DNA molecule during the process of transcription (e.g., in vitro transcription) is a form of amplification.
Devices, systems and methods disclosed herein are useful for detecting, counting and/or amplifying a few copies or even a single copy of a target polynucleotide sequence. Such polynucleotide sequences may be detected, for example, using Taqman® procedures. Fluid samples containing nucleic acids having target sequences are applied to devices herein, where primers, probes, and other PCR reagents may be used to amplify target nucleic acid sequences present in the samples. For example, in some embodiments, nucleic acids within a sample may be separated by flow within or along a channel. Barriers may be put into place to prevent mixing after separation. Amplification of the separated nucleic acids by PCR results in separated or isolated populations of amplified nucleic acids, where each nucleic acid population is derived from a small number of, or even only a single, nucleic acid molecule. In other embodiments, nucleic acids within a sample are allowed to separate by diffusion into a gel, resulting in separated or isolated populations of nucleic acids derived from a small number of, or even only a single, nucleic acid molecule. The nucleic acid molecule may be a target nucleic acid molecule. The gel may be thick, or may be a thin gel layer, as discussed in greater detail beow. In some embodiments, nucleic acids within a sample are separated into a well or wells, resulting in separate populations of nucleic acids derived from a small number of, or even only a single, nucleic acid molecule.
Such populations of copies of target polynucleotides may be detected by optical or by other methods. Detection may be after amplification by, e.g., PCR. Polynucleotides comprising copies of a single or a small number of target polynucleotide(s) may be recovered from such regions for analysis, sequencing, or further amplification or processing. Devices, systems and methods herein offer the advantages of detection of a small number of copies, or even of a single copy of a target nucleic acid molecule. Thus, devices, systems and methods herein may be used to amplify and to detect less than about 10 target nucleic acid molecules, or less than about 5 target nucleic acid molecules, or a single target nucleic acid molecule. Devices, systems and methods herein may be used to amplify and to detect between about 5 and about 10 target nucleic acid molecules, or between about 3 and about 5 target nucleic acid molecules, or between about 1 and about 3 nucleic acid molecules, or a single target nucleic acid molecule.
Amplification of single nucleic acid molecules and of populations of nucleic acid molecules is typically performed by polymerase chain reaction (PCR) in which repetitive thermal cycles of heating and cooling of solutions containing nucleic acid molecules in the presence of a thermostable polymerase (e.g., Taq polymerase), primers, nucleotides, and other reagents results in the production of multiple copies of target nucleic acid molecules. For example, thermal cycles may comprise a denaturation portion, having a temperature typically between about 73° C. and about 99° C., or between about 85° C. and about 98° C., or between about 90° C. and about 97° C., or between about 93° C. and about 96° C.; an extension portion having a temperature typically between about 55° C. and about 72° C., or between about 60° C. and about 70° C., or between about 62° C. and about 68° C.; and an annealing portion having a temperature between about 22° C. and about 55° C., or between about 37° C. and about 55° C.
Methods for a wide variety of PCR applications are known in the art, and described in many sources, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, Section 15, John Wiley & Sons, Inc., New York (1994); Mullis et al. (Methods in Enzymology 155:335-350 (1987); Martin et al., (Methods in Enzymology 305:466-476 (2000); U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159 and 4,965,188.
Reverse transcriptase PCR (RT-PCR) is a PCR reaction that uses RNA template and a reverse transcriptase to first generate a DNA template molecule prior to the multiple cycles of DNA-dependent DNA polymerase primer elongation. Multiplex PCR refers to PCR reactions that produce more than one amplified product in a single reaction, typically by the inclusion of more than two primers in a single reaction. “Real-Time PCR” refers to PCR methods which allow monitoring of the progress of the reaction as the reaction proceeds. Such monitoring may occur between thermal cycles, or during a thermal cycle, and typically uses optical methods for detecting the presence of double-stranded nucleic acids. For example, see Klein, Trends in Molecular Medicine, 8(6):257-260 (2002). “Quantitative PCR” refers to PCR methods that provide an indication or measurement of the actual numbers of copies of nucleic acids produced by the amplification procedures, as opposed to relative numbers commonly obtained with other PCR methods. See, for example, Gilliland et al., PNAS 87:2725-2729 (1990) and methods discussed in Jung et al., Clin. Chem. Lab. Med. 38(9):833-839 (2000); Martin et al., Meth. Enzymol. 305:466-476 (2000); and Klein, supra.
In some embodiments, detection methods employ a labeled probe with the amplified DNA in a hybridization assay. For example, one method (termed “in situ hybridization”) uses a complementary single stranded DNA probe to which a label molecule has been attached. This probe is hybridized to the specific DNA sequences in the amplified sample, if there are any, and the excess probe and label is washed away. Then the locations of the remaining label molecules are rendered visible by treatment with developer reagents. For example, a label molecule on the amplified DNA may be biotin, and the binding molecule, coupled to the enzyme, avidin; or, the label molecule may be digoxigenin, and the binding molecule an anti-digoxigenin antibody. The labels on the label molecules may also be colored, fluorescent, or radioactive.
Another suitable PCR method is the “TAQMAN®” 5′ nuclease method. A “Taqman®” probe is a probe having a fluorescent indicator moiety attached internally or at one end (typically the 5′ end of the probe), and a quencher moiety attached internally or at an end (e.g., where a fluorescent indicator is attached at one end, a quencher moiety may be attached at the other end). When attached to the probe the proximity of the fluorescent indicator to the quencher prevents significant fluorescence from being emitted (due to fluorescence resonance energy transfer (FRET), or Forster-type energy transfer) or by non-resonance mechanisms. The probe is designed to be complementary to a sequence on a target polynucleotide, so that it anneals to a portion of a nucleic acid strand that is duplicated by PCR. As the primer is extended by DNA polymerase during PCR, the probe is cleaved so that the fluorescent moiety and the quencher moiety are no longer bound together by an intact probe. Thus, after the probe has been degraded by the action of DNA polymerase, the fluorescent moiety and the quencher moiety become separated, allowing emission of fluorescence. A threshold level of fluorescence may be determined or assigned as a measure of the progress of PCR amplification. Typically, a threshold level is defined at a level above which a logarithmic plot of fluorescence increases linearly with cycle number.
In some embodiments, 5′ nuclease assays and other assays are useful in “real-time” PCR techniques. Such techniques and methods allow the detection of amplified target nucleic acid molecules and the measurement of the progress of the PCR reaction. Such techniques typically do not require opening the reaction vessel in order to monitor PCR progress. By, for example, monitoring fluorescence emitted from the reactants, non-invasive and potentially continuous measurement is possible (see, e.g., Klein, Trends in Molecular Medicine, 8(6):257-260 June 2002, or Real-Time PCR—An Essential Guide, K. Edwards et al., Eds, Horizon Bioscience, Norfolk, UK (2004)). Exemplary methods for 5′ nuclease-mediated cleavage of Taqman®-type probes can be found, for example, in PCT Publication No. WO 96/15270 (Livak et al.), wherein fluorescence can be sampled during the denaturation step of each thermal cycle. Other real time PCR protocols are described for example in WO 96/34983 (Mayrand), WO 99/37670 (Coull et al.), WO 95/13399 (Tyagi et al.), and WO 01/94638 (Chen et al.). In such methods, a probe (containing a fluorescer moiety and a quencher moiety at opposite ends of the probe) is annealed to a target strand prior to the extension step of PCR (at an annealing temperature that is less than the primer extension temperature in the PCR cycle), and the resultant fluorescence can be measured as an indication of the amount of amplification at a particular thermal cycle. Typical “real-time PCR” indicators that are not sequence specific comprise, e.g., ethidium bromide, propidium iodide, SYBR™ Green I and II, PicoGreen™, and the Hoechst 33258 Dye. Ethidium bromide is a fluorescent compound that fluoresces while bound to double-stranded DNA. Other exemplary “real time” methods are described in, e.g., Holland, et al., PNAS 88:7276-7280 (1991), Higuchi et al. (Biotechnology 10:413-417 (1992), Higuchi et al. U.S. Pat. No. 6,171,785, Gelfand et al., U.S. Pat. No. 5,210,015, and Fisher, et al. (U.S. Pat. No. 5,491,063).
All patents and publications referred to herein, both supra and infra, are hereby incorporated herein by reference in their entireties.
Fisher et al. (U.S. Pat. No. 5,491,063) provides an assay that allows the simultaneous detection of the accumulation of amplified target and the sequence-specific detection of the target sequence. The method of Fisher et al. provides a reaction that results in the cleavage of single-stranded oligonucleotide probes labeled with a light-emitting label. The reaction is carried out in the presence of a DNA binding compound that interacts with the label to modify the light emission of the label. The method utilizes the change in light emission of the labeled probe that results from degradation of the probe to detect the presence of target molecules and of copies of target molecules. The methods are applicable in general to assays that utilize a reaction that results in cleavage of oligonucleotide probes, and in particular, to homogeneous amplification/detection assays where hybridized probe is cleaved concomitant with primer extension.
PCR can be used to amplify and/or detect a small number of target nucleic acid molecules. As used herein with reference to the novel systems, devices and methods, a small number of target nucleic acid molecules refers to less than about 10 target nucleic acid molecules, or to less than about 5 target nucleic acid molecules, or to a single target nucleic acid molecule. Thus, a small number of target nucleic acid molecules may be between about 5 and about 10 target nucleic acid molecules, or between about 3 and about 5 target nucleic acid molecules, or between about 1 and about 3 nucleic acid molecules, or may be a single target nucleic acid molecule.
Several views of the channel device 12 are illustrated in
Channel device 12 has multiple channels 42 connecting with probe/primer wells 44 and with a target well 46. All probe/primer wells 44 connect with at least one channel 42. Some, but not necessarily all, channels 42 connect with a particular probe/primer well 44. All channels 42 connect with target well 46, so that a connection exists via a channel 42 between target well 46 and each probe/primer well 44.
As indicated in
Channels 42 and wells 44 and 46 may be formed by cutting, etching, or otherwise removing material from a substrate 48, or by pressing or compressing a substrate 48 (which may first be heated or softened) to obtain the desired configuration. Channels 42 and wells 44 and 46 of a device 12 may also be formed at the same time as the substrate 48, by molding or casting a material to have such depressions. Alternatively, a channel 42 may be a tube or other structure having a bore. Thus, for example, a channel 42 may be formed by placing a tube or other material having a bore onto or into a substrate 48, or combining such material to form a substrate 48 with a bore or bores. Such tubes may be capillary tubes. Alternatively, channels 42 and wells 44 and 46 may be formed on top of a substrate 48, or by placing a wall or walls onto a substrate 48, or may be formed in part by cutting into a substrate 48 and in part by building up at least a portion of a substrate 48. A substrate 48 may be translucent (i.e., allowing the passage of electromagnetic radiation) or may be transparent (i.e., allowing the passage of electromagnetic radiation effective to allow localization of a source of electromagnetic radiation within or on the other side of the substrate 48, such as allowing the formation of an image from electromagnetic radiation passing through the material). Alternatively (e.g., where a detector 20 is disposed opposite a channel 42 or an uncovered face of a well 44 or 46), the substrate 48 need not be transparent, while the channel 42 may be transparent to at least a portion of the electromagnetic spectrum or may be open on a portion disposed opposite a detector 20.
The channels 42 may have small dimensions so that fluid within the channels flows slowly, limiting the rate of dispersion of target nucleic acids present in liquids within the channels. However, although slow, such fluid flow is enhanced by capillary action due to the size and configuration of the channels. In some embodiments, an elongated channel has a width of less than about 10 mm, or less than about 5 mm, or less than about 2 mm, or less than about 1 mm, or less than about 0.5 mm, or less than about 0.2 mm. Thus, in some embodiments, an elongated channel may have a width of between about 5 mm and about 10 mm; or between about 2 mm and about 5 mm; or between 1 mm and about 2 mm; or between about 0.5 mm and about 1 mm, or between about 0.2 mm and about 0.5 mm, or more than 0 mm and less than about 0.2 mm. In some embodiments, an elongated channel has a depth of less than about 5 mm, or less than about 2 mm, or less than about 1 mm, or less than about 0.5 mm. Thus, in some embodiments, an elongated channel may have a depth of between about 2 mm and about 5 mm; or between about 2 mm and about 2 mm; or between 0.5 mm and about 1 mm; or less than about 0.5 mm.
In some embodiments, an elongated channel may have a width of about 0.3 mm and a depth of about 0.15 mm. In some embodiments, a device has multiple elongated channels, each having a width of about 0.3 mm and depths of about 0.15 mm. Channels 42 of channel device 12 may be about 0.3 mm wide by about 0.15 mm deep. Probe/primer wells 44 may be about 2 mm by about 2 mm; and a target well 46 may be about 2 mm by about 9 mm. In some embodiments, a channel 42 may have a length of up to about 30 cm, or may have a length of up to about 20 cm, or may have a length of up to about 10 cm, or may have a length of up to about 5 cm, or may have length of less than about 5 cm. Thus, a channel 42 may have a length of between about 20 cm and about 30 cm; or may have a length of between about 10 cm and about 20 cm; or may have a length of between about 5 cm and about 10 cm; or may have a length of less than about 5 cm.
As illustrated schematically in
The presence of probes 14, primers 16, and target nucleic acid molecules 18 within channels 42 in a solution suitable for PCR provides conditions in which amplification of target nucleic acid molecules 18 may be performed. Such a solution may comprise buffers, salts, and/or other constituents useful for performing PCR. A device 12 and a system 10 are each configured for thermal cycling suitable for PCR amplification of target nucleic acid molecules 18. The number of copies of target nucleic acid molecules 18 increases with each thermal cycle. The rate of this increase may decline if the availability of nucleotides, primers or probes becomes limiting. Since within the channels 42 target nucleic acid molecules 18 are typically found in isolated and separate regions 52, initially (i.e., before thermal cycling) with one, or with a small number of copies of target nucleic acid molecules 18 in each region 52, with succeeding PCR thermal cycles multiple copies of target nucleic acid molecules 18 increase within such separate regions 18. Thus, within each separate region 52, all copies of target nucleic acid molecules 18 will be derived from a single original copy of a target nucleic acid molecule 18, or from a small number of original copies of target nucleic acid molecules 18.
Amplification of copies of target nucleic acid molecules is often plotted on an “amplification plot” in which the fluorescence signal detected from amplified nucleic acid molecules is plotted against the cycle number. The thermal cycle at which the fluorescence signal passes a set threshold may be termed the “threshold cycle” and is often denoted “CT.” A useful threshold is where the fluorescence signal becomes detectable (indicating that amplification of target nucleic acid molecules is detectable). Fluorescence (or other signal) from copies of target nucleic acid molecules 18 may be measured within each separate region 52 to measure the amount of amplification and the progress of the amplification of target molecules. An amplification plot of the signal may be made, and the signal compared to a CT effective that the amplification of target nucleic acid molecules may be quantified. Other methods of detecting and analyzing the amplification of target molecules may also be employed.
Copies of target nucleic acid molecules 18 and indicator (e.g., dyes) will tend to diffuse away over time from their initial location within a region 52. With multiple cycles, such diffusion might lead to overlap between different regions 52 and to mixing of copies of target nucleic acid molecules 18 that are derived from different original copies of the target nucleic acid molecule 18. It is desirable to reduce such diffusion in order to reduce the overlap or mixing of copies of target nucleic acid molecules 18 within a channel 42. In order to reduce the diffusion distance, thermal cycles may be fast, or few, or fast and few. Thus, the duration of the heating phase of a thermal cycle may be short, or the duration of the cooling phase of a thermal cycle may be short, or both. The numbers of thermal cycles may be kept to a minimum in order to reduce the dispersal of copies of target nucleic acid molecules 18 and of indicator dyes. Use of a viscous solution and/or matrix may also be effective to reduce the diffusion distance, either alone or in combination with fast and/or few thermal cycles. A viscous buffer has a viscosity greater than that of water, that is, greater than about 1 centipoise (g/cm-s) at room temperature. A viscous buffer thus has a viscosity of greater than 1 centipoise, or greater than about 2 centipoise, or greater than about 10 centipoise, and may be greater than about 100 centipoise, greater than about 500 centipoise, or greater than about 5000 centipoise at room temperature.
Real-time PCR techniques may be used to allow the detection of amplified target nucleic acid molecules 18. For example, amplification of target nucleic acid molecules 18 may cause degradation of probes 14 and thereby release of indicator dyes. Release of fluorescent indicator dyes is detectable by a detector 20 of a system 58 after amplifying by PCR. The indicator dye may be from Taqman® probes for use in a PCR system to indicate amplification of target molecules 18 complementary to probes 14.
As illustrated in
A detector 20 may comprise a camera. A camera image of the entire portion 54 may be used in order to count the number of amplified spots, i.e. the number of regions 52 containing amplified target nucleic acid molecules 18. The camera image may be taken as soon as possible after the last thermal cycle in order to limit diffusion distance. Multiple assays may be performed and detected in a portion 54 using different probe/primer sets including different color fluorescent molecules for each probe type. For example, for each color, a separate picture may be obtained with an appropriate color filter for detection by an optical detection device, or separate colors may be detected simultaneously with different detection devices sensitive to different optical wavelengths, or a single detector sensitive to multiple wavelengths. Suitable optical detection devices comprise photomultiplier tubes, charge-coupled detector devices (CCDs), video cameras, and other optical devices. A detector 20 may comprise a microscope (e.g., a confocal microscope) and will typically comprise a lens, mirror or other optical collector, an optical detector, and associated power, controlling elements, input and output elements, and may also comprise a filter, a diaphragm or shutter, scanner, and other elements as well. A detector may comprise fiber optic components.
In some embodiments of a device 12 of a system 10, channels 42 may be segmented into small wells 51 after target molecules 18 have been added, as illustrated in
For example, barriers 53 may be used to segment channels 42 into small wells 51 so that regions of amplified target matches 52 remain separate and do not mix together. This is illustrated in
After deposition of a droplet 72 onto gel layer 68, probes 14 and primers 16 diffuse into the gel layer 68 on the bottom 66 of a well 62 of a barrier device 60, as illustrated in
Following deposition of a droplet 72 (containing probes 14 and primers 16) into a well 62 and diffusion of probes 14 and primers 16 into a gel layer 68 on a bottom 66 of a well 62, a droplet 74 (containing target nucleic acid molecules 18) may be placed on a barrier device 60, as illustrated in
Thermal cycling applied to a barrier device 60 having probes 14, primers 16 and target molecules 18 diffused into thin gel layers 68 of wells 62 is effective to amplify target molecules by PCR. As indicated in the Figures, the target nucleic acid molecules 18 are well separated, so that a single target nucleic acid molecule 18, or a small number of target nucleic acid molecules 18, are found at any one site in a well 62. The number of copies of target nucleic acid molecules 18 increases with each cycle (at least in the absence of depletion of primers 14, probes 16, or other necessary component of the PCR reaction), so that copies of target molecules 18 tend to diffuse away from the initial site where the initial target nucleic acid molecule 18. The thin gel layer 68 is configured to reduce the spread of copies of the target nucleic acid molecule 18 and of indicator dyes away from any one of the sites of initiation of the PCR amplification. Thus, regions 76 of amplified copies of target nucleic acid molecules 18 derived from a single, or from as few as a small number of, target nucleic acid molecules 18 arise and dot the bottom 66 of the wells 62.
In some embodiments, at least a portion of each of bottom 66, thin gel layer 68, and substrate 70 is transparent or translucent to at least a portion of the electromagnetic spectrum. Bottom 66, thin gel layer 68, and substrate 70 are typically transparent to light, such as visible light, infrared light, ultraviolet light, or electromagnetic radiation of other wavelengths suitable for detection of amplification of target nucleic acid molecules 18. Alternatively, bottom 66 and substrate 70 need not be transparent, in some embodiments where imaging is performed only through the thin gel layer 68 (which must then be transparent or translucent), as by imaging taken from above the thin gel layer 68.
A system 82 having a device 84 with a deep well 86 containing a thick gel layer 88 is illustrated in
Thus, a system 82 and device 84 are suitable for mixing target molecules 18, probes 14, and primers 16 within the volume of a well 86. A device 84 may be subjected to thermal cycling to amplify target nucleic acid molecules 18 within a deep well 86 by PCR. Amplification of target nucleic acid molecules 18 (e.g., by PCR using Taqman® probes) will produce large numbers of copies of the individual target nucleic acid molecules 18 within, and release indicator dyes into, a localized region 90 around the initial locations of the target nucleic acid molecules 18. The thick gel layer 88 serves to reduce the spatial dispersion of copies of target molecules 18 and indicator dye released during the copying of target molecules 18, thereby limiting the size and spread of small regions 90. Amplification of target molecules 18 can then readily be detected in small regions 90 within a deep well 86. Regions 90 containing copies of target nucleic acid molecules 18 produced by PCR amplification are detectable by fluorophores released from probes 14 as primers 16 are extended during PCR cycles. PCR thermal cycling creates roughly spherical regions 90 of fluorescence within the volume of a well 86.
Such small regions 90 are illustrated in
Roughly spherical regions 90 produced by amplification of target nucleic acid molecules 18 within the thick gel layer 88 in a deep well 86 of a device 84 may be detected and counted by optical means. In some embodiments, portions of a device 84, a deep well 86, and the thick gel layer 88 are transparent or translucent to at least a portion of the electromagnetic spectrum. Alternatively (e.g., where a detector 20 is disposed opposite an uncovered face of a gel layer 88), only the thick gel layer 88 is transparent or translucent to at least a portion of the electromagnetic spectrum. For example, as illustrated in
A deep well 86 of a deep well device 84 is preferably deeper than a well 62, and a thick gel layer 88 is preferably thicker than a thin gel layer 68 of a device 60. Such a thick gel layer 82 may be in a device 84 having a plurality of wells 86 or may be in a device 84 having only a single well 86. Use of such an embodiment is similar to the use of a device 60, except that vertical diffusion of target nucleic acid molecules 18 and of indicator dyes is not substantially limited by the physical dimensions of the thick gel layer 82 or of the well 84.
For example, a thick gel layer 88 contained within a deep well 86 may comprise an acrylamide gel layer, or may be made from other materials including gelatin, agar, agarose, acrylamide, Sepharose®, Sephadex®, Sephacryl®, casein, unfixed gels and cross-linked gels. A thick gel layer 86 may be made from a mixture of materials. Gel layer 88 may be translucent, and is typically transparent to at least a portion of the electromagnetic spectrum, allowing imaging or scanning of small regions 90 within it.
The PCR solutions including amplification and wash solutions are typically water-based solutions so that upon contact with wells 100 and hydrophobic regions 102, such solutions will tend to remain in contact with wells 100 and will tend to drain or flow away from hydrophobic regions 102. As illustrated in
Following a desired number of thermal cycles, the end 104 of post 98 is removed from the oil 110 and may be inspected by eye or imaged to detect the presence of and/or quantify amplification of target nucleic acid molecules 18.
In some embodiments, a post device 98 may have wells 100 having depths or less than about 10 mm, or of less than about 5 mm, or less than about 2 mm, or of less than about 1 mm. Thus, wells 100 of a post device 98 may have depths of between about 5 mm and about 10 mm, or between about 2 mm and about 5 mm, or between about 1 mm and about 2 mm, or of between about 0 mm and about 1 mm. Wells 100 of a post device 98 may have widths of less than about 5 mm, or less than about 2 mm. A hydrophobic region 102 may be made of, or covered with, for example, a hydrophobic material such as plastic, hydrophobic polymers, glass, metal, or other hydrophobic material. A well 100 may be made or, or covered with, for example, a hydrophilic material such as silanized glass, polysaccharides, hydrophilic polymers such as polyester terephthalate (PET) and glycol-modified polyethylene terephthalate (PETG).
In an alternative embodiment, a hydrophobic surface configured as the bottom of a container for holding a solution may have multiple hydrophilic holes or depressions on the surface, forming hydrophilic wells separated by hydrophobic surfaces. For example, as illustrated in
As illustrated in
Probes 14 and primers 16 dried in a well 126 are illustrated in
The shallow walls 124 are effective to prevent the spread of probes 14 or primers 16 between wells 126 upon deposition of a solution 128 into a well 126. Thus, where different probes 14 or primers 16 are present in different wells 126, different assays may be performed simultaneously on a single plate to detect different target nucleic acid molecules 18 to which probes 14 and/or primers 16 are directed. Hydrophobic surfaces 122 also prevent the spread of solution between hydrophilic depressions 118, so that, after PCR, indicator dye or other means for identifying the presence of a target nucleic acid molecule 18 present in one hydrophilic depression 118 does not spread to a neighboring hydrophilic depression 118.
The plate 120 containing mixed probes 14, primers 16 and target nucleic acid molecules 18 in the hydrophilic depressions 118, may be inserted into oil 134 contained in an oil trough 136, allowing the mixture of targets 18, probes 14, and primers 16 to be thermal cycled without evaporation (
As illustrated in
In some embodiments, hydrophilic depressions 118 may have depths of less than about 10 mm, or of less than about 5 mm, or less than about 2 mm, or of less than about 1 mm. Thus, hydrophilic depressions 118 may have depths of between about 5 mm and about 10 mm, or between about 2 mm and about 5 mm, or between about 1 mm and about 2 mm, or between about 0 mm and about 1 mm. Hydrophilic depressions 118 may have widths of less than about 10 mm, or less than about 5 mm, or less than about 2 mm. Wells 126 may have lengths and widths of up to about 5 cm, or of up to about 2 cm, or of up to about 1 cm or less. Thus, wells 126 may have lengths and widths of between about 2 cm and about 5 cm, or between about 1 cm and about 2 cm, or less than about 1 cm.
Also provided are methods for amplifying and/or detecting small numbers of nucleic acid molecules. Methods herein comprise contacting a first solution having probe and/or primer molecules with a channel, or chamber, effective that the solution is conducted along the channel or into the chamber, the probe and/or primer molecules being configured to hybridize with at least a portion of a target nucleic acid molecule. Solution flow may be by capillary action, although it may be produced or aided by pressure or suction. The solution is dried so that the primer and/or probe molecules are retained within the channel or chamber. A second solution containing a target nucleic acid molecule is contacted with the channel or chamber so that the second solution is conducted along the channel, or into the chamber, effective to mix the target nucleic acid molecule, dried probe and primer molecules into the second solution at an initial temperature. The initial temperature may be, for example, room temperature. Where the first solution has probes but not primers, the second solution may include primers. Where the first solution has primers but not probes, the second solution may include probes. Alternatively, where the first solution has only one of probes and primers, but no both probes and primers, the other reagent (primers or probes) may be provided by a third solution, included with the sample, or by other means.
Valves may be used to regulate the flow of one or more solutions. For example, valves may be polyethylene glycol (e.g., of a molecular weight selected to have the desired melting temperature and degradation characteristics), wax, polymer, or other material which may be removed or altered to allow passage of material by e.g., heat, or light, electrical power, or other controlling signal. In some embodiments of the methods, barriers may be raised or placed to limit diffusion along the channels after target nucleic acid molecules have passed along them.
Heat is then applied to raise the temperature of the mixed target nucleic acid, probe and primer molecules, separating nucleotide dimers, and then the temperature is allowed to become reduced, completing a thermal cycle. PCR amplification is allowed to occur, and then the temperature is raised again, to separate complementary stands. Upon reduction of temperature, amplification of target nucleic acid molecules again occurs, and the process is repeated a sufficient number of times effective to produce nucleic acid copies of said target nucleic acid molecule effective to amplify and/or detect the target nucleic acid molecules.
In some embodiments of methods disclosed herein, the second solution comprising a target nucleic acid molecule is a dilute solution containing a plurality of target nucleic acid molecules, effective that individual target nucleic acid molecules are separated from one another within the channel. It will be understood, however, that solutions may be applied in any order, and that designation of a first solution and of a second solution is not meant to limit the order of application of solutions in methods disclosed herein. In some methods, indicators are cleaved from probes by the extending primers to generate fluorescence around targets that match the probes. The indicators may be fluorescent indicators. In some methods, Taqman® probes are used. Multiple indicators, such as fluorescent dyes with different fluorescence wavelengths, may be used to indicate different target molecules or different conditions.
Thermal cycles may be repeated after a short interval of time so as to prevent diffusion of target nucleic acid molecule copies greater than a short distance from the individual target nucleic acid molecules that were copied. The number of cycles may be greater than about fifty, or fewer than about fifty, or fewer than about thirty, or fewer than about twenty cycles, or may be fewer than about ten cycles. The second solution may be, but need not be, a viscous buffer solution.
In some embodiments of the methods, amplification of the target nucleic acid molecule is detected by detection of an optical signal, such as a fluorescence signal. In some embodiments of the methods, detection is effected as soon as possible after the last thermal cycle in order to limit the distance that nucleic acid copies or indicators travel away from their point of origin or release. Viscous buffers may also be used to reduce or slow the diffusion of nucleic acid copies and indicator molecules. Detection may be by eye, and may be by optical methods, using, for example, optical devices such as cameras, scanners, microscopes, such as a confocal microscope, charge-coupled devices, and photomultiplier tubes. Multiple signals (such as multiple fluorescent wavelengths) may be detected, simultaneously or sequentially (e.g., by the use of multiple detectors simultaneously, or by a single detector and multiple filters used sequentially). Detection of indicators may be ongoing during the amplification process, or may be performed at intervals during the process. For example, fluorescence measurements may be taken soon after the completion of each thermal cycle.
In further methods, a small number of nucleic acid molecules may be amplified and/or detected by contacting a first solution containing a primer molecule and a probe molecule with a gel within a well and allowing at least some of the primers and probes to diffuse into the gel. A second solution containing a target nucleic acid molecule is contacted with the gel, so as to mix together the target nucleic acid molecule with probe and primer molecules within the gel. The mixing occurs at an initial temperature. Heat is then applied to raise the temperature of the mixed target nucleic acid, probe and primer molecules to a raised temperature above the initial temperature. Target nucleic acid dimers will dissociate at raised temperatures. The temperature is then allowed to become reduced to a temperature closer to the initial temperature. Amplification of the target nucleic acid molecules by PCR occurs. The temperature is then raised, and PCR thermal cycles are repeated effective to produce nucleic acid copies effective to amplify and/or detect the target nucleic acid molecule. In some methods, the thermal cycles are repeated more than about thirty times, or less than about thirty times, or less than about twenty times, or less than about ten times.
Suitable PCR methods include 5′ nuclease methods, such as Taqman® methods, using probes having fluorescent indicators such as dyes that may be cleaved from probes during primer extension. In the practice of the methods disclosed herein, the gel may be a thin gel layer, which may have a thickness of less than about 1 mm, or less than about 0.5 mm, or a less than about 0.1 mm, or less than about 0.05 mm. Thus, the gel may be a thin gel layer having a thickness of between about 0.5 mm and about 1 mm, or between about 0.1 mm and about 0.5 mm, or between about 0.05 mm and about 0.1 mm, or between about 0 mm and about 0.05 mm. In some embodiments, the thin gel layer comprises a gel layer having a thickness of about 0.04 mm.
In some embodiments of methods, a thick gel layer is contacted with a solution containing primer molecules, probe molecules and target nucleic acid molecules. The gel is preferably within a well. The probe, primer and target molecules are allowed to diffuse into the gel at an initial temperature. Heat is then applied to raise the temperature of the mixed target nucleic acid, probe and primer molecules to a raised temperature above the initial temperature. Target nucleic acid dimers will dissociate at raised temperatures. The temperature is then allowed to become to a temperature closer to the initial temperature. Amplification of the target nucleic acid molecules by PCR occurs. The temperature is then raised, and PCR thermal cycles are repeated effective to produce nucleic acid copies effective to amplify and/or detect the target nucleic acid molecule. In some methods, the thermal cycles are repeated more than about fifty times, or less than about fifty times, or less than about thirty times, or less than about twenty times, or less than about ten times.
Another embodiment of the methods for amplifying and/or detecting small numbers of nucleic acid molecules comprises placing a hydrophilic well that is surrounded by a hydrophobic surface in contact with a solution containing a target nucleic acid molecule, a primer molecule and a probe molecule. Removing the hydrophilic well the solution allows a portion of the solution to remain in contact with the hydrophilic well, while substantially no solution remains in contact with the hydrophobic surface. This provides separate aliquots of solution in the separate hydrophilic wells. Placing a portion of the device having the hydrophilic wells into a hydrophobic liquid, such as an oil, so that the solution within the hydrophilic well remains in contact with that well. Application of PCR, including thermal cycling, is then performed to make copies of the target nucleic acid molecule within the solution in the well. Copies are detected, indicating the presence of the target nucleic acid molecule and its amplification. For example, fluorescence from indicators cleaved from probes as the copies are made may be detected by optical methods, and the presence and amount of target nucleic acid determined. Detection of electromagnetic radiation, such as infrared, visible, or ultraviolet light, may be by, e.g., a detector (such as a camera, charge-coupled device, photomultiplier, or other optical device) and may be by use of one or more optical fibers.
A further method for amplifying and/or detecting small numbers of nucleic acid molecules comprises applying a first solution containing a primer molecule and a probe molecule in contact with a hydrophilic well surrounded by a hydrophobic surface. The first solution is dried so that probe and primer molecules dry into or onto at least a portion of the hydrophilic well. The hydrophilic well is then contacted with a volume of a second solution containing a target nucleic acid molecule, and the well removed from contact with the volume of second solution, leaving a mixture of target nucleic acid molecules, probes, and primers in the second solution in contact with the well. At least a portion of the substrate is placed into contact with a hydrophobic liquid, such as an oil, so that the mixture remains in contact with the well. The oil is effective to prevent escape or dispersal of the second solution from the well. Copies of the target nucleic acid molecule are then made using PCR techniques within or adjacent the mixture. The presence of the target nucleic acid molecule or copies of it are then detected. Progress of the PCR amplification is detected and monitored, an may be detected by optical methods. Taqman® probes and techniques may be used. Alternatively, the order of application of the solutions may be reversed, with or without drying some elements prior to application of other elements.
Fluorescent indicators suitable for use in some embodiments of the methods comprise fluoroscein dyes (U.S. Pat. Nos. 5,188,934; 6,008,379; 6,020,481), rhodamine dyes (U.S. Pat. Nos. 5,366,860; 5,847,162; 5,936,087; 6,051,719; 6,191,278), benzophenoxazine dyes (U.S. Pat. No. 6,140,500), energy-transfer dye pairs of donors and acceptors (U.S. Pat. Nos. 5,863,727; 5,800,996; 5,945,526), cyanines (Kubista, WO 97/45539), ethidium bromide, propidium iodide, and other fluorescent molecules. Examples of fluorescein dyes comprise 6-carboxyfluorescein; 2′,4′,1,4,-tetrachlorofluorescein; and 2′,4′,5′,7′,1,4-hexachlorofluorescein (Menchen, U.S. Pat. No. 5,118,934).
The term “substrate” refers to a base surface which may support other elements or surfaces of a device. A substrate may be, in part or wholly, composed of metal, glass, plastic, ceramic, or other material. A substrate may be, for example, glass, silica, quartz, controlled-pore-glass (CPG), or reverse-phase silica. A substrate may comprise such materials as oligosaccharides, nitrocellulose, diazocellulose, dextran, agar, agarose, Sepharose®, Sephadex®, Sephacryl®, cellulose, starch, nylon, latex beads, magnetic beads, paramagnetic beads, superparamagnetic beads, and microtitre plates. Plastics, such as organic polymers, may comprise, for example, polyacrylamide, polycarbonate, polyimide, polymethylmethacrylate, polydimethylsiloxane, polyethylene, polyethyleneoxy, polyfluoroethylene (including polytetrafluoroethylene), polypropylene, polysulfone, polystyrene, polypropylene, polyurethane, and polyvinylchloride, as well as co-polymers and grafts thereof.
A substrate may be translucent (allowing the passage of optical radiation) or transparent (allowing the passage of optical radiation with little loss or distortion), or may have a portion that is translucent or transparent (e.g., a window). Translucent or transparent materials comprise glass, quartz, polycarbonate, polymethylmethacrylate, and other materials.
A substrate may form or support a well, depression or other container, vessel, feature or location. A substrate may have features such as channels, grooves, pathways, wells, barriers, or other features effective to contain a fluid and to direct and control the flow of fluid. Such features may be fabricated in a solid substrate by any suitable method, including molding (e.g., injection molding). Alternatively, or in addition, such features may be fabricated by microfabrication techniques such as lithographic techniques used in fabrication of semiconductor devices, (including, for example, photolithographic etching, plasma etching, and wet chemical etching). Such features may be fabricated by micromachining techniques such as laser drilling or laser ablation, micromilling, air abrasion, LIGA, reactive ion etching, embossing, and other techniques known in the art. LIGA (an acronym based on the first letters for the German words for lithography and electroplating) is a well-known process for fabricating features and devices with very small dimensions. A general review of the LIGA process is given in the article by W. Ehrfeld, et al., “LIGA Process: Sensor Construction Techniques Via X-Ray Lithography,” Technical Digest IEEE Solid State Sensor and Actuator Workshop, 1988, pp. 14-4. Fabrication methods suitable for preparing embodiments of devices described herein are described, for example, in U.S. Pat. Nos. 5,162,078, 5,378,583, 5,527,646, 5,631,514, 5,679,502, 5,571,410, 5,917,260, and 6,176,962 d and references cited therein.
Fluid flow within a microfluidic device is typically directed by the walls of a channel configured to contain the fluid. Design of such channels is described in, for example, U.S. Pat. No. 5,842,787 and references cited therein. Flow within a small capillary or thin gel layer may be by capillary action. Solutes within a fluid may flow by diffusion. Pathways configured for fluid flow may be treated or coated to reduce adsorption of solutes flowing within them. For example, a channel may be silanized or may be coated with bovine serum albumin (BSA), cytochrome C, or other protein or chemical to reduce non-specific adsorption of protein, nucleic acid, or other material to the walls of the channel. A fluid flowing within a channel may itself contain BSA for the same purpose.
Fluid flow may also be effected by pressure gradients, temperature gradients, voltage gradients, osmotic gradients, or by other means or combination of means. A pressure gradient may be provided by a pump, or by compression of all or part of a chamber or channel, or in other ways. Alternatively, fluid flow may be impelled by electrophoresis, or by electro-osmotic means. Movement of the device (e.g., rotation to create centrifugal force) may also be used to impel or direct fluid flow. Thus, hydraulic, electrokinetic, osmotic, or other means may be used to direct fluid flow along or within a channel or other such structure. Flow may be regulated or stopped by a valve, or a gate or barrier, or in other ways.
A substrate may have a plurality of locations, and be configured to define locations in an array. The various locations may be addressable for robotic delivery of reagents, or for detection of hybridization at the locations. Detection of hybridization may be by eye, and may be by detection means including camera, photomultiplier, scanning by laser illumination and confocal or deflective light gathering, or by other means.
A detector may be a device or system for sensing a signal provided by a target or indicator. A detector may be configured, for example, to detect radiation, fluorescence, phosphorescence, luminescence, pH, charge, current, voltage, redox potential, absorbance, temperature, and/or may comprise an electrical, magnetic, thermal, acoustic, or other sensor. A detector typically comprises an optical detector, such as, for example, a photomultiplier tube, a charge-coupled device (CCD), a scanning detector, a confocal device, or other device.
The methods, devices, assemblies and systems disclosed herein enable highly-quantitative counting of single copy events using Taqman® reagents and other assays for amplification of target nucleic acid molecules. Methods, devices, assemblies and systems disclosed herein are further illustrated in the following EXAMPLES which illustrate assemblies, devices, and methods for use in systems having features of the invention. Nucleic acid targets are distributed in such low concentration that single copies can be amplified and fluoresce without diffusing with other targets. In this way, as few as only a single copy of target can be amplified a million fold in a very small volume. For example, using Taqman® assay methods each target copy will generate an unquenched reporter. The reporters fluoresce when illuminated by a light source. A means of low noise excitation coupled with the highly concentrated signal enables detection by eye, avoiding the need for expensive photon sensors and optics. However, if desired, photon sensors and optics may be used in place of, or in addition to, observation by eye.
The methods, devices, assemblies and systems described above and in the Examples below, may be used to detect the presence of target nucleic acids indicative of the presence of target organisms. Such target organisms include pathogens, such as bacterial, viral, fungal, or other pathogens. A target pathogen may be, e.g., Mycobacterium Tuberculosis or Bacillus anthracis, which cause tuberculosis and anthrax, respectively. Thus, for example, a target pathogen may be one that causes tuberculosis, anthrax, diphtheria, meningitis, whooping cough, tetanus, pneumonia, rabies, influenza, smallpox, or other disease. Such pathogens may indicate disease in a sample taken from a host animal or a human patient or from a bodily fluid or waste from a host animal or a human patient. Detection of target nucleic acid from a target organism in a sample of food, raw material, effluent, water source, material used in the production of a pharmaceutical or of an industrial product, or other material may also be used to detect the presence of a target organism in the source of the sample.
For example, gram positive and gram negative bacteria can be detected. Gram positive bacteria to be detected include, for example, bacteria belonging to the genera Staphylococcus, Streptococcus, Listeria, Clostridium, and Corynebacteria. Gram negative bacteria to be detected include, for example, bacteria belonging to the family Enterobacteriaceae. Gram negative bacteria to be detected include, for example, gram negative bacteria belonging to the genera Haemophilus, Bacteroides, Pseudomonas, Neisseria, and Legionella can be detected. Target organisms to be detected may include fungi belonging to the genera Candida, Cryptococcus, Coccidiodes and Histoplasma. Viral pathogens may also be detected, e.g., viruses from Paramyxoviridae, Rhabdoviridae, Filoviridae, Boma Disease Virus, Orthomyxoviridae, Bunyaviridae and Arenaviridae, including viral pathogens such as nucleic acids derived from, and indicative of, influenza, herpes, polio, smallpox, hepatitis, human immunodeficiency virus (HIV), Ebola, hanta, or other viruses. Where the virus to be detected is an RNA virus (e.g., the virus has genetic material encoded by ribonucleic acid (RNA)), reverse transcriptase enzymes may be included with the reagents used so as to provide deoxyribonucleic acid (DNA) copies of the viral nucleic acid.
The human eye is a very sensitive detector, capable of detecting as few as 10 photons landing within a an area of about 50 micrometer in diameter. An example of a device having features disclosed herein in which the human eye may serve as a detector is shown in
A thermal assembly 154 may be configured to receive a reaction assembly 156, e.g., to hold a reaction assembly 156 in contact with thermal elements of the thermal assembly 154. As illustrated in
A detector 170 is used to detect light indicating the progress of the assay reactions and to detect the presence of target nucleic acid molecules and copies of such molecules. The human eye can be the detector 170, but a camera, photo-multiplier tube, a charge-coupled device, or other detector could also be used in addition to, or in place of, the eye of a human observer. Light is detected by a detector 170 via aperture 172 and may be aided by a lens or lenses 174 and a filter or filters 176. An aperture 172 may be an opening, or may be a window (e.g., a transparent or translucent covering across an opening), and may be an opening that may be covered by a shutter, a diaphragm, or other element. Where the detector 170 is a human eye, an eye-guard 178 may be helpful to protect the eye of the observer. An eye guard 178 may be a flexible eye-guard 178, and may assist in blocking external light from the eye. An eye guard 178 may also serve to position the eye of an observer, and may serve to attach other forms of detectors 170 as well.
The optical assembly 152 is designed to mechanically couple onto the thermal assembly 154, blocking external light and aligning its optical axis 180 to the reaction assembly 156. As shown, the optical assembly 152 may contain several lenses 174 (which may be positive and/or negative lenses) or none at all, depending on the light collection efficiency needed to detect an assay in the reaction assembly 156. A filter 176 or filters 176 may also be needed, particularly long pass filters 176 to block out light from the excitation source while passing the longer wavelength fluorescence. Arrow 181 indicates light traveling form the reaction assembly 156 towards lenses 174 and filter 176.
A device 150 as disclosed herein may be configured to be reusable, or to be disposable, and different portions or assemblies may individually be configured either for re-use or to be disposable. For example, the device 150 illustrated in
Assemblies as illustrated in
A sample may be introduced directly into a reaction assembly 156, or may be processed prior to introduction into a reaction assembly 156.
The top view (
After the reagent chamber 186 is filled with sample 198, the reaction assembly 156 is thermal cycled (
The reaction assembly 210 illustrated in
Cell debris and chemicals that do not electrophorese, and particles larger than the pores of matrix 226 will not enter the buffer chamber 212. The prevention of the entry of such cell debris, chemicals, and particles prevents the inhibition of PCR that might otherwise occur in the presence of such cell debris, chemicals, and particles. After sufficient time for most or all of the gDNA to be electrophoresed into the buffer chamber 212, the valves 228 open. Opening of valves 228 allows passage of material into reagent chamber 230 (as indicated by horizontal arrows shown in
In embodiments, a very small distance (e.g. ≦100 μm) separates the window 216 and the support 234 such that when the valves 228 open, capillary force draws the buffer 222 (including the gDNA from sample 238) from the buffer chamber 212 into the reagent chamber 230, displacing air in the reagent chamber 230 into the buffer chamber 212. Entry of sample solution including nucleic acids into reagent chamber 230 is indicated by arrows 243. All reagents 232 needed for the Taqman® reaction or other desired amplification reaction are available in the reagent chamber 230. Reagents 232 may be provided in the reagent chamber 230 by being pre-deposited in the reagent chamber 230 or may be provided by being pre-deposited in the buffer chamber 212 (from where they are drawn into the reagent chamber 230 by capillary action). Reagents 232 include, e.g., DNA probes, DNA primers, dNTPs, polymerase enzymes, magnesium, other salts, buffers, and other agents.
After the reagent chamber 230 is filled with sample 238, the reaction assembly 210 is thermal cycled (illustrated in
A further embodiment of devices and systems having features of the invention is illustrated in
A thermal assembly 304 is configured to receive or engage an assay cartridge 306, e.g., within a slot 322 or other receiving element, socket or receptacle. Thermal assembly 304 includes a thermal source 323, such as a peltier device, capable of heating and/or cooling an assay cartridge 306 received within or engaged with a thermal assembly 304. In embodiments, multiple assay cartridges 306 may be received within a thermal assembly 304, sequentially and/or simultaneously. A thermal assembly 304 may also include a power source (e.g., a battery 324), an on/off switch 325, and a controller 326. In embodiments, a thermal assembly 304 may be connected to an external power source in addition to, or instead of, including a battery 324. A controller 326 is typically configured to control the timing and temperature of thermal cycling of the thermal source 323, and may perform other functions as well (e.g., provide and/or record signals related to status or progress of a thermal cycle, or of a series of cycles, or of other operations). A cable 327 may connect to a light source 328. A thermal assembly may further include other features, including indicators (e.g., numerical or alphabetic displays, and light-emitting diodes (LEDs) for signaling status, progress or completion of an operation, occurrence of an unexpected event or malfunction), connectors suitable for data transfer.
Detection assembly 308 is a further element of a system 300. Detection assembly 308 is configured to engage and receive a light source 328, which, as illustrated in
An assay cartridge 306 as illustrated in
A detection assembly 308 engaged with an assay cartridge 306 is shown in
As shown in
Sample preparation within a preparation module 316 is further illustrated in
As illustrated in
Operation of a cross-electrophoresis preparation module 350 is illustrated in
Following the electrophoresis illustrated in
Opening third valve 384 allows nucleic acid molecules 394 in elution buffer 388 to flow out of output chamber 368 for analysis. For example, an output chamber 368 may be connected to input channel 342 and assay chambers 320 as illustrated in
As illustrated in
Following introduction of nucleic acid molecules 394 and reagents, assay chamber 320 and its contents are subjected to thermal cycling effective to amplify nucleic acid molecules 394 present in sample 354 within assay chamber 320. Amplified target nucleic acid molecules 410 shown in
Thus, as illustrated in
It will be understood that multiple target nucleic acid molecules may be detected with a single device. Different sets of probes and primers directed to different target nucleic acid molecules may be provided in different assay chambers 320, or different reaction chambers 186, or in different wells as discussed in the specification and the Examples above. In embodiments, different sets of probes and primers directed to different target nucleic acid molecules may be provided in a single assay chamber 320, or a single reaction chamber 186, or in a single well as discussed above. In such embodiments comprising different sets of probes and primers directed to different target nucleic acid molecules within a single chamber, well, or region, amplification of the different target nucleic acid molecules occurs at the same time within such single chambers, wells, or regions. Different target nucleic acid molecules may be detected and identified within a single chamber, well, or region, for example, where the probes and primers for different targets have different fluorescent molecules, allowing detection of each target by its distinctive identifying fluorescent signal.
Probes and/or primers 404 may be directed to target nucleic acid molecules found, for example, in pathogens, contaminants, or cancerous tissues. Pathogens whose presence may be tested for may be bacterial, viral, fungal, or other pathogens, and include, for example, tuberculosis, anthrax, streptococcus, staphylococcus, and other bacterial pathogens, viral, fungal and other pathogens, including other pathogens listed elsewhere in the specification.
It will be understood that the description of the methods, devices and systems herein is not meant to be limiting. Moreover, although individual features of the embodiments may be shown in some of the drawings and not in others, or described in conjunction with some devices and methods and not with others, those skilled in the art will recognize that individual features of one embodiment may be combined with any or all of the features of another embodiment.
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional application Ser. No. 60/534,095 filed Dec. 31, 2003, the contents of which application is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60534095 | Dec 2003 | US |
