WO2018081666A1 - Procédés de comptage de molécules simples d'adn/arn - Google Patents

Procédés de comptage de molécules simples d'adn/arn Download PDF

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WO2018081666A1
WO2018081666A1 PCT/US2017/058912 US2017058912W WO2018081666A1 WO 2018081666 A1 WO2018081666 A1 WO 2018081666A1 US 2017058912 W US2017058912 W US 2017058912W WO 2018081666 A1 WO2018081666 A1 WO 2018081666A1
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stranded
polynucleotide
double
strand
circular
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PCT/US2017/058912
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English (en)
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Meihong Lin
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Silgentech Inc.
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Priority claimed from US15/358,076 external-priority patent/US20180100180A1/en
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Publication of WO2018081666A1 publication Critical patent/WO2018081666A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Definitions

  • the present invention relates to DNA and RNA identification and quantification.
  • Single molecule detection or digital counting technologies such as digital PCR and single molecule sequencing have great advantages over conventional methods for genetic mutation detection and quantification since the former can identify and quantitate genes at a single molecule resolution.
  • the present invention provides for methods of generating DNA
  • nanorings/nanoballs which may be used for counting DNA or RNA molecules at a single molecule resolution on different gene detection or sequencing platforms.
  • polynucleotide molecules is provided.
  • the method includes: (a) obtaining a plurality of double-stranded polynucleotide fragments from original polynucleotide molecules; (b) optionally, modifying the ends of the polynucleotide fragments obtained in (a) such that each of the double-stranded polynucleotide fragments has an overhang at the 3' end and a phosphate group at the 5' end of each strand; (c) ligating two single- stranded
  • polynucleotide adaptors or a double- stranded polynucleotide linker, to two ends of each of the double- stranded polynucleotide fragments obtained in (a) or (b), respectively, to form single- or double-stranded circular polynucleotide molecules; (d) optionally, linearly amplifying each of the single- or double-stranded circular polynucleotide molecules to form single- stranded nanoballs; and (e) identifying and quantifying the circular polynucleotide molecules obtained in (c), or the single- stranded nanoballs in (d) if (d) is performed, thereby obtaining a counting of the original polynucleotide molecules.
  • the original polynucleotide molecules can comprise DNA and/or RNA.
  • DNA molecules can be counted using the above outlined procedure.
  • double-stranded cDNA can be first synthesized using the RNA molecules as templates, followed by the fragmentation of the cDNA and the subsequent procedure.
  • the ligation in (c) comprises ligating a first single-stranded polynucleotide adaptor onto one of the two ends of each polynucleotide fragment, and ligating a second single-stranded polynucleotide adaptor to the other of the two ends of each polynucleotide fragment obtained in (a) or (b), to thereby form a single-stranded circular polynucleotide molecule.
  • first single-stranded polynucleotide adaptor and the second single-stranded polynucleotide adaptor have the same 3' end overhang, or the first single-stranded polynucleotide adaptor and the second single-stranded polynucleotide adaptor have different 3' end overhangs.
  • first and second single-stranded polynucleotide adaptors each comprise a 3' chain end on which a topoisomerase enzyme is covalently attached.
  • the amplification in (d) is performed and comprises performing rolling circle amplification using the single-stranded circular polynucleotide molecules obtained in (c) as templates to form single- stranded nanoballs, and performing the rolling circle amplification comprises using a primer complementary to a region of the first or the second single- stranded polynucleotide adaptor.
  • the amplification in (d) is performed and comprises performing rolling circle amplification using the single-stranded circular polynucleotide molecules obtained in (c) as templates, and one or multiple primers complementary to one or multiple regions of a strand of the double-stranded polynucleotide fragments obtained in (a) or (b) to form single-stranded nanoballs.
  • the ligation in (c) comprises ligating two ends of one double-stranded polynucleotide linker to two ends of each polynucleotide fragment obtained in (a) or (b), respectively, to form a double- stranded circular polynucleotide molecule.
  • the two ends of the double-stranded polynucleotide linker have the same 3' end overhang. In other embodiments, two ends of the double- stranded polynucleotide linker have different 3' end overhangs.
  • the ligation in (c) comprises using at least a first double-stranded polynucleotide linker and a second double-stranded polynucleotide linker, at least one 3' end overhang on one strand of the first double- stranded polynucleotide linker is different from at least one 3' end overhang on one strand of the second double- stranded polynucleotide linker.
  • the ligation in (c) comprises using a double-stranded polynucleotide linker that includes that has a phosphate group at the 5' end of both strands.
  • the ligation in (c) comprises using a double-stranded polynucleotide linker that includes one strand having a 5' end with a phosphate group and the other strand having a 5' end without a phosphate group, whereby the ligation produces a circular double-stranded polynucleotide molecule having a continuous circular strand and a nicked circular strand.
  • the amplification in (d) can comprise performing rolling circle amplification using the nicked circular strand as a primer to amplify the continuous circular strand as a template to form single-stranded nanoballs.
  • the amplification in (d) is performed and comprises performing rolling circle amplification using at least one strand of the double- stranded circular polynucleotide molecules obtained in (c) as templates to form single-stranded nanoballs, and performing the rolling circle amplification comprises using a primer complementary to a region of a strand of the double-stranded polynucleotide linker.
  • the amplification in (d) is performed and comprises performing rolling circle amplification using at least one strand of the double- stranded circular polynucleotide molecules obtained in (c) as templates to form single-stranded nanoballs, and performing the rolling circle amplification comprises using one or multiple primers complementary to one or multiple regions of a strand of the double- stranded polynucleotide fragments obtained in (a) or (b).
  • the method further comprises: prior to (d), preferentially digesting circular polynucleotide molecules containing methylated nucleotides, if present, in the double-stranded circular polynucleotide molecules, to thereby produce non-circular polynucleotide segments, and then removing the non-circular polynucleotide segments.
  • the identification and quantification in (e) can comprises hybridizing the circular polynucleotide molecules obtained in (c), or hybridizing the single-stranded molecules obtained in (d) if (d) is performed, to microarrays.
  • the hybridization can also be done against fluorescence dye-conjugated molecular beacons or scorpions.
  • the identification and quantification in (e) comprises sequencing the circular polynucleotide molecules obtained in (c), or sequencing the single-stranded nanoballs obtained in (d) if (d) is performed.
  • Figure 1 depicts an overview of a process for counting of polynucleotide molecules in accordance with some embodiments of the present invention.
  • Figures 2 and 3 each depict a flowchart for counting of polynucleotide molecules in accordance with some embodiments of the present invention.
  • FIGS 4 and 5 depict example processes for counting polynucleotide molecules in accordance with some embodiments of the present invention.
  • embodiments of the present invention provide methods to manipulate polynucleotides for obtaining single-molecule counting of the number of the
  • polynucleotides The disclosed methods are simple and low cost, have high sensitivity, specificity, and precision, allow a very small amount of input samples to be tested, and offer short sample to data turnaround time, and is therefore applicable to single gene detection or targeted multiplexing or genome-wide applications.
  • nucleotide refers to a subunit of a nucleic acid.
  • polynucleotide includes a single- stranded (ss) or double-stranded (ds) DNA and RNA.
  • ss single- stranded
  • ds double-stranded
  • cDNA is a type of DNA synthesized from an ss-RNA.
  • nucleotide analogue as used herein is in general a compound in which one or more of the three moieties of a nucleotide (a phosphate backbone, a pentose sugar, either ribose or deoxyribose, and one of four nucleobases) is modified, for example, by attachment of one or more substituents and/or by replacement of one or more of the skeletal atoms.
  • a nucleotide analogue functions in a manner similar or analogous to a naturally occurring nucleotide.
  • FIGS 1-3 illustrate an overview of a general process for counting single polynucleotide molecules according to some embodiments of the present invention.
  • a plurality of double- stranded polynucleotide fragments are obtained from original single- or double- stranded polynucleotide molecules.
  • the ends of the double-stranded polynucleotide fragments can be modified or repaired to incorporate phosphate groups and appropriate overhangs which improve efficiency for the subsequent ligation.
  • the process branches into two alternative strategies (a first strategy and a second strategy) based on how the polynucleotide fragments are made into circular polynucleotides. The details of the steps according to the first strategy and the second strategy are illustrated in Figure 2 and Figure 3, respectively.
  • Figures 4 and 5 depict example processes according to the first strategy and second strategy, respectively. As discussed above in connection with Figure 1, it is understood that not all steps illustrated in Figures 4 and 5 are needed, and different reagents may be used and different steps may be taken. Hereinbelow, detailed description of the processes and variations in the reagents used in the process as well as the steps performed in the processes are provided in conjunction with Figures 1-5.
  • each double- stranded polynucleotide fragment obtained at step 100 or 200 are ligated to two identical or different single-stranded polynucleotide adaptors each containing a 5' phosphate, thereby forming a single- stranded circular polynucleotide molecule.
  • the single- stranded circular polynucleotide molecules obtained from step 310 are identified and quantified on any suitable gene detection platform, from which the counting of the original double- stranded polynucleotide molecules can be obtained.
  • the single- stranded circular polynucleotide molecules are linearly amplified to form single- stranded polynucleotide nanoballs.
  • linear amplification it is meant the amplicon grows along the replication template in a linear fashion, rather than the template being replicated in multiple cycles of repeated heating and cooling which results in an exponential growth of the number of copies of the templates.
  • the thus obtained amplification products, the single- stranded nanoballs are then subject to a suitable gene detection platform for identification and quantification (at step 710), from which the counting of the original double-stranded polynucleotide molecules can be obtained.
  • methylated circular polynucleotides can be preferentially removed at step 510.
  • step 320 two ends of a double- stranded polynucleotide linker (each containing a 5' phosphate on each of the two strands as exemplified in Example 3) are ligated to the two ends of a single double-stranded polynucleotide fragment (obtained at step 100 or 200), respectively, to form a double-stranded circular polynucleotide molecule.
  • the double- stranded circular polynucleotide molecules obtained from step 320 are identified and quantified on any suitable gene detection platform, from which the counting of the original
  • double-stranded polynucleotide molecules can be obtained.
  • the double-stranded circular polynucleotide molecules are linearly amplified to form single-stranded polynucleotide nanoballs using one of the strands as amplification template, and a primer or multiple primers complementary to any region of the circular
  • amplification products the single-stranded nanoballs
  • a suitable gene detection platform for identification and quantification from which the counting of the original double-stranded polynucleotide molecules can be obtained.
  • methylated circular polynucleotides can be preferentially removed at step 520.
  • two ends of a double-stranded polynucleotide linker where only one of the two strands contains a 5' phosphate are ligated to link the two ends of one strand of the double- stranded polynucleotide fragment (obtained at step 100 or 200), and ligated to only one end of the other strand, to form a double-stranded circular polynucleotide molecule with one circular strand having complete continuity while the other strand contains a unlinked nick at the location where the 5' phosphate group is missing.
  • either the continuously circular strand or the nicked strand of the circular polynucleotide molecules obtained from step 320 are identified and quantified on any suitable gene detection platform, from which the counting of the original
  • double-stranded polynucleotide molecules can be obtained.
  • the continuously circular strand of the circular polynucleotide molecules is linearly amplified to form single-stranded polynucleotide nanoballs using the continuously circular strand (circle) as amplification template, and the other strand that has the nick as the primer.
  • the thus obtained amplification products, the single-stranded nanoballs are then subject to a suitable gene detection platform for identification and quantification (at step 720), from which the counting of the original double-stranded polynucleotide molecules can be obtained.
  • methylated circular polynucleotides can be preferentially removed at step 520.
  • Obtaining double-stranded polynucleotide fragments from original polynucleotide molecules at step 100 can be performed by cell lysis, DNA/RNA purification from cells or any tissues; for counting RNA, double-stranded cDNA synthesis based on the RNA as templates; DNA/cDNA fragmentation using physical disruption, enzyme digestion or chemical treatment which includes but is not limited to bioruptors, heating, CviRI, Maell, Alul, DNase I or any other enzymes, fractionation/isolation from any organism (e.g. human), or any sample originated or derived from an organism (e.g.
  • the obtained fragments may have blunted ends, or have an overhang of various nucleotides at the 3' end and a phosphate group at the 5' end of each strand.
  • Modifying the ends (or end repairs) of the polynucleotide fragments at step 200 may include addition of a phosphate at the 5' ends of both strands by using a kinase, and blunt ending the fragments with enzymes such as T4 DNA polymerase.
  • enzymes such as T4 DNA polymerase.
  • terminal unpaired nucleotides may be removed from DNA ends by using an enzyme with exonuclease activity, which hydrolyzes a terminal phosphodiester bond, thereby removing the overhang one base at a time.
  • DNA fragments with 5' overhangs may be blunted by filling in a recessed 3' terminus with DNA polymerase in the presence of dNTPs.
  • End removal or fill-in can be accomplished using a number of enzymes, including DNA Polymerase I Large (Klenow) Fragment (NEB #M0210), T4 DNA Polymerase (NEB #M0203) or Mung Bean Nuclease (NEB #M0250).
  • Overhangs having one or more nucleotide units created by restriction digestion may directly be used in the ligation.
  • overhang(s) can be added to the 3' ends of double- stranded DNA/cDNA fragments by KlenTaq/Taq DNA polymerase or any other enzymatic or chemical reaction (optional when TOPO ligation is used). As a result, each fragment may have the same or different overhangs on either end.
  • the end repair techniques of the polynucleotide fragments often result in polynucleotide fragments having a distribution of overhangs on either end (e.g., a fragment may have a dA overhang on the 3' end of both strands, a dA overhang on 3' end of one strand and a dG overhang on 3' end of the other strand, or a dG overhang on the 3' end of both strands).
  • the end repair described above is not performed, and the polynucleotide fragments obtained from the fragmentation of the original polynucleotides are directly ligated by single- stranded polynucleotide adaptors (as exemplified by 332 and 334 in Figure 4, self-annealed with or without loop structures) or double- stranded polynucleotide linkers which are further explained below.
  • both ends of each double- stranded polynucleotide fragment are ligated to a respective polynucleotide adaptor with the aid of T4 DNA ligase, topoisomerase (e.g. Vaccinia topoisomerase I), ampligase, E. coli ligase, or other suitable chemical linkage method to form single- stranded circular polynucleotides (also referred to as single- stranded dumbbells or nanorings).
  • topoisomerase e.g. Vaccinia topoisomerase I
  • ampligase e.g. Vaccinia topoisomerase I
  • E. coli ligase e.g. Vaccinia topoisomerase I
  • the turn may take the form of a loop structure consisting of unpaired nucleotides.
  • an adaptor does not have a loop structure.
  • An adaptor can include a barcode sequence for sample indexing and multiplexing of different samples, and/or include a sequence complimentary to a polynucleotide suitable for rolling circle amplification and sequencing.
  • the 3' end region of the adaptor may have an overhang composed of one or more nucleotides of the same or different (e.g., single or multiple dT or dC) or their analogues complementary to the overhangs of the
  • the two adaptors ligated on either end of one polynucleotide fragment can be the same or different.
  • the two adaptors can have identical sequences throughout, including the end overhangs, or different sequences elsewhere except the same end overhangs, or the same sequences elsewhere but different overhangs.
  • a single adaptor or a mixture of adaptors that have different overhangs complementary to the overhangs to those of the polynucleotide fragments may be used.
  • the adaptors used in the ligation can be a mixture of both adaptors having a dT overhang on the 3' end and adaptors having a dC overhang on the 3' end.
  • the 5' end of each adaptor can have a phosphate
  • the components of each adaptor can be any type of nucleotides or their analogues.
  • the bonds connecting the components of each adaptor can be phosphodiester bonds or any other type of bonds.
  • the adaptors can each include a 3' chain end overhang (such as dT or dC) on which a topoisomerase enzyme is covalently attached (see 334 in Figure 4). In this case, the ligation reaction may not need additional T4 NDA ligase or another ligase.
  • the single- stranded nanorings are processed by a suitable gene detection platform for identification and quantification, e.g., by hybridization to microarrays or by sequencing.
  • a suitable gene detection platform for identification and quantification e.g., by hybridization to microarrays or by sequencing.
  • the single- stranded nanorings can be hybridized to microarrays, the microarrays are washed, and scanned.
  • the single- stranded nanorings can be hybridized to fluorescence dye-conjugated molecular beacons or scorpions.
  • the single-stranded nanorings can be processed by a molecular counting platform including but not limited to next generation sequencing, real time PCR, and bead reader.
  • the amplification can be a rolling circle amplification in the presence or absence of one or more fluorescent DNA binding dyes, with one or more than one primer, such as a universal labeled/unlabelled
  • Each of such single- stranded DNA nanoball is composed of tandem repeats of ⁇ (adaptor)-(positive strand of the original
  • the polynucleotide fragments are ligated to two identical adaptors on either end, wherein the primers attach to both adaptors to initiate amplification at two different locations.
  • the two ends are ligated to different adaptors and a primer complimentary to only one of the adaptors is used in amplification, only one ball is produced on each nanoring template.
  • the amplification of step 610 can use a single primer complementary to a single region of a strand of a particular double-stranded polynucleotide fragment. In some embodiments, the amplification of step 610 can use a mixture of multiple primers each complementary to a single region of a strand of a particular double-stranded polynucleotide fragment to be analyzed/counted.
  • the amplification products, the single-stranded nanoballs are processed by a suitable gene detection platform for identification and quantification, e.g., by hybridization to microarrays or by sequencing.
  • a suitable gene detection platform for identification and quantification, e.g., by hybridization to microarrays or by sequencing.
  • the nanoballs can be hybridized to microarrays, the microarrays are washed, and scanned.
  • the nanoballs can be fed into a molecular counting platform including but not limited to next generation sequencing, real time PCR, and bead reader.
  • the two ends of a double- stranded DNA linker are respectively connected to the two ends of a double-stranded DNA fragment with T4 DNA ligase, topoisomerase (e.g. Vaccinia topoisomerase I), ampligase, E. coli ligase, or other suitable chemical linkage method to form double-stranded circular polynucleotides (also referred to as double-stranded nanorings).
  • T4 DNA ligase e.g. Vaccinia topoisomerase I
  • ampligase e.g. Vaccinia topoisomerase I
  • E. coli ligase E. coli ligase
  • the polynucleotide linker can include a 3' chain end overhang (such as dT or dC) on each strand on which a topoisomerase enzyme is covalently attached (see 352, 354), in which case the ligation reaction may not need additional T4 NDA ligase or another ligase.
  • the 3' overhangs of the linker can be single or multiple nucleotides and/or nucleotide analogues of any type that are complementary to the 3' overhangs of the double-stranded polynucleotide fragments to be counted.
  • the double-stranded DNA linker can have single dT or dC overhang at the 3' end of each strand, e.g., it can have a single dT overhang at the 3' end of each strand, a single dC overhang at the 3' end of each strand, or a single dC overhang at the 3' end of one strand and a single dT overhang at the 3' end of the other strand.
  • any of these double-stranded DNA linkers can be used alone or their mixtures can be used.
  • a double- stranded DNA linker can have a phosphate group at the 5' end of each strand, or a phosphate group at the 5' end of only one of the two strands.
  • the ligation product will be a double stranded circular polynucleotide molecule or nanoring with a nick at the ligation location where there is no phosphate group on the double- stranded DNA linker.
  • the nanoring thus formed will have one strand that is completely continuously circular and the other strand having a nick (or discontinuity) at the ligation site where a 5' phosphate group is missing.
  • the phosphate group can be introduced during oligonucleotide synthesis or any other approaches that are not specified here.
  • the double-stranded DNA linker can include two polynucleotide strands that are perfectly matched except the end overhangs (354 or 358), or two polynucleotide strands that include at least one secondary structure formed by a plurality of mismatched nucleotide residues on at least one strand (352 or 356).
  • the two ends of the polynucleotide linker can include an overhang of a single or multiple dC or dT on the 3' end on each strand (352, 354, 356, 358).
  • the components of each linker can be any type of nucleotides or their analogues.
  • the bonds connecting the components of each strand of the linker can be phosphodiester bonds or any other type of bonds.
  • the two ends of the polynucleotide linker can be each covalently attached with a topoisomerase enzyme (352 or 354).
  • the ligation reaction may not need additional T4 NDA ligase or another ligase.
  • the polynucleotide linker can include a barcode sequence for sample indexing multiplexing of different samples, and/or include a sequence on one of its strands that is complimentary to a polynucleotide suitable for rolling circle amplification and sequencing.
  • the double- stranded nanorings are processed by a suitable gene detection platform for identification and quantification, e.g., by hybridization to microarrays or by sequencing.
  • a suitable gene detection platform for identification and quantification, e.g., by hybridization to microarrays or by sequencing.
  • the double- stranded nanorings can be hybridized to microarrays, the microarrays are washed, and scanned.
  • the double- stranded nanorings can be hybridized to fluorescence dye-conjugated molecular beacons or scorpions.
  • the double-stranded nanorings can be processed by a molecular counting platform, which includes but is not limited to next generation sequencing, real time PCR, and bead reader.
  • the amplification can be a rolling circle amplification in the presence or absence of one or more fluorescent DNA binding dyes, with one or more than one primer, such as a universal labeled/unlabelled primer or multiplex (more than one) primers with secondary structures (664, 666) or without secondary structures (662), nucleotides and/or analogues thereof (such as dNTPs, ribonucleotides, locked nucleic acid (LNA), biotinylated dNTPs, dNTPs conjugated with various reporter dyes) and Phi29 or any other DNA polymerases (e.g., Bst) to form single-stranded DNA nanoballs.
  • this amplification can use one or multiple primers complementary to one or multiple regions of a strand of the
  • this amplification can use one or multiple primers complementary to one or multiple regions of a strand of the double-stranded polynucleotide fragments to be analyzed/counted.
  • the amplification can use the nicked strand as a primer, and the other strand as the template for amplification.
  • the amplification products, the single-stranded nanoballs are processed by a suitable gene detection platform for identification and quantification, e.g., by hybridization to microarrays or by sequencing.
  • the nanoballs can be hybridized to microarrays, the microarrays are washed, and scanned.
  • the nanoballs can be fed into a molecular counting platform including but not limited to next generation sequencing, real time PCR, and bead reader.
  • circular polynucleotide molecules with higher degree of methylation can be preferentially removed, e.g., by using methylation- sensitive enzyme(s) to break down the circular polynucleotide molecules, and remove the resulting non-circular polynucleotide segments, e.g., by using exonuclease(s).
  • the single-stranded circular polynucleotide molecules still retain a double- stranded configuration except at the two loop ends, and are therefore susceptible to the action of methylation- sensitive enzyme(s).
  • This step can be particularly important for gene analysis for a fetus by using circulating DNAs in the blood of the pregnant mother bearing the fetus, because the mother's DNA has higher degree/frequency of methylation, and therefore removal of at least portions of the mother's DNA from the sample can improve the efficiency of the detection.
  • the primer sequence(s) for the rolling circle amplification can have any suitable adaptor sequence(s) at the 5' end of the primer for sample indexing and molecule counting (e.g. sequencing) on platform including but not limited to next generation sequencer.
  • the primers can be composed of units including but not limited to deoxyribonucleotide, ribonucleotide, Locked Nucleic Acid (LNA), and any of their modified analogues.
  • the DNA-binding reporter dyes include but not limited to SYBR Green, Propidium iodide, YO-PRO-1, and TOTO-3, or molecular beacon or scorpion probes with different reporter dye and quencher combination for DNA identification and counting on platforms including but not limited to microarrays, flow cytometers, bead counters, and droplet readers. Incorporation of biotin-conjugated dNTP into the nanoballs can allow the nanoballs to be visualized with reagents such as streptavidin-conjugated antibodies and SAPE.
  • the single- stranded or double- stranded nanorings may be sequenced in a rolling circle manner for sequencing error correction on single molecule sequencing platforms from companies including but not limited to Pacific Biosciences, or a nanopore-based platform offered by Genia, where a protein pore embedded in a lipid bilayer membrane constitute the nanopore.
  • the nanoballs can be sequenced on the platforms including but not limited to MinlON from Oxford Nanopore, and the instruments from other companies including but not limited to Pacific Biosciences and BGI, where one nanoball can be completely sequenced in a nanopore or a nanoring can be sequenced repeatedly in a rolling circle manner for sequencing error correction.
  • the existence of the tandem repeat sequences of fragments in the nanoball allows multiple scan of the same sequence, thereby correcting reading errors by correlating the results from the multiple scans.
  • Example 1 Creation of a genome- wide single-stranded DNA nanoring library by ligating mixtures of single-stranded adaptors onto both ends of each genomic DNA fragment of the genome
  • a PCR tube add 10 ng end-repaired human genomic DNA fragments (200 - 500 bp in length) each containing a 5' phosphate and a single 3' dA or dG overhang, 2 ⁇ T4 DNA Ligase Buffer (10X), 10 ng single-stranded adaptor oligonucleotide 1 (SEQ ID No. 1) and 10 ng single-stranded adaptor oligonucleotide 2 (SEQ ID No. 2) and 1 ⁇ T4 DNA Ligase. Bring the total volume to 20 ⁇ with Nuclease-free water. Incubate the reaction at room temperature for 10 minutes. Heat inactivate at 65 °C for 10 minutes.
  • SEQ ID No. 1 Single-stranded adaptor oligonucleotide 1
  • SEQ ID No. 2 Single-stranded adaptor oligonucleotide 2
  • Example 2 Creation of a genome- wide double-stranded DNA nanoring library (with a nick on one of the two strands of each nanoring) by ligating mixtures of
  • double-stranded polynucleotide linkers having a hairpin secondary structure to connect both ends of one strand and one end of the other strand of each genomic DNA fragment
  • a PCR tube add 10 ng end-repaired human genomic DNA fragments (200 - 500 bp in length) each containing a 5' phosphate and a single 3' dA or dG overhang, 2 ⁇ T4
  • Double-stranded polynucleotide linker having a hairpin secondary structure in one strand
  • Both linker LI and linker L2 have two double-stranded arms formed by paired
  • Linker LI has a single dT overhang at the 3' end of each strand
  • linker L2 has a single dC overhang at the 3' end of each strand.
  • linker LI and linker L2 each have a phosphate group on a 5' end of its lower strand, and the 5' end of its upper strand does not have a phosphate group.
  • Such a structure results in the ligation product (circular double- stranded polynucleotide or nanoring) having one strand with complete continuity throughout the circle or nanoring and the other strand having a discontinuity or nick at the position of the ligation where the phosphate group is missing.
  • Example 3 Creation of a genome- wide double-stranded DNA nanoring library by ligating mixtures of double-stranded adaptors to connect both ends of each genomic
  • DNA Ligase Buffer (10X), 10 ng double-stranded polynucleotide linker having a hairpin secondary structure, L3 (composed of SEQ ID Nos. 7 and 8) and 10 ng Double- stranded polynucleotide linker having a hairpin secondary structure, L4 (composed of SEQ ID Nos. 9 and 10) and 1 ⁇ T4 DNA Ligase. Bring the total volume to 20 ⁇ with Nuclease-free water. Incubate the reaction at room temperature for 10 minutes. Heat inactivate at 65 °C for 10 minutes.
  • Double-stranded polynucleotide linker L3 (with a hairpin secondary structure in one
  • Double-stranded polynucleotide linker L4 (with a hairpin secondary structure
  • both linker L3 and linker L4 have two double- stranded arms formed by paired nucleotides, and a hairpin secondary structure formed by a segment of polynucleotides in the middle of the lower strand.
  • Linker L3 has a single dT overhang at the 3' end of each strand
  • linker L4 has a single dC overhang at the 3' end of each strand.
  • linker L3 and linker L4 each have a phosphate group on a 5' end of each of the strands.
  • Example 4 Creation of a genome- wide single-stranded DNA nanoball library by rolling circle amplification using the genome-wide single-stranded nanoring library generated in Example 1 and a polynucleotide primer
  • SEQ ID No. 11 Oligonucleotide used as the primer for rolling circle amplification 5 X GAGCGTCGTGTAGGGAAAGAGT3 '
  • Example 5 Creation of a genome- wide single-stranded DNA nanoball library by rolling circle amplification using the genome-wide single-stranded nanoring library generated in Example 2 and nicked complementary strand of the nanorings as primers
  • the nanorings in the nanoring library in Example 2 include a nicked structure in the circular double-stranded DNA nanoring.
  • the rolling circle amplification can use the strand that does not have the nick as a template for amplification.
  • the nicked strand can be used as a primer for such amplification.
  • Example 6 Creation of a genome- wide single-stranded DNA nanoball library by rolling circle amplification using the genome-wide double-stranded nanoring library generated in Example 3 and a polynucleotide primer.
  • SEQ ID No. 12 Oligonucleotide used as a primer for rolling circle amplification
  • Example 7 Preferential removal of methylated nanorings from the genome-wide nanoring libraries generated in Examples 1 through 3

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Abstract

L'invention concerne un procédé de comptage de molécules polynucléotidiques simple brin ou double brin. Une pluralité de fragments polynucléotidiques double brin est obtenue à partir de molécules initiales polynucléotidiques simple brin ou double brin. Les extrémités des fragments polynucléotidiques peuvent être modifiées pour présenter des parties qui dépassent, appropriées pour la ligature. Deux adaptateurs simple brin ou un lieur polynucléotidique double brin sont ligaturés aux deux extrémités de chacun des fragments polynucléotidiques double brin pour former des molécules polynucléotidiques circulaires simple ou double brin, qui peuvent être amplifiées de manière linéaire pour former des nanobilles simple brin. Les molécules polynucléotidiques circulaires avant l'amplification, ou les nanobilles simple brin obtenues dans l'amplification, peuvent être identifiées et quantifiées sur une plate-forme de détection de gène, ce qui permet d'obtenir un comptage des molécules initiales polynucléotidiques simple ou double brin.
PCT/US2017/058912 2016-10-28 2017-10-28 Procédés de comptage de molécules simples d'adn/arn WO2018081666A1 (fr)

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US15/358,076 US20180100180A1 (en) 2016-01-05 2016-11-21 Methods of single dna/rna molecule counting
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Publication number Priority date Publication date Assignee Title
WO2022141061A1 (fr) * 2020-12-29 2022-07-07 深圳华大智造科技股份有限公司 Procédé de construction rapide de bibliothèque cyclisée et de lieur formant un cycle
WO2023050968A1 (fr) * 2021-09-30 2023-04-06 深圳华大智造科技股份有限公司 Lieur d'adn double brin pour la préparation d'une nanobille d'adn et procédé de préparation à cet effet, un kit et ses utilisations.

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US20090203551A1 (en) * 2007-11-05 2009-08-13 Complete Genomics, Inc. Methods and Oligonucleotide Designs for Insertion of Multiple Adaptors Employing Selective Methylation
WO2015157747A1 (fr) * 2014-04-11 2015-10-15 Redvault Biosciences Lp Systemes et procedes de replication clonale et d'amplification de molecules d'acide nucleique pour des applications genomiques et therapeutiques
US20160194686A1 (en) * 2006-10-27 2016-07-07 Complete Genomics, Inc. Efficient Arrays of Amplified Polynucleotides
US20160265031A1 (en) * 2015-03-13 2016-09-15 Life Technologies Corporation Methods, compositions and kits for small rna capture, detection and quantification

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US20070072297A1 (en) * 2000-02-25 2007-03-29 Stratagene California Method for ligating nucleic acids and molecular cloning
US20160194686A1 (en) * 2006-10-27 2016-07-07 Complete Genomics, Inc. Efficient Arrays of Amplified Polynucleotides
US20090203551A1 (en) * 2007-11-05 2009-08-13 Complete Genomics, Inc. Methods and Oligonucleotide Designs for Insertion of Multiple Adaptors Employing Selective Methylation
WO2015157747A1 (fr) * 2014-04-11 2015-10-15 Redvault Biosciences Lp Systemes et procedes de replication clonale et d'amplification de molecules d'acide nucleique pour des applications genomiques et therapeutiques
US20160265031A1 (en) * 2015-03-13 2016-09-15 Life Technologies Corporation Methods, compositions and kits for small rna capture, detection and quantification

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022141061A1 (fr) * 2020-12-29 2022-07-07 深圳华大智造科技股份有限公司 Procédé de construction rapide de bibliothèque cyclisée et de lieur formant un cycle
WO2023050968A1 (fr) * 2021-09-30 2023-04-06 深圳华大智造科技股份有限公司 Lieur d'adn double brin pour la préparation d'une nanobille d'adn et procédé de préparation à cet effet, un kit et ses utilisations.

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