WO2017044651A2 - Oligonucléotides courts extincteurs pour réduire la fluorescence de ligne de base de sonde taqman - Google Patents

Oligonucléotides courts extincteurs pour réduire la fluorescence de ligne de base de sonde taqman Download PDF

Info

Publication number
WO2017044651A2
WO2017044651A2 PCT/US2016/050811 US2016050811W WO2017044651A2 WO 2017044651 A2 WO2017044651 A2 WO 2017044651A2 US 2016050811 W US2016050811 W US 2016050811W WO 2017044651 A2 WO2017044651 A2 WO 2017044651A2
Authority
WO
WIPO (PCT)
Prior art keywords
oligonucleotide
quencher
oligonucleotide probe
linked
fluorophore
Prior art date
Application number
PCT/US2016/050811
Other languages
English (en)
Other versions
WO2017044651A3 (fr
Inventor
Erik Gustafson
Xin Zeng
Antonio Reyes
Jessica Smith
Original Assignee
Beckman Coulter, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beckman Coulter, Inc. filed Critical Beckman Coulter, Inc.
Publication of WO2017044651A2 publication Critical patent/WO2017044651A2/fr
Publication of WO2017044651A3 publication Critical patent/WO2017044651A3/fr

Links

Classifications

    • 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
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

  • This invention relates to compositions and methods for detecting and quantifying a target nucleic acid sequence.
  • PCR polymerase chain reaction
  • the TaqMan ® assay is one of such assays for quantifying polynucleotides (see e.g., U.S. Patent No. 5,723,591).
  • two PCR primers flank a central oligonucleotide probe.
  • the probe contains a fluorophore and a quencher moiety that are in close proximity to each other. This allows the fluorescence energy from the fluorophore to be transferred to the quencher and become undetectable.
  • the polymerase cleaves the oligonucleotide probe.
  • the cleavage causes the fluorophore and the quencher moiety to become physically separated, and thus allows the fluorescent emission from the fluorophore to be detected. As more PCR product is created, the intensity of the fluorescent emission increases.
  • a defined signal threshold is determined for reactions comprising the target nucleic acid and reactions comprising the reference nucleic acid and the number of cycles required to reach this threshold value (Ct) is determined.
  • the absolute or relative copy numbers of the target molecule can be determined on the basis of the Ct values obtained for the target nucleic acid and the reference nucleic acid.
  • the amount of the target molecule is determined by comparing the signal with a calibration curve, e.g., the VERIS viral load assays.
  • Molecular beacons are oligonucleotide hairpins which undergo a
  • the conformational change of the oligonucleotide increases the physical distance between a fluorophore and a quencher moiety present on the oligonucleotide. This increase in physical distance causes the effect of the quencher to be diminished, thus increasing the signal derived from the fluorophore.
  • compositions comprising quencher oligonucleotides that are useful for reduction of the baseline fluorescence of real time PCR reactions.
  • the invention can be used to increase the analytical sensitivity of real time PCR assays.
  • the invention provides for a composition
  • a composition comprising: a) an oligonucleotide probe linked to a fluorophore and a first quencher moiety; and b) a quencher oligonucleotide, which hybridizes to a subsequence of the oligonucleotide probe and which is linked to a second quencher moiety which quenches the fluorophore when the
  • oligonucleotide probe and the quencher oligonucleotide are hybridized to each other.
  • the invention provides for a composition
  • a composition comprising: a) an oligonucleotide probe linked to a fluorophore and a first quencher moiety, b) a quencher oligonucleotide linked to a second quencher moiety, and c) an adapter oligonucleotide comprising a first subsequence which hybridizes to the quencher oligonucleotide and a second subsequence which hybridizes to the oligonucleotide probe, such that the second quencher moiety quenches the fluorophore when the oligonucleotide probe and the quencher oligonucleotide are both hybridized to the adapter oligonucleotide.
  • the invention provides a method of monitoring amplification of a target nucleic sequence in a sample.
  • the method steps comprise:
  • the invention provides a method of monitoring amplification of a target nucleic sequence in a sample.
  • the method steps comprise the step of: a) contacting the sample with: i) an oligonucleotide probe, which hybridizes specifically to the target nucleic acid sequence and is linked to a fluorophore and a first quencher moiety; ii) a quencher oligonucleotide linked to a second quencher moiety; iii) an adapter oligonucleotide comprising a subsequence which hybridizes to the quencher oligonucleotide and a
  • the method steps also comprise the step of detecting fluorescence from the fluorophore. The increase in the fluorescence correlates with amplification of the target nucleic acid sequence, thereby monitoring the amplification.
  • the invention provides a kit comprising: a) an oligonucleotide probe linked to a fluorophore and a first quencher moiety; b) a quencher oligonucleotide, which hybridizes to a subsequence of the oligonucleotide probe and which is linked to a second quencher moiety which quenches the fluorophore when the oligonucleotide probe and the quencher oligonucleotide are hybridized to each other; and c) primers suitable for amplification of a target nucleic acid.
  • the invention provides a kit comprising: a) an oligonucleotide probe linked to a fluorophore and a first quencher moiety; b) a quencher oligonucleotide linked to a second quencher moiety; c) an adapter oligonucleotide comprising a subsequence which hybridizes to the quencher oligonucleotide and a subsequence which hybridizes to the oligonucleotide probe, such that the second quencher moiety quenches the fluorophore when the oligonucleotide probe and the quencher oligonucleotide are both hybridized to the adapter oligonucleotide; and d) primers suitable for amplification of a target nucleic acid.
  • the kit may further comprise dNTPs and buffers and other reagents suitable for amplification of the target nucleic acid.
  • the fluorophore is linked to the 5' end of the oligonucleotide probe and the first quencher moiety is linked to the 3' end of the oligonucleotide probe.
  • the second quencher moiety is linked to the 3' end of the quencher oligonucleotide.
  • the quencher oligonucleotide is shorter than the oligonucleotide probe.
  • the oligonucleotide probe can be between about 15 and about 60 nucleotides in length. In one embodiment, the oligonucleotide probe is between about 10 and 20 nucleotides in length.
  • the fluorophore is CAL Fluor Gold 540 or CAL Fluor Orange 560, and the first and second quenchers are Black Hole Quencher-1 (BHQ-1).
  • the fluorophore is Quasar 670 or Quasar 705, and the first and second quenchers are Black Hole Quencher-2 (BHQ-2) or Black Hole Quencher-3 (BHQ-3).
  • the first and second quenchers are Black Hole Quencher-1 (BHQ-1).
  • the method provided by the invention uses Thermus species Z05 for amplification.
  • the target nucleic acid is derived from a bacterium or a virus.
  • the target nucleic acid used in the kit is from human immunodeficiency virus (HIV).
  • the target nucleic acid is Hepatitis C Virus (HCV).
  • Figure 1 is a schematic representation of a specific quencher oligonucleotide and its interaction with an oligonucleotide probe.
  • Figures 2A-2D are schematic representations of universal quencher oligonucleotides and their interactions with adaptor oligonucleotides and oligonucleotide probes.
  • Figure 3 shows that quencher oligonucleotides Q3 and Q4 increase the gain of the HIV Taqman® assays.
  • Figure 4 shows that quencher oligonucleotides Q3 and Q4 increase the gain of the HIV Taqman® assays in a concentration dependent manner.
  • Figure 5 shows that the relationship between the gain increase and the concentration of the quencher oligonucleotide is non-linear.
  • Figure 6 shows that using quencher oligonucleotides in Taqman® assays does not affect Ct determination.
  • Figures 7A-7B show that using quencher oligonucleotides does not affect the linearity of the HIV Taqman® assays.
  • Figure 8 shows that a specific quencher oligonucleotide improved gain of Taqman® assays using probes having different fluorophores.
  • Figure 9 shows a quencher oligonucleotide increases the gain of the HCV Taqman® assays.
  • Figure 10 shows the effect of four different universal quenchers on the gain of the HIV Taqman® assays.
  • Figures 11 A-B show that adding a universal quencher oligonucleotide with its matching adaptor oligonucleotide increased the gain while adding the adaptor oligonucleotide without the universal quencher oligonucleotide decreased gain and increased baseline fluorescence.
  • Figure 12 shows that gain increase is dependent on the relative concentrations between the adaptor, quencher oligonucleotide, and the oligonucleotide probe.
  • Figure 13 shows that a universal quencher oligonucleotide improved gain of HIV Taqman® assays using oligonucleotide probes having different fluorophores.
  • Quantification of a target nucleic acid sequence relies on the detection of fluorescence emitted during PCR amplification when the polymerase cleaves the
  • oligonucleotide probe fluorescence detection in a given experiment can be obscured by high baseline fluorescence, i.e., the fluorescence emitted from the intact probe. This problem is most commonly caused by insufficient quenching of the fluorophore by the quencher moiety on the intact probe.
  • This invention provides compositions of quencher oligonucleotides and methods to use them to reduce the baseline fluorescence in order to improve accuracy and sensitivity of the assay.
  • a quencher oligonucleotide of the invention contains a polynucleotide sequence that directly hybridizes to the oligonucleotide probe in the real time PCR reaction, or hybridizes to an adaptor, which in turn hybridizes to the oligonucleotide probe. These interactions bring the quencher moiety of the quencher oligonucleotide to be within the distance that allows Fluorescence Resonance Energy Transfer (FRET) to occur.
  • FRET Fluorescence Resonance Energy Transfer
  • the quenching of the fluorophore is released during amplification.
  • the polymerase used in the PCR reactions can have 5 '-3' exonuclease activity and breaks the oligonucleotide probe during elongation, which results in the separation of the fluorophore from the quencher moiety on the oligonucleotide probe and also from the quencher moiety on the quencher oligonucleotide.
  • the separation allows detection of unquenched emission of fluorescence that is proportional to the amount of amplification product of the target nucleic acid that is produced in the PCR.
  • the fluorophore of the intact oligonucleotide probe is quenched by both quencher moiety on the quencher oligonucleotide and the quencher moiety on the oligonucleotide probe. This reduces the baseline fluorescence and increases the assay sensitivity.
  • a "polynucleotide” refers to a covalently linked sequence of nucleotides (i.e., ribonucleotides for RNA and deoxyribonucleotides for DNA) in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next.
  • the term “polynucleotide” includes, without limitation, single- and double- stranded polynucleotide.
  • the term "polynucleotide” as it is employed herein embraces chemically, enzymatically or metabolically modified forms of
  • Polynucleotide also embraces a short polynucleotide, often referred to as an oligonucleotide (e.g., a primer or a probe).
  • a polynucleotide has two opposite ends, the 5' end and the 3' end. The end of a polynucleotide at which a new linkage would be to a 5' carbon is its 5' terminal nucleotide. The end of a polynucleotide at which a new linkage would be to a 3' carbon is its 3' terminal nucleotide.
  • a terminal nucleotide is the nucleotide at the end position of the 3' end or the 5 ' end.
  • a polynucleotide sequence even if internal to a larger polynucleotide (e.g., a sequence region within a polynucleotide), also can be said to have 5' and 3' ends.
  • oligonucleotide refers to a short polynucleotide, typically less than or equal to 150 nucleotides long (e.g., between 5 and 150, preferably between 10 to 100, more preferably between 15 to 50 nucleotides in length). However, as used herein, the term is also intended to encompass longer or shorter polynucleotide chains.
  • oligonucleotide may hybridize to other polynucleotides, therefore serving as a probe for polynucleotide detection.
  • a biological sample is a sample obtained from a patient.
  • the sample can be whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid or urine.
  • target polynucleotide and “target nucleic acid” refer to a polynucleotide whose amount is to be determined in a sample.
  • a target-binding sequence refers to a sequence which hybridizes to a target nucleic acid.
  • the term “complementary” refers to the concept of sequence complementarity between regions of two polynucleotide strands or between two regions of the same polynucleotide strand. It is known that an adenine base of a first polynucleotide region is capable of forming specific hydrogen bonds ("base pairing") with a base of a second polynucleotide region which is antiparallel to the first region if the base is thymine or uracil.
  • a cytosine base of a first polynucleotide strand is capable of base pairing with a base of a second polynucleotide strand which is antiparallel to the first strand if the base is guanine.
  • a first region of a polynucleotide is complementary to a second region of the same or a different polynucleotide if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide of the first region is capable of base pairing with a base of the second region. Therefore, it is not required for two complementary polynucleotides to base pair at every nucleotide position.
  • “Complementary” refers to a first polynucleotide that is 100% or "fully" complementary to a second polynucleotide and thus forms a base pair at every nucleotide position.
  • “Complementary” also refers to a first polynucleotide that is not 100%, but is substantially complementary (e.g., 90%, or 80% or 70% complementary) to a second polynucleotide and contains mismatched nucleotides at one or more nucleotide positions, such that hybridization between two regions or two polynucleotide strands occurs under defined conditions.
  • hybridization refers to the pairing of complementary, including partially complementary polynucleotide strands.
  • Hybridization and the strength of hybridization is impacted by many factors well known in the art including the degree of complementarity between the polynucleotides, stringency of the conditions involved affected by such conditions as the concentration of salts, the melting temperature (Tm) of the formed hybrid, the presence of other components (e.g., the presence or absence of polyethylene glycol), the molarity of the hybridizing strands and the G:C content of the polynucleotide strands.
  • Various hybridizations may occur in the real time PCR reactions using the invention, such as, hybridization of the primers to the template DNA, hybridization of the target-binding sequence of the oligonucleotide probes to the target nucleic acid, and hybridization of the quencher-binding sequence of the oligonucleotide probes to the quencher oligonucleotide.
  • hybridization of the primers to the template DNA hybridization of the target-binding sequence of the oligonucleotide probes to the target nucleic acid
  • hybridization of the quencher-binding sequence of the oligonucleotide probes to the quencher oligonucleotide.
  • hybridization may occur despite of some degree of mismatches. Usually, hybridization occurs in two polynucleotides having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementarity to each other. It is within the skill of one of ordinary skill in the art to design primers and oligonucleotide sequences in the
  • 5' to 3' exonuclease activity or “5' ⁇ 3' exonuclease activity” refers to that activity of a template-specific nucleic acid polymerase e.g. a 5' ⁇ 3' exonuclease activity traditionally associated with some DNA polymerases whereby mononucleotides or oligonucleotides are removed from the 5' end of a polynucleotide in a sequential manner, (i.e., E. coli DNA polymerase I has this activity whereas the Klenow (Klenow et al, 1970, Proc. Natl. Acad. Sci., USA, 65: 168) fragment does not, (Klenow et al, 1971, Eur. J.
  • a template-specific nucleic acid polymerase e.g. a 5' ⁇ 3' exonuclease activity traditionally associated with some DNA polymerases whereby mononucleotides or oligonucleotides are removed from
  • polynucleotides are removed from the 5' end by an endonucleolytic activity that may be inherently present in a 5' to 3' exonuclease activity.
  • PCR polymerase chain reaction
  • the PCR reaction involves a repetitive series of temperature cycles and is typically performed in a volume of 20-100 ⁇ .
  • the reaction mix comprises dNTPs (each of the four deoxynucleotides dATP, dCTP, dGTP, and dTTP), primers, buffers, DNA polymerase, and polynucleotide template.
  • dNTPs deoxynucleotides dATP, dCTP, dGTP, and dTTP
  • primer refers to an oligonucleotide or oligonucleotides having a sequence complementary to a DNA sequence used as template DNA in PCR reactions. Primers are annealed to a complementary region of the template DNA and extended along the template DNA by a polymerase. The complementary portion of a primer can be any length that supports specific and stable hybridization between the primer and the target sequence under the reaction conditions.
  • the primers used in the invention may have also one or more modified nucleotides that contain modifications to the base, sugar and/or phosphate moieties.
  • the term "monitoring” means monitoring the fluorescence emitted by the oligonucleotide probes. This may also include detecting and measuring the intensity of the fluorescence, collecting and recording the fluorescence intensity data, for example on a computer readable medium of the types known in the art. Further steps include mathematical treatment of the data, analyses and interpretations thereof either with or without a computer.
  • T m is used in reference to the "melting temperature.”
  • the melting temperature is the temperature at which one half of a population of double-stranded polynucleotides or nucleobase oligomers (e.g., hybridization complexes), in homoduplexes or heteroduplexes, become dissociated into single strands.
  • the prediction of a T m of a duplex polynucleotide takes into account the base sequence as well as other factors including structural and sequence characteristics and nature of the oligomeric linkages. Methods for predicting and experimentally determining T m are known in the art.
  • a T m is traditionally determined by a melting curve, wherein a duplex nucleic acid molecule is heated in a controlled temperature program, and the state of association/dissociation of the two single strands in the duplex is monitored and plotted until reaching a temperature where the two strands are completely dissociated. The T m is read from this melting curve.
  • a T m can be determined by an annealing curve, wherein a duplex nucleic acid molecule is heated to a temperature where the two strands are completely dissociated.
  • the temperature is then lowered in a controlled temperature program, and the state of association/dissociation of the two single strands in the duplex is monitored and plotted until reaching a temperature where the two strands are completely annealed.
  • the T m is read from this annealing curve.
  • the present invention is directed to probe-based PCR quantitation or qualitative methods, which rely on sequence-specific oligonucleotide probes that bind to the target nucleic acid sequences specifically.
  • the oligonucleotide probe used in this approach typically contains a fluorescent molecule (the fluorophore) and a quencher moiety.
  • the quencher moiety accepts energy from the fluorophore and dissipates it by either proximal quenching or by Fluorescence Resonance Energy Transfer (FRET).
  • FRET Fluorescence Resonance Energy Transfer
  • Fluorescence Resonance Energy Transfer is an important technique for investigating a variety of biological phenomena that are characterized by changes in molecular proximity. FRET techniques have been also adapted for use in a variety of realtime PCR assays to quantify DNA amplification.
  • FRET is a distance-dependent interaction between two molecules where the excited donor molecule (e.g., a flurophore) transfers energy to an acceptor molecule (e.g., a quencher moiety).
  • the energy transfer occurs without emission of photons, and is based on dipole- dipole interactions between the two molecules.
  • the emission spectrum of the donor molecule must overlap the absorption spectrum of the acceptor molecule; the transition dipole orientations of the donor and acceptor molecules must be approximately parallel; and the donor and acceptor molecule must be in close proximity to each other (typically 10-100 A). If the donor and acceptor are spaced apart by too great a distance, then the donor fluorophore cannot transfer resonance energy to the acceptor.
  • FRET FRET
  • a quencher moiety as the acceptor molecule, the emission spectra of which overlaps with the fluorescent donor molecule.
  • the efficiency of quenching is directly correlated with the distance between the donor (the fluorophore) and the acceptor (the quencher moiety).
  • the quencher moiety itself may or may not be a molecule that can emit fluorescence.
  • the fluorophore-quencher moiety pair prefferably has sufficient spectral overlap in order to achieve sufficient quenching.
  • Fluorophores with an emission maximum between 500 and 550 nm such as FAM, TET and HEX, are best quenched by quenchers with absorption maxima between 450 and 550 nm, such as DABCYL and BHQ-1.
  • Fluorophores with an emission maximum above 550 nm such as rhodamines (including TMR, ROX and Texas red) and Cy dyes (including Cy3 and Cy5) are best quenched by quenchers with absorption maxima above 550 nm (including BHQ-2).
  • real time PCRs using the invention employ an additional FRET, which occurs between the fluorophore on the oligonucleotide probe and the quencher moiety on the quencher oligonucleotide.
  • the energy of the fluorophore thus is transferred not only to the quencher moiety on the oligonucleotide probe, but also the one on the quencher oligonucleotide.
  • the "double quenching" effectively reduces the baseline fluorescence and increases assay sensitivity.
  • the configurations of various oligonucleotide probes and quencher oligonucleotides - which are designed to engage the double quenching - are described below.
  • the additional quenching does not compromise detection of the fluorescence produced due to the amplification of the template DNA in the PCR reaction; the cleavage of the oligonucleotide probe by the polymerase during the process breaks the proximity between the fluorophore and both quencher moieties, thus allowing unquenched emission of fluorescence.
  • One of the probe-based quantitation methods employs the 5' exonuclease activity of polymerases such as Taq.
  • An oligonucleotide probe that is complementary to the PCR product comprising the target nucleic acid sequence, yet distinct from the PCR amplification primers is labelled with a FRET pair comprising a flurorophore and a quencher moiety.
  • the two molecules are situated within close proximity to each other such that the fluorescence from the fluorophore is quenched by the quencher moiety.
  • the 5' exonuclease proceeds to digest the probe, separating the FRET pair and leading to increased fluorescence.
  • a variation on this technology uses a nucleic acid wherein the FRET pair is internally quenched, for example, by having a hairpin conformation.
  • the FRET pair Upon hybridization to a sequence of interest, the FRET pair is separated and the donor molecule emits fluorescence.
  • the fluorescence values are recorded during each cycle of the amplification process.
  • the fluorescence signal is directly proportional to DNA concentration over a broad range, and the linear correlation between PCR product and fluorescence intensity is used to calculate the amount of template DNA (comprising the target nucleic acid sequence) present at the beginning of the reaction.
  • the threshold cycle or Ct Value is the most important parameter for quantitative PCR. This threshold must be established to quantify the amount of DNA in the samples. It is inversely correlated to the logarithm of the initial copy number. The threshold should be set above the amplification baseline and within the exponential increase phase (which looks linear in the log phase). Most assay systems automatically calculate the threshold level of fluorescence signal by determining the baseline (background) average signal and setting a threshold 10-fold higher than this average. Fluorophore
  • a fluorophore is a chemical compound that absorbs light energy at one wavelength and nearly instantaneously emits light at another, longer wavelength of lower energy.
  • Fluorophores as referred to in the invention can also be compounds that produce
  • fluorophores are either heterolytic or polyaromatic hydrocarbons.
  • the fluorescence signature of each individual fluorophore is unique in that it provides the wavelengths and amount of light absorbed and emitted. During fluorescence, the absorption of light excites electrons to a higher electronic state where they remain for about 1-10 x 10-8 seconds and then they return to the ground state by emitting a photon of energy.
  • fluorescent light is emitted. The light intensity can be measured by flurometer or a pixel-by-pixel digital image of the sample.
  • Fluorescence intensity depends on the efficiency with which fluorophores absorb and emit photons, and their ability to undergo repeated excitation/emission cycles.
  • the intensity of the emitted fluorescent light is a linear function of the amount of fluorophores present. The signal becomes nonlinear at very high fluorophore concentrations.
  • fluorophores can be used in the oligonucleotide probe, including but not limited to: CAL Fluor® Gold 540, CAL Fluor® Orange 560, Quasar® 670, Quasar® 705, 5 - FAM (also called 5 - carboxyfluorescein; also called Spiro(isobenzofuran - 1(3H), 9'
  • the quencher moiety used in the invention can be any material that can quench detectable emission of radiation, for example, fluorescent or luminescent. Quenching can involve any type of energy transfer, including but not limited to, photoelectron transfer, proton coupled electron transfer, dimer formation between closely situated fluorophores, transient excited state interactions, collisional quenching, or formation of non-fluorescent ground state species.
  • a quencher moiety is joined with a flurophore in a configuration that permits energy transfer from the fluorophore to the quencher moiety to result in a reduction of the flurorescence by FRET, as described above. It is not intended that that the term "quencher moiety" be limited to one that participates in FRET.
  • the quencher moieties used in the invention may or may not emit fluorescence themselves upon energy transfer from the fluorophore.
  • Some quencher moieties for example, tetramethyl-6-carboxyrhodamine (TAMRA), can re-emit the energy absorbed from the fluorophore at a wavelength or using a signal type that is also detectable but distinguishable from the fluorophore emission.
  • TAMRA tetramethyl-6-carboxyrhodamine
  • quencher moieties such as the Black Hole Quenchers (BHQs), including Black Hole Quencher- 1 (BHQ-1) , Black Hole Quencher-2 (BHQ-2), Black Hole Quencher-3 (BHQ-3) have no native fluorescence, thus can virtually eliminate background problems seen with other quencher moieties.
  • BHQs Black Hole Quenchers
  • BHQ-2 Black Hole Quencher-2
  • BHQ-3 Black Hole Quencher-3
  • Additional quencher moieties include but are not limited to, DABCYL and rhodamine dyes, such as tetrapropano-6-carboxyrhodamine (ROX).
  • the degree of the reduction of fluorescence of a fluorescent by the quencher moiety is not limited, per se, except that a quenching effect should minimally be detectable by whatever detection instrumentation is used. Fluorescence is "quenched" when the fluorescence emitted by the fluorophore is reduced as compared with the fluorescence in the absence of the quencher by at least 10%, for example, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9% or more.
  • the oligonucleotide probe of the invention comprises a polynucleotide sequence, a fluorophore, and a quencher moiety.
  • the fluorophore and the quencher may be attached to any nucleotide of the oligonucleotide sequence, so long as the fluorophore and quencher are in close proximity such that FRET results in quenching of the fluorophore.
  • the fluorophore and the quencher are attached to the two terminal nucleotides at the opposite ends of the polynucleotide of the oligonucleotide probe.
  • the fluorophore is linked close to or at the 5' end, e.g., the 5' terminal nucleotide, of the polynucleotide
  • the quencher moiety is linked close to or at the 3' end, e.g., the 3' terminal nucleotide, of the polynucleotide.
  • the fluorophore is linked close to or at the 3' end, e.g., the 3' terminal nucleotide, of the polynucleotide and the quencher moiety is linked close to or at the 5' end, e.g., the 5' terminal nucleotide, of the polynucleotide.
  • the fluorophore and the quencher are attached to two nucleotides of the polynucleotide sequence that are between 5 and 60 nucleotides apart.
  • the fluorophore and quencher are attached to two nucleotides of the polynucleotide sequence that are between 10 and 25 nucleotides apart.
  • the fluorophore and quencher are attached to two nucleotides of the
  • polynucleotide sequence that are between 20 and 24 nucleotides apart.
  • An exemplary oligonucleotide probe is shown in Table 1, i.e., the oligonucleotide probe "HIV LTR PIP".
  • the oligonucleotide probe can be of any length. In one embodiment, it is 5-60 nucleotides in length.
  • the polynucleotide sequence comprises two non-overlapping sequences: a quencher-binding sequence that hybridizes to the quencher oligonucleotide or the adapter oligonucleotide, and a target-binding sequence that hybridizes to the target nucleic acid.
  • the gap region may have a length that is within the range of 0-5 nucleotides. In one embodiment, the gap region has a length of 0-2 nucleotides.
  • the quencher-binding sequence and the target-binding sequence may have different lengths, for example, a length that is within the range of be 5-30 nucleotides, or 5-15 nucleotides, or 9-15 nucleotides.
  • the quencher-binding sequence and the target-binding sequence may occupy different percentages of the entire length of the polynucleotide sequence.
  • the target binding sequence may occupy at least 20%, 25%, 30%, 40%, 50%, 60%, 70%), 80%) or 90% of the polynucleotide sequence of the oligonucleotide probe.
  • the quencher oligonucleotide provided by the invention comprises a polynucleotide sequence that is linked to a quencher moiety.
  • the quencher moiety can be linked to any nucleotide on the polynucleotide sequence of the oligonucleotide quencher.
  • the quencher moiety is linked close to or at the 3' end of the polynucleotide sequence of the quencher oligonucleotide, e.g., the 3' terminal nucleotide.
  • the quencher moiety is linked close to or at the 5' end, e.g., the 5' terminal nucleotide, of the polynucleotide sequence.
  • the quencher oligonucleotide may be shorter or longer than the oligonucleotide probe and the quencher moiety on the quencher
  • oligonucleotide may be the same as or different from the quencher moiety on the
  • the quencher oligonucleotide is a specific quencher oligonucleotide. In another embodiment of the invention, the quencher
  • oligonucleotide is a universal quencher oligonucleotide.
  • a specific quencher oligonucleotide comprises a polynucleotide sequence complementary to the quencher-binding sequence on the oligonucleotide probe.
  • Hybridization between these two complementary sequences brings the quencher moiety on the quencher oligonucleotide to be in close proximity to the fluorophore on the
  • FRET occurs and results in quenching of the fluorescence of the fluorophore.
  • the oligonucleotide probe (e.g., a Taqman® probe) comprises a target-binding sequence that hybridizes to a target nucleic acid sequence and a quencher-binding sequence that hybridizes to the quencher oligonucleotide.
  • the oligonucleotide probe has a fluorophore at the 5' end and a quencher at the 3' end.
  • the quencher oligonucleotide comprises a polynucleotide sequence that hybridizes to the quencher-binding sequence of the oligonucleotide probe and a quencher moiety at the 3' end.
  • quencher moiety of the oligonucleotide probe and the quencher moiety of the quencher oligonucleotide probe are represented by "Q", they may or may not be the same molecule or the same type of quencher moiety.
  • a specific quencher oligonucleotide used in the invention may comprise any number of nucleotides.
  • the quencher oligonucleotide may be 5-60 nucleotides in length. In one embodiment, the quencher oligonucleotide is between 5 and 30 nucleotides in length. In other embodiments, the quencher oligonucleotide is 10-20 nucleotides in length. In one embodiment, the quencher oligonucleotide is 13-14 nucleotides in length.
  • Table 1 shows some exemplary oligonucleotide quenchers ("Q1"-"Q4") and the oligonucleotide probe ("HIV LTR P1P”) that can be used in the real time PCR assays for HIV detection.
  • a specific quencher oligonucleotide and the oligonucleotide probe can be in any concentration for sufficient quenching to occur in order to reduce the baseline fluorescence.
  • the molar ratio between the quencher oligonucleotide and the oligonucleotide probe in a real time PCR reaction may be any value that is within the range of 1 : 1 to 1 :50. In one preferred embodiment, the ratio is 1 :5 or 1 : 10, or any value in between. It is within the skill of one of ordinary skill in the art to determine the optimal ratios suitable for any particular reaction.
  • a universal quencher oligonucleotide is used together with an adaptor
  • the adaptor oligonucleotide used in the invention comprises a quencher-binding sequence complementary to the quencher oligonucleotide, and a probe-binding sequence complementary to the oligonucleotide probe.
  • the adaptor oligonucleotide acts as an anchor to bring the quencher moiety on the quencher oligonucleotide and the fluorophore on the oligonucleotide probe to be in close proximity of each other for quenching to occur.
  • oligonucleotide are non-overlapping and each may be 5-30 nucleotides in length. In one embodiment, they are 10-20 nucleotides in length each.
  • the adaptor oligonucleotide may also comprise a gap region between the two sequences, which has a length within the range of 0- 10 nucleotides, or 0-5 nucleotides, or 0-2 nucleotides.
  • Figures 2A-2D illustrate four exemplary embodiments of the invention which use universal quencher oligonucleotides, adaptor oligonucleotides, and oligonucleotide probes.
  • the universal quencher oligonucleotide comprises a quencher moiety at the 5'.
  • the oligonucleotide probe comprises a fluorophore at the 5' and a quencher moiety at the 3'.
  • the adaptor oligonucleotide comprises a quencher-binding sequence at the 3', which hybridizes to the quencher oligonucleotide, and a probe-binding sequence at the 5', which hybridizes to the oligonucleotide probe.
  • the universal quencher oligonucleotide comprises a quencher moiety at the 5'.
  • the oligonucleotide probe comprises a fluorophore at the 3' and a quencher moiety at the 5'.
  • the adaptor oligonucleotide comprises a quencher-binding sequence at the 5', which hybridizes to the quencher oligonucleotide, and a probe-binding sequence at 3', which hybridizes to the oligonucleotide probe.
  • the universal quencher oligonucleotide comprises a quencher moiety at the 3'.
  • the oligonucleotide probe comprises a fluorophore at the 3' and a quencher moiety at the 5'.
  • the adaptor oligonucleotide comprises a quencher-binding sequence at the 5', which hybridizes to the quencher oligonucleotide, and a probe-binding sequence at 3', which hybridizes to the oligonucleotide probe.
  • the universal quencher oligonucleotide comprises a quencher moiety at the 3'.
  • the oligonucleotide probe comprises a fluorophore at the 5' and a quencher moiety at the 3'.
  • the adaptor oligonucleotide comprises a quencher-binding sequence at the 3', which hybridizes to the quencher oligonucleotide, and a probe-binding sequence at 5', which hybridizes to the oligonucleotide probe.
  • the universal quencher oligos used in this invention may have a length that is within the range of 11 - 60 nucleotides, or 10-20 nucleotides. In some embodiments, the universal quencher oligo is 13 -14 nucleotides in length. Exemplary quenchers and probes and adaptor oligonucleotide sequences are shown in Table 2.
  • the universal quencher oligonucleotide, adaptor oligonucleotide, or oligonucleotide probe used in the real time PCR reaction can be at any concentrations and they can be mixed at any ratios for sufficient quenching to occur in order to reduce the baseline fluorescence.
  • the molar ratio between the oligonucleotide probe and the adaptor is within the range of from 1 : 1 to 1 : 15. In one embodiment, the molar ratio is 1 :5.
  • the molar ratio between the adaptor and the universal quencher oligonucleotide may be within the range of from 1 : 1 to 1 : 10. In a particular embodiment, the molar ratio between the adaptor and the quencher is 1 :4.
  • the invention may be used to detect or measure the presence of any target nucleic acid sequence in a sample.
  • the target nucleic acid may be from a biological sample, or a secondary target such as a product of an amplification reaction, and the like.
  • a sample as used in the invention is an aliquot of material, frequently an aqueous solution or an aqueous suspension derived from biological material containing DNA.
  • Samples to be assayed for the presence of the target nucleic acid by the methods of the present invention include, for example, cells, tissues, e.g., tumors, homogenates, lysates, extracts, and other biological molecules and mixtures thereof.
  • samples typically used in the methods of the invention include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, sputum, bronchial washings, bronchial aspirates, urine, lymph fluids and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; tissue specimens which may or may not be fixed; and cell specimens which may or may not be fixed.
  • human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, sputum, bronchial washings, bronchial aspirates, urine, lymph fluids and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like
  • biological fluids such as cell culture supernatants
  • tissue specimens which may or may not be fixed and cell specimens which may or may not be fixed
  • the target nucleic acid that can be detected using the invention may be derived from any organisms, for example, human, Protists (Trichomonas), viruses (e.g., Adenovirus, Herpes viruses, Pox viruses, Retroviruses, (such as Human Immunodeficiency Virus (HIV)), Hepatitis virus (such as Hepatitis A, B, and C), and Papilloma virus), bacteria (e.g.,
  • Corynebacteria Pneumococci, Streptococci, Staphylococci, Neisseria, Enterobacteriaciae, Coliform, Salmonellae, Shigellae, other enteric bacilli, hemophilus-Bordetella, Pateurellae, Brucellae, Aerobic Spore-forming Bacilli, Anaerobic Spore-forming Bacilli, Mycobacteria, Actinomycetes, Spirochetes, Mycoplasmas, Rickettsiae, Chlamydia), fungi (e.g.,
  • Cryptococcus Blastomyces, Hisoplasma, Coccidioides, Paracoccidioides, and Candida
  • plant insect or animal cells.
  • a target nucleic acid of the invention may be a naturally occurring polynucleotide (i.e., one existing in nature without human intervention), or a recombinant polynucleotide (i.e., one existing only with human intervention), including but not limited to genomic DNA, cDNA, plasmid DNA, total RNA, mRNA, tRNA, rRNA.
  • a "target polynucleotide” or “target nucleic acid” may contain a modified nucleotide which include phosphorothioate, phosphite, ring atom modified derivatives, and the like.
  • a target nucleic acid may be of any length.
  • a target nucleic acid of the present invention contains a known sequence of at least 10 nucleotides, at least 20 nucleotides, 50 nucleotides, at least 100 or more nucleotides, for example, 500 or more nucleotides.
  • the target nucleic acid is a DNA sequence from the Human Immunodeficiency Virus (HIV). In another embodiment of the invention, the target nucleic acid sequence is a DNA sequence from the Hepatitis C Virus (HCV).
  • HCV Hepatitis C Virus
  • Fluorophores and quencher moieties used in the invention are commercially available or can be synthesized by techniques known in the art. Methodologies for attachment of many dyes to oligonucleotides can be performed via direct coupling or using a spacer molecule of between 1 and 5 atoms in length. Methodologies for attachment of many dyes to oligonucleotides are described in many references, for example, Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Inc., Eugene, 1989) and U. S. Pat. No. 5, 188,934. Kits
  • the present invention also provides a kit for measurement of the amount of target nucleic acid using a real time PCR assay.
  • the kit comprises a) an oligonucleotide probe linked to a fluorophore and a quencher moiety fluorescent probe that contains a sequence that can hybridize to the target nucleic acid sequence. The sequence is linked to a fluorophore and linked to a quencher, b) a specific quencher oligonucleotide that comprises a sequence that hybridizes to the probe and is linked to a quencher at one end, and c) at least one primer pair that is adapted to amplify the target nucleic acid.
  • the kit of the invention comprises a) one fluorescent probe that contains a sequence that can hybridize to the target nucleic acid sequence.
  • the sequence is linked to a fluorophore and linked to a quencher, b) a universal quencher and an adaptor sequence, where the one part of the adaptor can hybridize to the target nucleic acid nucleic acid and one part of the adaptor can hybridize to the universal quencher oligonucleotide, and c) at least one primer pair that is adapted to amplify the target nucleic acid.
  • the kit of the invention may optionally include polymerases and buffers suitable for real time PCR reactions.
  • the kit may also include a protocol for performing real time PCR reactions.
  • PCR reactions using the invention can be performed in any thermocycler commonly used for PCR.
  • cyclers with real-time fluorescence measurement capabilities including instruments capable of measuring real-time including VERIS MDx system (Beckman Coulter) Taq Man 7700 AB (Applied Biosystems, Foster City, CA), Rotorgene 2000 (Corbett Research, Sydney, Australia), LightCycler (Roche Diagnostics Corp, Indianapolis, IN), iCycler (Biorad Laboratories, Hercules, CA) and Mx4000TM
  • oligonucleotide probes and adaptor oligonucleotides were used in accordance with the disclosure to achieve the objective of increasing the sensitivity of real time PCR reactions.
  • the sensitivity of the PCR reactions in the following examples was measured by gain, which is the extent to which the fluorescent signal generated by the amplification of target sequence rises above the baseline fluorescence in the absence of amplified target.
  • Gain is calculated as the ratio of the signal detected at the last PCR cycle compared to the signal detected at the beginning of cycle 1, as shown in the formula below.
  • EXAMPLE 1 SHORT QUENCHER OLIGONUCLEOTIDE IMPROVES THE GAIN FOR HIV ASSAY a.
  • a specific oligonucleotide quencher Q4 improved the gain of the HIV assay.
  • Figure 3 shows that a specific quencher oligonucleotide improved the gain of the HIV reverse transcriptase-PCR (RT-PCR) assay.
  • Real time RT-PCRs were performed to detect the HIV RNA.
  • Two primer pairs, HIV R 014 primer, HIV_R_028_RO primer, HIV LTR S6 primer, and HIV LTR S7 Primer, were used at a concentration of .45uM to amplify the HIV RNA.
  • the PCR reaction mixture also comprises the oligonucleotide probe HIV LTR PIP (shown in Table 1) at a concentration of .20uM and the specific quencher
  • oligonucleotide Q4 (also shown in Table 1) at a concentration of l .OuM.
  • the control PCR reaction was set up in the same manner as the experimental group except that water was used in place of the oligonucleotide quencher Q4.
  • the thermal regimen was set at 42°C for 2min, 65°C for 6min, 92°C for 1 min, 60°C for 15sec, 50°C for 15sec, 95°C for 5sec. The last three steps were repeated 45 times. The fluorescence at the first cycle and the last cycle were measured and gain for each reaction was calculated using the formula described above.
  • FIG. 4 shows that oligonucleotide quencher's increasing gain of the PCR reactions is concentration dependent.
  • Two specific quencher oligonucleotides Q3 and Q4 (Table 1), three different concentrations of each, were used in the experiment. The three different concentrations correspond to three different ratios between the amount of the oligonucleotide probe and the amount of the quencher oligonucleotide in the reactions (hereinafter "probe-quencher ratio"): 1 : 1, 1 :2, or 1 :5.
  • Probe-quencher ratio the amount of the oligonucleotide probe and the amount of the quencher oligonucleotide in the reactions
  • Replicate reactions were set up for each concentration.
  • the control reactions were set up the same manner except that no quencher oligonucleotides were added. Fluorescence signals from the PCR reactions were detected and the gain calculated.
  • Figure 5 shows that the relationship between the increase in gain and relative concentration of the specific oligonucleotide quenchers is non-linear.
  • oligonucleotide probe HIV LTR PIP and specific quencher oligonucleotide Q3 were used in the PCR reactions at a probe-quencher ratio of 1 : 1, 1 :2, 1 :5, 1 : 10, 1 :25, or 1 :50.
  • Gain of each reaction was calculated using the method described as above. The result showed that the gain increased as the concentration of the specific quencher oligonucleotide increased, reaching the highest level when the probe-quencher ratio was 1 :5, and then decreased when the ratio continued to increase to 1 :25 or higher.
  • Addition of specific quencher oligonucleotides 03 or 04 did not affect Ct calling or the linearity of HIV Taqman assay.
  • Figure 6 shows that adding the specific quencher oligonucleotide to the PCR reactions did not affect Ct determination. Except for the control reactions (labeled "PIP" on the plot), Q3 or Q4 was added to the PCR reactions at a probe-quencher ratio of 1 : 1, 1 :2 or 1 :5. Multiple replicate reactions were set up for each probe-quencher ratio. The Ct values of all reactions were recorded. The result shows that the average Ct values of PCR reactions occurring in the presence of Q3 or Q4 were within the range of 24.8 to 25.1, which were agreeable with the average Ct value of the control reactions: 24.94.
  • Figure 7A shows that quencher oligonucleotides do not affect the linearity of the real time PCR assay.
  • Q3 was added at a probe-quencher ratio of 1 :5 and the PCR reactions contain 27, 270, 2700, or 27000 HIV RNA molecules.
  • the control PCR reactions were performed on the template DNA of the same serial dilutions in the absence of Q3. Gain from the PCR reactions were calculated. The result shows that for each template DNA concentration, the reactions containing Q3 had higher gain on average than the control group. This indicates that the quencher
  • oligonucleotide can improve gain over a broad range of template DNA concentrations.
  • Figure 8 shows that quencher oligonucleotide's effect on gain increase is not limited to any unique fluorophore on the oligonucleotide probe.
  • the probe in the first group (“C540") had CAL Fluoro® Gold 540 at the 5' and BHQ-1 at the 3' end.
  • the probe in the second group (“C560”) had CAL Fluor® Orange 560 at the 5' and BHQ-1 at the 3' end.
  • the probe in the third group (“Q670”) had Quasar® 670 at the 5' end and BHQ-2 at the 3' end.
  • the probe in the fourth group (“Q679”) had Quasar® 679 at the 5' end and BHQ-2 at the 3' end.
  • FIG. 9 shows that the use of a specific oligonucleotide quencher increased gain for the HCV real time PCR assays.
  • the HCV quencher oligonucleotide has a sequence of 5'-GCACGCCCAAAT-3' (SEQ ID NO: 15) that has a BHQ-1 linked at the 3' end.
  • the HCV oligonucleotide probe used in the assay has a sequence of 5'-ATTTGGGCGTGCCCCCG-3' (SEQ ID NO: 14), a fluorophore at the 5' end, and a quencher moiety at the 3' end. Except for the control reactions, the HCV quencher oligonucleotide was added to reactions at a probe-quencher ratio of 1 :5.
  • Figure 10 shows a universal quencher oligonucleotide increased the gain of the HIV real time PCR assays. This experiment uses the same oligonucleotide probe as the one used in
  • Example 1 HIV LTR P1P. Universal quencher oligonucleotides Jess 1, Jess 2, Tony 1, or
  • quencher oligonucleotide was 1 : 1 : 2 in each of these reactions.
  • the sequences and the modifications of these oligonucleotides are shown in Table 2.
  • Two additional PCR reaction groups were set up: one is the control group, which contains neither a quencher
  • oligonucleotide without its matching quencher oligonucleotide adversely affects the sensitivity of the real time PCR assay.
  • the universal quencher oligonucleotide's effect on gain increase is dependent on the relative concentrations of the adaptor, the quencher oligonucleotide and the oligonucleotide probe in the reaction.
  • Figure 12 shows that the universal oligonucleotide quencher's effect on gain increase is concentration-dependent. In this experiment, the universal quencher
  • oligonucleotide Tony 1 and its matching adaptor ATI were added to all reactions containing the oligonucleotide probe HIV LTR PIP except for the controls.
  • the ratio of the amount of adaptor and quencher is 1 :4 across all experimental groups.
  • the ratio of the amount of oligonucleotide probe relative to the adaptor is 1 :2, 1 :5, 1 : 10 or 1 :20.
  • the reactions having the probe-adaptor ratio of 1 :5 showed the highest gain on average. This indicates that, similar to the specific quencher oligonucleotide, the universal quencher oligonucleotide's effect on gain increase is also concentration dependent.
  • the universal oligonucleotide quencher Tony 1 (aka "J14") can also be used to improve reaction gain in real time PCR assays using other fluorophore systems.
  • Figure 13 shows a universal quencher oligonucleotide improved gain in reactions using CAL Fluor® Orange 560, Quasar® 670, or Quasar® 705 as the fluorophore.
  • CAL Fluor® Orange 560 Quasar® 670
  • Quasar® 705 the fluorophore
  • C540 had CAL Fluoro® Gold 540 at the 5' and BHQ-1 at the 3' end.
  • the probe in the second group (“C560”) had CAL Fluor® Orange 560 at the 5' and BHQ-1 at the 3' end.
  • the probe in the third group (“Q670”) had Quasar® 670 at the 5' end and BHQ-2 at the 3' end.
  • the probe in the fourth group (“Q679") had Quasar® 679 at the 5' end and BHQ-2 at the 3' end.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des oligonucléotides extincteurs et des procédés pour utiliser ces oligonucléotides pour réduire une fluorescence de ligne de base et augmenter la sensibilité d'analyse pour des analyses PCR en temps réel. L'invention concerne également des kits qui peuvent être utilisés à cet effet.
PCT/US2016/050811 2015-09-10 2016-09-08 Oligonucléotides courts extincteurs pour réduire la fluorescence de ligne de base de sonde taqman WO2017044651A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562216794P 2015-09-10 2015-09-10
US62/216,794 2015-09-10

Publications (2)

Publication Number Publication Date
WO2017044651A2 true WO2017044651A2 (fr) 2017-03-16
WO2017044651A3 WO2017044651A3 (fr) 2017-04-06

Family

ID=56940442

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/050811 WO2017044651A2 (fr) 2015-09-10 2016-09-08 Oligonucléotides courts extincteurs pour réduire la fluorescence de ligne de base de sonde taqman

Country Status (1)

Country Link
WO (1) WO2017044651A2 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5188934A (en) 1989-11-14 1993-02-23 Applied Biosystems, Inc. 4,7-dichlorofluorescein dyes as molecular probes
US5723591A (en) 1994-11-16 1998-03-03 Perkin-Elmer Corporation Self-quenching fluorescence probe

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5455170A (en) * 1986-08-22 1995-10-03 Hoffmann-La Roche Inc. Mutated thermostable nucleic acid polymerase enzyme from Thermus species Z05
WO2004074447A2 (fr) * 2003-02-18 2004-09-02 Applera Corporation Compositions et procedes pour l'analyse multiplex de polynucleotides
WO2006045009A2 (fr) * 2004-10-20 2006-04-27 Stratagene California Compositions de sonde triplex et procedes de detection de polynucleotide
EP2116614A1 (fr) * 2008-05-06 2009-11-11 Qiagen GmbH Détection simultanée de plusieurs séquences d'acide nucléique dans une réaction
US9637790B2 (en) * 2010-12-03 2017-05-02 Brandeis University Detecting mutations in DNA

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5188934A (en) 1989-11-14 1993-02-23 Applied Biosystems, Inc. 4,7-dichlorofluorescein dyes as molecular probes
US5723591A (en) 1994-11-16 1998-03-03 Perkin-Elmer Corporation Self-quenching fluorescence probe

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HAUGLAND: "Handbook of Fluorescent Probes and Research Chemicals", 1989, MOLECULAR PROBES, INC.
KLENOW ET AL., EUR. J. BIOCHEM., vol. 22, 1971, pages 371
KLENOW ET AL., PROC. NATL. ACAD. SCI., vol. 65, 1970, pages 168

Also Published As

Publication number Publication date
WO2017044651A3 (fr) 2017-04-06

Similar Documents

Publication Publication Date Title
AU2018204665B2 (en) Detection of target nucleic acid sequences using different detection temperatures
US10400276B2 (en) Multiplex amplification and detection
US20110224417A1 (en) Novel mixtures for assaying nucleic acid, novel method of assaying nucleic acid with the use of the same and nucleic acid probe to be used therefore
JP2014110793A (ja) 核酸増幅のためのプライマー、プローブおよび方法
NZ521593A (en) Hybridisation beacon and method of rapid sequence detection and discrimination
WO2010013017A1 (fr) Amplification et détection multiplex
EP2438189B1 (fr) Amplification multiplexe et détection
JP2012513191A (ja) 試料中の標的核酸を検出するための方法
US6461817B1 (en) Non-competitive co-amplification methods
US20140274779A1 (en) Multiplex allele detection
US20240011083A1 (en) Looped primer and loop-de-loop method for detecting target nucleic acid
WO2022034303A1 (fr) Oligonucléotide switch
WO2017044651A2 (fr) Oligonucléotides courts extincteurs pour réduire la fluorescence de ligne de base de sonde taqman
US20160024563A1 (en) Method for performing a melting curve analysis
CN114250286B (zh) 用于核酸检测的组合、试剂盒及其应用
WO2022136023A1 (fr) Solution permettant de détecter une séquence d'arn viral
WO2023056345A1 (fr) Signalisation de cassettes fret sélectionnables en fonction de la température
ei C ycler iQ D etection S ystem

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16766790

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16766790

Country of ref document: EP

Kind code of ref document: A2