WO2024123672A1 - Suppression de signal hors cible dans des réactions d'amplification d'acide nucléique - Google Patents

Suppression de signal hors cible dans des réactions d'amplification d'acide nucléique Download PDF

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Publication number
WO2024123672A1
WO2024123672A1 PCT/US2023/082292 US2023082292W WO2024123672A1 WO 2024123672 A1 WO2024123672 A1 WO 2024123672A1 US 2023082292 W US2023082292 W US 2023082292W WO 2024123672 A1 WO2024123672 A1 WO 2024123672A1
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probe
target
composition
dark
nucleic acid
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PCT/US2023/082292
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English (en)
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Noah ELDER
MinGin KIM
Rounak FEIGELMAN
Junko Stevens
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Life Technologies Corporation
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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
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • This disclosure is directed to compositions and methods for suppressing interaction of a target nucleic acid probe with off-target nucleic acids in the reaction mixture.
  • amplification of a particular nucleic acid sequence is essential to allow its detection in, or isolation from, a sample in which it is present in very low amounts.
  • PCR Polymerase chain reaction
  • the target nucleic acid is mixed with similar nucleic acid sequences in the test sample.
  • the PCR components intended for interacting with the target nucleic acid instead interact with the similar, off-target sequences. This can detrimentally introduce error into the associated detection and quantification analyses.
  • compositions and methods for suppressing off-target signal generation in a nucleic acid amplification process utilize a detector probe with a detectable label and a dark probe that omits a detectable label.
  • the detector probe is configured to specifically interact with a target nucleic acid target.
  • the dark probe is configured to specifically interact with an off-target sequence that is similar to the target nucleic acid, the dark probe thereby limiting off- target interaction between the detector probe and the off-target sequence.
  • a composition for suppressing off-target signal generation in a nucleic acid amplification process comprises a detector probe comprising a detectable label.
  • the detector probe is configured to specifically interact with a nucleic acid target to enable detection and/or quantification of the nucleic acid target.
  • the composition also includes a dark probe that omits a detectable label.
  • the dark probe is configured to specifically interact with an off-target sequence to thereby block or limit interaction between the detectable label and the off-target sequence.
  • the detector probe is configured to target a first allele of a particular single nucleotide polymorphism (SNP) location and the dark probe is configured to target a second, different allele at the SNP location.
  • SNP location is associated with an oncogenic mutation.
  • the concentration of dark probe provided in the composition is at least as much as an amount of detector probe.
  • a method for suppressing off-target signal generation in a nucleic acid amplification process comprises providing a composition that includes a detector probe with a detectable label, the detector probe configured to specifically interact with a nucleic acid target to enable detection and/or quantification of the nucleic acid target, and a dark probe that omits a detectable label and is configured to specifically interact with an off-target sequence to thereby block or limit interaction between the detectable label and the off-target sequence.
  • the method further comprises forming a reaction mixture comprising a sample and the composition, subjecting the reaction mixture to an amplification process, and detecting or quantifying the nucleic acid target.
  • the amplification process is digital PCR (dPCR).
  • Figure 1 A is an overview of a conventional nucleic acid amplification process in which a detector probe is intended to specifically interact with a nucleic acid target and generate a corresponding signal as a result, the schematic showing that the detector probe or some portion of detector probes may instead interact with off- target sequences, falsely adding to the resulting signal;
  • Figure IB is an overview of an improved nucleic acid amplification process in which a “dark probe” is also provided in the reaction mixture, the dark probe being configured to specifically interact with the off-target sequence and thereby block the detector probe from interacting with the off-target sequence, thus suppressing cross-reactivity of the detector probe;
  • Figures 2A-2C illustrate examples of detector probe and dark probe pairs having different types of overlapping portions
  • Figures 3 A and 3B compare results of separate conventional KRAS 516 assays, Figure 3A showing an assay in which KRAS 516 was provided as template, and Figure 3B showing an assay in which KRAS 518 was provided as template, the results showing that the KRAS 516 assay probe has substantial cross-reactivity with the off-target KRAS 518 template and can thereby lead to false positives and/or falsely inflated measurement values for KRAS 516;
  • Figures 4A and 4B compare results of separate KRAS 516 assays enhanced to include dark probes for blocking the off-target KRAS 518 template, Figure 4 A showing an assay in which KRAS 516 was provided as template and Figure 4B showing an assay in which KRAS 518 was provided as template, the results showing that the inclusion of dark probes for blocking the off-target KRAS 518 template effectively suppressed cross-reactivity of the KRAS 516 target probe with the off-target KRAS 518 template; and
  • Figures 5A-5D show results of a dark probe titration test for two example dark probes.
  • Figure 1A illustrates a conventional nucleic acid amplification process in which a labelled detector probe is intended to specifically interact with a nucleic acid target and generate a corresponding signal.
  • the detector probe (or some portion of a plurality of detector probes) may instead interact with an off-target sequence, adding to the resulting signal.
  • This cross-reactivity can lead to, for example, false positive detection of the target and/or an inflated calculated amount of the target.
  • Figure IB illustrates an improved nucleic acid amplification process in which a “dark probe” is also provided in the reaction mixture.
  • the dark probe is configured to specifically interact with the off-target sequence and thereby block the detector probe from interacting with the off-target sequence. This beneficially reduces the amount of cross-reactivity between the detector probe and the off-target sequence, which in turn enables the signal to better correspond to the amount of target.
  • the amount of signal associated with the detectable label will better correspond to the amount of nucleic acid target in the sample and will therefore enable more accurate detection and/or quantification of the target nucleic acid.
  • an off-target suppression embodiment such as shown in Figure IB is beneficial where a test sample has (or is suspected of having) one or more sequences that are similar enough to the target nucleic acid to cause some amount of cross-reactivity with the detector probe.
  • one or more dark probes may be used in assays that are designed to amplify and detect a specific allele of a nucleic acid target that is often present with other alleles in the sample.
  • the detector probe is designed to specifically interact with the targeted specific allele, while each dark probe is designed to specifically interact with other alleles to thereby reduce interaction between the detector probe and the off-target nucleic acids of the other alleles.
  • the problem of cross-reactivity with off-target sequences is particularly acute in rare mutation assays designed to detect and/or quantify rare alleles that may be present with other, more abundant alleles.
  • the targeted allele may be present within a background of wild type and other alleles.
  • even limited cross-reactivity with off-target sequences can have a substantial effect on the results simply because the amount of off-target nucleic acid is significantly greater than the amount of target nucleic acid containing the intended allele. That is, even if the detector probe interacts with only a small percentage of the off-target sequences, the abundance of off-target nucleic acid templates relative to target nucleic acid templates in the reaction mixture can lead to excessive disruption of the results.
  • Rare mutation assays may include assays designed to detect and/or quantify oncogenic mutations, for example.
  • the sample will often include relatively high levels of other alleles (e.g., wild type or other alleles) that can affect the results even where the detector probe is designed to specifically interact with the target allele.
  • the sample comprises genomic DNA with a target locus having a mutant allele frequency (MAF) of 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.75% or less, 0.5% or less, 0.25% or less, 0.15% or less, or 0.1 % or less, and the method is capable of effectively detecting the rare mutant allele with greater accuracy than otherwise similar assays omitting dark probes, and/or is capable of effectively quantifying the rare mutant allele with greater accuracy than otherwise similar assays omitting dark probes.
  • MAF mutant allele frequency
  • the off-target nucleic acid sequences are similar to the target sequences except for a few nucleotides (e.g., 2-3 nucleotides). Often, the target sequence and the off-target sequence(s) differ by only a single nucleotide.
  • the target may be a first allele of a particular single nucleotide polymorphism (SNP) location, and the off-target sequence(s) are other alleles of the same SNP location.
  • the detector probe is configured to target the first allele of the SNP location, and a dark probe is configured to target a second, different allele at the SNP location.
  • Some embodiments may include additional dark probes, such as a dark probe for targeting a third allele at the SNP location.
  • additional dark probes such as a dark probe for targeting a third allele at the SNP location.
  • an off-target sequence is not necessarily a different allele of the target sequence, but still resembles the target enough to cause crossreactivity with the detector probe.
  • the detector probe may be configured to target a particular allele at a specified nucleotide location.
  • An off-target nucleic acid may match the target nucleic acid at the mutation locus but have another nucleotide that differs from the target located some number of nucleotides (e.g., 1 to 20) away from the mutation locus.
  • a detector probe and a corresponding dark probe will have similar sequences.
  • the dark probe is adjusted relative to the detector probe to specifically target an off-target sequence rather than the target sequence itself. Often, a detector probe and a corresponding dark probe are the same size, though this need not be the case in all embodiments.
  • Figures 2A-2C illustrate example detector probe and dark probe pairs.
  • a detector probe and a corresponding dark probe will each have an “overlapping sequence portion” representing the respective sequence portions that overlap when the detector probe and dark probe are aligned.
  • the detector probe and the dark probe are the same size and will fully overlap one another, as shown in Figure 2A.
  • the dark probe is shorter than the detector probe, so the overlapping portion is the size of the dark probe (i.e., the detector probe has a 5’ portion not within the overlapping portion).
  • the probes are substantially the same size, but the dark probe is shifted relative to the detector probe such that the detector probe includes a 5’ section not within the overlapping portion and the dark probe includes a 3’ section not within the overlapping portion.
  • the differentiating feature(s) of the probes lie within the overlapping sequence portion.
  • the overlapping sequence portions are preferably about 10-30 nucleotides in length to ensure the dark probes can properly block interaction between the detector probes and the off-target sequences.
  • the overlapping portions of the detector probe and the dark probe differ by only a single nucleotide, with the detector probe including an A (as an example) and the dark probe including a G (as an example) at the corresponding location. This will be the case where the target and off- target sequences are different alleles of a particular SNP.
  • the overlapping sequence portions of the detector probe and the dark probe may differ at more than one nucleotide, may include one or more deletions, and/or may include one or more insertions relative to the opposing probe.
  • the detector probe and dark probe will have about 80%, about 85%, about 90%, or about 95% identity, or will have identity within a range defined by any two of the foregoing values.
  • Some embodiments may include a single detector probe and a single dark probe. Such embodiments may be considered single-plex with respect to the detector probe and single-plex with respect to the dark probe. Other embodiments may include one or more additional detector probes (each typically uniquely labelled and directed to a specific target), and thus be multi-plex with respect to the detector probes, and/or may include one or more additional dark probes directed to other off-target sequences, and thus be multi-plex with respect to the dark probes.
  • each detector probe may have a single corresponding dark probe or multiple corresponding dark probes.
  • the assay may include two dark probes (i.e., dark probe “la” and dark probe “lb”) associated with the first detector probe and each designed to block a different off-target sequence that could cross-react with the first detector probe, and include a single dark probe (i.e., dark probe 2) associated with the second detector probe and designed to block an off-target sequence that could cross-react with the second detector probe.
  • the number of dark probes associated with each different detector probe need not be consistent, and the number of dark probes utilized with each detector probe can instead be chosen based on the number of off-target sequences to be blocked from that particular detector probe.
  • Some embodiments may include one or more detector probes without a corresponding dark probe.
  • the assay may include two dark probes (i.e., dark probe “la” and dark probe “lb”) associated with the first detector probe and each designed to block a different off-target sequence that could cross-react with the first detector probe, a single dark probe (i.e., dark probe 2) associated with the second detector probe and designed to block an off-target sequence that could cross-react with the second detector probe, and may omit any dark probes associated with the third detector probe. This may be the case where significant off-target interaction of the third detector probe is not expected.
  • the dark probe is provided at a concentration at least as great as the concentration of detector probe. In some embodiments, the dark probe is provided at a concentration greater than the concentration of the detector probe. In embodiments that include multiple dark probes each associated with a single detector probe, each of those dark probes may be provided at a concentration that is equal to or greater than the concentration of the detector probe.
  • the detector probe includes a detectable label, whereas the dark probe omits a detectable label.
  • the detectable label of the detector probe may be a fluorescent label. Examples of fluorescent labels are known in the art and include, for example, VIC, FAM, JUN, ABY, Alexa Fluor dye labels (e.g., AF647 and AF676), and combinations thereof.
  • Exemplary detectable labels that may be utilized with the embodiments described herein include, for example:
  • Fluoresceins e.g., 5-carboxy-2,7-dichlorofluorescein, 5 Carboxyfluorescein (5-FAM), 6-JOE, 6-carboxyfhiorescein (6-FAM), VIC, FITC, 6- carboxy-4’,5’-dichloro-2’,7’-dimethoxy-fluorescein (JOE)), 5 and 6-carboxy-l,4- dichloro-2’,7’-dichloro-fluorescein (TET), 5 and 6-carboxy-l,4-dichloro-2’,4’,5’,7’- tetra-chlorofhiorescein, HEX, PET, NED, Oregon Green (e.g. 488, 500, 514));
  • Cyanine Dyes e.g. Cy dyes such as Cy3, Cy3.18, Cy3.5, Cy5, Cy5.18, Cy5.5, Cy7;
  • Rhodamines e.g., 110, 123, B, B 200, BB, BG, B extra, 5 and 6- carboxytetramethylrhodamine (5-TAMRA, 6-TAMRA), 5 and 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Rhod-2, ROX (6-carboxy-X-rhodamine), 5 and 6-ROX (carboxy-X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, 5 and 6 TAMRA (carboxytetramethyl-rhodamine), (TRITC), ABY, JUN, LIZ, RAD, RXJ, Texas Red; and Texas Red-X);
  • Alexa Fluor fhiorophores which is a broad class including many dye types such as cyanines
  • FRET donor/acceptor pairs e.g., fluorescein/fluorescein, fluorescein/rhodamine, fluorescein/cyanine, rhodamine/cyanine, fluorescein/ Alexa Fluor, Alexa Fluor/rhodamine
  • FRET donor/acceptor pairs e.g., fluorescein/fluorescein, fluorescein/rhodamine, fluorescein/cyanine, rhodamine/cyanine, fluorescein/ Alexa Fluor, Alexa Fluor/rhodamine
  • Fluorophore labels may be associated with quenchers such as dark fluorescent quencher (DFQ), black hole quenchers (BHQ), Iowa Black, QSY7, QSY21 quencher, Dabsyl and Dabcel sulfonate/carboxylate quenchers, and MGB- NFQ quenchers.
  • quenchers such as dark fluorescent quencher (DFQ), black hole quenchers (BHQ), Iowa Black, QSY7, QSY21 quencher, Dabsyl and Dabcel sulfonate/carboxylate quenchers, and MGB- NFQ quenchers.
  • Fluorophore labels may also include sulfonate derivatives of fluorescein dyes with SO3 instead of the carboxylate group, phosphoramidite forms of fluorescein, and/or phosphoramidite forms of Cy5, for example.
  • Detector probes may be configured as TaqMan probes, which are known in the art and described in greater detail below. Such probes are able to hybridize to a target downstream from a primer such that exonuclease activity of the polymerase during subsequent primer extension separates a dye label from a quencher to increase the dye signal.
  • Detector probes and dark probes may be about 10 to about 40 nucleotides in length, more preferably about 15 to about 35 nucleotides in length, more preferably about 18 to about 30 nucleotides in length.
  • Dark probes as disclosed herein preferably include a 3 ’ block to prevent or limit the dark probes from acting as primers.
  • the 3’ block may include, for example, an alkyl spacer, a terminal 3 ’ phosphate, a dideoxy nucleotide, an inverted 3 ’ end, or other extension blocks as known in the art.
  • KRAS is a proto-oncogene, and certain KRAS mutations are implicated in a variety of cancers.
  • the KRAS 516 mutation is a 34 G>T nucleotide mutation which causes a G12C amino acid change.
  • assays for detecting and/or quantifying the KRAS 5156 mutation can be obscured by similar off-target sequences, including the KRAS 517 (34 G>A) and/or KRAS 518 (34 G>C) alleles.
  • the dark probes include one or both of those shown in Table 1.
  • An example detector probe is also shown.
  • Other detector probes for KRAS 516 are known in the art (e.g., the detector probe of Assay ID Hs000000047_rm, available from Thermo Fisher Scientific). The reverse complements of these sequences may alternatively be utilized.
  • Figures 3 A and 3B compare results of separate conventional KRAS 516 dPCR assays.
  • Figure 3A shows an assay in which KRAS 516 was provided as template (at 0.5% MAF)
  • Figure 3B shows an assay in which KRAS 518 was provided as template (at 1.75% MAF).
  • VIC fluorescence corresponds to the wild type allele
  • FAM fluorescence corresponds to the mutant allele.
  • Table 2 The quantification results are tabulated in Table 2.
  • Figures 4A and 4B compare results of separate KRAS 516 dPCR assays enhanced to include dark probes (at 1 pM) for blocking the off-target KRAS 518 template, with conditions otherwise held the same as in Example 1.
  • Figure 4A shows the assay in which KRAS 516 was provided as template
  • Figure 4B shows the assay in which KRAS 518 was provided as the template.
  • the results show that the inclusion of dark probes for blocking the off-target KRAS 518 template effectively suppressed cross-reactivity of the KRAS 516 target probe with the off-target KRAS 518 template.
  • the quantification results are tabulated in Table 3.
  • Figures 5 A-5D show results of a dark probe dPCR titration test.
  • Figure 5A shows results using the dark probe of SEQ ID NO: 1 against a KRAS 517 template (5% MAF).
  • Figure 5B shows results using the dark probe of SEQ ID NO:2 against the KRAS 517 template (5% MAF).
  • Figure 5C shows results using the dark probe of SEQ ID NO: 1 against a KRAS 518 template (18% MAF).
  • Figure 5D shows results using the dark probe of SEQ ID NO:2 against a KRAS 518 template (18% MAF).
  • the dark probe of SEQ ID NO:2 was provided at 2 pM in a reaction mixture including 10X higher concentration of off- target template relative to target template. Two pools of template were formed. Pool 1 included target templates KRAS 516, 520, and 521 at 0.5% MAF each. Pool 2 included the same target templates at the same MAF and included off-target templates KRAS 517 and 518 at 5% MAF each. These pools were subjected to a multiplex dPCR assay targeting the KRAS 516, 520, and 521 mutations. The results are tabulated in Table 4. [0057] The results were substantially similar whether Pool 1 or Pool 2 was used, indicating that the dark probe beneficially limited cross-reactivity while also not disrupting the quantification of the target templates.
  • Assays utilizing the off-target suppression embodiments described herein may additionally include one or more sets of primers to enable amplification of target nucleic acids.
  • an assay may include at least one pair of primers configured to amplify the nucleic acid target.
  • Multiplex embodiments may include additional sets of primers, each set designed to enable amplification of a different target.
  • amplification reaction mixture components known in the art may also be included in an assay composition and/or in an assay kit. Such components may include, for example, polymerase, nucleotides, one or more buffers, and/or one or more salts to promote amplification of the target when the mixture and a sample combined therewith are exposed to amplification conditions.
  • Dark probes may be provided at concentrations of at least about 0.5 M, or at least about 0.75 pM, or at least about 1 pM, or at least about 1.25 pM, or at least about 1.5 pM, or at least about 1.75 pM, or at least about 2 pM.
  • the source for the sample will often be a clinical sample, such as a blood sample.
  • Other sources for the sample include, but are not limited to, forensic or environmental samples (e.g., clothing, soil, paper, surfaces, water), plants, human and/or animal skin, hair, blood, serum, feces, milk, saliva, urine, and/or other secretory fluids.
  • Amplified products resulting from use of one or more embodiments described herein can be generated, detected, and/or analyzed on any suitable platform.
  • the nucleic acid targets may be single-stranded, doublestranded, or any other nucleic acid molecule of any size or conformation.
  • the amplification processes described herein can include PCR (see, e.g., U.S. Pat. No. 4,683,202).
  • the PCR is real time or quantitative PCR (qPCR).
  • the PCR is an end point PCR.
  • the PCR is digital PCR (dPCR).
  • Other amplification methods such as, e.g., loop-mediated isothermal amplification (“LAMP”), and other isothermal methods are also contemplated for use with the assay embodiments described herein.
  • LAMP loop-mediated isothermal amplification
  • dPCR In rare mutation assays, dPCR is commonly utilized.
  • the reaction mixture is partitioned into many small reaction volumes (i.e., partitions) so that the target nucleic acid is in some, but not all, of the reaction volumes / partitions.
  • the reaction volumes are subjected to thermal cycling, and the proportion of “positive” partitions that generate a signal (usually a fluorescence signal) indicative of the presence of the target is determined.
  • Quantitation is based on application of Poisson statistics, using the number of negative/non-reactive reaction volumes and assuming a Poisson distribution to establish the number of initial copies that were distributed across all the reaction volumes.
  • Embodiments that include dPCR may utilize a variety of partitioning mechanisms or devices as known in the art or as may be developed in the future.
  • some conventional dPCR systems utilize a plurality of droplets encapsulated by an oil phase to form the plurality of parti tions/reaction volumes.
  • Other embodiments may utilize an array of microchambers.
  • QuantStudio Absolute Q system available from Thermo Fisher Scientific, which uses a microfluidic array plate to perform the compartmentalizing/partitioning of sample.
  • the nucleic acid amplification assays as described herein are performed using a qPCR instrument, including for example a QuantStudio Real-Time PCR system, such as the QuantStudio 5 RealTime PCR System (QS5), QuantStudio 7 RealTime PCR System (QS7), and/or QuantStudio 12K Flex System (QS12K), or a 7500 Real-Time PCR system, such as the 7500 Fast Dx system, from Thermo Fisher Scientific.
  • a QuantStudio Real-Time PCR system such as the QuantStudio 5 RealTime PCR System (QS5), QuantStudio 7 RealTime PCR System (QS7), and/or QuantStudio 12K Flex System (QS12K
  • a 7500 Real-Time PCR system such as the 7500 Fast Dx system, from Thermo Fisher Scientific.
  • nucleic acid includes compounds having a plurality of natural nucleotides and/or non-natural (or “derivative”) nucleotide units.
  • a “nucleic acid” can further comprise non-nucleotide units, for example peptides.
  • Nucleic acid therefore encompasses compounds such as DNA, RNA, peptide nucleic acids, phosphothioate-containing nucleic acids, phosphonate-containing nucleic acids and the like. There is no particular limit as to the number of units in a nucleic acid, provided that the nucleic acid contains 2 more nucleotides, nucleotide derivatives, or combinations thereof, specifically 5, 10, 15, 25, 50, 100, or more. Nucleic acids can encompass both single and double-stranded forms, and fully or partially duplex hybrids (e.g., RNA-DNA, RNA-PNA, or DNA-PNA).
  • primer may refer to more than one primer and refers to an oligonucleotide, whether occurring naturally, as in a purified restriction digest, or produced synthetically, which is capable of acting as a point of initiation of synthesis along a complementary strand when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is catalyzed. Such conditions include the presence of four different deoxyribonucleoside triphosphates and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer and at a suitable temperature.
  • a primer is typically 11 bases or longer; more specifically, a primer is 17 bases or longer, although shorter or longer primers may be used depending on particular application needs.
  • target refers to a region of a nucleic acid which is to be either amplified, detected, or both.
  • the target sequence resides between the two primer sequences used for amplification.
  • any of the possible alternatives listed for that element or component may generally be used individually or in combination with one another, unless implicitly or explicitly stated otherwise.
  • embodiments described herein may also include properties and/or features (e.g., ingredients, components, members, elements, parts, and/or portions) described in one or more separate embodiments and are not necessarily limited strictly to the features expressly described for that particular embodiment. Accordingly, the various features of a given embodiment can be combined with and/or incorporated into other embodiments of the present disclosure. Thus, disclosure of certain features relative to a specific embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment. Rather, it will be appreciated that other embodiments can also include such features.

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Abstract

L'invention concerne des compositions et des procédés pour supprimer la génération de signal hors cible dans un processus d'amplification d'acide nucléique. Les compositions et les procédés utilisent une sonde de détection avec un marqueur détectable et une sonde sombre omettant un marqueur détectable. La sonde de détection est conçue pour interagir spécifiquement avec un acide nucléique cible. La sonde sombre est conçue pour interagir spécifiquement avec une séquence hors cible similaire à l'acide nucléique cible, la sonde sombre limitant ainsi l'interaction hors cible entre la sonde de détection et la séquence hors cible.
PCT/US2023/082292 2022-12-05 2023-12-04 Suppression de signal hors cible dans des réactions d'amplification d'acide nucléique WO2024123672A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
WO2017062613A1 (fr) * 2015-10-07 2017-04-13 Illumina, Inc. Réduction de capture hors cible dans des techniques de séquençage

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
WO2017062613A1 (fr) * 2015-10-07 2017-04-13 Illumina, Inc. Réduction de capture hors cible dans des techniques de séquençage

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