WO2013022807A1 - Procédés et compositions pour détection d'acides nucléiques à l'aide de clivage par 5'-nucléase et d'amplification - Google Patents

Procédés et compositions pour détection d'acides nucléiques à l'aide de clivage par 5'-nucléase et d'amplification Download PDF

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WO2013022807A1
WO2013022807A1 PCT/US2012/049672 US2012049672W WO2013022807A1 WO 2013022807 A1 WO2013022807 A1 WO 2013022807A1 US 2012049672 W US2012049672 W US 2012049672W WO 2013022807 A1 WO2013022807 A1 WO 2013022807A1
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primer
probe
amplicon
digestion
driving
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PCT/US2012/049672
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English (en)
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Eugene Spier
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Unitaq Bio
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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
    • C12Q1/6858—Allele-specific amplification
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/30—Oligonucleotides characterised by their secondary structure
    • C12Q2525/301—Hairpin oligonucleotides

Definitions

  • PCR polymerase chain reaction
  • a probe molecule that includes a fluorescent label and a quencher hybridizes to a PCR amplification product, and is digested by the 5' exonuclease activity of a polymerase. The 5' exonuclease activity releases the fluorescent label from the probe, thereby separating the fluorescent label from the quencher and permitting detection of the fluorescent signal.
  • U.S. Patent 5,210,015 to Gelfand et al. discloses 5 ' nuclease (TaqMan) assays. It teaches in claim 1 that the 3' end of the probe is annealed upstream of the 5 '-end of the probe: "...a labeled oligonucleotide containing a sequence complementary to a second region of the same target nucleic acid sequence strand, but not including the nucleic acid sequence defined by the first oligonucleotide, to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first oligonucleotide and to the labeled oligonucleotide such that the 3' end of the first oligonucleotide is upstream of the 5' end of the labeled oligonucleotide".
  • U.S. Patent 5,487,972 to Gelfand et al. describes the 3' end of the primer being "adjacent" to the 5' end of the probe and "labeled oligonucleotide containing a sequence complementary to a second region of the same target nucleic acid sequence strand, but not including the nucleic acid sequence defined by the fist oligonucleotide" (primer).
  • U.S. Patent 5,804,375 to Gelfand et al. describes a "reaction mixture” (composition) that includes two primers and a probe, the probe being “between” the two primers. It also defines the term “adjacent” as follows: “The term 'adjacent' as used herein refers to the positioning of the primer with respect to the probe on its complementary strand of the template nucleic acid.
  • the primer and probe may be separated by 1 to about 20 nucleotides, more preferably, about 1 to 10 nucleotides, or may directly abut one another, as may be desirable for detection with a polymerization-independent process.
  • the 'adjacency' may be anywhere within the sequence to be amplified, anywhere downstream of a primer such that primer extension will position the polymerase so that cleavage of the probe occurs.”
  • Primer3 is the most widely used software to design PCR primers and probes (Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist
  • the prior art mentioned above explicitly teaches to avoid overlap between the probe sequence and the extendable primer sequence in PCR. Intuitively this makes sense, since any overlap would create a competition between the primer and the probe: if the 3 ' end of the primer is annealed, the 5 ' end of the probe must be off the template or vice versa.
  • the prior art teaches that polymerase extension is the first step and that it precedes 5'nuclease cleavage.
  • Described herein are methods and compositions related to 5 '-nuclease during PCR that (a) increase signal generation in qPCR or (b) are using to improve mutation-specific primer specificity using a blocking oligo.
  • the method includes forming an amplicon; annealing a probe comprising a 5 ' portion, and a probe, comprising a 5 ' end region, and a digestion-driving primer, comprising a 3 ' end, to the amplicon; wherein the 3 ' end of the digestion-driving primer overlaps the 5 ' portion of the probe by at least one nucleotide when both are annealed to the amplicon, wherein a 5 'nuclease reaction can precede the primer extension of said digestion-driving primer.
  • the overlap is one nucleotide.
  • the digestion-driving primer and the probe comprise two parts of a single molecule, wherein the single molecule forms a circular structure when the two parts anneal to the amplicon.
  • the method includes providing a second primer with a 5 '-probe portion; extending the second primer to form the amplicon, wherein said amplicon comprises a region complementary to the 5' - probe portion; said amplicon forming an intramolecular hairpin stem structure during PCR by annealing of the 5 '-probe portion and said region complementary to the 5 '-probe portion; and, annealing the digestion-driving primer to the amplicon, so that the 3 ' end of the digestion-driving primer overlaps the 5 '-portion of the stem by at least one nucleotide.
  • a PCR reaction mixture comprising (i) an enzyme having 5 'nuclease activity; (ii) a digestion-driving primer comprising a 3' end, and (iii) a probe comprising a 5 ' region, wherein the 3 ' end of the digestion-driving primer and the 5 ' region of the probe overlap by at least one nucleotide when both the primer and the probe are annealed to an amplicon formed in said mixture.
  • the digestion- driving primer and the probe comprise two parts of a single molecule, wherein the single molecule is capable of forming a circular structure when the two parts anneal to the amplicon.
  • the reaction mixture includes a second primer comprising a 5 '-probe portion, said second primer capable of generating an amplicon having a region complementary to the 5' probe portion; wherein said amplicon is capable of forming an intramolecular hairpin stem structure by annealing of the 5 '-probe portion and the region complementary to the 5 '-probe portion, wherein the digestion-driving primer can anneal to said amplicon so that the 3 ' end of the digestion-driving primer overlaps the 5 ' portion of the stem.
  • the 5 ' region of the probe when annealed to the amplicon is more thermodynamically stable as compared to the 3 ' region of the digestion-driving primer when it is annealed to the amplicon, so that the structure with 5' probe portion being annealed to the amplicon, favored by 5 'nuclease, is predominant.
  • modified nucleotides are used to alter the thermodynamic stability of the 3' region of the digestion driving primer and 5' region of the probe when the primer and probe are annealed to the amplicon.
  • the 3 ' end of the primer has a mismatch (non- Watson-Crick pair) or blocked non-extendable 3 ' end when annealed to the amplicon.
  • the (a) probe, (b) primer-probe after extension or (c) digestion-driving primer comprise a label and label quencher that are capable of being separated from each other by said 5 'nuclease reaction and generating a detectable signal that allows detection of the presence and/or the amount of target nucleic acid.
  • the 5 ' end of the probe comprises a flap sequence tag comprising a label quencher or a label configured to generate a fluorescent or
  • a software tool comprising instructions to design primers and probes for methods as described above or for preparing PCR mixtures as described above, said instructions comprising choosing a probe and a digestion-driving primer that match a target nucleotide sequence, or tags in an encoded target sequence, so that there is an overlap of at least one nucleotide between the 5 ' end of the probe and the 3 ' end of the digestion-driving primer.
  • a kit includes a digestion-driving primer, a probe or second primer-probe and second primer for use in mixtures as described above, wherein all primers and probes required for a specific application are delivered to customers mixed together or separately and primers and probes can be inventoried, pre-designed or designed when customers submit their target sequences.
  • we provide methods to increase specificity of mutation- specific PCR comprising providing a template comprising a putative mutated allele; annealing to said template a blocking oligo having a 5 ' base which matches the mutated allele; and annealing to said template a mutation-specific primer having a 3' end which matches said mutated allele; wherein a 5 'nuclease reaction cleaves the 5' end of the blocking oligo when the mutation-specific primer and the oligo are annealed to the mutated template, but wherein the blocking oligo is cleaved very inefficiently if the template is wild type, thereby further slowing mutation-specific primer extension on the wild-type template.
  • a PCR reaction mixture used to detect SNPs or mutations comprising (i) a polymerase with 5 'nuclease activity; (ii) a mutation-specific primer; (iii) a blocking oligo with blocked or otherwise un-extendable 3 ' end; and (iv) template comprising a putative mutated allele, such that the 3 ' end of the primer and at the 5 ' end of the blocking oligo when both are annealed to the template match the mutated allele, and wherein the 5 'nuclease activity is capable of cleaving the 5 ' end of the blocking oligonucleotide so that the primer can extend on a mutant allele, but the 5 'nuclease cleavage is substantially reduced if the template is wild-type, thus providing additional blocking for primer extension.
  • the mutation-specific primer and blocking oligonucleotide comprises a single molecule that forms a circular
  • the digestion-driving primer, probe and/or amplicon (after surface-bound primer extends) and optionally the second primer are attached to a surface during PCR or a bridge amplification and contain at least one label and optionally least one label quencher.
  • Fig. 1 schematically illustrates a traditional 5' nuclease (TaqMan) cleavage reaction.
  • Fig. 2 schematically illustrates some embodiments of a primer extension reaction.
  • Fig. 3 schematically illustrates an exemplary universal detection format.
  • Fig. 4 schematically illustrates an exemplary universal circle detection format.
  • Fig. 5 schematically illustrates an exemplary surface-based universal detection format that utilizes bridge PCR and circular primer-probe topology.
  • Fig. 6 schematically illustrates an exemplary universal circle detection format using primers incorporating flaps.
  • Fig. 7 shows quantitative PCR data.
  • Fig. 8 schematically illustrates some embodiments of mutation [allele] -specific
  • Fig. 9 illustrates a flow diagram of some embodiments of a program for primer and probe design.
  • the present invention is broadly applicable to any application in which one desires to detect or measure the amount of one or more target nucleic acids in a sample of interest.
  • overlap 5 ' nuclease assay methods and compositions used in PCR in which the sequence at the 3 ' end of a digestion-driving primer overlaps with the sequence at the 5' end of the detection probe.
  • the present invention is broadly applicable to any application in which one desires to detect or measure the amount of one or more target nucleic acids in a sample of interest.
  • the present invention is based in part on the surprising observation by Applicant, as further described below (Example 1 and Fig. 7), that when primers and probes which were intentionally designed to incorporate a single-base overlap between the sequence of the 3 ' end of the primer and the sequence of the 5 ' end of the detection probe were utilized in a quantitative PCR (qPCR) reaction (also referred to as real-time PCR), the amplification signals improved as compared with control reactions using conventional, non-overlapping primers and probes.
  • qPCR quantitative PCR
  • the current dogma for qPCR detection is that the method always produces relative data: delta C t values are used to compare samples to each other or to the "standard curve” and quantification is different for each assay.
  • Conventional TaqMan methodology see U.S.
  • Patent 5,487,972 and 5,804,375 is considered the "gold standard" for qPCR.
  • intramolecular detection methods described previously, see, e.g., U.S. Patent 6,326,145 to Whitcombe et al.
  • DNA polymerases used in PCR are bi-functional molecules having both polymerization (P) and 5 ' nuclease (N) active sites, which are contained in separable domains.
  • a polymerase may be depicted as having a polymerase active center (shown as a dashed circle in Fig. 1 and 2) and a 5' nuclease active center (shown as a solid circle in Fig. 1 and 2) oriented towards the junction of the 3' end of the primer and 5' end of the probe. Referring to Fig.
  • the polymerase (i) has to displace the 5 ' most matching base of the probe and make a base extension to generate a one -base overlap (Fig. 1(a) and 1(b)), (ii) the 3' end of the primer needs to move away from the template so that the 5 ' base of the probe can anneal back to the template (Fig. 1(d)) and (iii) the polymerase has to reorient so that its nuclease active center faces the 5' end of the probe (Fig. 1(d)) in order to perform the cleavage (not shown). Cleavage can only occur if the 5' end nucleotide is annealed to the template. In some circumstances, the polymerase does not have sufficient strand
  • step (i) would decrease the overall efficiency of PCR.
  • the polymerase would have an easier time by merely continuing the extension, with
  • one or more nucleotides are designed such that at the 5 ' end of the probe is more thermodynamically stable in binding to the template than the nucleotides at the 3' end of the primer (the two are competing to anneal to the same base[s] on the template) in order to promote the structure in Fig. 2(a) and thus promote 5 'nuclease activity over extension.
  • a dinucleotide at the 5 ' end of the probe can be designed to be more thermodynamically stable than the dinucleotide at the 3 ' end of the primer.
  • duplex stabilizing bases in the 5' region of the probe where nuclease cleavage is to occur and/or introduce duplex destabilizing bases close to the 3 ' end of the primer.
  • duplex-stabilizing bases include, but are not limited to, C-5 propynyl-dC (pdC), 5-methyl-dC or AP-dC that can substitute for "C", pdU for "T” and 2-amino-dA for "A”.
  • pdC C-5 propynyl-dC
  • 5-methyl-dC or AP-dC that can substitute for "C", pdU for "T”
  • 2-amino-dA for "A”.
  • probes that overlap with primers can use modified bases, for example, LNA (locked nucleic acids) are currently used by UPL (universal probe library) from Exiqon/Roche.
  • probe-stabilizing moieties may be used, such as, MGB (minor groove binder from Epoch/Life technologies) or BHQplus (BioSearch).
  • a modification is introduced at the 3 ' end of the primer which overlaps the 5 ' end of the probe, such that polymerase cannot extend or extends
  • a mismatch base at the 3 ' end of a primer would cause the 3 ' end of the primer to be less thermodynamically stable than the 5' end of the probe thus promoting the structure shown in Fig. 2(a).
  • the polymerase would be very inefficient in extending such a mismatch, while the 5 'nuclease activity would not be affected by the 3' mismatch (Lyamichev, 1999). After the 5 'nuclease cleavage, the 3 '-exonuclease
  • a base modification can be incorporated at the 3' end of the primer, e.g., pyrophosphate, that prevents primer extension until the blocking moiety is removed.
  • Probes can include one or more than one quencher.
  • ZEN probes from IDT include both a 3 '-quencher and a second internal ZEN quencher closer to the middle of the probe that decreases the background fluorescence.
  • step (b) maintaining the mixture of step (a) with a template-dependent nucleic acid polymerase having a 5' to 3' nuclease activity under conditions sufficient to permit the 5' to 3' nuclease activity of the polymerase to cleave the annealed, labeled oligonucleotide and release labeled fragments; and
  • step (c) detecting and/or measuring the release of labeled fragments.
  • the 3' end of the first oligonucleotide in the annealed duplex of step (a) overlaps the 5' end of the annealed, labeled oligonucleotide by one nucleotide.
  • nucleotide overlaps described herein may be readily introduced in universal assays such as described in our Published Application No. US 2011/0212846 (herein incorporated by reference in its entirety), as well as traditional target-specific 5 'nuclease (aka TaqMan) assays described above.
  • the present invention is directed to methods and
  • compositions for detecting and/or measuring the amount of a target nucleic acid in a sample using a set of universal PCR primers with sequences that do not hybridize to the target nucleic acid of interest provides a method for detecting a target nucleic acid that includes tagging the target nucleic by linking first and second universal segments at the two ends of a molecule in a way that depends on the target nucleic acid, and PCR amplifying the tagged target nucleic acid using universal primers that hybridize to the universal segments.
  • regions of the PCR amplicons corresponding to the first and second universal segments form intramolecular hairpin stems, where hairpin stem formation is a prerequisite for detection of the nucleic acid, e.g., by removal of a label, label quencher and/or FRET dye from the amplicon.
  • nucleic acids that utilize artificial universal tagging sequences rather than traditional direct detection of DNA/RNA using primers and probes matching the target nucleic acids.
  • Target nucleic acids are only used to specifically connect distinct universal tagging sequences into a single molecule during the first encoding step.
  • the detection is based on the intra-molecular Watson-Crick base-pairing (a hairpin stem) between the two universal tags in the amplicon.
  • the base-pairing between the two universal tagging sequences enables detection of nucleic acids that preserves "three tag" detection specificity: spurious amplifications and primer dimers have a low probability of forming hairpins.
  • universal surface solid-phase detection for encoded nucleic acid targets are also provided.
  • step (a) includes priming on both strands of a first tagged nucleic acid using first universal primer 300 and second universal primer 302.
  • a 3' portion (Ai) of the first universal primer anneals to first universal segment 304, while a 3' portion (Ci) of the second universal primer anneals to portion C'i of second universal segment 306.
  • the first universal primer includes detectable label Dl at or near the 5' end of the first universal primer, 5' portion (Bi) and a label quencher or FRET dye (Q).
  • a polymerase blocking unit e.g., HEG (hexethylene glycol), or any other blocker known in the field in case the presence of the quencher or FRET dye is not sufficient to stop the polymerase extension.
  • HEG hexethylene glycol
  • primers with a HEG or Sp-18 polymerase blocking unit are commercially available, e.g., from BioSearch Technologies.
  • Some quenchers can also serve as polymerase blockers, e.g., ZEN from IDT.
  • polymerase blocking units are not shown.
  • a polymerase blocking unit is optionally positioned in proximity to quencher "Q" in the middle portion of the universal primers.
  • a polymerase blocker may not be required if the 5 '-tail that folds into a stem has one or more bases at the 5' end that are not complementary to the middle universal tag sequence, so that the hairpin formed by the opposite strand of DNA (with the 3 '-end at the end of the stem) is not extendable during PCR.
  • PCR amplification results in double stranded product 308.
  • a polymerase blocking unit positioned proximal to the label quencher or FRET dye prevents a polymerase from copying the 5' portion (Bi) of the first universal primer, such that the bottom strand of product 308 cannot form a hairpin when it becomes single-stranded.
  • Hairpin formation is shown at step (c) of Fig. 3.
  • Product 308 is melted (e.g., by raising the temperature to approximately 95°C.) to separate the upper strand from the lower strand, and when the temperature is subsequently decreased, the upper strand of product 308 forms a hairpin having a stem between 5' portion (Bi) of the first universal primer and portion B'i at the opposite end of the strand.
  • the second universal primer Ci anneals to a complementary portion of the upper strand.
  • the 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one or more nucleotides. In some embodiments, the 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one nucleotide.
  • Intra-molecular hairpin formation occurs rapidly and is driven by thermodynamics: the free energy is determined by stem length, GC-content and loop length. It is important that the melting temperature (Tm) of the hairpin be significantly higher (e.g., approximately 10°C. or higher) than the Tm of the second universal primer 302. This way, when the temperature is decreased, nearly 100% of the molecules will form the hairpin before the second universal primer anneals.
  • Tm melting temperature
  • 5' nuclease activity cleaves the detectable label Dl from the 5' end of the amplicon, thereby increasing the distance between the label and the quencher or FRET dye and permitting detection of the label.
  • fluorescent dyes are known in the art and commercially available, e.g., FAM, TET, JOE, VIC, HEX, CY3, TAMRA, TexasRed, CY5, ROX and many other dyes and quenchers can be used, e.g., Dabsyl, MGB-NFQ, BHQ-[0123], Iowa Black, ZEN quencher from IDT, and others.
  • all primers in Fig. 3 are target- specific, and the formation of an intramolecular hairpin stem is similar to that described in U.S. Pat. No. 6,326,145.
  • a detection format involving the formation of a universal circle are schematically illustrated in Fig. 4.
  • the tagged target nucleic acid is amplified using universal primer 400 (including Dl-Bi-Q-spacer-x-Ci-3') and second universal primer 402 (including Ai), where Dl is a detectable label, Q is a quencher and "x" is a polymerase blocking unit.
  • a 3' portion (Ci) of the first universal primer anneals to first universal segment 404, while a 3' portion (Ai) of the second universal primer anneals to second universal segment 406.
  • primer 400 is designed such that the 3' end of the primer overlaps with its own 5' tail (forming a circular structure).
  • the first universal primer Ci at step (b) generates a signal by 5' exonuclease cleavage of Dl and separation of Dl from the quencher, and then extends.
  • a surface-based universal detection format provided by the present invention are schematically illustrated in Fig. 5.
  • universal detection is accomplished via "bridge PCR" or bridge amplification, where the universal primers are attached to the surface of a bead.
  • first universal primer 500 and second universal primer 502 are attached to the surface of bead 504.
  • the first universal primer includes Q-Bi-Dl-spacer-[surface]-spacer-Ci-3' and the second universal primer includes Ai, where Q is a label quencher and Dl is a detectable label, e.g., a fluorescent dye.
  • First tagged target nucleic acid (including Ai-B'i-C'i) anneals to both the Bi and Ci portions of the first universal primer, bringing the 3 '-end of Ci close to the 5 '-quencher of Bi.
  • the 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one or more nucleotides. In some embodiments, the 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one nucleotide.
  • the Ci at step (c) cleaves the quencher and generates fluorescent signal Dl .
  • the extended amplicon forms a bridge by looping back onto Ai on the surface, and Ai extends to form a complementary strand, thus continuing amplification on the surface.
  • the first universal primer on the surface is shown attached in the middle.
  • An alternative configuration in accordance with the present invention would be to have a spacer-Ci-3' primer attached to the bead at the 5' end and Q-Bi-Dl-spacer-5' attached at the 5' end. The ends of the two attached primers would come into proximity with each other due to their attachment to the same bead surface.
  • the two parts of the first universal primer are formally two separate oligonucleotides, but being bound to the same bead surface they are effectively linked into a single molecule where the surface provides a link.
  • Ai primer can be in solution (not surface bound), so that only a single strand with the label is bound to the surface.
  • the same Ai solution primer can be used by surfaces with different B- and Ci tags.
  • beads with different intrinsic bead properties can be used for multiplex detection in Fig. 5.
  • bead color Luminex microspheres
  • holographic images Illumina VERACODETM
  • barcodes Applied BIOCODETM beads, Affymetrix liquid arrays
  • Bead encoding can be performed by the bead vendor: each type of bead with different colors or barcodes is combined with specific tags Bi, Ci and optionally Ai. All encoded beads are pooled together for multiplex detection and the universal pool can be used to detect or measure any nucleic acid target. Users can mix their samples containing target nucleic acids, encoding primers, master mix and encoded beads and PCR cycle the mixture. First, encoded target molecules will be generated and then these molecules anneal to the surface bound universal primers and generate a signal on the surface (see, e.g., Fig. 5).
  • targets are first tagged by linking universal segments together.
  • the tagged/encoded sample is optionally diluted and added to the pooled decoding beads for detection.
  • bridge amplification or PCR generates signal on the bead surface.
  • the beads can be laid on a surface (e.g., VERACODETM), streamed past a detector (e.g., Luminex microspheres) or directly imaged in a well (e.g., Applied BIOCODETM).
  • the barcode on a bead or color of a microsphere determines the target and the fluorescent signal on the surface measures the amount of this nucleic acid target in the sample.
  • multiplex surface detection can measure the amounts of multiple targets in a well using a set of universal beads or microspheres.
  • the universal detection formats described above are exemplary approaches. Additional universal detection formats are possible and within the scope of the present invention. It will also be appreciated that any of the exemplary universal detection formats described above can be combined with any of the universal segment linking strategies and universal detection applications described herein. Further, it will be understood that the universal detection formats of the present invention can occur in multiplex, where 2 or more, e.g., 4 or more, 6 or more, 8 or more, 10 or more, hundreds or more, or even thousands or more different target nucleic acids of interest can be detected simultaneously in wells (e.g., nano-wells), on beads, on an array, using integrated fluidics chips (IFC), and the like.
  • wells e.g., nano-wells
  • IFC integrated fluidics chips
  • step (a) includes priming on both strands of a first tagged nucleic acid using first universal primer 66 and second universal primer 68.
  • a 3' portion (Ai) of the first universal primer anneals to portion Ai of a first universal segment 62, while a 3' portion (Ci) of the second universal primer 68 anneals to portion C'i of second universal segment 64.
  • the first universal primer includes flap Fli and detectable label La at or near the 5' end of the flap, 5' portion (Bi) and a label quencher or FRET dye (Q).
  • the second universal primer includes flap F2i and detectable label La at or near the 5' end of the flap, 5' portion (Ei) and a label quencher or FRET dye (Q).
  • PCR product is melted (e.g., by raising the temperature to approximately 95°C.) to separate the strands, and when the temperature is subsequently decreased, one strand of product forms a hairpin having a stem between 5' portion (Bi) of the first universal primer and portion B'i at the opposite end of the strand and the other strand having a stem between 5' portion (Ei) of the first universal primer and portion E'i at the opposite end of the strand.
  • the universal primer Ci anneals to a complementary portion of one strand.
  • the 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one or more nucleotides.
  • the 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one nucleotide.
  • the 3' terminus of primer Ai overlaps the 5' terminus of portion Ei by one or more nucleotides.
  • 5' nuclease activity cleaves the flaps allowing signal generation.
  • improved methods for enrichment and detection of rare alleles which take advantage of the interplay between the 5 ' nuclease activity and the polymerse activity of DNA polymerase in order to increase the specificity of mutation (allele)-specific PCR.
  • the present methods provide a novel means to prevent mutation specific primers from occasionally extending a 3 ' mismatched nucleotide on the "wild-type" alleles, and thereby substantially avoid false positive readings.
  • a blocking oligonucleotide is used with its 5 ' nucleotide matching the mutation in the same reaction with an allele-specific blocking oligonucleotide.
  • an allele-specific primer has an arrow at the 3 '-end.
  • the blocking oligonucleotide or tag is depicted as a double-line.
  • Fig. 8(a) shows blocking on a wild-type amplicon
  • Figs. 8(b) and 8(c) show 5 'nuclease cleavage followed by primer extension on a mutated amplicon.
  • Fig. 8(a) shows blocking on a wild-type amplicon
  • Figs. 8(b) and 8(c) show 5 'nuclease cleavage followed by primer extension on a mutated amplicon.
  • the 5 'nuclease reaction is inhibited because of the 5' nucleotide mismatch in the blocking oligonucleotide.
  • the 5 'nuclease reaction will readily cleave off the 5' nucleotide on DNA templates with mutations, and polymerase will then extend the primer, displacing the rest of the blocking oligonucleotide off of the template (Fig. 8(c)).
  • the blocking oligonucleotide has a non-extendable 3' end; either a blocking moiety, e.g., 3 'phosphate, amine, a quencher, C3, etc. or mismatch bases can be used (shown as "X" in Fig. 8(a)-(c)).
  • the blocking oligo has a higher Tm that the mutation-specific primer, so that it anneals before the primer.
  • the blocking methods as disclosed herein differ from other blocking methods (e.g., Zhou et al., 2011 and Tan et al., 2010) in at least two ways: (a) both allele-specific primer and blocking oligonucleotide are bound to the template concurrently rather than competing with each other, so that only one of the two can bind at any given time; (b) the blocking oligonucleotide matches the mutation, rather than the wild- type as in the other methods.
  • Some embodiments utilize a unimolecular format, in which the function of the blocking oligonucleotide is performed by the 5 '-tail part of the allele-specific primer itself, when it anneals to the template forming a circle, so that the 3 ' and 5 ' ends of the allele- specific primer match the same mutated allele on the template (Figs. 8d, 8e).
  • a blocking moiety e.g., HEG (AKA SP18) is present in the region of the spacer of circular allele-specific primer ("X" in Fig 8d-f), so that it continues forming a circle, rather than priming, during later cycles of PCR.
  • Overlap 5 'nuclease assays can be used for all nucleic acid detection applications, including, but not limited to, (1) SNP/indel/MNP genotyping or allelic expression, (2) mRNA (gene expression) including splice junctions and fusions, (3) copy number variation, (4) miRNA or other non-coding RNA, (5) methylation, (6) bacteria, viral or fungal or any species, (7) mutations, including somatic (8) translocations, inversions, insertions, deletions or other large-scale genomic variations, and (9) nucleic acids treated with bisulphate, nucleases or restriction enzymes.
  • the read-out for qPCR reaction can be real-time: signal measured at many or every cycle or end-point signal detected at the end of qPCR.
  • the latter can also be used for digital counting, so called digitalPCR: counting number of positive (1, 2, 3 etc. copies of [encoded] target DNA) and negative droplets, beads or wells, and applying Poisson statistics to accurately approximate the number of [encoded] DNA target molecules in the sample.
  • a new software tool is provided herein which allows the user to incorporate overlaps between the probe and the associated primer.
  • the methods include applying criteria for designing a candidate primer and a candidate probe for amplifying the target, wherein the criteria include designing the 3 ' end of the primer to overlap the 5 ' end of the probe by at least one nucleotide.
  • the methods can also include receiving and storing a sequence of a target nucleic acid to be amplified by the primer and probe; and storing and/or displaying the sequence of the candidate primer and probe.
  • the methods can include transmitting the sequence of the candidate primer and probe.
  • Fig. 9 is a flow diagram of some embodiments of a computer implemented process 900 for generating candidate PCR primers and probes for use in the methods disclosed herein.
  • a user interface e.g., form
  • the target sequence is received (i.e., entered by the user) at step 920 and stored.
  • the parameters, including start and stop, are received at step 930.
  • primer parameters are optionally received.
  • primer and probe overlap parameters are received.
  • step 960 the user clicks a "Pick Primer" key or similar selection key, and the program scores and displays candidate forward primers, candidate reverse primers, and probe sequences.
  • step 970 the program scores and displays candidate primer and probe sequences.
  • primer/probe design software tools pick multiple forward, reverse primer and probe candidates independently and then find combinations that provide better scores based on multiple criteria: primer/probe-Tm, complementarity between oligos, primer/probe/amplicon lengths, etc.
  • Primer3 software documentation describes multiple criteria used for primer/probe design.
  • the overlap primer/probes design methods as disclosed herein, are different: first multiple candidate digestion-driving primer and probe pairs with a desired overlap are picked and scored. Then multiple 2 nd primer candidates are scored by themselves and against primer/probe pairs based on the criteria that are similar to those traditionally used for primer probe design.
  • the 2 nd primers are spliced with the probe with an optional polymerase blocker in-between.
  • the probe can match either of the two DNA strands and thus it can be paired with either the forward or the reverse primer.
  • the reverse primer primes on the strand different from the probe, so this is only a "computational" and not a real overlap.
  • kits suitable for carrying out an amplification as described herein can include reagents suitable for carrying out qPCR amplification reactions.
  • the kit comprises forward and reverse amplification primers suitable, as described herein, for amplifying a target polynucleotide of interest and a detection probe.
  • the 3' end of the forward primer and the 5 ' end of the probe are designed to have an at least one nucleotide overlap when bound to a template as described herein.
  • the overlap is two nucleotides.
  • the overlap is one nucleotide.
  • a kit includes at least one DNA polymerase, dNTPs, and/or buffer, suitable for carrying out template-dependent primer extension.
  • the probe is labeled with a labeling system suitable for monitoring amplification of a target polynucleotide as a function of time.
  • a kit can include written directions for carrying out methods as described herein.
  • a kit can include software for designing primers and probes as disclosed herein and/or instructions for accessing and using website implementing such software.
  • a kit can include means for detecting amplification products.
  • Target-specific overlap 5 'nuclease assays using probes, hairpins, or circles can be offered to customers as (a) designed when customer submits targets (AKA "assays-by- design"); (b) pre-designed for a set of popular targets, e.g., RefSeq mRNAs, and (c) inventoried assays, e.g., for miRNA targets. Assays can be provided to customers preloaded in plates, cartridges, or beads / surface-attached.
  • the universal overlap 5' nuclease assays e.g., as described in Published Application No. US 2011/0212846) would be inventoried and can be offered preloaded in plates, cartridges, and beads or other surfaces.
  • Overlap 5 'nuclease assays can be used for research use, applied markets, e.g., pathogen detection in food, bio-production reactors, human identification, forensics, environmental testing, epidemiology, etc., veterinary, and human diagnostic use.
  • target nucleic acids that find use in the invention can be obtained from a wide variety of sources.
  • target nucleic acids can be obtained from biological or laboratory samples including cells, tissues, lysates, and the like.
  • the source of target nucleic acids includes cells or tissues from an individual with a disease, e.g., cancer or any other disease of particular interest to the user.
  • a disease e.g., cancer or any other disease of particular interest to the user.
  • kits are commercially available for the purification of target nucleic acids from cells or tissues, if desired (see, e.g., EASYPREPTM, FLEXIPREPTM, both from
  • any target nucleic acid can be custom or standard ordered from any of a variety of commercial sources.
  • Labeling strategies for labeling nucleic acids and corresponding detection strategies can be found, e.g., in Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals Sixth Edition by Molecular Probes, Inc. (Eugene Oreg.); or Haugland (2001) Handbook of Fluorescent Probes and Research Chemicals Eighth Edition by Molecular Probes, Inc. (Eugene Oreg.) (Available on CD ROM).
  • PCR polymerase chain reaction
  • PCR methods and reagents, as well as optimization of PCR reaction conditions are well known in the art. Details regarding PCR and its uses are described, e.g., in Van Pelt-Verkuil et al. (2010) Principles and Technical Aspects of PCR Amplification Springer; 1st Edition ISBN-10: 9048175798, ISBN-13: 978- 9048175796; Bustin (Ed) (2009) The PCR Revolution: Basic Technologies and
  • thermostable polymerases include, but are not limited to, Taq and Tth polymerase (commercially available from Life Technologies).
  • RT-PCR amplification reactions may be carried out with a variety of different reverse transcriptases (or mixture of reverse transcriptases), although in some embodiments thermostable reverse- transcriptions are preferred.
  • Suitable thermostable reverse transcriptases include, but are not limited to, reverse transcriptases such as AMV reverse transcriptase, MuLV, and Tth reverse transcriptase.
  • the polymerase is selected from the group consisting of Taq DNA polymerase, Taql DNA polymerase, Thr DNA polymerase, Tfl DNA polymerase, Tru DNA polymerase, Tli DNA polymerase, Tac DNA polymerase, Tne DNA polymerase, Tma DNA polymerase, rTth DNA polymerase, Pfu DNA polymerase, Pho DNA polymerase, Pwo DNA polymerase, Kod DNA polymerase, Bst DNA polymerase, Sac DNA
  • KlenTaq-1 polymerse E. Coli DNA polymerase I
  • Klenow fragment of E. Coli DNA polymerase I SEQUENASETM 1.0 DNA polymerase (Amersham Biosciences)
  • SEQUENASE 2.0 DNA polymerase and mixtures thereof.
  • Temperatures suitable for carrying out the various denaturation, annealing and primer extension reactions with the polymerases and reverse transcriptases are well-known in the art and/or can be determined empirically.
  • the universal detection steps of the present invention can be performed in real-time, e.g., where one or more detectable signals (if any) corresponding to the presence or amount of one or more target nucleic acids are detected at the conclusion of one or more PCR cycles prior to completion of thermal cycling.
  • Real-time/quantitative PCR techniques are known in the art. Detailed guidance can be found in, e.g., Clementi M. et al (1993) PCR Methods Appl, 2: 191-196; Freeman W. M. et al (1999) Biotechniques, 26: 112- 122, 124-125; Lutfalla G. and Uze G. (2006) Methods Enzymol, 410: 386-400; Diviacco S.
  • Fig, 7 compares 5 'nuclease amplification curve signals for (a) probe-based
  • amplification curves correspond to three dilutions (10 3 , 10 4 and 10 5 copies) and "overlap” ("o”; corresponding to curves 700', 710' and 720', respectively) and "non-overlap” ("n”; corresponding to curves 700, 710 and 720, respectively) assay designs.
  • the two designs were identical, except the "overlap" designs had an additional base at the 3' end of the primer: compare SEQ ID NO: 2 for the probe assays (a) and SEQ ID NO: 7 and SEQ ID NO: 8 for the hairpin. In order to keep the same primer lengths, an extra base was added at the 5' end of the "non-overlapping" primer.
  • the "n” and “o” assays shared the same opposite primer (SEQ ID NO: 3), and probe (SEQ ID NO: 4), and used artificial template SEQ ID NO: 5.
  • the hairpin assays in 7(b) used the same dual-labeled primer (SEQ ID NO: 6) and artificial template (SEQ ID NO: 9).
  • the six amplification curves correspond to three dilutions (10 3 , 10 4 and 10 5 copies) and "overlap" ("o”; corresponding to curves 730', 740' and 750', respectively) and “non-overlap” ("n”; corresponding to curves 730, 740 and 750, respectively) assay designs.
  • target nucleic acid or “target polynucleotide” is a nucleic acid sequence of interest to be detected potentially present in a sample prior to any changes in the nucleic acid sequence composition made during sample processing, and may be either single- stranded or double stranded.
  • primer refers to a polynucleotide that is serving as an initiation site for polymerase extension during PCR.
  • primers can have one or several labels and are called “primer-probes”.
  • a "5 '-probe portion" of a primer- probe includes at least one label that is used to generate a detectable signal.
  • a “digestion-driving primer” is a primer that drives the 5 'nuclease cleavage activity of a polymerase during PCR.
  • a "universal tag” is a part of the primer or amplicon with an artificial sequence that does not match the target nucleic acid, with the possible exception of very short tags that either intentionally or accidentally match target nucleic acid.
  • Primer extension is the enzymatic addition, i.e. polymerization, of monomeric nucleotide units to a primer while the primer is hybridized (annealed) to a template polynucleotide.
  • An "amplicon” is a DNA sequence generated in an amplification reaction.
  • Amplicons may include both target DNA sequences, and universal tagging sequences.
  • a "probe” is an oligonucleotide that carries at least one label that is used to generate a detectable signal. Probes usually have a blocked 3' end to prevent their extension by a polymerase.
  • a "universal tag” is a part of the primer, probe or amplicon (after primer extends) comprising an artificial sequence that does not match the target nucleic acid, with the possible exception of very short tags that either intentionally or accidentally match the target nucleic acid.
  • Primer extension is the enzymatic addition, i.e. polymerization, of monomeric nucleotide units to a primer while the primer is hybridized (annealed) to a template polynucleotide.
  • Amplicons is a DNA sequence generated in PCR reaction. Amplicons may include both target DNA sequences, and universal tagging sequences.
  • qPCR refers to quantitative Polymerase Chain Reaction, where the signal is measured during and/or at the end of PCR.
  • C t (also referred to as C p and C q ) is defined as the cycle at which fluorescence is determined to be statistically significant above background.
  • the threshold cycle is inversely proportional to the log of the initial copy number. The more target molecules are present to begin with, the fewer the number of cycles it takes to get to a point where the fluorescent signal is detectable above background.
  • a "universal primer/probe” is a primer and/or probe that has one or several universal tags that do not match the target nucleic acid and may optionally contain a short stretch of target nucleic acid that occurs at the locus that is detected.
  • a "5 '-flap” is a universal tag at the 5' end of a primer, probe or amplicon. In some embodiments, it is cleavable by an enzyme with 5 ' nuclease activity.
  • allele means a particular genetic variant or
  • polymorphism in the sequence of a gene representing an alternative form of the gene.
  • allele-specific PCR amplification refers to
  • allele-specific when used in reference to probes or primers, means that a particular position of the probe or primer is complementary to an allele of a target polynucleotide sequence in the strand this primer or probe is to anneal.
  • a “surface attached oligonucleotide (primer or probe”) indicates that the oligonucleotide does not diffuse in the reactor volume, but stays at a certain predefined location. Examples include flat surfaces, e.g., microarrays, beads, blots on paper, gel, etc.
  • An “encoding or linking reaction” is a step of target nucleic acid detection that generates "encoded DNA molecules with universal tags" that correspond to target nucleic acids. PCR pre-amplification with 5' tailed (tagged) primers is an example of an encoding or linking reaction.
  • An “encoded target” is target DNA with attached universal tags after the encoding reaction.
  • wild-type refers to a gene or gene product that has the characteristics of that gene or gene product that is most frequently observed in a population and is, thus, arbitrarily designated the “normal” or “wild-type” form of the gene.
  • modified or mutant refers to a gene or gene product that displays modifications in sequence when compared to the wild-type gene or gene product.

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Abstract

Dans certains modes de réalisation, l'invention concerne des procédés et des compositions pour améliorer l'efficacité d'un clivage par 5'-nucléase au cours d'une PCR en incorporant une superposition à base courte, typiquement unique, d'une séquence entre l'extrémité 3' d'une amorce déclenchant la digestion et la partie 5' d'une sonde. Une superposition similaire peut également être utilisée lorsque l'amorce possède une partie supplémentaire en 5' et un espaceur entre ses extrémités 5' et 3', de telle sorte qu'elle peut former une configuration circulaire unimoléculaire lorsqu'elle est hybridée à l'amplicon. Dans certains modes de réalisation, un amplicon forme une structure en épingle à cheveux intramoléculaire de telle sorte que l'extrémité 5' d'une tige se superpose à l'extrémité 3' d'une amorce déclenchant la digestion, lorsque la dernière s'hybride à l'amplicon. Les réactions d'amplification peuvent être exécutées en solution ou à l'aide d'une amorce et/ou des sondes liées à une surface. Dans certains modes de réalisation, l'invention concerne des procédés et des compositions qui permettent d'augmenter la spécificité d'une mutation (allèle) spécifique d'une PCR à l'aide d'oligos de blocage pouvant être clivés par une réaction de 5'-nucléase.
PCT/US2012/049672 2011-08-05 2012-08-05 Procédés et compositions pour détection d'acides nucléiques à l'aide de clivage par 5'-nucléase et d'amplification WO2013022807A1 (fr)

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CN109486912A (zh) * 2018-12-07 2019-03-19 领航基因科技（杭州）有限公司 一种用于数字pcr扩增的探针引物组合及设计方法
CN113195734A (zh) * 2018-12-20 2021-07-30 豪夫迈·罗氏有限公司 通过固相分子成像检测靶核酸
CN116083561A (zh) * 2023-02-14 2023-05-09 山东鲁抗好丽友生物技术开发有限公司 一种核酸检测试剂盒及其在核酸检测中的应用

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