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WO2010111776A1 - Procédé et système destinés à l'amplification et à la quantification de multiples acides nucléiques - Google Patents

Procédé et système destinés à l'amplification et à la quantification de multiples acides nucléiques Download PDF

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WO2010111776A1
WO2010111776A1 PCT/CA2010/000466 CA2010000466W WO2010111776A1 WO 2010111776 A1 WO2010111776 A1 WO 2010111776A1 CA 2010000466 W CA2010000466 W CA 2010000466W WO 2010111776 A1 WO2010111776 A1 WO 2010111776A1
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distinct
microsphere
nucleic acid
bound
beads
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Yuanmin Wu
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FUNGLYN BIOTECH Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/45—Magnetic mixers; Mixers with magnetically driven stirrers
    • B01F33/452—Magnetic mixers; Mixers with magnetically driven stirrers using independent floating stirring elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples

Definitions

  • TITLE METHOD AND SYSTEM FOR MULTIPLE NUCLEIC ACID AMPLIFICATION AND QUANTITATION
  • FIELD [0001] The application relates to a method, system and kits for amplifying and quantitating multiple distinct nucleic acids in a single chamber.
  • Mullis et al. 4683202, Mullis
  • PCR can increase the number of copies of target molecules with high specificity.
  • Multiplex PCR is a variant of PCR which enables simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. Since its first description in 1988 by Chamberlain et al [Nucleic Acids Res. 1988 Dec 9;16(23):11141-56, Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification, Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT.], this method has been applied in many areas of DNA testing, including analyses of deletions, mutations, and polymorphisms, or quantitative assays and reverse transcription PCR.
  • qPCR is available for amplifying and detecting a limited number of targets, such as one or two amplicons, in a single tube.
  • targets such as one or two amplicons
  • the template is divided into equal-sizes and put into separate wells (typically 48, 96, 192, or 384). This type of method creates the issues of rising costs and is labor consuming.
  • Q-PCR provides a convenient and accurate method for measuring the amount of nucleic acid sequences in a sample, it is limited to a single sequence or a small number of sequences in a single fluid volume.
  • a DNA microarray is a multiplex technology that consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles of a specific DNA sequence. This can be a short section of a gene or other DNA element that are used as probes to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions.
  • Probe-target hybridization is usually detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target.
  • fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target.
  • probes are attached to a solid surface by a covalent bond to a chemical matrix (via epoxy-silane, amino-silane, lysine, polyacrylamide or others).
  • the solid surface can be glass or a silicon chip, in which case they are commonly known as gene chip.
  • Variations in data obtained from microarray analysis is usually caused in three ways: measurement error associated with the reading of fluorescent signals; natural variability in the biological system, and technical variations due to the extraction, labelling and hybridisation of samples (Fundamentals of experimental design for cDNA microarrays, Nature Genetics 32, 490 - 495, Churchill, 2002) .
  • Single-molecule PCR using water-in-oil emulsion is a technology used for clonal amplification of a single molecule in an oil/water emulsion.
  • PCR millions of clonally amplified molecules are bound to a sequencing bead.
  • clonal bead populations are generated in water-in-oil microreactors containing primer coupled beads, template, PCR components, and primers. Templates (either a fragment or mate-paired library) are amplified in about 10- ⁇ m microreactors.
  • a polymerase extends from the primer adapter after the template anneals to the primer-coupled beads.
  • a complementary sequence is extended off the bead surface followed by template dissociation.
  • the method of ePCR has been applied for providing high copies of DNA sequencing involving variations of cloning or in vitro amplification, and tens of millions for a human genome, necessary for sequencing a whole genome economically.
  • United States Patent, 7,323,305 (Leamon, John H. et al.) describe a large-scale parallel pyrosequencing system capable of sequencing roughly 400-600 megabases of DNA per 10-hour run on the Genome Sequencer FLX with GS FLX Titanium series reagents.
  • the technology is known for its unbiased sample preparation and long, highly accurate sequence reads (400-500 base pairs in length), including paired reads.
  • Software analysis tools including an assembler, mapper and amplicon variant analyzer, are included with the system. The system relies on fixing nebulized and adapter-ligated DNA fragments to small DNA-capture beads in a water- in-oil emulsion.
  • Each DNA-bound bead is placed into a -29 ⁇ m well on a PicoTiterPlate, a fiber optic chip. A mix of enzymes such as DNA polymerase, ATP sulfurylase, and luciferase are also packed into the well. The PicoTiterPlate is then placed into the GS FLX System for sequencing.
  • United States Patent, 7,425,431 describe bead- based arrays and methods of making bead-based arrays.
  • the arrays have a plurality of beads wherein an individual bead has a population of substantially identical nucleic acid sequences attached to them and wherein the population of substantially identical nucleic acid sequences differs in sequence from the population of substantially identical nucleic acid sequences attached to other beads.
  • Emulsion PCR is applied for amplifying.
  • the plurality of beads is immobilized in a semi-solid medium to form an array.
  • the semi-solid medium can be made from polyacrylamide, cellulose, polyamide, cross-linked agarose, cross-linked dextran or cross-linked polyethylene glycol.
  • the semi-solid medium has x, y and z axes, and the plurality of beads is randomly arranged relative to the x and y axes.
  • the beads can be immobilized as a monolayer, for example, near the top surface of the semisolid medium.
  • the present application provides a method of amplifying and quantitating two or more distinct nucleic acid sequences in a single chamber comprising: a) adding a test sample to a solution in the single chamber; said solution comprising: i) two or more microspheres; each microsphere comprising a distinct detectable label and at least one bound copy of a distinct forward primer for amplifying a distinct nucleic acid; ii) two or more distinct reverse primers; each reverse primer related to the distinct forward primer on each microsphere for amplifying each distinct nucleic acid; and iii) (a) two or more nucleic acid probes; each probe specific to each distinct nucleic acid bound to the microsphere and each probe comprising a quencher and a reporter; or (b) a double-stranded DNA-specific dye; b) amplifying the two or more distinct nucleic acid sequences under continual motion for at least one cycle; wherein the cycle comprises a denaturation phase, a primer annealing phase, and an
  • steps b)-d) are repeated at least one time.
  • 10 or more distinct nucleic acids are amplified.
  • 20 or more distinct nucleic acids are amplified.
  • 100 or more distinct nucleic acids are amplified.
  • step d) comprises two or more optical measurements. In an embodiment, a first optical measurement detects the fluorescence of each distinct bead and a second optical measurement detects the fluorescence from the hybridized probes or intercalated dye.
  • the forward primer is attached to the microsphere by a coupling group, such as NH 2 , COOH or an affinity protein, such as streptavidin or biotin.
  • a coupling group such as NH 2 , COOH or an affinity protein, such as streptavidin or biotin.
  • the forward primer is covalently attached to the microsphere by a coupling group.
  • the forward primer is attached to the microsphere via a coupling group and a spacer, such as a (dT)n oligomer. In an embodiment, the spacer is labeled.
  • the microsphere can be any suitable material, such as an inorganic material, a natural polymer or a synthetic polymer.
  • the microsphere is from 0.1 microns to 100 microns.
  • the microsphere is from 1 to 25 microns.
  • the microsphere is from 5 to 10 microns.
  • the probe is hairpin shaped with an internally quenched fluorophore which is restored when the probe hybridizes to target sequence.
  • the application provides a system for amplifying and quantifying nucleic acids in a sample comprising: a) an amplification chamber for receiving (i) a fluorescently- labeled bead array comprising a surface coupled with an array of nucleic acid forward primers and (ii) a solution to be held in contact with the beads, the solution comprising reverse primers; b) a temperature controller for carrying out multiple temperature phases and cycles comprising (i) a heating and cooling module and (ii) a temperature sensor; c) a force controller for controlling the motion of the beads in the solution; d) a detector house comprising (i) a light source; (ii) a lens; (iii) an optical filter; and (iv) a detector; wherein the detector house is capable of delivering light to excite the solution and the beads
  • the system further comprises a system control block comprising a computer and software capable of signal collection and image analysis.
  • the image analysis distinguishes the beads and determines the amounts of amplified products hybridized to a probe or intercalated with a double-stranded DNA-specific dye using the detected light.
  • kits comprising two or more distinct microspheres, each microsphere comprising a distinct detectable label.
  • a kit comprising (i) two or more distinct microspheres, each microsphere comprising at least one copy of a bound distinct forward primer and (ii) a solution comprising two or more reverse primers and (a) two or more labeled nucleic acid probes or (b) a double-stranded DNA-specific dye.
  • Figure 1 shows a schematic of the amplification phase of an embodiment described herein.
  • Figure 2 shows a schematic of the fluorescence detection phase of an embodiment described herein.
  • Figure 3 shows a schematic of an optical system used in an embodiment described herein.
  • Figure 4 shows a schematic of primers bound on the surfaces of the beads and hybridized with templates.
  • Figure 5 shows a schematic of the extension phase of an embodiment described herein.
  • Figure 6a shows probes bound to amplified nucleic acid sequence.
  • Figure 6b shows double stranded DNA specific dye intercalated with amplified nucleic acid sequence.
  • Figure 7 shows a molecular beacon probe that can be used in an embodiment described herein.
  • Figure 8 shows results of a calibration of microspheres.
  • Figure 8a shows an image of five different bead with the scale of 10000.
  • Figure 8b shows an image of five different bead with the scale of 2000.
  • Figure 8c shows results of fluorescence intensity of five different bead.
  • Figure 9 shows the results of amplification with two pairs of primers and molecular beacon probes.
  • Figure 10 shows the results of amplification with five pairs of primers and molecular beacon probes.
  • Figure 11 shows the results of amplification with two pairs of primers and a double-stranded DNA-specific dye: EvaGreen.
  • Figure 12 shows the results of amplification with five pairs of primers and a double-stranded DNA-specific dye: EvaGreen.
  • the present application provides novel methods, systems and kits for amplifying and quantitating nucleic acids from a sample.
  • the application provides a method of detecting multiple nucleotide sequences in a single compartment by performing real-time measurements of amplicon bound on microspheres.
  • the method involves performing multiplex PCR amplifications in a solution and the amplicons hybridized with a probe or intercalated with a dye, wherein the solution contains the nucleic acid sequences of interest.
  • a forward primer is bound to one bead which is distinguishable while the reverse primer is solved in solution.
  • the microspheres are perpetually moving in solution during amplification but form a monolayer during measurement of optical signals ( Figures 1 and 2).
  • each distinct forward primer hybridizes to the complementary distinct nucleic acid ( Figure 4) and the DNA is extended ( Figure 5). Once extended, each distinct extended nucleic acid is bound by a distinct probe or intercalated with a dye ( Figure 6a and 6b).
  • the application provides a method of amplifying and quantitating two or more distinct nucleic acid sequences in a single chamber comprising: a) adding a test sample to a solution in the single chamber, said solution comprising: i) two or more microspheres; each microsphere comprising a distinct detectable label and at least one bound copy of a distinct forward primer for amplifying a distinct nucleic acid; ii) two or more distinct reverse primers; each reverse primer related to the distinct forward primer on each microsphere for amplifying each distinct nucleic acid; and iii) (a) two or more nucleic acid probes; each probe specific to each distinct nucleic acid bound on the microsphere and each probe comprising a quencher and a reporter; or (b) a double-stranded DNA specific dye; b) amplifying the two or more distinct nucleic acid sequences under continual motion for at least one cycle; wherein the cycle comprises a denaturation phase, a primer annea
  • Amplification in step b) may be performed by any method of amplification.
  • Methods of amplification include, without limitation, polymerase chain reaction (PCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA).
  • amplification in step b) is performed by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Polymerase chain reaction refers to a process for amplifying a target nucleic acid sequence as generally described in lnnis et al, Academic Press, 1990 in Mullis el al., U.S. Pat. No. 4,863,195 and Mullis, U.S. Patent No. 4,683,202.
  • Conditions for amplifying a nucleic acid template are described in M.A. lnnis and D. H. Gelfand, PCR Protocols, A Guide to Methods and Applications M.A. Innis, D. H. Gelfand, JJ. Sninsky and T.J. White eds, pp3-12, Academic Press 1989.
  • Conditions which may be employed in the methods of the disclosure using PCR are those which permit hybridization and amplification reactions to proceed in the presence of DNA in a sample and appropriate complementary hybridization primers.
  • Conditions suitable for a polymerase chain reaction are generally known in the art. For example, see M.A. Innis and D. H. Gelfand, PCR Protocols, A guide to Methods and Applications M.A. Innis, D. H. Gelfand, JJ. Sninsky and TJ. White eds, pp3-12, Academic Press 1989.
  • the PCR utilizes polymerase obtained from the thermophilic bacterium Thermus aquatics (Taq polymerase, GeneAmp Kit, Perkin Elmer Cetus) or other thermostable polymerase.
  • Typical PCR conditions include a pre-amplification step at a high temperature, for example 94-95 0 C for 2-10 minutes; followed by an amplification cycle including a denaturation phase, optionally at 90-98 0 C, an annealing phase, optionally at 37-65 0 C, and an extension phase, optionally at 70-75 0 C.
  • the amplification method can use temperature cycling or be isothermal.
  • the amplification method can be exponential or linear.
  • a temperature cycle generally corresponds to an amplification cycle.
  • Isothermal amplifications can in some cases have amplification cycles, such as denaturing cycles, and in other cases, the isothermal amplification reaction will occur monotonically without any specific amplification cycle.
  • the PCR generally requires several components:
  • a DNA polymerase e.g. Taq polymerase or another DNA polymerase, used to synthesize a DNA copy of the region to be amplified;
  • dNTPs Deoxynucleotide triphosphates
  • a buffer solution which provides a suitable chemical environment for optimum activity and stability of the DNA polymerase.
  • a divalent cation such as magnesium or manganese ions.
  • the reaction Prior to the first cycle, the reaction can be subjected to a hold step during an initialization step, the PCR reaction can be heated to a temperature of 93-98°C, and this temperature is then held for 1-10 minutes. This first hold is employed to ensure that most of the DNA template and primers are denatured, i.e., that the DNA is melted by disrupting the hydrogen bonds between complementary bases of the DNA strands.
  • Temperature cycling can then begin with one step at, for example, 90-98 0 C, optionally 93-98 0 C, for e.g. 5-30 seconds (denaturation step).
  • the denaturation is followed by the annealing step.
  • the reaction temperature is lowered so that the primers can anneal to the single-stranded DNA template and also the probes can be hybridized on the amplicons which are attached on the beads.
  • the temperature at this step depends on the melting temperature of the primers, and is usually between 37 and 65 0 C, optionally 50-60 0 C, for 20-40 seconds.
  • the annealing step is followed by an extension step during which the DNA polymerase synthesizes new DNA strands complementary to the DNA template strands.
  • the temperature at this step depends on the DNA polymerase used. For example, Taq polymerase has a temperature optimum of about 70-75 0 C; thus, a temperature of 72°C may be used.
  • the DNA polymerase condenses the 5'-phosphate group of the dNTPs with the 3'- hydroxyl group at the end of the nascent (extending) DNA strand, i.e., the polymerase adds dNTP's that are complementary to the template in 5' to 3' direction, thus reading the template in 3' to 5' direction.
  • the extension time may depend on both the DNA polymerase used and on the length of the DNA fragment to be amplified.
  • each temperature cycle has three phases; denaturation, annealing, and elongation.
  • the amplified products from the amplification are referred to as amplicons.
  • Step d) allows quantitation of the amplicons.
  • the amount of amplicons produced can be measured during or at the end of each amplification cycle. After a number of amplification cycles, the amount of nucleic acid in the original sample can be determined by analyzing the build- up of amplicon over the number of amplification cycles.
  • Step d) allows for the determination of the presence or amount of very small amounts of sample, in some cases, down to the detection of a single molecule.
  • the present application provides a bead array of multiple nucleic acids, thus allowing for the measurement of the presence or amount of multiple nucleic acid sequences in the same sample during the same amplification reaction.
  • the present application provides for the determination of more than about 3, 5, 10, 20, 30, 40, 50, 75, 100, 1000, 10,000, or more nucleic acid sequences in the same sample during the same amplification reaction. Accordingly, in one embodiment, 10 or more distinct nucleic acids are amplified. In yet another embodiment, 20 or more distinct nucleic acids are amplified. In a further embodiment, 100 or more distinct nucleic acids are amplified.
  • amplicon is a molecular species that is created from the amplification of a nucleotide sequence.
  • An amplicon is typically a polynucleotide such as RNA or DNA or mixtures thereof, in which the sequence of nucleotides in the amplicon correlates with the sequence of the nucleotide sequence from which it was generated (i.e. either corresponding to or complementary to the sequence).
  • the amplicon can be either single stranded or double stranded.
  • the amplicon being measured is single stranded and bound to the microsphere.
  • the amplicon that is being measured is double-stranded and bound to the microsphere.
  • isolated refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
  • nucleic acid molecule is intended to include unmodified DNA or RNA or modified DNA or RNA.
  • the nucleic acid molecules or polynucleotides of the disclosure can be composed of single- and double stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically double-stranded or a mixture of single- and double-stranded regions.
  • the nucleic acid molecules can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the nucleic acid molecules of the disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritiated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus “nucleic acid molecule” embraces chemically, enzymatically, or metabolically modified forms.
  • polynucleotide shall have a corresponding meaning.
  • nucleotide sequence or “nucleic acid sequence” as used in this context refers to a sequence of nucleotides of which it is desired to know the presence or amount.
  • the length of the sequence can be any length that can be amplified into amplicons, for example up to about 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or more than 1000 nucleotides in length. It will be understood by those of skill in the art that as the number of nucleotides gets very large, the amplification can be less effective.
  • the general quantitative PCR method can be described as follows.
  • a PCR reaction is carried out with a pair of primers designed to amplify a given nucleic acid sequence in a sample.
  • the appropriate enzymes and dNTP's are added to the reaction, and the reaction is subjected to a number of amplification cycles.
  • the amount of amplicon generated from each cycle is detected, but in the early cycles, the amount of amplicon can be below the detection threshold.
  • the amplification can be seen as occurring in two phases, an exponential phase, followed by a non-exponential plateau phase. During the exponential phase, the amount of PCR product approximately doubles in each cycle.
  • reaction components are consumed, and ultimately one or more of the components becomes limiting. At this point, the reaction slows and enters the plateau phase.
  • the amount of amplicon remains at or below background levels, and increases are not detectable, even though amplicon product accumulates exponentially. Eventually, enough amplified product accumulates to yield a detectable signal.
  • the cycle number at which this occurs is called the threshold cycle, or CT. Since the CT value is measured in the exponential phase when reagents are not limited, detection at step d) can be used to reliably and accurately calculate the initial amount of template present in the reaction.
  • the CT of a reaction is determined mainly by the amount of nucleic acid sequence corresponding to amplicon present at the start of the amplification reaction. If a large amount of template is present at the start of the reaction, relatively few amplification cycles will be required to accumulate enough products to give a fluorescent signal above background.
  • steps b)-d) can be repeated at least 1 , 2, 3, 4, 5, 7,
  • steps b)-d) are repeated at least one time. In another embodiment, steps b)-d) are repeated at least 10 times. In yet another embodiment, steps b)-d) are repeated 20-40 times.
  • the microspheres are kept under continual motion, for example, by a magnetic stirrer under a magnetic force or by exposure to an ultrasonic force. In one embodiment, the continual motion is perpetual motion.
  • the solution is stabilized in step c) which allows the microspheres to form a monolayer.
  • the monolayer may be formed by a force that attracts the microspheres to the bottom of the solution chamber.
  • the force is a magnetic, electrical or centrifugal force. These types of forces can be precisely adjusted and controlled.
  • a second force works with the force in step b) to form the monolayer.
  • a distinct primer refers to a primer that amplifies a particular or distinct nucleic acid sequence and includes, without limitation, a primer or more than one primer. Each distinct forward and reverse primer is designed to amplify a distinct nucleic acid sequence.
  • the length and bases of primers for use in PCR are selected so that they will hybridize to different strands of the desired sequence and at relative positions along the sequence such that an extension product synthesized from one primer when it is separated from its template can serve as a template for extension of the other primer into a nucleic acid of defined length.
  • Primers which may be used in the disclosure are oligonucleotides, i.e., molecules containing two or more deoxyribonucleotides which occur naturally as in a purified restriction endonuclease digest or are produced synthetically using techniques known in the art such as for example phosphotriester and phosphodiester methods (See Good et al. Nucl. Acid Res 4:2157, 1977) or automated techniques (See for example, Conolly, B.A. Nucleic Acids Res. 15:15(7): 3131 , 1987).
  • the primers are capable of acting as a point of initiation of synthesis when placed under conditions which permit the synthesis of a primer extension product which is complementary to a distinct nucleic acid sequence, i.e., in the presence of nucleotide substrates, an agent for polymerization such as DNA polymerase and at suitable temperature and pH.
  • the primers are sequences that do not form secondary structures by base pairing with other copies of the primer or sequences that form a hairpin configuration.
  • the primers are usually short, chemically synthesized DNA molecules with a length of about 10 to about 30 bases.
  • the length of primers can be for example about 20-30 nucleotides, and the sequence of the primers are complementary to the beginning and the end of the DNA fragment to be amplified. They anneal to the DNA template at these starting and ending points, where DNA polymerase binds and begins the synthesis of the new DNA strand.
  • Pairs of primers should generally have the similar melting temperatures (Tm).
  • Tm melting temperatures
  • a primer with a Tm significantly higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence, while Tm significantly lower than the annealing temperature may fail to anneal and extend at all.
  • the primers may contain non- complementary sequences provided that a sufficient amount of the primer contains a sequence which is complementary to the nucleic acid of interest or oligonucleotide fragment thereof, which is to be amplified.
  • the forward primer is attached to the microsphere.
  • the forward primer is attached by a coupling group.
  • the term "coupling group" as used herein refers to a moiety that binds directly to the bead and binds to a nucleotide sequence.
  • the coupling group is NH 2 , COOH or an affinity binding protein, such as biotin or streptavidin.
  • the coupling group is covalently attached to the microsphere.
  • the forward primer is attached to the microsphere by a coupling group and a spacer.
  • the spacer comprises a (dT)n oligomer, wherein "n" can be any length of nucleotides, for example, 10-100 nucleotides, optionally 40-60 nucleotides, and wherein (dT) can be a T nucleotide or any other nucleotide or combinations of nucleotides.
  • the spacer is a chemical, such as dodecyl.
  • the distinct detectable label on the microsphere is attached to the spacer. The spacer is utilized to enhance the efficiency of hybridization of the terminally anchored oligonucleotide primer to its template.
  • microsphere refers to diameter particles ranging in size from 0.1 microns to 100 microns. In an embodiment, the microsphere is 1 to 25 microns. In another embodiment, the microsphere is 5 to 10 microns.
  • beam herein is used interchangeably with the term microsphere.
  • the microsphere can be made of any suitable material. In one embodiment, the microsphere is an inorganic material, a natural polymer or a synthetic polymer. For example, these materials include, without limitation, uniform polymeric, silica and magnetic microsphere particles.
  • the microsphere may be coupled to a large number of the same distinct forward primers, which is complementary to a region of a template DNA or distinct nucleic acid of interest, for example, at least 1 , 10, 100, 1000, 10,000 or 100,000 copies of the same distinct forward primer can be coupled to each microsphere.
  • the number of copies coupled on the microsphere will depend on the exciting light source and the sensitivity of the detector.
  • test sample refers to a sample comprising nucleic acids.
  • sample types including, without limitation, cDNA, genomic DNA, RNA, cells, or viruses may be used.
  • Method for extracting the nucleic acids from a sample are known in the art, for example, nucleic acids of the sample may be extracted following the protocol in Molecular Cloning [Molecular cloning: a laboratory manual. 2 J Sambrook, EF Fritsch, T Maniatis - 1989 - Cold Spring Harbor Laboratory Press Cold Spring Harbor, New York].
  • probe refers to a sequence that hybridizes to the target distinct nucleic acid sequence.
  • a probe would generally be 20 nucleotides long or be at least 20 nucleotides long.
  • the probe could also be 25, 30, 35, 40, 45, 50, 55, 60 or more nucleotides in length and the probe can include the full length of the complement to the sequence to which it is intended to bind.
  • the probes used herein are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in solutions.
  • the probes comprise a reporter and a quencher and are measured by measuring the optical signals from the amplified sequences.
  • reporter refers to a moiety attached, optionally covalently, to the probe that produces a signal, such as a light or fluorescent signal.
  • signal such as a light or fluorescent signal.
  • fluorescent molecules can be utilized including, without limitation, small molecules, fluorescent proteins and quantum dots.
  • quencher refers to a moiety attached, optionally covalently, to the probe that quenches or decreases the signal intensity of a reporter.
  • the probe is hairpin shaped with an internally quenched fluorophore which is restored when the probe hybridizes to target sequence.
  • This type of probe is typically referred to as a molecular beacon (see Figure 7).
  • these molecules are non-fluorescent, because the stem hybrid keeps the fluorophore close to the quencher.
  • the probe sequence in the loop hybridizes to its target, forming a rigid double helix, a conformational reorganization occurs that separates the quencher from the fluorophore, restoring fluorescence such that the optical signals from the fluorophore can be measured.
  • Molecular beacons can be utilized that are complementary to a sequence in the middle of the expected amplicon bound to the beads.
  • the length of their arm sequences should be chosen so that a stem is formed at the annealing temperature of the polymerase chain reaction.
  • the length of the loop sequence should be chosen so that the probe-target hybrid is stable at the annealing temperature. Whether a molecular beacon actually exhibits these designed features is determined by obtaining thermal denaturation profiles.
  • the molecular beacons with appropriate thermal denaturation characteristics are included in each reaction at a concentration similar to the concentration of the primers. During the denaturation step, the molecular beacons assume a random coil configuration and fluorescence can be emitted.
  • molecular beacons As the temperature is lowered to allow annealing of the primers, stem hybrids form rapidly, preventing fluorescence. However, at the annealing temperature, molecular beacons also bind to the amplicons and generate fluorescence when a specific light is excited on the hybrid of the probe and amplicon bound on the bead. When the temperature is raised to allow primer extension, the molecular beacons dissociate from their targets and do not interfere with polymerization. A new hybridization takes place in the annealing step of every cycle, and the intensity of the resulting fluorescence indicates the amount of accumulated amplicon.
  • quenching of molecular beacons has been shown to occur through a direct transfer of energy from the fluorophore to quencher. Consequently, a common quencher molecule can be used, increasing the number of possible fluorophores that can easily be used as reporters.
  • An oligonucleotide probe will generally hybridize, bind, or duplex, with a particular nucleotide sequence of an amplicon under stringent conditions even when that sequence is present in a complex mixture.
  • Appropriate stringency conditions which promote nucleic acid hybridization are known to those skilled in the art, or may be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • stringent hybridization conditions as used herein means that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule.
  • the hybridizing portion is at least 50% the length with respect to one of the polynucleotide sequences encoding a polypeptide.
  • a double-stranded DNA-specific dye is used that is able to intercalate with double-stranded amplicons and the fluorescence from the dye indicates the amount of the amplicon.
  • dyes are known in the art and include, without limitation, SYBR Green I and EvaGreen.
  • step d) comprises two or more optical measurements.
  • a first optical measurement detects the distinct detectable label of each bead and a second optical measurement detects the fluorescence from the hybridized probes or intercalated dyes.
  • the two or more optical measurements are carried out for different wavelength fluorescent signals. The time of the measurements will be dependent on how many colors of dyes are coated on the beads.
  • the probes or dyes will emit signal from each addressable location which can be detected using, for example, a series of lenses and a light detector in Fig. 4 (e.g., a charge-couple device (CCD) camera or a complementary metal-oxide-semiconductor (CMOS) image sensor and or a
  • Photomultiplier Tube When the reporter does not, itself, emit a light signal, there is measurement of fluorescence background from the solution.
  • the signal at each addressable location is measured in real time, for example, by a CCD camera focused on the monolayer surface. As the signal at any location changes as a result of binding, the change is measured.
  • binding kinetics or “hybridization kinetics” as used herein refers to the rate at which the binding of the amplicon to the probe occurs in a binding/hybridization reaction.
  • binding reaction as used herein describes the reaction between probes and amplicons.
  • hybridization reaction is a binding reaction wherein the binding comprises hybridization, for example of complementary nucleic acids. In some cases, binding reaction refers to the concurrent binding reactions of multiple amplicons and probes, and in other cases, the term binding reaction refers to the reaction between a single probe with a single amplicon.
  • the binding or hybridization kinetics can provide information about the characteristics of the probe-amplicon binding such as the strength of binding, the concentration of amplicon, the competitive binding of an amplicon, the density of the probes, or the existence and amount of cross-hybridization.
  • the binding conditions can be changed in order to explore multiple sets of binding conditions during the same binding experiment.
  • the condition which is changed can be, for example, any condition that affects the rate of binding of amplicon to probe.
  • the condition which is changed includes, without limitation, temperature, pH, stringency, amplicon concentration, ionic strength, an electric field, or the addition of a competitive binding compound.
  • the signal or optical measurement detected can be correlated with the binding of amplicons to the plurality of probes or of intercalation of dye.
  • the type of signals appropriate for the application is any signal that correlates to the amount of amplicon.
  • Appropriate signals include, for example, light signals with the wavelength of 200 to 1000 nm or higher.
  • Examples of optical signals useful in the present application are signals from fluorescence and luminescence.
  • the terms "optical” and "light” are used interchangeably. While some are described with reference to visible (optical) light, the descriptions are not meant to limit to those particular electromagnetic frequencies.
  • the signal measured is generally the signal from the region around the beads.
  • the measurements from the beads provide information to distinguish the beads from each other.
  • the measurement of hybridization of the probes to the amplicons attached on the beads or to intercalation of the dye with the amplicons attached on the beads provides the information of amplicon measurement.
  • the measurement can be correlated with the amount of amplicon present.
  • the "distinct detectable label” as used herein refers to a detectable label on one bead which is distinct or differentially detectable compared to a detectable label on another bead. In one embodiment, fluorescence is applied for distinguishing the different beads.
  • the beads can be coated with different color and/or content of dyes and are coupled with different primers to produce amplicons with different DNA sequences bound to microsphere.
  • Dyes with different wavelengths can be used to distinguish the different microspheres, thus, the microspheres are addressable by the wavelength and the number of copies is detected by intensity at each addressable location.
  • the probe is labeled with the same dye and intensity is measured, whereas the microspheres are labeled with different dyes so that they are addressable or the microspheres are labeled with a different dye than the double-stranded DNA-specific dye so that the microspheres are addressable.
  • the fluorescence signal emitted surrounding a particular addressable microsphere can be measured.
  • fluorescence refers to the process wherein a molecule relaxes to its ground state from an electronically excited state by emission of a photon.
  • fluorescence also encompasses phosphorescence.
  • a molecule is promoted to an electronically excited state generally by the absorption of ultraviolet, visible, or near infrared radiation. The excited molecule then decays back to the ground state, or to a lower-lying excited electronic state, by emission of light.
  • An advantage of fluorescence for the methods described herein is its high sensitivity. Fluorimetry may achieve limits of detection several orders of magnitude lower than for absorption. Limits of detection will depend on many factors, such as the intensity of exciting light source and the sensitivity of the detection.
  • the limit of detection is 10 "10 M.
  • a lower limit of detection is possible for intensely fluorescent molecules; in favorable cases under stringently controlled conditions, the ultimate limit of detection (a single molecule) may be reached.
  • the use of an image of the fluorescently labeled probe may be obtained before binding has occurred in order to effectively establish a baseline signal for the state where no binding of amplicon to probe has occurred.
  • the application provides a system for amplifying and quantifying nucleic acids in a sample comprising: a) an amplification chamber capable of receiving (i) a fluorescently-labeled bead array comprising a surface coupled with an array of nucleic acid forward primers and (ii) a solution to be held in contact with the beads, the solution comprising reverse primers; b) a temperature controller capable of carrying out multiple temperature phases and cycles comprising (i) a heating and cooling module and (ii) a temperature sensor; c) a force controller for controlling the motion of the beads in the solution; d) a detector house comprising (i) a light source; (ii) a lens; (iii) an optical filter; and (iv) a detector; wherein the detector house is capable of delivering light to excite the solution and the beads to allow detection of fluorophores.
  • the "chamber” as used herein refers to a vessel that is made of a material that allows light and emission through, including, without limitation, any chamber that has a flat base, such as a flat-based tube or well and includes, without limitation, a chamber made of quartz, glass or a transparent plastic material.
  • the system further comprises a system control block comprising a computer and software capable of signal collection and image analysis.
  • the image analysis distinguishes the beads and determines the amounts of amplified products hybridized to the probe or intercalated with the dye using the detected light. Images may be acquired by a CCD-based detector mounted on a microscope.
  • the temperature controller can control the temperature at any place within the system that controls the temperature of the solution.
  • any means can be used for controlling the temperature including, without limitation, resistive heaters, Peltier devices, infrared, fluid or gas flow.
  • the temperature is controlled to within 0.1 0 C.
  • the controller is capable of changing the temperature during the amplification in order to define the phases of the temperature cycles.
  • the temperature sensor measures temperature at one or multiple locations in the solution. The temperature can be measured by any means including, without limitation, by thermistor or thermocouple.
  • Any force controller for controlling the motion of the beads in the solution can be used, including, without limitation, a set of magnetic forces.
  • the set of magnetic forces may be from different directions that permits a magnetic stirrer to shake the beads during amplification but allows a monolayer to form during optical measurement.
  • the exciting light source includes, without limitation, a halogen lamp, a mercury arc lamp, a xenon arc lamp, a mercury halide arc lamp, LED and lasers.
  • the detector includes, without limitation, a CCD or CMOS camera, a PMT, or a Photodiode.
  • the present application provides a kit comprising two or more distinct microspheres for use in the methods and systems disclosed herein, each microsphere comprising a distinct detectable label.
  • the user could design and attach primers for amplification and prepare a labeled nucleic acid probe or use a double- stranded DNA-specific dye for quantitation of a desired nucleic acid.
  • the application provides a kit comprising (i) two or more distinct microspheres for use in the methods and systems disclosed herein, each microsphere comprising at least one copy of a bound distinct forward primer and (ii) a solution comprising two or more reverse primers and (a) two or more labeled nucleic acid probes or (b) a double- stranded DNA-specific dye.
  • the microsphere comprises at least 1 , 10, 100, 1000, 10,000 or 100,000 copies of bound distinct forward primers.
  • the microspheres further comprise a spacer.
  • the microspheres comprise a spacer and a (dT)n oligomer.
  • the kit may further comprise instructions for use.
  • kits and systems described herein are useful for the determination of the presence and amount of multiple nucleotide sequences in a sample.
  • the methods, systems and kits may be used for example, in expression monitoring and for measuring genetic information.
  • the application allows for many nucleotide sequences relating to genes, e.g. 10, 100, 1 ,000, 10,000 or more genes to be analyzed at once.
  • expression monitoring refers to the determination of levels of expression of particular, typically preselected, genes. For example, amplicons derived from nucleic acid samples such as messenger RNA that reflect the amount of expression of genes are hybridized to the arrays during or after amplification cycles, and the resulting hybridization signal as a function of time provides an indication of the amount of amplicon which can then be used to determine the level of expression of each gene of interest.
  • the whole transcriptome can be measured comprising all or a substantial portion of the expression in a cell, or group of cells.
  • the expression of only a few genes, such as 5 to 100 genes is measured, for example, to diagnose a specific condition.
  • the array has a high degree of probe redundancy (multiple probes per gene) and the expression monitoring methods provide accurate measurement and do not require comparison to a reference nucleic acid.
  • the methods, systems and kits described herein may be used in a wide variety of circumstances including, without limitation, for determining the presence and amount of multiple nucleotide sequences in a sample, expression monitoring, measuring genetic information, for detection of disease, identification of differential gene expression between two samples (e.g., a pathological as compared to a healthy sample), screening for compositions that up regulate or down regulate the expression of particular genes, for the analysis of genetic DNA including determination of single- nucleotide polymorphisms (SNPs) for genotyping and allele discrimination assays, for the detection of DNA and RNA viruses and for the detection of genetically modified organisms, for genetic testing and for diagnostics.
  • SNPs single- nucleotide polymorphisms
  • the above disclosure generally describes the present application.
  • Step 1 Synthesis of primer array
  • the primer array was commercially synthesized from Bio Basic Inc. (Markham, Ontario). One pair of primers is required for every target sequence. One primer was designed to hybridize to the target, and attached on the bead as a forward primer, the other primer is the reverse primer, which corresponds to the other strand of the target. The reverse primer was not coupled to the bead.
  • the forward primer was designed and synthesized with the structure: 5'biotin-(dT)n-(forward primer) or 5'NH 2 (dT)n-(forward primer), where, the forward primer was attached to a bead; (dT)n represents repeats of nucleotide of dT (or any nucleotide or combination of nucleotides) as a linker (spacer) between the coupling group and the forward primer; one or more fluorescent moieties were labeled on the middle of this spacer; n is the number with the range of 20-80; and the forward primer is a sequence with length of 20-50 bases, optionally 30 to 50 bases.
  • the reverse primer was designed and synthesized with the structure of 5'nucleotides, where primer(S) represented a reverse primer which was not coupled to the bead.
  • A/B/C 5'CCTGC TGGTG GCTCC AGTTC (SEQ ID NO:6)
  • a paramagnetic bead (Bangs Lab. Fishers, IN) was used with the following features: 1) Size: 5.90 ⁇ m (can be in range of 1 to 25 ⁇ m).
  • Step 3 Coupling the primer to the bead
  • the incubating reagent (Coupling Kit) was also purchased from Bangs Lab. (Fishers, IN, USA). The incubation procedure from Bangs Lab. was used:
  • PolyLink Coupling Buffer 55mM MES, pH5.2, 0.05% Proclin R300 PolyLink Wash/Storage Buffer: 1OmM Tris, pH8.0, 0.05% Bovine Serum Albumin, 0.05% Proclin R300 PolyLink EDAC (Carbodiimide).
  • the efficiency of coupling was calculated by comparing with a prepared standard sample by using UV-visible spectrophotometer at 280nm.
  • Step 4 Synthesis of Molecular Beacon Probe Array
  • the molecular beacon probe is designed with the following features:
  • Loop a 15-30 base pair region of the molecular beacon which is complementary to the target sequence.
  • the beacon stem sequence lies on both the ends of the loop. It is typically 5-7 bp long at the sequences at both the ends are complementary to each other.
  • 5' fluorophore Towards the 3' end of the molecular beacon, is attached a dye as a reporter that fluoresces in presence of a complementary target.
  • 3' quencher (non fluorescent): The quencher dye is covalently attached to the 3' end of the molecular beacon and when the beacon is in closed loop shape, prevents the fluorophore from emitting light.
  • the same reporter and quencher is attached on all probes in the probe array, i.e. only one color of signal is measured for all probes hybridized on the amplicons bound on the beads.
  • FAM represents the fluorophore 6-carboxyfluorescein
  • DABCYL represents a quenching chromophore
  • the DNA or RNA template to be prepared can be extracted following the protocol in Molecular Cloning [Molecular cloning: a laboratory manual. 2 J Sambrook, EF Fritsch, T Maniatis - 1989 - Cold Spring Harbor Laboratory Press Cold Spring Harbor, New York].
  • PCR master mix (about 1 - 15 micro-liter) including Taq-polymerase, dNTPs, etc.
  • the bead array was added by using a microscope or special designed tools.
  • One single particle of the bead coupled with a forward primer was used for a single PCR, the total number of the beads added was dependent on the size of the array.
  • the solution was placed into an instrument for amplification.
  • the amplification was thermo-cycled according to the following protocol: a) 94. degree. C. 3 - 10 minutes b) 94. degree. C. 5-30 seconds (denaturing) c) 55. degree. C. 30 - 90 second (annealing and measurement) d) 72. degree. C. 20 - 60 second (extension) e) Repeat steps b)-d), 20 - 40 times f) 72. degree. C. 2 minute Monolayer of random beads [00117] A magnetic stirrer was applied for shaking the beads all the time except the annealing and measurement phase. The motion of the beads was controlled by a set of precisely adjusted magnetic forces from different directions until a monolayer of the beads was formed during the annealing phase.
  • the monolayer of the beads was immobilized on the bottom of the reaction chamber.
  • the chamber made of quartz, glass or transparent plastic material, let both the exciting light source and emission through.
  • a microscope and a focus control were used. Exciting light source was either by a mercury arc lamp, a xenon arc lamp, a mercury halide arc lamp, LED or Lasers. Images were acquired by a CCD-based detector mounted on the microscope.
  • Step 1 Measurements to confirm a monolayer
  • Measurement was carried out immediately at the annealing step.
  • a focused beam of exciting light with a specific wavelength was shone on the bottom of the chamber. Images were continuously recorded to confirm whether or not a monolayer had formed. The images were immediately analyzed by software and compared to a pre-set standard. Precisely controlled forces were applied to form a monolayer. The magnitudes of the forces were adjusted to ensure there was no overlap between the beads. If there was any overlap, the force was increased to ensure separation. The force was minimized or stopped to ensure the monolayer was immobilized after the recorded image showed that the monolayer was formed without any overlap between the beads.
  • Step 2 Measurements for distinguishing and addressing the bead array
  • the beads are immobilized, they serve as addressable markers. Thus the disordered beads have an advantage which can perform precise image alignment.
  • One exciting light with a specific wavelength was shone on the monolayer and an image was recorded. The second exciting light with another specific wavelength was then shone on the monolayer and the second image was recorded. The two images were aligned and analyzed by the software for distinguishing and addressing the bead array where the beads had been labeled with fluorescent molecules.
  • Step 3 Measurement for qPCR array
  • Step 1 Calibration of the Beads:
  • Steps 1&2 were repeated, to add Bead C (coupled with forward primer C) into the tube
  • Step 2 Preparation for PCR solution:
  • Reverse Primer A (around 2pmol) was added into 98 ⁇ l of PCR Master Mix (PCR Master Mix: 1.5mmol MgCb, 0.1 mmol/L dNTP).
  • Step 3 Amplification and measurement The solution was placed into a special designed instrument for amplification and measurement.
  • the amplification was thermo-cycled according to the following protocol: a) 95. degree. C. 10 minutes b) 94. degree. C. 20 seconds (denaturing) c) 55. degree. C. 45 second (annealing), d) 55. degree. C. 30 second (fluorescence measurement) e) 72. degree. C. 60 second (extension) f) Repeat steps b)-e), 40 times g) 72. degree. C. 2 minute
  • Fig.9 shows the results of the amplification. The results were very consistent with regular qPCR. All the points of the data were obtained according to the following procedure: 1. Confirm the monolayer was formed
  • Excitation Source blue LED lamp
  • Emission filter 685nm
  • Excitation Source blue LED lamp
  • Emission filter 525nm
  • Step 1 Calibration of the Beads:
  • the tube was put on a permanent magnet and then the suspension was tipped out.
  • Step 2 Preparation for PCR solution 1. 0.5 ⁇ l of Reverse Primer A (around 3pmol), 0.5 ⁇ l of Reverse Primer D (around 1pmol) and 0.5 ⁇ l of Reverse Primer E (around 1pmol) were added into 96 ⁇ l of PCR Master Mix.
  • Template A 1000 copies of TemplateB, 10000 copies of Template C, 1000 copies of Template D and 10000 copies of Template E) were added into the quartz tube.
  • Step 3 Amplification and measurement The solution was placed into a special designed instrument for amplification and measurement.
  • the amplification was thermo-cycled according to the following protocol: a) 95. degree. C. 10 minutes b) 94. degree. C. 20 seconds (denaturing) c) 55. degree. C. 45 second (annealing), d) 55. degree. C. 30 second (fluorescence measurement) e) 72. degree. C. 60 second (extension) f) Repeat steps b)-e), 40 times g) 72. degree. C. 2 minute
  • Fig.10. shows the results of the amplification. All the points of the data were obtained according to the following procedure.
  • Excitation Source blue LED lamp
  • Emission filter 685nm
  • Excitation Source blue LED lamp, Optical filter 470nm Emission filter: 525nm
  • the tube was put on a permanent magnet and the suspension was tipped out.
  • Step 3 Amplification and measurement The solution was placed into a special designed instrument for amplification and measurement. The amplification was thermo-cycled according to the following protocol: a) 95. degree. C. 10 minutes b) 94. degree. C. 20 seconds (denaturing) c) 60. degree. C. 45 second (annealing), d) 60. degree. C. 30 second (fluorescence measurement) e) 72. degree. C. 60 second (extension) f) Repeat steps b)-e), 40 times g) 72. degree. C. 2 minute Where, both Reverse Primer A and C have same sequences.
  • Fig.11 shows the results of the amplification. The results show very consistent with regular qPCR. All the points of the data were obtained according to the following procedure:
  • Excitation Source blue LED lamp
  • Emission filter 525nm
  • the tube was put on a permanent magnet and the suspension was tipped out.
  • Step 3 Amplification and measurement The solution was placed into a special designed instrument for amplification and measurement. The amplification was thermo-cycled according to the following protocol: a) 95. degree. C. 10 minutes b) 94. degree. C. 20 seconds (denaturing) c) 60. degree. C. 45 second (annealing), d) 60. degree. C. 30 second (fluorescence measurement) e) 72. degree. C. 60 second (extension) f) Repeat steps b)-e), 40 times g) 72. degree. C. 2 minute
  • Reverse Primer A, B and C have same sequences.
  • Fig.12 shows the results of the amplification. All the points of the data were obtained according to the following procedure:
  • Excitation Source blue LED lamp, Optical filter 470nm Emission filter: 525nm

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Abstract

La présente invention concerne un procédé d'amplification et de quantification d'au moins deux séquences d'acide nucléique distinctes dans un unique compartiment comprenant des microsphères liées à une amorce sens distincte destinée à l'amplification d'un acide nucléique distinct et des amorces antisens et des sondes ou des colorants en solution. L'invention concerne également des systèmes et des trousses destinés à être utilisés dans les procédés décrits dans la description.
PCT/CA2010/000466 2009-04-01 2010-03-30 Procédé et système destinés à l'amplification et à la quantification de multiples acides nucléiques Ceased WO2010111776A1 (fr)

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WO2008074023A2 (fr) * 2006-12-13 2008-06-19 Luminex Corporation Systèmes et procédés d'analyse multiplex de pcr en temps réel

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CN117229902A (zh) * 2023-11-16 2023-12-15 北京大学 一种空气或水中病毒核酸的实时在线快速检测装置
CN117229902B (zh) * 2023-11-16 2024-01-30 北京大学 一种空气或水中病毒核酸的实时在线快速检测装置

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