WO2022219408A1 - Nucleic acid enrichment and detection - Google Patents

Nucleic acid enrichment and detection Download PDF

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Publication number
WO2022219408A1
WO2022219408A1 PCT/IB2022/000217 IB2022000217W WO2022219408A1 WO 2022219408 A1 WO2022219408 A1 WO 2022219408A1 IB 2022000217 W IB2022000217 W IB 2022000217W WO 2022219408 A1 WO2022219408 A1 WO 2022219408A1
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Prior art keywords
nucleic acid
kit
sequence
sample
sequences
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PCT/IB2022/000217
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English (en)
French (fr)
Inventor
Robert Osborne
Magdalena STOLAREK-JANUSZKIEWICZ
Barnaby BALMFORTH
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Biofidelity Ltd
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Biofidelity Ltd
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Priority claimed from GBGB2105405.1A external-priority patent/GB202105405D0/en
Priority claimed from GBGB2105388.9A external-priority patent/GB202105388D0/en
Priority to KR1020237038978A priority Critical patent/KR102923285B1/ko
Application filed by Biofidelity Ltd filed Critical Biofidelity Ltd
Priority to CN202280041767.1A priority patent/CN117940581A/zh
Priority to BR112023021384A priority patent/BR112023021384A2/pt
Priority to EP22727973.4A priority patent/EP4143334A1/en
Priority to JP2023564030A priority patent/JP7760607B2/ja
Priority to IL307733A priority patent/IL307733A/en
Publication of WO2022219408A1 publication Critical patent/WO2022219408A1/en
Anticipated expiration legal-status Critical
Priority to ZA2023/09778A priority patent/ZA202309778B/en
Ceased legal-status Critical Current

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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/125—Allele specific primer extension
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/30—Detection characterised by liberation or release of label
    • C12Q2565/301—Pyrophosphate (PPi)
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 601_SEQUENCE_LISTING_ST25 created April 15, 2022, having a file size of 15,464 bytes, is hereby incorporated by reference in its entirety.
  • Targeted detection of low frequency variants in a pool of wild-type molecules is clinically important for early detection of cancer, monitoring of cancer progression, targeting of cancer therapies, non- invasive prenatal testing, monitoring of T-cell populations that target particular (neo-)antigens, and for early warning of organ transplant rejection.
  • a combination of hybridisation-capture and nextgeneration sequencing (NGS) is the most commonly applied method for targeted and multiplex detection of low frequency variants but has several suboptimal characteristics.
  • hybridisation capture enriches for target regions of interest but not for variant molecules. This results in the vast majority of sequencing reads deriving from wild-type rather than variant molecules. This is wasteful, adds cost, and makes it difficult to detect rare variants against the background of errors from storage, library preparation and sequencing.
  • Modified library preparation methods can increase specificity, yielding accurate sequencing data from single molecules.
  • methods like duplex sequencing also reduce sensitivity (for example, by modifying library preparation methods to avoid end-repair and by deliberately imposing molecular bottlenecks).
  • the method described here allows enrichment of variant molecules located within target regions of interest, using, for example, a modified hybridisation capture method based on pyrophosphorolysis (PPL). This both reduces the number of sequencing reads that are required but also opens the door to multiplexed detection of low frequency variant molecules below the current limit of detection of NGS.
  • PPL pyrophosphorolysis
  • a hybridisation method based on a pyrophosphorolysis reaction harness the double-strand specificity of pyrophosphorolysis; a reaction which will not proceed efficiently with single-stranded oligonucleotide substrates or double- stranded substrates which include blocking groups of nucleotide mismatches.
  • methods that comprise contacting a sample (e.g., containing two or more different nucleic acid molecules) with a probe and pyrophosphorolysis reaction reagents and enriching for or depleting a first nucleic acid molecule relative to a second nucleic acid molecule based on different complementarity of the probe to the first and second nucleic acid molecules, resulting in different levels of pyrophosphorolysis of the probe when hybridized to the first and second nucleic acid molecules.
  • a sample e.g., containing two or more different nucleic acid molecules
  • a probe and pyrophosphorolysis reaction reagents enriching for or depleting a first nucleic acid molecule relative to a second nucleic acid molecule based on different complementarity of the probe to the first and second nucleic acid molecules, resulting in different levels of pyrophosphorolysis of the probe when hybridized to the first and second nucleic acid molecules.
  • the method comprises enriching for or depleting a first nucleic acid molecule in a sample that comprises a mixture of nucleic acid molecules, by contacting the first nucleic acid molecule with a probe that differs in complementary to a target region on the first nucleic acid molecule relative to other nucleic acid molecules in the sample; conducting a pyrophosphorolysis reaction; and enriching for or depleting the first nucleic acid molecule.
  • the probe has greater complementary to the target region of the first nucleic acid molecule than it does to a corresponding target region of a second nucleic acid in the sample.
  • the probe has lesser complementary to the target region of the first nucleic molecule than it does to a corresponding target region of a second nucleic acid in the sample.
  • the first nucleic acid and the second nucleic acid differ by a sequence variation
  • the probe includes a sequence that is perfectly complementary to the target region of the sequence of greater complementarity and has one or more mismatches to a sequence variation found in the corresponding target region of the sequence of lesser complementarity.
  • the probe contains one or more mismatches to the target regions of both the first and second nucleic acid molecules, but contains more mismatches to the target region of the nucleic acid of lesser complementarity.
  • the probe is designed such that the degree of pyrophosphorolysis differs when the probe is hybridized to a first nucleic acid relative to a second nucleic acid, permitting selective enrichment or depletion of the first nucleic acid relative to the second nucleic acid based on the different reaction product generated by the pyrophosphorolysis.
  • provided herein are methods for increasing or decreasing the ratio of a first nucleic acid sequence to a second nucleic acid sequence in a sample, comprising: a) exposing a sample, comprising the first and second nucleic acid sequences, to a probe that differs in complementarity to the first and second nucleic acid sequences; b) conducting a pyrophosphorolysis reaction; and c) enriching for or depleting the first nucleic acid sequence relative to the second nucleic acid sequence.
  • a method for increasing the ratio of a first nucleic acid sequence to second nucleic acid sequence in a sample comprising the steps of: a. introducing the sample comprising one or more nucleic acid analytes to a first reaction mixture comprising: i. a single-stranded probe oligonucleotide Ao having differential complementarity to the first and second nucleic acid sequences (e.g., wherein the 3' end of said probe is perfectly complementary to one of the first or second sequence but imperfectly complementary to the other); b. introducing the reaction mixture produced by step (a) to a second reaction mixture comprising: ii.
  • a pyrophosphorolysing enzyme iii. a source of pyrophosphate ion wherein Ao anneals (e.g., perfectly) to one of the nucleic acid sequences to create an at least partially double-stranded intermediate product in which Ao (e.g., the 3' end of Ao) forms a doublestranded complex with said sequence and Ao is pyrophosphorolysed in the 3'-5' direction from its 3'- end, whilst any Ao that has annealed less perfectly (e.g., imperfectly) to the other sequence is pyrophosphorolysed in the 3'-5' direction to a lesser extent due to said less perfect (e.g., imperfect) annealing; c.
  • Ao anneals (e.g., perfectly) to one of the nucleic acid sequences to create an at least partially double-stranded intermediate product in which Ao (e.g., the 3' end of Ao) forms a doublestranded complex with said
  • any Ao sequence complexes which were more perfectly (e.g., perfectly) annealed by: iv. allowing the strands of said complex to separate (e.g., melt apart) as a consequence of the pyrophosphorolysis reaction; or v. heating the reaction mixture to a temperature sufficient for the strands of said complex to separate (e.g., melt apart) but which is below the temperature required for the strands of any Ao which less perfectly (e.g., imperfectly) annealed to separate (e.g., melt apart); and d. separating Ao, and thereby any nucleic acid sequences remaining annealed thereto, from any nucleic acid sequences not annealed to Ao.
  • Probes may be provided with one or more components that are removed prior to or during the pyrophosphorolysis reaction.
  • probes may be provided with a non-complementary flap or other blocking group at their 3' end that prevents initiation of a pyrophosphorolysis or polymerase reaction until the blocking group is removed.
  • the blocking group may be removed by any suitable mechanism (e.g., enzymatic cleavage, chemical reaction, temperature shift, etc.).
  • the sequence of the probe that provides the differential pyrophosphorolysis products, when hybridized to distinct nucleic acid molecules may be positioned at any suitable location in the initial probe. For example, a mismatch sequence may be positioned at the 3' terminal base of the 3' end of the probe.
  • a mismatch sequence may be positioned internally in the probe at the 3' end.
  • a mismatch sequence may be positioned centrally in the probe.
  • a mismatch sequence may be positioned within the 5' half of the probe.
  • the 5' end of the probe may comprise a region (e.g., a 5' tail) that acts as an identifier (e.g., a sample identifier).
  • a region e.g., a 5' tail
  • an identifier e.g., a sample identifier
  • the analytes/sequences to which the method of the invention can be applied are those nucleic acids, such as naturally-occurring or synthetic DNA or RNA molecules, which include the target polynucleotide sequence(s) being sought.
  • the analytes/sequences will typically be present in an aqueous solution containing it and other biological material and in some embodiments the analytes/sequences will be present along with other background nucleic acid molecules which are not of interest for the purposes of the test. In some embodiments, the analytes/sequences will be present in low amounts relative to these other nucleic acid components.
  • the analyte is derived from a biological specimen containing cellular material
  • sample-preparation techniques such as filtration, centrifuging, chromatography or electrophoresis.
  • compositions and methods of the invention may be employed against any type of sample, including, but not limited to environmental (e.g., water, soil, air, etc.) samples and biological samples.
  • Biological samples may be from any source including plants, animals, infectious disease agents, and the like.
  • the analytes/sequences are derived from a biological sample taken from a mammalian subject (especially a human patient) such as blood, plasma, sputum, urine, skin, biopsy or surgical resection.
  • the biological sample will be subjected to lysis in order that the analytes/sequences are released by disrupting any cells present.
  • the analytes/sequences may already be present in free form within the sample itself; for example cell-free DNA circulating in blood or plasma.
  • the compositions and methods of the invention find particular use with historically challenging sample types that may have low allele fractions of the analyte of interest.
  • samples include blood, urine, cytosponge- collected samples (e.g., oesophageal samples), bronchoalveolar lavage (BAL) derived samples, pleural fluid, and cerebrospinal fluid (CSF).
  • samples are pooled samples. Pooled samples involve mixing multiple samples togethers in a batch where the pooled collection is tested. This approach increases the number of individual samples that can be tested using a more limited amount of resources. Pooled samples of interest include, but are not limited to, donated blood samples, agricultural samples. food samples, sperm samples, and biological samples tested for the presence of infectious disease agents (e.g., SARS-CoV-2, HIV, HCV, etc.).
  • the pooled sample is an environmentally collected sample (e.g., wastewater sample) that has, by the nature of its generation, pooled samples from multiple different sources.
  • pooling of samples may reduce the allele fraction of variants as the samples dilute each other, it can provide a dramatic increase in efficiency of screening. Because the technology provided herein enables detection at very low allele fractions, it is particularly well suited for analysis of pooled samples. In some embodiments, a fraction of each initial sample is pooled without use of barcodes or other complex preparation steps and the pooled sample is tested. If a positive result is obtained, remaining fractions of the unpooled samples may be tested individually.
  • compositions e.g., reagents, kits, reactions mixture, instruments, software
  • compositions comprising one or more reagents necessary, sufficient, or useful for conducting a method as described herein.
  • compositions comprise: one or more oligonucleotides AO, wherein AO comprises: a sequence (e.g., 3' end) which is differentially complementary to a known first sequence and a known second sequence (e.g., perfectly complementary to a known first sequence but imperfectly complementary to a known second sequence); one or more pyrophosphorolysing enzymes; and one or more sources of pyrophosphate ions.
  • the compositions further comprise a target nucleic acid isolation component that segregates target nucleic acid molecules that are more perfectly (e.g., perfectly) complementary to said probe (e.g., 3' end of said probe) from nucleic acid molecules less perfectly (e.g., imperfectly) complementary to said probe (e.g., 3' end of said probe) following a pyrophosphorolysis reaction.
  • the compositions comprise one or more solid supports.
  • the compositions comprise one or more buffers.
  • AO comprises a 5' tail region which is not complementary to either a known first or known second sequence.
  • AO further comprises a capture moiety.
  • the composition further comprises one or more capture oligonucleotide Co which comprises a capture moiety and wherein a 5' tail region of AO is complementary to a region of Co-
  • the capture moiety is biotin and the solid support comprises streptavidin.
  • the solid support comprises a capture oligonucleotide Co and wherein a 5' tail region of Ao is complementary to a region of Co.
  • the solid support is a bead
  • the composition further comprises one or more epigenetic modification sensitive or dependent restriction enzymes. In some embodiments, the composition further comprises one or more restriction endonucleases. In some embodiments, the composition further comprises one or more transposomes. In some embodiments, the composition comprises a Cas protein (e.g., Cas9). In some embodiments, the composition further comprises one or more transposases. In some embodiments, the composition further comprises one or more ligases. In some embodiments, the composition further comprises one or more blocking oligonucleotides. In some embodiments, the one or more blocking oligonucleotides are resistant to pyrophosphorolysis.
  • the composition further comprises reagents for conducting an amplification (e.g., PCR), sequencing (e.g., next generation sequencing), or detection reaction.
  • the composition further comprises one or more molecular probes.
  • the one or more molecular probes are fluorescently labelled.
  • the Ao e.g., 3' end of the Ao
  • the Ao is more complementary (e.g., perfectly complementary) to a wild type sequence of the human genome and less complementary (e.g., imperfectly complementary) to a mutant allele of said wild type sequence.
  • the Ao (e.g., 3' end of the Ao) is less complementary (e.g., imperfectly complementary) to a wild type sequence of the human genome and more complementary (e.g., perfectly complementary) to a mutant allele of said wild type sequence.
  • the composition further comprises components for the transcription of RNA into cDNA.
  • the composition is a reaction mixture comprising a reaction, at a particular time point, of any of the methods described herein.
  • the reaction mixture is present prior to pyrophosphorolysis.
  • the reaction mixture is present during pyrophosphorolysis.
  • the reaction mixture is present after pyrophosphorolysis.
  • the reaction mixture comprises probe/nucleic acid hybridization complexes of the methods described herein.
  • the reaction mixture comprises captured nucleic acid molecules of the method described herein.
  • the reaction mixture comprises regions comprising concentrations of a desired target nucleic that are higher or lower than the concentration of the desired target nucleic that was present in a sample that underwent a pyrophosphorolysis reaction.
  • reaction mixtures comprising: a sample; pyrophosphorolysis reagents at pyrophosphorolyzing concentrations (i.e., reagents concentrations that favor pyrophosphorolysis); a first nucleic acid molecule from the sample hybridized to a probe having a sequence, wherein a discrimination region of the probe is complementary to the first nucleic acid molecule; and a second nucleic acid molecule from the sample hybridized a probe having said sequence, wherein the discrimination region of the probe is not perfectly complementary to the second nucleic acid molecule.
  • a reaction mixture comprising a sample; pyrophosphorolysis reagents at pyrophosphorolyzing concentrations; and a first nucleic acid molecule in a first region of the reaction mixture, wherein the first nucleic acid has a higher or lower concentration in the first region than the concentration of the first nucleic acid in the sample.
  • compositions e.g., uses of the kits, uses of the reaction mixtures, uses of the reagents, uses of the instruments, uses of the software.
  • uses of the composition for enriching or depleting a target nucleic acid in a sample e.g., uses of the kits, uses of the reaction mixtures, uses of the reagents, uses of the instruments, uses of the software.
  • the devices and instruments find use in the methods described herein.
  • the devices and instrument find use to collect and distribute samples into reaction vessels.
  • the devices and instruments provide reaction chambers for conducting the methods.
  • the devices and instruments provide multiple zones or regions (e.g., wells, channels, etc) for housing a reaction and/or for isolating enriched desired target nucleic acids or depleting desired target nucleic acids.
  • the devices and instruments find use to amplify or sequence nucleic acid molecules.
  • the devices and instruments find use to detect nucleic acid molecules.
  • the devices and instruments find use to receive or transmit information from a user.
  • the devices and instrument may comprise a user interface to receive user instructions and a display to visually present results to a user.
  • computing devices find use to control instruments or devices to facilitate the methods described herein.
  • the computing devices collect, analyse, and report data.
  • the computing devices comprise one or more processors that run a computer program.
  • the computing devices comprise non-transitory computer readable media (e.g., software) comprising instructions that direct a processor to carry out one or more of the computing steps.
  • Figure 1 A schematic representation of one embodiment of the invention wherein biotinylated probes are pre-bound to streptavidin coated paramagnetic beads.
  • the bead-bound probes are then hybridised to target DNA and undergo pyrophosphorolysis.
  • bead-bound probes are perfectly complementary to a wild-type sequence and imperfectly complementary/mismatched to a variant sequence.
  • Probes hybridised to wild-type sequences are pyrophosphorolysed and the wild- type DNA is released from beads into solution, whilst probes hybridised to variant sequences are mismatched and so are not pyrophosphorolysed to the same extent as wild-type sequences, resulting in the variant sequences remaining bead bound.
  • Variant sequences may then then be identified, in one example by sequencing.
  • FIG. 2 A schematic representation of one embodiment of the invention wherein biotinylated probes are hybridised to target DNA, undergo pyrophosphorolysis and are then captured onto streptavidin paramagnetic beads.
  • probes are perfectly complementary to a wildtype sequence and imperfectly complementary/mismatched to a variant sequence.
  • Probes hybridised to wild-type sequences are pyrophosphorolysed and the wild-type DNA is released from the probe whilst probes hybridised to mismatched variant sequences are not pyrophosphorolysed to the same extent as wild-type sequences and the variant sequences thus remain probe bound.
  • Probes are then captured onto streptavidin paramagnetic beads, with only variant sequences thereforebecoming bead-bound. Variant sequences may then then be identified, in one example by sequencing.
  • Figure 3 A schematic representation of one embodiment of the invention according to that described in Figure 1 wherein prior to hybridisation of probe to target DNA, the target DNA undergoes adaptor tagging and amplification by PCR. Following hybridisation, probes undergo pyrophosphorolysis and adaptor tagged variant sequences remain bead-bound. Bead-bound variant sequences then undergo amplification by PCR.
  • Figure 4 Pyrophosphorolysis dependent release of target molecule.
  • the lower the Cq value the more target sequence that has been released into the supernatant from beads.
  • the Cq value is lower for a target sequence that is fully complementary to the enrichment probe when the pyrophosphorolysis reaction is performed. This indicates that the target of interest was released from beads. There was no difference in the Cq values for the mismatched target sequence, regardless of whether the pyrophosphorolysis reaction is performed. This indicates that the mismatched target stayed on beads and wasn't released to supernatant.
  • FIG. 5 Detection of EGFR exon 20 T790M variant.
  • the graph shows PPi dependent detection of
  • T790M By increasing the temperature of the hybridisation step to 60oC, more T790M variant molecules are recovered. Increasing the temperature of the hybridisation step to 6O0C reduces recovery of the WT (Wild type) molecules. In the given conditions 0.2fM of the T790M variant molecules can be detected.
  • Figure 6 Detection of the EGFR exon 20T790M variant at a different variant allele fraction (VAF).
  • FIG. 6A Showing PPi-dependent detection as low as 0.1%VAF.
  • Figure 6A Graph shows detection of T790M variant from 0-50% VAF.
  • Figure 6B Zoom in on detection of 0.1% VAF.
  • T790M variant at 0.1%VAF T790M variant at 0.1%VAF.
  • one hour of hybridisation is not enough to detect variants of interest.
  • the best performance when using a three hour hybridizations step is achieved with buffer 2 (see Example 3).
  • FIG. 8 Enrichment factor for the EGFR exon 20 T790M variant. Graph shows the enrichment factor for 0.1 and 1%VAF and its dependency on which buffer is used during the hybridisation step.
  • Increase in the enrichment factor is dependent on the presence of PPi.
  • the highest enrichment factor is achieved with Buffer 2 and buffer 5 (see Example 3).
  • the present method uses pyrophosphorolysis as way of enriching for or depleting desired nucleic acid sequences.
  • the pyrophosphorolysis reaction relies on complementarity between hybridised strands, and hence only digests strands without or with fewer mismatches.
  • the reaction selectively shortens certain sequences which are complementary, whilst leaving mismatched sequences less digested.
  • the reaction can be performed such that molecules hybridized to the shortened sequences are recovered and analysed. Alternatively, the reaction can be performed such that less shortened sequences are analysed. Alternatively, the reaction can be performed so that the sequences which have not undergone any pyrophosphorolysis are analysed.
  • the enrichment or depletion may be repeated one or more times to further enrich a sample for the sequence of interest. For example, in some embodiments, a second round of enrichment or depletion occurs after the first round is completed using the same reagents as the first round. In other embodiments, a different probe is used that is selective for a different sequence of the target nucleic acid that is to be enriched for or removed. This approach is particularly well suited in instances where the sequence to be enriched or depleted differs from the sequence to be eliminated by at least two base positions.
  • a target nucleic acid containing two polymorphisms relative to wild type may undergo a first round of enrichment or depletion based on the first polymorphism and a second round of enrichment or depletion based on the second polymorphism. Exponential levels of enrichment or depletion may be achieved by employing multiple rounds.
  • a target nucleic acid is modified to generate a synthetic sequence (e.g., addition of a polymorphism), prior to enrichment or depletion, such that the synthetic sequence is targeted for enrichment or depletion relative to sequences not containing the synthetic sequence.
  • two or more nucleic acids may be enriched for or depleted in any given round of the reaction by use of multiple probes.
  • a particular nucleic acid may be enriched for while a second nucleic acid may be depleted in one or more rounds of the reaction.
  • a method for altering the ratio of a first nucleic acid sequence to a second nucleic acid sequence in a sample comprising at least a first and second sequence
  • the method comprising the steps of: a. introducing the sample comprising one or more nucleic acid analytes to a first reaction mixture comprising: i. a single-stranded probe oligonucleotide Ao wherein the probe is differentially complementary to first and second sequences (e.g., the 3' end or other region of said probe is perfectly complementary to one of the first or second sequence but imperfectly complementary to the other); ii. a pyrophosphorolysing enzyme; and
  • Hi. a source of pyrophosphate ion; wherein Ao anneals (e.g., perfectly) to the first nucleic acid sequence to create an at least partially double-stranded intermediate product in which the Ao(e.g., the 3' end of Ao) forms a double- stranded complex with said sequence and Ao is pyrophosphorolysed in the 3'-5' direction from its 3' end, whilst any Ao that has annealed less well (e.g., imperfectly) to the second sequence is pyrophosphorolysed in the 3'-5' direction to a lesser extent due to said lesser
  • a method for increasing the ratio of a first nucleic acid sequence to a second nucleic acid sequence in a sample comprising at least a first and second sequence
  • the method comprising the steps of: a. introducing the sample comprising one or more nucleic acid analytes to a first reaction mixture comprising: i. a single-stranded probe oligonucleotide Ao wherein said probe (e.g., the 3' end of or other region of said probe) is more complementary (e.g., perfectly complementary) to one of the first or second sequence but less complementary (e.g., imperfectly complementary) to the other; b.
  • reaction mixture produced by step (a) to a second reaction mixture comprising: ii. a pyrophosphorolysing enzyme; and iii. a source of pyrophosphate ion wherein Ao anneals (e.g., perfectly) to one of the nucleic acid sequences to create an at least partially double-stranded intermediate product in which the Ao(e.g., the 3' end of Ao) forms a double-stranded complex with said sequence and Ao is pyrophosphorolysed in the 3'-5' direction from its 3'-end, whilst any Ao that has annealed less well (e.g., imperfectly) to the other sequence is pyrophosphorolysed in the 3'-5' direction to a lesser extent due to said lower (e.g., imperfect) annealing; c.
  • a second reaction mixture comprising: ii. a pyrophosphorolysing enzyme; and iii. a source of pyrophosphate
  • any Ao sequence complexes which were better (e.g., perfectly) annealed by: i. allowing the strands of said complex to separate (e.g., melt apart) as a consequence of the pyrophosphorolysis reaction; or ii. heating the reaction mixture to a temperature sufficient for the strands of said complex to separate (e.g., melt apart) but which is below the temperature required for the strands of any Ao which annealed less well
  • the separation of any Ao sequence complexes which were better (e.g., perfectly) annealed occurs by using reaction conditions that favour Ao sequence complexes which were better (e.g., perfectly) annealed over Ao sequence complexes with less (e.g., imperfect) annealing. This could take the form of changes to the reaction mixture temperature and/or changes to the pH of the reaction mixture and/or changes to the salinity of the reaction mixture.
  • the first and second reaction mixtures are combined such that the first reaction mixture of step (a) further comprises a pyrophosphorolysing enzyme and a source of pyrophosphate ion.
  • Ao which anneals better (e.g., perfectly) to one of the nucleic acid sequences to form a double-stranded complex is pyrophosphorolysed to such an extent that the doublestranded complex separates (e.g., melts), separating the shortened Ao from the nucleic acid sequence.
  • Ao which less well (e.g., imperfectly) annealed to the other sequence remains in a doublestranded complex with said sequence.
  • the reaction mixture can be heated to denature.
  • double-stranded complexes comprising Ao sequences which were better (e.g., perfectly) annealed, and thus more completely pyrophosphorolysed, separate (e.g., melt apart) whilst those double-stranded complexes comprising Ao which were less well (e.g., imperfectly) annealed, and pyrophosphorolysed less, remain double-stranded due to such complexes possessing a higher melting temperature as a result of more complementary base-pairs remaining.
  • the pH of the reaction mixture can be raised to denature.
  • doublestranded complexes comprising Ao sequences which were better (e.g., perfectly) annealed, and thus more completely pyrophosphorolysed, denature whilst those double-stranded complexes comprising Ao which were less well (e.g., imperfectly) annealed, and pyrophosphorolysed less, remain double-stranded due to such complexes possessing more complementary base-pairs and requiring an even higher pH to become denatured.
  • the salt concentration of the reaction mixture is lowered.
  • doublestranded complexes comprising Ao sequences which were better (e.g., perfectly) annealed, and thus more completely pyrophosphorolysed, denature whilst those double-stranded complexes comprising Ao which were less well (e.g., imperfectly) annealed, and pyrophosphorolysed less, remain double-stranded due to such complexes possessing more complementary base-pairs and requiring an even lower salt concentration to become denatured.
  • chemical agents e.g., dimethylsulfoxide (DMSO), formamide, etc.
  • DMSO dimethylsulfoxide
  • formamide formamide
  • nucleic acid molecules (displacing oligonucleotides) and enzymes or proteins are used separate hybridized nucleic acid molecules.
  • two or more of the following characteristics of the reaction mixture are altered such that separation of double-stranded complexes comprising Ao sequences which were better (e.g., perfectly) annealed occurs whilst double-stranded complexes comprising Ao which were less well (e.g., imperfectly) annealed remain hybridised: the pH of the reaction mixture; the temperature of the reaction mixture; the salt concentration of the reaction mixture; addition of chemical denaturing agents.
  • capture of Ao onto a solid support occurs prior to, or following, step (a).
  • the separation in step (d) is performed through capture of Ao onto a solid support prior to, or following, step (a).
  • Ao further comprises a 5' tail region which is not complementary to either of the sequences.
  • two probes are employed, one for the forward strand of a target and one for the reverse stand.
  • the capture onto the solid support is performed through hybridisation of the
  • Ao further comprises a capture moiety through which it is bound to the solid support.
  • the capture moiety is biotin and the solid support comprises streptavidin.
  • Ao is extended in the reaction with biotinylated nucleotides for subsequent capture.
  • a diphosphohydrolase enzyme e.g., Apyrase
  • the diphosphohydrolase enzyme hydrolyses released nucleotides from the pyrophosphorolysis reaction, maintaining optimal pyrophosphorolysis reaction conditions.
  • inorganic pyrophosphatase enzyme is provided in the methods or compositions of the inventions.
  • Inorganic pyrophosphatase removes pyrophosphate ions after pyrophosphorolysis and before subsequent steps where the presence of pyrophosphate ions may be undesired or suboptimal.
  • the technology is not limited to the use of capture to partition or enrich for nucleic acid molecules of interest.
  • Molecules may be partitioned or enriched, for example, based on differences in size, charge, or shape or other physical or chemical properties.
  • a moiety is added to the nucleic acid of interest (e.g., via click chemistry modification), whereby the added moiety imparts a selectable distinguishing characteristic to the nucleic acid of interest.
  • the solid support is a bead.
  • the bead is a magnetic or paramagnetic bead.
  • one or more wash steps are performed between one or more of the steps.
  • wash and hybridisation steps are performed at elevated temperatures between 25-95 °C.
  • the sample is an adaptor tagged library of nucleic acid analytes.
  • step (d) either the nucleic acids that remain annealed to Ao, or those not annealed to Ao, are identified.
  • the sequences are identified by an amplification reaction (e.g., polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), strand displacement amplification
  • amplification reaction e.g., polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), strand displacement amplification
  • sequences are identified by isothermal identification.
  • sequences are identified by PCR.
  • sequences are identified by microarray analysis. In some embodiments, the sequences are identified by sequencing.
  • the sequences are identified by Next Generation Sequencing (NGS) (e.g.. bridge amplification sequencing (Illumina), SMRT sequencing (PacBio), Ion Torrent sequencing, nanopore sequencing, pyrosequencing, and the like).
  • NGS Next Generation Sequencing
  • multiple different probe oligonucleotides Ao are employed, each with different sequences (e.g., 3' end sequences) designed to anneal (e.g., perfectly anneal) to a different target sequence, and wherein the concentrations of multiple target sequences are simultaneously increased relative to non-target sequences.
  • sequences e.g., 3' end sequences
  • anneal e.g., perfectly anneal
  • the different probe oligonucleotides Ao have sequences (e.g., 3' end sequences) which anneal (e.g., perfectly anneal) to sequences of the human genome that include genetic variants associated with the presence, treatment, or monitoring of disease.
  • step (d) the presence or absence of said genetic variants in the sample analyte is inferred through the identification of nucleic acid sequences which have annealed to, and subsequently been released from, Ao, and the presence or absence of disease is thereby inferred.
  • step (d) the presence or absence of said genetic variants in the sample analyte is inferred through the identification of nucleic acid sequences which have annealed to, and subsequently been released from, Ao, and the appropriate treatment for a disease is thereby inferred.
  • step (d) the presence or absence of said genetic variants in the sample analyte is inferred through the identification of nucleic acid sequences which have annealed to, and subsequently been released from, Ao, and the response of a patient to a specific disease treatment is thereby inferred.
  • step (d) the presence or absence of said genetic variants in the sample analyte is inferred through the identification of nucleic acid sequences which have annealed to, and subsequently been released from, Ao, and one or more patient treatment decisions are made based on the presence of absence of said genetic variants.
  • the sample is a human blood or tissue sample.
  • the disease is a cancer.
  • the cancer is lung cancer.
  • one or more hybridisation steps are performed between one or more of the steps.
  • the nucleic acid analysed is a methylated sequence or is derived from a methylated sequence.
  • the technology is used to differentiate methylation status at any particular or multiple locations in a target nucleic acid.
  • Methylated sequences may first be modified using chemical treatments (e.g., oxidation, reduction, bisulfite treatment) or by exposure to methylation dependent restriction enzymes or any other suitable approach followed by enrichment and/or identification of the modified sequence.
  • nucleic acid sequences present in the sample are bisulfite treated prior to step (a) of the method.
  • nucleic acid sequences present in the sample are bisulfite treated following step (a) of the method.
  • nucleic acid sequences present in the sample are treated enzymatically to convert cytosines to uracil prior to step (a) of the method.
  • nucleic acid sequences present in the sample are treated enzymatically to convert cytosines to uracil following step (a) of the method.
  • nucleic acid sequences present in the sample are exposed to epigenetic-dependent/sensitive restriction enzymes prior to step (a) of the method.
  • nucleic acid sequences present in the sample are exposed to epigenetic-dependent/sensitive restriction enzymes following step (a) of the method.
  • targeted regions of RNA present in the sample are transcribed into DNA prior to step (a) of the method.
  • targeted regions of RNA present in the sample are transcribed into DNA following step (a) of the method.
  • a method of capturing a target nucleic acid sequence of interest from a sample comprising at least a first and second sequence comprising the steps of: a. introducing the sample comprising one or more nucleic acid analytes to a first reaction mixture comprising: i. a single-stranded probe oligonucleotide Ao wherein said probe contains a sequence
  • step (a) introducing the reaction mixture produced by step (a) to a second reaction mixture comprising: i. a pyrophosphorolysing enzyme; and ii. a source of pyrophosphate ion wherein Ao anneals (e.g., perfectly anneals) to one of the nucleic acid sequences to create an at least partially double-stranded intermediate product in which Ao (e.g., the 3' end of Ao) forms a double-stranded complex with said sequence and Ao is pyrophosphorolysed in the 3'-5' direction from its 3'-end, whilst any Ao that has annealed to a lesser extent (e.g., imperfectly) to the other sequence is pyrophosphorolysed in the 3'-5' direction to a lesser extent due to said lesser (e.g., imperfect) annealing; c.
  • Ao anneals e.g., perfectly anneals
  • Ao e.g., perfectly anneals
  • any Ao sequence complexes which were annealed by: i. allowing the strands of said complex to separate (e.g., melt apart) as a consequence of the pyrophosphorolysis reaction; or ii. heating the reaction mixture to a temperature sufficient for the stands of said complex to separate (e.g., melt apart) but which is below the temperature required for the stands of any Ao which is less well annealed (e.g., imperfectly annealed) to separate (e.g., melt apart); and d. separating Ao, and thereby any nucleic acid sequences remaining annealed thereto, from any nucleic acid sequences not annealed to Ao.
  • the solid support is a polymer and/or resin coated solid surface.
  • the solid support is a polystyrene solid support.
  • polystyrene (CgHg)n is a polymer wherein n is 10, 20, 30, 40, 50, 100, 150, 200,
  • the polystyrene solid support is a particle, micro particle, magnetic bead, magnetic microparticle, paramagnetic bead, paramagnetic microparticle, resin or any particulate that comprises polystyrene polymers.
  • the polystyrene support is modified to include one or more of the following functional groups: amine, carboxylate, sulfonate, trimethylamine and/or epoxide.
  • the solid support is a magnetic or paramagnetic bead.
  • the magnetic or paramagnetic bead is a shape which maximises the surface area of said bead.
  • the magnetic or paramagnetic bead is a regular shape.
  • the magnetic or paramagnetic bead is an irregular shape.
  • the magnetic or paramagnetic bead has a diameter of less than, or equal to:
  • the solid support is a magnetic or paramagnetic polystyrene bead.
  • the magnetic or paramagnetic polystyrene bead comprises iron oxide.
  • the magnetic or paramagnetic polystyrene bead is Streptavidin-coupled.
  • the solid support is a Streptavidin-coupled Dynabead (RIM).
  • RIM Streptavidin-coupled Dynabead
  • the solid support is a dextran-modified surface.
  • the dextran-modified surface is a particle, micro particle, magnetic or paramagnetic bead, resin or any particulate that comprises dextran polymers.
  • the dextran polymers have an approximate molecular weight from 1000 to
  • the dextran polymers have an approximate molecular weight from 25000 to about 100000.
  • the dextran-surface modified is further modified to include one or more functional groups.
  • the dextran-modified surface is modified to include one or more of the following functional groups: amine, carboxylate, sulfonate, trimethylamine and/or epoxide.
  • the solid support is a magnetic or paramagnetic bead.
  • the magnetic or paramagnetic bead is a shape which maximises the surface area of said bead.
  • the magnetic or paramagnetic bead is a regular shape.
  • the magnetic or paramagnetic bead is an irregular shape.
  • the magnetic or paramagnetic bead has a diameter of less than, or equal to:
  • the magnetic or paramagnetic bead is a dextran magnetic bead selected from:
  • Nanomag* Dextran ND
  • Nanomag* Dextran-SO3H ND-SO3H
  • BioMag* Dextran-Coated Charcoal or BioMag* Plus Dextran.
  • the solid support is a Polyethylene Glycol (PEG) or PEG-modified surface.
  • PEG Polyethylene Glycol
  • the Polyethylene Glycol (PEG) or PEG-modified surface is a particle, micro particle, magnetic or paramagnetic bead, resin or any particulate that comprises PEG.
  • PEG (CHiOjn) is a polymer wherein n is 10, 20, 30, 40, 50, 100,
  • n is any integer between any of these points, or n is within any range derivable between any two of these points, is utilised.
  • the PEG utilised is PEG-200, PEG-300, PEG-400, PEG-600, PEG-1000, PEG-1300-
  • the microparticle or bead is selected from Nanomag* PEG-300
  • the solid support is a Polyvinylpyrrolidone (PVP) or PVP-modified surface.
  • the PVP or PVP-modified surface is a particle, micro particle, magnetic or paramagnetic bead, resin or any particulate that comprises PVP.
  • PVP n-vinyl pyrrolidone
  • n-vinyl pyrrolidone is a polymer wherein n is 10, 20, 30,
  • n is any integer between any of these points, or n is within any range derivable between any two of these points, is utilised.
  • the solid support is a polysaccharide or polysaccharide-modified surface.
  • the polysaccharide or polysaccharide-modified surface is a particle, micro particle, magnetic or paramagnetic bead, resin or any particulate that comprises a polysaccharide.
  • the polysaccharide is selected from one or more of dextran, ficoll, glycogen, gum arable, xanthan gum, carageenan, amylose, agar, amylopectin, xylans and/or beta-glucans.
  • the solid support is a chemical resin or chemical resin-modified surface.
  • the chemical resin or chemical-resin modified surface is selected from one or more of the following resins: isocyanate, glycerol, piperidino-methyl, polyDMAP (polymer-bound dimethyl 4-aminopyridine), DIPAM (Diisopropylaminomethyl, aminomethyl, polystyrene aldehyde, tris(2-aminomethyl) amine, morpholino-methyl, BOBA (3-Benzyloxybenzaldehyde), triphenylphosphine or benzylthio-methyl.
  • the capture moiety is covalently attached to the solid support via a chemically-cleavable linker, such as a disulfide, allyl, or azide-masked hemiaminal ether linker.
  • a chemically-cleavable linker such as a disulfide, allyl, or azide-masked hemiaminal ether linker.
  • the capture moiety is covalently attached to the solid support via amide or phosphorothioate bonds.
  • the capture moiety comprises an oligonucleotide sequence and the solid support comprises oligonucleotides bearing the complementary sequence.
  • the oligonucleotide sequence comprises one or more modified bases and/or other such modifications known to the person skilled in the art, to change the melting temperature.
  • the presence of one or more modified bases and/or other such modifications known to the person skilled in the art leads to a decrease in the melting temperature.
  • the presence of one or more modified bases and/or other such modifications known to the person skilled in the art leads to an increase in the melting temperature.
  • the length of the complementary sequence is between 10, 20, 30, 40, 50, 100,
  • the length of the complementary sequence is between 10, 20, 30, 40, 50, and
  • the length of the complementary sequence is between 10-20, 10- 30, 10-40 and 10-50 bases.
  • the length of the complementary sequence is between 10-20, 10-30 and 10-
  • the length of the complementary sequence is between 10-20 and 10-30 bases.
  • the length of the complementary sequence is between 10 - 20 bases.
  • the capture moiety comprises a chemical modification and is attached to the solid support via an interaction between the chemical modification and the solid support.
  • the chemical modification is biotin and the solid support further comprises streptavidin.
  • captured oligonucleotide sequences are released from the solid support.
  • captured oligonucleotide sequences are released from the solid support by chemical denaturation.
  • chemical denaturation is achieved by the use of suitable concentration of base.
  • 0.1M of NaOH may be used.
  • oligonucleotide sequences are released from the solid support by the cleavage of a chemical linker through the addition of tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) for a disulfide linker; palladium complexes or an allyl linker; or TCEP for an azide- masked hemiaminal ether linker.
  • TCEP tris(2-carboxyethyl)phosphine
  • DTT dithiothreitol
  • oligonucleotide sequences are released from the solid support by removing a non-canonical base, from oligonucleotide sequences, and cleavage at the resultant abasic site.
  • the non-canonical base is uracil, which is removed by uracil DNA glycosylase.
  • the non-canonical base is 8-oxoguanine, which is removed by formamidopyrimidine DNA glycosylase (Fpg).
  • the capture moiety is an oligonucleotide region and release is performed through heating of the reaction mixture.
  • the reaction mixture is heated to 37°C - 100°C over 1 -20 minutes.
  • the reaction mixture is heated over 1 - 15 minutes.
  • the reaction mixture is heated over 1 - 10 minutes.
  • the reaction mixture is heated over 1 -5 minutes.
  • the reaction mixture is heated over 5 minutes.
  • the reaction mixture is heated to 37°C-85°C.
  • the reaction mixture is heated to 37°C-75°C.
  • the reaction mixture is heated to 37°C-65°C.
  • the reaction mixture is heated to 37°C-55°C.
  • the reaction mixture is heated to 37°C-45°C.
  • the temperature to which the reaction mixture is heated so enact release of complementary oligonucleotide regions depends on the length of said regions.
  • release is achieved through the cleavage of one or more oligonucleotide sequences. This cleavage can be achieved by any of the means previously, or subsequently, described or by any of the means known to the person skilled in the art.
  • oligonucleotide sequences are cleaved chemically.
  • oligonucleotide sequences are cleaved enzymatically.
  • oligonucleotide sequences are cleaved by a restriction enzyme.
  • oligonucleotide sequences are cleaved by epigenetic modification sensitive or dependent restriction enzymes.
  • oligonucleotide sequences are cleaved by methylation sensitive or dependent restriction enzymes.
  • oligonucleotide sequences are cleaved by hydroxymethylation sensitive or dependent restriction enzymes.
  • sequences prior to, or after, enrichment of variant or wild-type sequences, are enzymatically or chemically converted to allow detection of their methylation status.
  • enrichment refers to the selective isolation of target sequences from a mixture of target and non-target sequences, as described previously or subsequently.
  • the restriction enzymes are endonucleases.
  • oligonucleotide sequences are cleaved by a flap endonuclease.
  • oligonucleotide sequences comprise a photocleavable linker and oligonucleotide sequences are released from the solid support by cleavage of this linker.
  • oligonucleotide sequences are released from the solid support by cleavage of this linker.
  • the digestion continues until Ao lacks sufficient complementarity with the sequence for the pyrophosphorolysing enzyme to bind or for the pyrophosphorolysing reaction to continue. This typically occurs when there are between 6 and 20 complementary nucleotides remaining between the sequence and probe. In some embodiments, this occurs when there are between 6 and 40 complementary nucleotides remaining. Without being constrained by theory, there are a number of different ways in which the pyrophosphorolysis reaction may be stopped, in addition to those described previously or elsewhere.
  • the digestion may stop 3' of a mismatch or base modification. Such activity has been observed in archaeal DNA polymerases.
  • the pyrophosphorolysis reaction may stop due to the presence of a modification in the backbone of Ao-
  • This modification may be a modified base.
  • the base may be resistant to pyrophosphorolysis.
  • This modification may be a chemical backbone modification.
  • the pyrophosphorolysis reaction may stop due to the presence of mismatch in
  • the temperature of the reaction mixture may be increased to heat-inactivate the pyrophosphorolysis enzyme. In some embodiments, the temperature is increased to cause the probe-target duplex to melt apart.
  • any reagent that could cause the inactivation of the pyrophosphorolysis enzyme may be added to the reaction mixture.
  • the pH concentration may be modified to inactivate pyrophosphorolysis enzyme.
  • the salt concentration may be modified to inactivate pyrophosphorolysis enzyme.
  • the detergent concentration may be modified to inactivate pyrophosphorolysis enzyme.
  • the ion concentration may be modified to inactivate pyrophosphorolysis enzyme.
  • pyrophosphorolysis is carried out in the reaction medium at a temperature in the range 20 to 90°C in the presence of at least a polymerase exhibiting pyrophosphorolysis activity and a source of pyrophosphate ion.
  • a polymerase exhibiting pyrophosphorolysis activity
  • a source of pyrophosphate ion a source of pyrophosphate ion.
  • the pyrophosphorolysis step is driven by the presence of a source of excess polypyrophosphate, suitable sources including those compounds containing 3 or more phosphorous atoms.
  • the pyrophosphorolysis step is driven by the presence of a source of excess modified pyrophosphate.
  • Suitable modified pyrophosphates include those with other atoms or groups substituted in place of the bridging oxygen, or pyrophosphate (or poly-pyrophosphate) with substitutions or modifying groups on the other oxygens.
  • pyrophosphate or poly-pyrophosphate with substitutions or modifying groups on the other oxygens.
  • the source of pyrophosphate ion is PNP, PCP or Tripolyphoshoric Acid
  • sources of pyrophosphate ion for use in the pyrophosphorolysis step may be found in W02014/165210 and WO00/49180.
  • the X groups are independently selected from -H, -Na, -K, alkyl, alkenyl, or a heterocyclic group with the proviso that when both Z and B correspond to -O- and when n is 1 at least one X group is not H.
  • P( O)(O-X2) wherein Z is 0, NH or CH 2 and (a) XI is y,y-dimethylallyl, and X2 is -H; or (b) XI and X2 are both methyl; or (c) XI and X2 are both ethyl; or (d) XI is methyl and X2 is ethyl or vice versa.
  • fragmentation is achieved by sonication. In some embodiments, The
  • Bioruptor* (Denville, NJ) device may be used.
  • fragmentation is achieved by acoustic shearing. In some embodiments, the
  • Covaris* instrument (Woburn, MA) may be used.
  • fragmentation is achieved by nebulisation. Nebulization forces DNA through a small hole in a nebulizer unit, which results in the formation of a fine mist that is collected.
  • Fragment size is determined by the pressure of the gas used to push the DNA through the nebulizer, the speed at which the DNA solution passes through the hole, the viscosity of the solution, and the temperature.
  • fragmentation is achieved by hydrodynamic shear. In some embodiments,
  • the Hydroshear from Digilab (Marlborough, MA) may be used.
  • fragmentation is achieved by point-sink shearing. In some embodiments, fragmentation is achieved by needle shearing.
  • fragmentation is achieved via use of a French press.
  • fragmentation is achieved by enzymatic fragmentation.
  • fragmentation is achieved by restriction endonuclease digestion.
  • fragmentation is transposome mediated fragmentation.
  • fragmentation is achieved by Cas9.
  • fragmentation is achieved by Cas9, as described in US10577644, herein incorporated by reference in its entirety.
  • one or more different fragmentation techniques may be used.
  • one or more of the same or different fragmentation techniques may be used at one or more different points of the method.
  • fragmentation and adaptor tagging of sequences occurs at the same time or in the same step of the method.
  • One such example is Nextera DNA Library Prep Kit by Illumina.
  • the ends of nucleic acids may be polished and A- tailed prior to ligation to one or more adaptors.
  • the ends of nucleic acids may be polished and ligated to adaptors in a blunt-end ligation reaction.
  • adaptors are ligated to single-stranded DNA.
  • a terminal transferase enzyme is used to add nontemplated bases to the 3' end of fragments, providing a site for priming to make fragments double- stranded.
  • topoisomerase may be used in lieu of a DNA ligase.
  • TOPO cloning may be used to add adaptors to fragmented DNA.
  • transposases can be used to add adaptor sequences to nucleic acids.
  • standard transposons can be used but then modified to create a Y-shaped adaptor using oligonucleotide replacement.
  • blocking oligonucleotides are used to prevent cross-hybridisation of library molecules (so called 'daisy chaining').
  • blocking oligonucleotides are resistant to pyrophosphorolysis. This resistance may be as previously or subsequently described.
  • double-stranded molecules have strands 5'- adaptorl - insert - adaptor2' -3' and 5' -adaptor2 - insert - adaptorl' -3'.
  • the 5'- adaptorl -3' sequence on one strand could hybridise to a 5' - adaptorl' -3' sequence on a second strand.
  • the 5'-adaptor2'-3' sequence on one strand can hybridise to a 5'-adaptor2-3' sequence on a second strand.
  • pyrophosphorolysis could remove bases from the 3' of a target molecule, preventing its amplification during PCR.
  • Blocking oligonucleotides are typically used in hybridisation capture to mitigate against daisy-chaining.
  • these could be sequence complementary to adaptorl' and sequence complementary to adaptor2'.
  • the blocking oligonucleotides can be protected from pyrophosphorolysis by addition of 3' phosphorothioate bonds or by ensuring that the 3' of the adaptor tagged molecules is non-complementary to the blocking oligonucleotide.
  • alpha-thio-dNTPs that introduce phosphorothioate bonds that inhibit pyrophosphorolysis could be included in a PCR, or primer extension reaction, prior to pyrophosphorolysis.
  • the sample comprises one or more blocking oligonucleotides.
  • the first reaction mixture comprises one or more blocking oligonucleotides.
  • the second reaction mixture comprises one or more blocking oligonucleotides.
  • one or more blocking oligonucleotides are introduced to the first reaction mixture prior to step (b).
  • the sequencing of the method is Maxam-Gilbert sequencing.
  • the sequencing of the method is Sanger sequencing.
  • the sequencing of the method is shotgun sequencing.
  • the sequencing of the method is single-molecule real-time sequencing.
  • the sequencing of the method is ion semiconductor sequencing.
  • the sequencing of the method is pyrosequencing.
  • the sequencing of the method is sequencing by synthesis.
  • the sequencing of the method is combinatorial probe anchor synthesis
  • the sequencing of the method is sequencing by ligation.
  • the sequencing of the method is nanopore sequencing.
  • the sequencing of the method is GenapSys sequencing.
  • the sequencing is Next Generation Sequencing (NGS).
  • NGS Next Generation Sequencing
  • a method for screening a patient comprising the use of any previously or subsequently described embodiment of the method to detect the presence or absence of one or more specific nucleic acid sequences in a sample derived from a patient.
  • the person skilled in the art will appreciate that such a screening will be useful for monitoring a patient receiving treatment for one or more conditions, the treatment status of which can be ascertained by the levels of one or more nucleic acid sequences in a patient sample.
  • the treatment status of patients receiving treatment for one or more cancers can be ascertained by the levels of one or more nucleic acid sequences in their blood and/or the presence and/or absence of one or more specific variants.
  • a high level of circulating tumour nucleic acid sequences and/or the presence and/or absence of one or more specific variants can be used to deduce whether or not a particular treatment is having the desired effect.
  • a method of monitoring a patient in remission to detect any recurrence of disease in some embodiments, there is provided a method of monitoring a patient in remission to detect any recurrence of disease.
  • a method for the diagnosis and/or monitoring of one or more cancers in a patient comprising the use of any previously or subsequently described embodiment of the method to detect the presence or absence of one or more specific nucleic acid sequences in a sample derived from a patient.
  • the one or more specific nucleic acid sequences may be specific to an individual (identified from a tissue biopsy or surgical resection, for example, by a method of identification such as sequencing) and that in such cases an individual patient specific panel may be used.
  • a panel will cover known hotspot regions of the human genome, those that are recurrently mutated in a given cancer type.
  • a method for non-invasive prenatal testing comprising the use of any previously or subsequently described embodiments of the method to detect the presence or absence of one or more specific nucleic acid sequences in a sample derived from a patient, wherein the patient is a pregnant patient.
  • the sample is the plasma and or serum of the blood of a pregnant patient.
  • methods provided herein are employed to enrich and/or quantify a fetal fraction of a sample using a panel of common
  • a method of treating a patient comprising the steps of:
  • the treatment decision is the initiation of a particular treatment.
  • the treatment decision is the cessation of a particular treatment.
  • the treatment decision is an increase in the dose of a particular treatment.
  • the treatment decision is a decrease in the dose of a particular treatment.
  • the treatment decision is an increase in the frequency of administration of a particular treatment.
  • the treatment decision is a decrease in the frequency of administration of a particular treatment.
  • the treatment decision is the addition of an additional drug to an existing treatment regimen.
  • the treatment decision is the removal of a drug from an existing treatment regimen.
  • a device for performing steps (a)-(c) of the method in some embodiments, there is provided a device for performing steps (a)-(c) of the method.
  • a device for performing steps (a)-(b) of the method in some embodiments, there is provided a device for performing steps (a)-(b) of the method.
  • kits comprising one or more oligonucleotides, enzymes, reagents or components as previously or subsequently described in one or more embodiments.
  • a kit comprising:
  • One or more oligonucleotides Ao wherein Ao comprises: o a sequence (e.g., at a 3' end of Ao or elsewhere) which is complementary to (e.g., perfectly complementary to) a known first sequence but less complementary to
  • One or more pyrophosphorolysing enzymes One or more pyrophosphorolysing enzymes.
  • One or more sources of pyrophosphate ions are provided.
  • the kit further comprises one or more solid supports. These solid supports may be as previously or subsequently described.
  • the kit further comprises one or more buffers. These buffers may be as previously or subsequently described.
  • the kit further comprises one or more crowing agents (e.g., polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), dextran sulphate, etc.).
  • crowing agents e.g., polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), dextran sulphate, etc.
  • the kit further comprises one or more detergents (e.g., sodium dodecyl sulfate (SDS), TWEEN20, etc.).
  • one or more detergents e.g., sodium dodecyl sulfate (SDS), TWEEN20, etc.
  • the kit further comprises one or more solvents (e.g., formamide, ethylene carbonate, etc.).
  • solvents e.g., formamide, ethylene carbonate, etc.
  • the kit further comprises a phosphohydrolase (e.g. Apyrase).
  • a phosphohydrolase e.g. Apyrase
  • the kit further comprises a pyrophosphatase (e.g. Thermostable Inorganic
  • TIPP Pyrophosphatase
  • the one or more buffers may include additives which hybridise to repetitive sequences.
  • the one or more buffers may include COt-1 DNA, salmon sperm
  • DNA DNA, oligonucleotides that block ribosomal RNA, and the like.
  • Ao further comprises a 5' tail region which is not complementary to either a known first or known second sequence.
  • Ao may be as previously or subsequently described.
  • Ao further comprises a capture moiety.
  • the kit further comprises one or more capture oligonucleotides (Co), as previously or subsequently described.
  • the kit further comprises one or more capture oligonucleotide Co which comprises a capture moiety and wherein a 5' tail region of Ao is complementary to a region of Co-
  • the capture moiety is biotin and the solid support comprises streptavidin.
  • the solid support comprises a capture oligonucleotide Co and wherein a 5' tail region of Ao is complementary to a region of Co.
  • the solid support is a bead.
  • the bead is a magnetic or paramagnetic bead.
  • the kit further comprises one or more epigenetic modification sensitive or dependent restriction enzymes.
  • the kit further comprises one or more transposomes.
  • the kit further comprises Cas9.
  • the kit further one or more transposases.
  • the kit further comprises one or more ligases.
  • the kit further comprises one or more metal ions.
  • the kit further comprises one or more blocking oligonucleotides.
  • the one or more blocking oligonucleotides are resistant to pyrophosphorolysis.
  • the kit further comprises isothermal amplification reagents.
  • the kit further comprises Polymerase Chain Reaction (PCR) reagents (e.g., thermostable DNA polymerases, primers, dNTPs, buffer solution).
  • PCR Polymerase Chain Reaction
  • the kit further comprises sequencing reagents.
  • the kit further comprises Next Generation Sequencing (NGS) reagents (e.g., polymerases, dNTPs, buffer solution).
  • NGS Next Generation Sequencing
  • the kit further comprises a DNA library preparation kit (e.g., containing, as desired, polymerases, ligases, adapters, dNTPs, buffer solutions).
  • the kit further comprises one or more molecular probes.
  • the kit further comprises one or more molecular probes which are fluorescently labelled.
  • a region of Ao (e.g., the 3' end of) is more complementary to (e.g., perfectly complementary to) a wild type sequence of the human genome and less complementary to (e.g., imperfectly complementary to) a mutant allele of said wild type sequence.
  • a region of Ao (e.g., the 3' end of) is less complementary to (e.g., imperfectly complementary to) a wild type sequence of the human genome and more complementary to (e.g., perfectly complementary to) a mutant allele of said wild type sequence.
  • the kit further comprises one or more components for the transcription of
  • the kit further comprises one or more kits for the preparation of adaptor tagged libraries for sequencing, as previously or subsequently described or otherwise known to the person skilled in the art.
  • the kit further comprises one or more components for the fragmentation of nucleic acids, as previously or subsequently described or otherwise known to the person skilled in the art.
  • the kit further comprises one or more devices for the physical fragmentation of nucleic acids, as previously or subsequently described or otherwise known to the person skilled in the art.
  • the kit alternatively further comprises one or more components for the enzymatic fragmentation of nucleic acids, as previously or subsequently described or otherwise known to the person skilled in the art.
  • the kit further comprises one or more components for the physical fragmentation of nucleic acids and one or more components for the enzymatic fragmentation of nucleic acids.
  • the kit comprising multiple Ao, as previously or subsequently described.
  • kits comprising 1-1,000,000 individual Ao probes, each of which have sequences (e.g., 3' ends) which are perfectly complementary to an individual mutation, wherein the same sequences (e.g., at 3' ends or elsewhere) are imperfectly complementary to the wild type sequence of said mutation.
  • sequences e.g., 3' ends
  • kit comprising 1-
  • kits comprising 1-10,000 individual Ao probes. In some embodiments, there is provided a kit comprising 1-1,000 individual Ao probes.
  • kits comprising 1-1,000,000 individual Ao probes, each of which have sequences (e.g., 3' ends or elsewhere) which are imperfectly complementary to an individual mutation, wherein the same sequences (e.g., at 3' ends or elsewhere) are perfectly complementary to the wild type sequence of said mutation.
  • a kit comprising 1-100,000 individual Ao probes.
  • a kit comprising 1-10,000 individual Ao probes.
  • a kit comprising 1-1,000 individual Ao probes.
  • kits comprising multiple capture oligonucleotides (Co) as previously or subsequently described.
  • kits comprising multiple solid supports as previously or subsequently described.
  • a panel comprising multiple Ao which have sequences (e.g., at their 3' ends) which are perfectly complementary to known sequences.
  • a panel comprising multiple Ao which have sequences (e.g., at their 3' ends) which are imperfectly complementary to known sequences.
  • a panel comprising multiple Ao which have sequences (e.g., at their 3' ends) which are perfectly complementary to known variants in a given cancer type or range of cancer types.
  • a panel comprising multiple Ao which have sequences (e.g., at their 3' ends) which are imperfectly complementary to known variants in a given cancer type or range of cancer types, wherein the sequences (e.g., at their 3' ends) are perfectly complementary to the wild-type sequences.
  • a panel comprising multiple Ao some of which have sequences (e.g., at their 3' ends) which are perfectly complementary to known variants in a given cancer type or range of cancer types whilst others are imperfectly complementary.
  • the panel further comprises one or more capture oligonucleotides (Co) as previously or subsequently described.
  • the panel further comprises one or more blocking oligonucleotides as previously or subsequently described.
  • a polymorphism may be selected from any polymorphism (e.g., mutation) previously, or subsequently, described or known.
  • the person skilled in the art will thus appreciate that within the scope of the invention are included panels which may be useful in the detection of one or more polymorphisms (e.g., mutations) to any of the proto-oncogenes, oncogenes, or genetic markers for one or more disease states previously, or subsequently, described or known.
  • panels which may be useful in the detection of more variants that could be used in determining the presence, or absence, of genetic markers for one or more disease states or that are specific for a given patient, tissue, or cell, for example for tumor-informed monitoring.
  • panels which may be useful in the detection of one or more variants which are as yet unknown but which nonetheless could be used in determining the presence, or absence, or one or more disease states.
  • mutational signatures for many different cancer types, these are preferential modes of mutagenesis such as excess C>T at CpG dinucleotides.
  • Panels may comprise probes that are designed to detect these types of events occurring.
  • the panel comprises 1-1,000,000 individual probe molecules, each of which has a sequence (e.g., at their 3' end) which may be complementary to a specific target region including a target mutation.
  • the panel comprises 10,000-1,000,000 individual probe molecules, each of which has a sequence (e.g., at their 3' end) which may be complementary to a specific target region including a target mutation.
  • the panel comprises 100,000-1,000,000 individual probe molecules, each of which has a sequence (e.g., at their 3' end) which may be complementary to a specific target region including a target mutation. In some embodiments, the panel comprises 200,000-1,000,000 individual probe molecules, each of which has a sequence (e.g., at their 3' end) which may be complementary to a specific target region including a target mutation.
  • the panel comprises 10,000-100,000 individual probe molecules, each of which has a sequence (e.g., at their 3' end) which may be complementary to a specific target region including a target mutation.
  • the panel comprises 1,000-100,000 individual probe molecules, each of which has a sequence (e.g., at their 3' end) which may be complementary to a specific target region including a target mutation.
  • the panel comprises 1,000-10,000 individual probe molecules, each of which has a sequence (e.g., at their 3' end) which may be complementary to a specific target region including a target mutation.
  • the panel comprises 500-10,000 individual probe molecules, each of which has a sequence (e.g., at their 3' end) which may be complementary to a specific target region including a target mutation.
  • the panel comprises 500-1,000 individual probe molecules, each of which has a sequence (e.g., at their 3' end) which may be complementary to a specific target region including a target mutation.
  • the panel comprises 5-500 individual probe molecules, each complementary to a specific target mutation. In some embodiments, the panel comprises 5-400 individual probe molecules, each complementary to a specific target mutation. In some embodiments, the panel comprises 5-300 individual probe molecules, each complementary to a specific target mutation. In some embodiments, the panel comprises 5-200 individual probe molecules, each complementary to a specific target mutation. In some embodiments, the panel comprises 5-100 individual probe molecules, each complementary to a specific target mutation. In some embodiments, the panel comprises 5-50 individual probe molecules, each complementary to a specific target mutation.
  • a panel comprising a plurality of probe molecules wherein one or more probes are complementary to an EGER mutation, one or more probes are complementary to a KRAS mutation, one or more probes are complementary to a ERBB2/HER2 mutation, one or more probes are complementary to a EML4-ALK mutation, one or more probes are complementary to a ROS1 mutation, one or more probes are complementary to a
  • RET mutation and one or more probes are complementary to a MET mutation.
  • a panel comprising a plurality of probe molecules wherein one or more probes may be complementary to an EGFR mutation, one or more probes may be complementary to a KRAS mutation, one or more probes may be complementary to a ERBB2/HER2 mutation, one or more probes may be complementary to a EML4-ALK mutation, one or more probes may be complementary to a ROS1 mutation, one or more probes may be complementary to a RET mutation and one or more probes may be complementary to a MET mutation.
  • a panel of probe molecules selective for EGFR mutations there is provided a panel of probe molecules selective for EGFR mutations.
  • a panel of probe molecules selective for ERBB2/HER2 mutations there is provided a panel of probe molecules selective for ERBB2/HER2 mutations.
  • a panel of probe molecules selective for RET mutations there is provided a panel of probe molecules selective for RET mutations.
  • a panel comprising a plurality of probe molecules selective for one or more coding sequences (CDSs).
  • CDSs coding sequences
  • kits comprising a panel, which may be as previously or subsequently described, in combination with one or more reagents, which may be as previously or subsequently described.
  • magnetic microparticles are magnetically responsive microparticles which are attracted by a magnetic field.
  • the magnetic microparticles which may be used in the methods of the present invention comprise a magnetic metal oxide core, which is generally surrounded by a polymer coat which creates a surface that can bind to DNA, RNA, or PNA.
  • the magnetic metal oxide core is preferably iron oxide, wherein iron is a mixture of Fe 2+ and Fe 3+ .
  • the preferred Fe 2+ /Fe 3+ ratio is preferably 2/1, but can vary from about 0.5/1 to about 4/1.
  • Bioinformatics approaches are used to analyse sequencing data. In some embodiments, the presence or absence of specific variants is called. In other embodiments, data from multiple variants are combined to derive a probabilistic estimate for the presence or absence of tumor DNA.
  • Illumina sequencing data analysis includes conversion and demultiplexing of BCL files into FASTQ format using tools such as bcl2fastq.
  • sequencing reads include molecular identifiers.
  • molecular identifiers can be extracted from sequencing reads, appended to FASTQ headers, and the sequencing reads clipped.
  • barcodes with non-canonical bases can be filtered. The resulting reads can then be aligned using a tool such as bwa mem, using the -C option to append barcode sequences to alignments.
  • Alignments can then be sorted by coordinate, duplicate reads marked, and reads annotated with read coordinate, mate coordinate and optical duplicate auxiliary tags using biobambam2 bamsormadup and bammarkduplicatesopt. Reads can be filtered if they are not marked as properpairs or were marked as optical duplicate, supplementary, QC fail, unmapped or secondary alignments. Each read can then be marked with an auxiliary tag comprised of reference name, sorted read and mate fragmentation breakpoints, forward and reverse read barcodes, and read strand.
  • the sequencing data is analysed using a variant calling algorithm that does not use auxiliary tag data.
  • analysis of sequencing data compares the probability of observing data under two models. The first is a null model specifying the distribution of sequencing artifacts. The second is a model allowing for true variants. In this case, a variant is called if the probability under the alternative model exceeds that of the null model.
  • a panel of pre-characterised samples can help to model the error distribution for (first model).
  • auxiliary tags can be used to identify reads that likely derive from the same input molecule, and/or same strand of the same input molecule.
  • consensus base quality scores can be derived from reads that share the same auxiliary tag.
  • variants are identified using an artificial intelligence algorithm such as convolutional neural networks.
  • sequencing data can be further filtered to remove artefacts.
  • Example filters include the number of mismatches present on a given sequencing read; the alignment score and next best alignment score; base quality scores or consensus base quality scores; the minimum number of reads covering a given variant site; the position of the variant within the sequencing read; whether reads are 5' clipped; whether reads are improper pairs; whether reads contain indels; and the variant allele fraction of a given variant.
  • regions of the genome that include common SNPs, or are prone to alignment artefacts are filtered. There are many other filters known to the person skilled in the art.
  • a control sample is sequenced to filter out variants.
  • DNA from buccal epithelial, or other tissue sources could be sequenced to remove germline variants.
  • buffy coat or leukocyte DNA can be sequenced to filter out somatic mutations that derive from clonal haematopoiesis.
  • compositions, methods, and kits of the invention find use in a diverse range of applications and setting. In some embodiments, they find use in any methodology where a sequence is desired to be detected in a sample. In some embodiments, they find use in any methodology where there is a desire to detect a minority (e.g., rare) sequence in a complicated sample. In addition to the exemplary uses described above, a number of additional illustrative uses are provided below.
  • the compositions, methods, and kits find use in the analysis and treatment of infectious diseases.
  • the technology is of particular value for detection of low frequency mutations that may be present in a sample.
  • the technology finds use for the detection of low frequency mutations associated with treatment resistant (e.g., antibiotic resistant, anti-viral resistant, etc.) infectious diseases (e.g., HIV, tuberculosis, etc.).
  • treatment resistant e.g., antibiotic resistant, anti-viral resistant, etc.
  • infectious diseases e.g., HIV, tuberculosis, etc.
  • the technology further finds use in selective pulldown of bacterial or viral DNA or RNA for sequencing.
  • the technology is particularly well suited to the analysis and/or enrichment of analytes in complicated samples.
  • One area of growing research and clinical interest is microbiome analysis where the technology finds use to provide much higher specificity selection of desired bacterial DNA for sequencing or other analysis.
  • the technology also finds use in high throughput analysis of many different samples as well as multiplex analysis. These benefits find use in a wide variety of genotyping applications, including forensic analysis, paternity/maternity testing, disease analysis (e.g., cancer, infectious disease), drug susceptibility testing, agriculture and food testing (e.g., to assist with selective breeding, to identify trace contaminants, etc.).
  • genotyping applications including forensic analysis, paternity/maternity testing, disease analysis (e.g., cancer, infectious disease), drug susceptibility testing, agriculture and food testing (e.g., to assist with selective breeding, to identify trace contaminants, etc.).
  • the technology finds use in synthetic nucleic acid (e.g., DNA) error correction.
  • Synthetic nucleic acid is used in research, diagnostic, and clinical indications. It is often important to avoid or minimizing use of nucleic acid molecules having unintended or undesired sequences.
  • the technology finds use in identification and isolation of desired molecules from undesired molecules.
  • Nucleic acid editing is emerging as an important process in research, synthetic biology, and clinical applications.
  • CRISPR/CAS editing of nucleic acids, and related processes are emerging as important processes.
  • Many of these editing techniques result in a mixed populations of molecules that include intended edited products, unedited products, and unintended edited products.
  • the technology provided herein facilitates identification, selection, and isolation if intended edited products.
  • the technology also finds use in environmental monitoring.
  • the technology is particularly well suited to the analysis of environmental samples that may contain trace amounts of an analyte of interest.
  • samples include, but are not limited to, analysis of native and invasive species of organisms, early detection of invasive species, air and water contamination, and ancient DNA analysis.
  • Sample types include, but are not limited to, soil, water, snow, feces, mucus, gametes, shed skin, carcasses, hair, and air.
  • the technology finds use in the isolation of a desired subset of nucleic acid from a particular sample from other subsets.
  • the technology finds use in the isolation and analysis of chloroplast and mitochondrial genomes.
  • cell line screening for engineered and natural cells.
  • Such cell lines include, but are not limited to, cell cultures (primary and immortalized), stems cells (embryonic, induced pluripotent, de-differentiated, etc.), differentiated cells intended for cell therapies, ex vivo modified cells for research or clinical applications (e.g., CAR T cells), and genetically engineered cells.
  • the technology finds use to removed damaged or other undesired nucleic acid away from undamaged or desired nucleic acid.
  • the technology may be used to remove damaged
  • DNA from a sample prior to methylation analysis DNA from a sample prior to methylation analysis.
  • the technology finds use in pre-implantation screening of cells (e.g., embryos, eggs, sperm), liposomes, exosomes, nucleic acid vectors (e.g., gene therapy vector), and the like prior to there administration to a subject.
  • cells e.g., embryos, eggs, sperm
  • liposomes e.g., liposomes
  • exosomes e.g., nucleic acid vectors
  • the technology finds use in drug toxicity screening.
  • the technology is particularly well suited to the identification of DNA damage, generation of mutations, methylation changes, and the like that may be associated with the use of particular drugs.
  • probes may be used that reside over or are aligned with a breakpoint that associates a particular sequence with relevant correlated information (e.g., tissue of origin, association with diseases such as cancer, etc.).
  • relevant correlated information e.g., tissue of origin, association with diseases such as cancer, etc.
  • the technology may be used in any application where nucleic acid complexity reduction is desired.
  • the technology may be used for whole genome complexity reduction.
  • a restriction enzyme digestion or other nucleic acid fragmenting process is used followed by the step of pulling out only cleaved molecules using probes that match known end sequences.
  • the technology finds use in assessing microsatellite instability (MSI).
  • MSI microsatellite instability
  • Target nucleic acid molecules that differ in the presence of, number or, or nature of repeated nucleotides (e.g., GT/CA repeats) are enriched and/or identified in a sample.
  • MSI is associated with a number of diseases and conditions including, but not limited to, colon cancer, gastric cancer, endometrium cancer, ovarian cancer, hepatobiliary tract cancer, urinary tract caner, brain cancer, and skin cancers.
  • TMB tumor mutational burden
  • TMB has emerged as a predictive biomarker for immune checkpoint therapy, among other uses.
  • nextgeneration, whole-exome sequencing is employed to assess TMB or a gene panel that provides sequences of a subset of genes is assessed.
  • Use of the technology provided herein allows for TMB assessments that are more sensitive and significantly less costly and burdensome.
  • the technology finds use in haplotying. Genomic information reported as haplotypes rather than genotypes is increasingly important for personalized medicine, as well as a wide variety of research applications. Haplotypes, that are more specific than less complex variants such as single nucleotide variants, also have applications in prognostics and diagnostics, in the analysis of tumors, and in typing tissue for transplantation.
  • sequencing is the most common form of molecular haplotyping. The error rate of sequencing technologies presents a barrier to obtaining accurate information.
  • the technology provided herein allows for efficient and highly accurate haplotying.
  • the ability of the technology to enrich for any desired sequence or interest allow the technology to enhance existing nucleic acid methodologies. For example, many nucleic acid sequencing approaches struggle when there are repetitive sequence regions in a target nucleic acid.
  • the technology provided herein permits removal of repetitive regions, to make such sequencing reactions more accurate and efficient.
  • Example 1 Pyrophosphorolysis dependent release of the target sequence of interest
  • the tube was then placed on a magnet followed by removal of the storage buffer. To the tube was then added 1 mL of blocking buffer (IxPBS with 1 ug/mLtRNA) followed by rotation (40 rpm) at room temperature (RT) for 30 minutes.
  • blocking buffer IxPBS with 1 ug/mLtRNA
  • the ratio of beads/oligos and buffer was as follows:
  • the tube was then rotated (40 rpm) for 30 minutes at room temperature.
  • the lxBFF60.01% Triton-X buffer was then removed and a PPL mixture (stored at 4 °C) was added to the beads.
  • the PPL mixture consisted of:
  • the resulting mixture was incubated at 45 °C for 30 minutes.
  • the TIPP mixture consisted of:
  • the resulting mixture was incubated at 37°C for 5 minutes.
  • the mixture from point 7 was heated up to 60 °C for 5 min. Tubes with the mixture were transferred to the magnetic rack placed at a hot plate set up at 60 °C in order to separate magnetic beads from supernatant.
  • the Q5 buffer composition is not publicly available.
  • the resulting mixtures were then incubated at 95oC for 5 minutes and then allowed to slowly cool to room temperature.
  • Probe Ao SEQ ID NO 8 4. dPCR quantification of targets pre-bead prep Concentrations of the Wild-Type and Mutant oligonucleotides in the annealed and diluted mixtures were quantified using dPCR. 1000-fold dilution of 1% MAP and 10% MAP samples (taken from point
  • the dPCR mixture consisted of: lx Q5U buffer
  • the Q5 buffer composition is not publicly available.
  • REV (SEQ ID NO 12): where * represents a phosphorothioate bond, /5Phos/ represent 5' end phosphate, and a different primer mix was used for quantification of the Wild-Type and Mutant oligonucleotides.
  • Fluorescent readings were taken after incubation, and the dPCR manufacturer software used for quantification of the different oligos.
  • the blocked beads were spun down for 5 seconds in mini-centrifuge at 2000 x g and placed on top of a magnet followed by removal of the blocking solution by aspiration. 10 uL of 2x Binding buffer
  • the ratio of beads/oligos and buffer was as follows:
  • the lx BFF60.01% TWEEN20 buffer was then removed and a PPL mixture (stored at 4°C) was added to the beads.
  • the PPL mixture consisted of: lx BFF6 with 0.1% TWEEN20
  • the TIPP mixture consisted of: lx BFF6 with 0.1% TWEEN20
  • the resulting mixture was incubated at 37°C for 5 minutes.
  • the mixture from point 9 was heated up to 60°C for 10 min. Tubes with the mixture were transferred to the magnetic rack placed on a hot plate set up at 60°C to separate magnetic beads from supernatant.
  • Alexa 700 0.0003 pg/mL
  • the Q5 buffer composition is not publicly available.
  • REV (SEQ ID NO 12): where * represents a phosphorothioate bond, /5Phos/ represent 5' end phosphate, and a different primer mix was used for quantification of the Wild-Type and Mutant oligonucleotide.
  • Beads preparation a. Bead blocking step
  • ImL blocking buffer 0.1% Tween-20, Ipg/mL t-RNA
  • IxSSC buffer 5x SSC, 5x Denhardts solution, 5mM EDTA, 0.1% SDS
  • PPL has the following composition: lxBFF6-0.1% Tween-20
  • Primer mix 1 has:
  • step 7 After preamplification from step 7 is finished. Add 2 ⁇ L of mixture from step 7 to the reaction containing the following: lx Q5U buffer dNTPs 0.4 mM
  • Primer mix 2 has:
  • Primer mix 3 has:
  • Example 4 Detection of the EGFR exon 20 T790M variant at a different variant allele fraction (VAF)
  • ImL blocking buffer 0.1% Tween-20, Ipg/mLt-RNA
  • Mutant oligonucleotide (SEQ ID 15): Where /5Biosg/ stands for biotin on the 5' end.
  • PPL has the following composition: lxBFF6-0.1% Tween-20
  • Primer mix 1 has:
  • step 7 After preamplification from step 7 is finished. Add 2 ⁇ L of mixture from step 7 to the reaction containing the following: lx Q5U buffer dNTPs 0.4 rniVI
  • Primer mix 2 has:
  • Primer mix 3 has:
  • Reverse primer (SEQ ID 21): -Place sample in the QlAcuity Digital PCR System with lid on IQSoC
  • Example 5 Effect of hybridisation buffer and time of hybridization on detection of EGFR exon 20 T790M variant at 0.1%VAF.
  • Beads preparation a. Bead blocking step
  • ImL blocking buffer 0.1% Tween-20, Ipg/mLt-RNA
  • Buffer 1 IxSSC buffer (5x SSC, 5x Denhardt's solution, 5mM EDTA, 0.1% SDS) or
  • Buffer 2 ULTRAhybTM Ultrasensitive Hybridization Buffer (cat no. AM8670) or Buffer 3: IxSSC+DS buffer (5x SSC, 5x Denhardts solution, 5mM EDTA, 0.1% SDS, 5% dextran sulphate)
  • WT oligonucleotides mix 198 fM
  • WT oligonucleotides mix has:
  • Reverse strand 2 (SEQ ID NO 25): Forward strand 3 (SEQ ID NO 26):
  • Mutant oligonucleotides mix has:
  • Reverse strand 1 (SEQ ID NO 39): Forward strand 2 (SEQ ID NO 40):
  • Reverse strand 8 (SEQ ID NO 53): 3. Attachment oligonucleotides to the beads
  • PPL consists of the following composition: lxBFF6-0.1% Tween-20
  • Primer mix 4 has:
  • step 7 After preamplification from step 7 is finished. Add 2 ⁇ L of mixture from step 7 to thereaction containing the following: lx Q5U buffer dNTPs 0.4 mM
  • Primer mix 5 has:
  • Primer mix 6 has:
  • Step 2-4 repeated 30x -Take a picture of the partitions in green and yellow channels with exposure duration 600 and 700 ms respectively and gain 6 and 8 respectively. Data obtained from such an experiment is shown in FIG. 7.
  • Example 6 Enrichment factor for the EGFR exon 20 T790M variant.
  • Beads preparation a. Bead blocking step
  • ImL blocking buffer 0.1% Tween-20, Ipg/mLt-RNA
  • Buffer 1 IxSSC buffer (5x SSC, 5x Denhardt's solution, 5mM EDTA, 0.1% SDS) or
  • Buffer 2 ULTRAhybTM Ultrasensitive Hybridization Buffer (Thermo Fisher cat no. AM8670) or
  • Buffer 3 ULTRAhybTM-Oligo (Thermo Fisher cat no. AM8663) or
  • Buffer 4 IxSSC + 10% Formide buffer (5x SSC, 5x Denhardt's solution, 5mM EDTA, 0.1% SDS, 10% Formide) or
  • Buffer 5 IxSSC + 25% Formide buffer (5x SSC, 5x Denhardt's solution, 5mM EDTA, 0.1% SDS, 25% Formide) or Buffer 6: IxSSC + 48% Formide buffer (5x SSC, 5x Denhardt's solution, 5mM EDTA, 0.1% SDS, 48% Formide)
  • WT oligonucleotides mix 198/199.8 fM
  • WT oligonucleotides mix has SEQ ID NOs 22-37.
  • Mutant oligonucleotides mix has SEQ ID Nos 38-53.
  • PPL consists of the following composition: lxBFF6-0.1% Tween-20
  • Primer mix 4 consists of:
  • step 7 After preamplification from step 7 is finished. Add 2 ⁇ L of mixture from step 7 to the reaction containing the following: lx Q5U buffer dNTPs 0.4 mM
  • Primer mix 5 has:
  • Primer mix 6 has:

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IL307733A IL307733A (en) 2021-04-15 2022-04-15 Enrichment and discovery of nucleic acids
JP2023564030A JP7760607B2 (ja) 2021-04-15 2022-04-15 核酸の濃縮及び検出
KR1020237038978A KR102923285B1 (ko) 2021-04-15 2022-04-15 핵산 농축 및 검출
CN202280041767.1A CN117940581A (zh) 2021-04-15 2022-04-15 核酸的富集和检测
BR112023021384A BR112023021384A2 (pt) 2021-04-15 2022-04-15 Enriquecimento e detecção de ácidos nucleicos
EP22727973.4A EP4143334A1 (en) 2021-04-15 2022-04-15 Nucleic acid enrichment and detection
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