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WO2021168097A1 - Procédés et systèmes de traitement d'acides nucléiques - Google Patents

Procédés et systèmes de traitement d'acides nucléiques Download PDF

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Publication number
WO2021168097A1
WO2021168097A1 PCT/US2021/018558 US2021018558W WO2021168097A1 WO 2021168097 A1 WO2021168097 A1 WO 2021168097A1 US 2021018558 W US2021018558 W US 2021018558W WO 2021168097 A1 WO2021168097 A1 WO 2021168097A1
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Prior art keywords
nucleic acid
target nucleic
acid molecule
acid molecules
reaction site
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Seth Stern
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Genapsys Inc
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Genapsys Inc
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Priority to EP21757375.7A priority Critical patent/EP4107286A4/fr
Publication of WO2021168097A1 publication Critical patent/WO2021168097A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Definitions

  • DNA amplification is an indispensable tool in a variety of genetic analysis. Amplification of DNA from small quantities is critical in collection of DNA from a crime scene, archeological analysis, identification of genes of interest, and in medical diagnostics. The preparation of the genetic material for testing and subsequent analysis in such a manner to capture an entire genome efficiently and maintain its integrity is a challenging and critical limitation in any genetic methodology. Further, many fundamental problems in these fields center around the inability to decrease indefinitely the time required to process a single sample. One way of increasing throughput is to perform many processes in parallel with simultaneous amplification of cloned fragments in high density arrays.
  • An aspect of the present disclosure comprises a method for nucleic acid amplification: (a) contacting an array of reaction sites with a solution comprising a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules different from target nucleic acid molecules of said plurality of target nucleic acid molecules, such that a reaction site of said array of reaction sites comprises a target nucleic acid molecule of said plurality of target nucleic acid molecules and a non-target nucleic acid molecule of said plurality of non-target nucleic acid molecules, wherein said target nucleic acid molecule and said non-target nucleic acid molecule are immobilized at said reaction site, and wherein said target nucleic acid molecules and said non target nucleic acid molecules are present in said solution at a first ratio of target nucleic acid molecules to non-target nucleic acid molecules of at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1
  • (b) comprises amplifying said non-target nucleic acid molecule to generate a copy or complement of said non target nucleic acid molecule immobilized at said reaction site.
  • a second ratio of amplicons of said target nucleic acid molecule immobilized at said reaction site to said copy or complement of said non-target nucleic acid molecule immobilized at said reaction site is at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or l:100 or at about least 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7: 1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
  • said reaction site comprises a plurality of oligonucleotides immobilized thereon. In some embodiments, at least a portion of said target nucleic acid molecule is hybridized with a sequence of an oligonucleotide of said plurality of oligonucleotides. In some embodiments, in (b), said amplifying comprises conducting a nucleic acid extension reaction using said oligonucleotide and said target nucleic acid molecule. In some embodiments, said plurality of oligonucleotides comprises at least about 2 oligonucleotides. In some embodiments, in (b), said amplicons of said target nucleic acid molecule are at least partially complementary to said target nucleic acid molecule.
  • said amplicons of said target nucleic acid molecule are a clonal population of nucleic acids.
  • said amplifying is performed with the aid of an enzyme.
  • said enzyme is a recombinase.
  • said enzyme is a polymerase.
  • said contact said array of reaction sites with said solution is performed via flow of said solution.
  • (b) occurs contemporaneous with flow of said solution.
  • (b) occurs absent flow of said solution.
  • said reaction site comprises a particle. In some embodiments, said reaction site is planar.
  • said reaction site comprises a sensor configured to detect a signal indicative of a reaction associated with said target nucleic acid molecule or said amplicons of said target nucleic acid molecule.
  • said sensor is an electronic sensor.
  • the method further comprises sequencing said amplicons of said target nucleic acid molecule.
  • said sequencing is performed via sequencing-by-synthesis.
  • said sequencing is performed by detecting a signal indicative of an impedance, an impedance change, a conductivity, a conductivity change, a charge or a charge change.
  • one target nucleic acid molecule of said plurality of target nucleic acid molecules is immobilized at said reaction site.
  • an additional target nucleic acid molecule of said plurality of target nucleic acid molecules is immobilized at another reaction site of said array of reaction sites.
  • the method further comprises repeating (b) for said additional target nucleic acid molecule of said plurality of target nucleic acid molecules.
  • Another aspect the present disclosure comprises a method for nucleic acid amplification involving (a) contacting an array of reaction sites with a flowing solution comprising a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules different from target nucleic acid molecules of said plurality of target nucleic acid molecules, such that a reaction site of said array of reaction sites comprises a target nucleic acid molecule of said plurality of target nucleic acid molecules and a non-target nucleic acid molecule of said plurality of non-target nucleic acid molecules, wherein said target nucleic acid molecule and said non-target nucleic acid molecule are immobilized at said reaction site, and wherein a flow rate of said flowing solution is at most 20 pL/min; and (b) at said reaction site, amplifying said target nucleic acid molecule to generate amplicons of said target nucleic acid molecule immobilized at said reaction site, wherein said amplifying occurs at a rate sufficient to generate said amplicons
  • a second ratio of amplicons of said target nucleic acid molecule immobilized at said reaction site to said copy or complement of said non-target nucleic acid molecule immobilized at said reaction site is at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
  • said reaction site comprises a plurality of oligonucleotides immobilized thereon.
  • (b) comprises amplifying said non-target nucleic acid molecule to generate a copy or complement of said non-target nucleic acid molecule immobilized at said reaction site.
  • said target nucleic acid molecules and said non-target nucleic acid molecules are present in said flowing solution at a first ratio of target nucleic acid molecules to non-target nucleic acid molecules of at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or at least about 1: 1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
  • a second ratio of amplicons of said target nucleic acid molecule immobilized at said reaction site to said copy or complement of said non-target nucleic acid molecule immobilized at said reaction site is at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
  • said reaction site comprises a plurality of oligonucleotides immobilized thereon. In some embodiments at least a portion of said target nucleic acid molecule is hybridized with a sequence of an oligonucleotide of said plurality of oligonucleotides.
  • said amplifying comprises conducting a nucleic acid extension reaction using said oligonucleotide and said target nucleic acid molecule. In some embodiments, said plurality of oligonucleotides comprises at least about 2 oligonucleotides. In some embodiments in (b), said amplicons of said target nucleic acid molecule are at least partially complementary to said target nucleic acid molecule.
  • said amplicons of said target nucleic acid molecule are a clonal population of nucleic acids.
  • said amplifying is performed with the aid of an enzyme.
  • said enzyme is a recombinase.
  • said enzyme is a polymerase.
  • said reaction site comprises a particle.
  • said reaction site is planar.
  • said reaction site comprises a sensor configured to detect a signal indicative of a reaction associated with said target nucleic acid molecule or said amplicons of said target nucleic acid molecule.
  • said sensor is an electronic sensor. In some embodiments further comprising sequencing said amplicons of said target nucleic acid molecule.
  • said sequencing is performed via sequencing-by-synthesis. In some embodiments said sequencing is performed by detecting a signal indicative of an impedance, an impedance change, a conductivity, a conductivity change, a charge or a charge change. In some embodiments wherein one target nucleic acid molecule of said plurality of target nucleic acid molecules is immobilized at said reaction site. In some embodiments wherein an additional target nucleic acid molecule of said plurality of target nucleic acid molecules is immobilized at another reaction site of said array of reaction sites. In some embodiments further comprising repeating (b) for said additional target nucleic acid molecule of said plurality of target nucleic acid molecules. In some embodiments (b) occurs contemporaneous with flow of said solution. In some embodiments (b) occurs absent flow of said solution.
  • Another aspect of the present disclosure comprises a method for nucleic acid amplification: (a) contacting an array of reaction sites with a solution comprising a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules different from target nucleic acid molecules of said plurality of target nucleic acid molecules, such that a reaction site of said array of reaction sites comprises a target nucleic acid molecule of said plurality of target nucleic acid molecules and a non-target nucleic acid molecule of said plurality of non-target nucleic acid molecules, wherein said target nucleic acid molecule and said non-target nucleic acid molecule are immobilized at said reaction site, and wherein said reaction site comprises an electronic sensor; and (b) at said reaction site, amplifying said target nucleic acid molecule to generate amplicons of said target nucleic acid molecule immobilized at said reaction site, wherein said amplifying occurs at a rate sufficient to generate said amplicons of said target nucleic acid molecule without
  • (b) comprises amplifying said non-target nucleic acid molecule to generate a copy or complement of said non-target nucleic acid molecule immobilized at said reaction site.
  • said target nucleic acid molecules and said non-target nucleic acid molecules are present in said solution at a first ratio of target nucleic acid molecules to non-target nucleic acid molecules of at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90: 1, or 100:1.
  • a second ratio of amplicons of said target nucleic acid molecule immobilized at said reaction site to said copy or complement of said non-target nucleic acid molecule immobilized at said reaction site is at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
  • said reaction site comprises a plurality of oligonucleotides immobilized thereon. In some embodiments at least a portion of said target nucleic acid molecule is hybridized with a sequence of an oligonucleotide of said plurality of oligonucleotides. In some embodiments said amplifying comprises conducting a nucleic acid extension reaction using said oligonucleotide and said target nucleic acid molecule. In some embodiments said plurality of oligonucleotides comprises at least about 2 oligonucleotides. In some embodiments said amplicons of said target nucleic acid molecule are at least partially complementary to said target nucleic acid molecule.
  • said amplicons of said target nucleic acid molecule are a clonal population of nucleic acids.
  • said amplifying is performed with the aid of an enzyme.
  • said enzyme is a recombinase.
  • said enzyme is a polymerase.
  • said reaction site comprises a particle.
  • said reaction site is planar.
  • said electronic sensor is configured to detect a signal indicative of a reaction associated with said target nucleic acid molecule or said amplicons of said target nucleic acid molecule.
  • said sequencing is performed via sequencing-by-synthesis.
  • said sequencing is performed by using said electronic sensor to detect a signal indicative of an impedance, an impedance change, a conductivity, a conductivity change, a charge or a charge change.
  • one target nucleic acid molecule of said plurality of target nucleic acid molecules is immobilized at said reaction site.
  • an additional target nucleic acid molecule of said plurality of target nucleic acid molecules is immobilized at another reaction site of said array of reaction sites.
  • said contact said array of reaction sites with said solution is performed via fluid flow of said solution.
  • said electronic sensor comprises at least one electrode. In some embodiments said electronic sensor comprises a pair of electrodes. In some embodiments at least one electrode is within a Debye length of a surface said reaction site or said target nucleic acid molecule.
  • Another aspect of the disclosure described herein comprises a method for nucleic acid amplification comprising (a) contacting an array of reaction sites with a solution comprising a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules different from target nucleic acid molecules of said plurality of target nucleic acid molecules, such that a reaction site of said array of reaction sites comprises a target nucleic acid molecule of said plurality of target nucleic acid molecules and a non-target nucleic acid molecule of said plurality of non-target nucleic acid molecules, and wherein said target nucleic acid molecule and said non target nucleic acid molecule are immobilized at said reaction site; and (b) at said reaction site, amplifying said target nucleic acid molecule to generate amplicons of said target nucleic acid molecule immobilized at said reaction site, wherein said amplifying occurs at a rate sufficient to generate said amplicons of said target nucleic acid molecule without amplification of other target nucleic
  • (b) comprises amplifying said non target nucleic acid molecule to generate a copy or complement of said non-target nucleic acid molecule immobilized at said reaction site.
  • said target nucleic acid molecules and said non-target nucleic acid molecules are present in said solution at a first ratio of target nucleic acid molecules to non-target nucleic acid molecules of at most 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
  • a second ratio of amplicons of said target nucleic acid molecule immobilized at said reaction site to said copy or complement of said non-target nucleic acid molecule immobilized at said reaction site is at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or atleast about 1: 1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45: 1, 50: 1, 60:1, 70:1, 80:1, 90:1, or 100:1.
  • said reaction site comprises a plurality of oligonucleotides immobilized thereon. In some embodiments at least a portion of said target nucleic acid molecule is hybridized with a sequence of an oligonucleotide of said plurality of oligonucleotides. In some embodiments said amplifying comprises conducting a nucleic acid extension reaction using said oligonucleotide and said target nucleic acid molecule. In some embodiments said plurality of oligonucleotides comprises at least about 2 oligonucleotides. In some embodiments said amplicons of said target nucleic acid molecule are at least partially complementary to said target nucleic acid molecule.
  • said amplicons of said target nucleic acid molecule are a clonal population of nucleic acids.
  • said amplifying is performed with the aid of an enzyme.
  • said enzyme is a recombinase.
  • said enzyme is a polymerase.
  • said reaction site comprises a particle.
  • said reaction site is planar.
  • said reaction site comprises a sensor configured to detect a signal indicative of a reaction associated with said target nucleic acid molecule or said amplicons of said target nucleic acid molecule.
  • said sensor is an electronic sensor.
  • said sequencing is performed via sequencing-by-synthesis.
  • said sequencing is performed by detecting a signal indicative of an impedance, an impedance change, a conductivity, a conductivity change, a charge or a charge change.
  • one target nucleic acid molecule of said plurality of target nucleic acid molecules is immobilized at said reaction site.
  • an additional target nucleic acid molecule of said plurality of target nucleic acid molecules is immobilized at another reaction site of said array of reaction sites.
  • the method comprises repeating (b) for said additional target nucleic acid molecule of said plurality of target nucleic acid molecules.
  • said contact said array of reaction sites with said solution is performed via fluid flow of said solution.
  • (b) occurs contemporaneous with flow of said solution.
  • (b) occurs absent flow of said solution.
  • Another aspect of the present disclosure comprises of a method of amplifying nucleic acids, comprising: (a) contacting an array of reaction sites with a solution comprising a plurality of nucleic acid molecules, wherein said plurality of nucleic acid molecules comprises at least a first subset of nucleic acid molecules and a second subset of nucleic acid molecules, and wherein a reaction site of said array binds at least one first nucleic acid molecule from said first subset of nucleic acid molecules and at least one second nucleic acid molecule from said second subset of nucleic acid molecules; and (b) subjecting said reaction site to conditions sufficient to amplify (i) said at least one first nucleic acid molecule, (ii) said at least one second nucleic acid molecule, or (iii) said at least one first nucleic acid molecule and said at least one second nucleic acid molecule, to generate a population of amplification products at said reaction site, which population of amplification products corresponds to said at least
  • said first subset of nucleic acid molecules and said second subset of nucleic acid molecules are present in said population of amplification products at a ratio of first subset of nucleic acid molecules to second subset of nucleic acid molecules of at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or at least about 1: 1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
  • said array binds at least one first nucleic acid molecule from said first subset of nucleic acid molecules and at least one second nucleic acid molecule from said second subset of nucleic acid molecules at a ratio of first subset of nucleic acid molecules to second subset of nucleic acid molecules of at most about 1:1, 1:2, 1 :3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
  • said array comprises a plurality of electrically or magnetically immobilized probes.
  • at least one nucleic acid molecule of said plurality of nucleic acid molecules comprises an adapter sequence.
  • at least one nucleic acid molecule of said plurality of nucleic acid molecules comprises a primer sequence.
  • at least one nucleic acid molecule of said first subset of nucleic acid molecules comprises a first primer sequence and at least one nucleic acid molecule of said second subset of nucleic acid molecules comprises a second primer sequence.
  • said population of amplification products is generated by a nucleic acid extension reaction.
  • said population of amplification products corresponds to said at least one first nucleic acid molecule and said at least one second nucleic acid molecule, and said population of amplification products corresponding to said at least one first nucleic acid molecule or said population of amplification products corresponding to said at least one second nucleic acid molecule is blocked from a nucleic acid extension reaction.
  • at least one of said population of amplification products corresponding to said at least one first nucleic acid molecule or said population of amplification products corresponding to said at least one second nucleic acid molecule comprises a blocking sequence thereby blocking said nucleic acid extension reaction.
  • said immobilized probes comprise an oligonucleotide(s).
  • said array of reaction sites is among a plurality of arrays of reaction sites.
  • the method further comprises repeating (a) - (b) at another array of said reaction sites.
  • FIG. 1 illustrates a computer system that is programmed or otherwise configured to implement methods provided herein.
  • FIG. 2 illustrates a workflow for nucleic acid processing.
  • FIG. 3 illustrates a workflow for blocking of amplification of non-target sequences using non-target nucleic acid molecule(s).
  • nucleic acid generally refers to a polymeric form of nucleotides of any length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 500, 1000 or more nucleotides), either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a nucleic acid may include one or more subunits selected from adenosine (A), cytosine (C), guanine (G), thymine (TO, and uracil (U), or variants thereof.
  • a nucleotide can include A, C, G, T, or U, or variants thereof.
  • a nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be A, C, G, T, or U, or any other subunit that is specific to one of more complementary A, C, G, T, or U, or complementary to a purine (e.g., A or G, or variant thereof) or pyrimidine (e.g., C, T, or U, or variant thereof).
  • a nucleic acid may be single-stranded or double stranded, in some cases, a nucleic acid molecule is circular.
  • Non-limiting examples of nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • Nucleic acids can include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a nucleic acid molecule may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • oligonucleotide generally refers to a nucleic acid molecule comprising at least one nucleotide that may have various lengths such as either deoxyribonucleotides or ribonucleotides or analogs thereof.
  • An oligonucleotide may comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000, 50,000, 100,000 or more nucleotides.
  • An oligonucleotide may comprise at most about 100,000, 50,000, 10,000, 5,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less nucleotides.
  • An oligonucleotide may be unbound (e.g., in solution) or bound (e.g., chemically bonded to a substrate).
  • Oligonucleotides may include one or more nonstandard nucleotide(s), nucleotide analog(s), modified nucleotides, or any combination thereof.
  • target sequence generally refers to a particular nucleotide sequence.
  • target nucleic acid molecule generally refers to a nucleic acid molecule comprising a target sequence and serves as a template for the nucleic acid amplification methods and systems described herein.
  • non-target nucleic acid molecule generally refers to a nucleic acid molecule that is not the target sequence(s) for the nucleic acid amplification methods and systems described herein.
  • the term “poisoned sequence”, as used herein, generally refers to a non-target nucleic acid molecule(s) that is targeted to the array, immobilized, and copies generated alongside the target nucleic acid molecule(s). Amplicons of the target sequence may be immobilized to a copy of the non-target nucleic acid molecule at the reaction site. The poisoned amplification products may be blocked through an adapter sequence from amplifying alongside the target sequence.
  • active oligonucleotide generally refers to a nucleic acid molecule comprising at least one nucleotide that may have various lengths such as either deoxyribonucleotides or ribonucleotides or analogs thereof that is targeted to a non-poisoned sequence.
  • polymerase generally refers to any enzyme capable of catalyzing a polymerization reaction.
  • examples of polymerases include, without limitation, a nucleic acid polymerase.
  • the polymerase can be naturally occurring or synthesized.
  • a polymerase can be a polymerization enzyme.
  • a transcriptase or a ligase is used (e.g. enzymes which catalyze the formation of a bond).
  • Examples of polymerases include but are not limited to DNA polymerase, RNA polymerase, a thermostable polymerase, a wild-type polymerase, a modified polymerase, Taq polymerase, and variants, modified products, and derivatives thereof.
  • the polymerase is a single subunit polymerase.
  • the polymerase can have high processivity, namely the capability of the polymerase to consecutively incorporate nucleotides into a nucleic acid template without releasing the nucleic acid template.
  • DNase as used herein, generally refers to an enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, degrading DNA.
  • RNase as used herein generally refers to an enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the RNA backbone, degrading RNA.
  • the term “probe”, as used herein, generally refers to a first moiety configured to bind to a second moiety the presence or absence of which may be used to detect the presence or absence of an amplified product.
  • the first moiety may be an antibody, a ligand (e.g., a small molecule ligand), an oligonucleotide at least partially complimentary to an oligonucleotide of the second moiety, or any combination thereof.
  • detectable moieties may include radiolabels, stable isotope labels, fluorescent labels, chemiluminescent labels, enzymatic labels, colorimetric labels, or any combination thereof.
  • sample generally refers to any sample containing or suspected of containing a nucleic acid molecule.
  • a sample can be a biological sample containing one or more nucleic acid molecules.
  • the biological sample can be obtained (e.g., extracted or isolated) from or include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears.
  • the biological sample can be a fluid or tissue sample (e.g., skin sample).
  • the sample is obtained from a cell-free bodily fluid, such as whole blood.
  • the sample may include cell-free DNA and/or cell-free RNA.
  • the sample can include circulating tumor cells.
  • the sample is an environmental sample (e.g., soil, waste, ambient air and etc.), industrial sample (e.g., samples from any industrial processes), and food samples (e.g., dairy products, vegetable products, and meat products).
  • the sample may be processed prior to loading into the microfluidic device.
  • the sample may be processed to lyse cells, purify the nucleic acid molecules, and/or to include reagents.
  • template generally refers to a complementary, non-coding single strand of nucleic acid that is used by DNA polymerase enzyme as a basis of copying DNA.
  • hybridization generally refers to the phenomenon in which single stranded nucleic acids anneal to complementary DNA or RNA.
  • Electrode generally refers to an electric conductor that carries electric current into non-metallic solids, liquids, gases, plasmas, or vacuums.
  • An electrical conductor may be but is not always a solid.
  • the electrode from which electrons emerge is the cathode and is designated negative while the electrode that receives electrons is the positively designated anode.
  • a substrate used for an array can include a layer of transparent electrical conductor.
  • the layer of electrical conductor may be used as an electrode to connect an electrical source such as a battery or a signal generator.
  • a voltage across the conductive layers can be used to manipulate the force on a nucleic acid and/or amplification reagent to control the rate of transport to the site, capture at the site, removal from the site, amplification at the site, or any combination thereof.
  • an electric field can be applied on the outer surfaces of a well such that the electric field that penetrates the vessel walls induces an electrical force on reagents within the vessel, providing a degree of control over the rates of transport, capture, removal, amplification, or any combination thereof.
  • kinetic exclusion generally refers to when a process occurs a sufficiently rapid rate to effectively exclude another event or process from occurring.
  • the seeding and amplification can proceed simultaneously under conditions where the amplification rate exceeds the seeding rate. In such a condition the rapid rate at which copies are made at a site seeded by a first nucleic acid will effectively exclude a second nucleic acid from seeding the site for amplification.
  • amplification and “amplify” may be used interchangeably and generally refer to generating one or more copies or “amplified product” of a nucleic acid. Such amplification may be using polymerase chain reaction (PCR) or isothermal amplification, for example.
  • PCR polymerase chain reaction
  • amplicon generally refers to the product of copying the nucleic acid.
  • the product may have a nucleotide sequence that is the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid.
  • An amplicon can be produced by any of a variety of amplification methods that use the nucleic acid, or an amplicon thereof, as a template including, for example, polymerase extension, polymerase chain reaction, rolling circle amplification, ligation extension, or ligation chain reaction.
  • An amplicon can be a nucleic acid molecule having a single copy of a particular nucleotide sequence or multiple copies of the nucleotide sequence.
  • a first amplicon of a target nucleic acid is may be a complementary copy.
  • Subsequent amplicons are copies that are created, after generation of the first amplicon, from the target nucleic acid or from the first amplicon.
  • a subsequent amplicon can have a sequence that is substantially complementary to the target nucleic acid or substantially identical to the target nucleic acid.
  • amplification site generally refers to a site in or on an array where one or more amplicons can be generated.
  • An amplification site can be further configured to contain, hold or attach at least one amplicon that is generated at the site.
  • the term "array” refers to a population of sites that can be differentiated from each other according to relative location. Different molecules that are at different sites of an array can be differentiated from each other according to the locations of the sites in the array.
  • An individual site of an array can include one or more molecules of a particular type. For example, a site can include a single target nucleic acid molecule having a particular sequence or a site can include several nucleic acid molecules having the same sequence (or complementary sequence thereof). The sites of an array can be different features located on the same substrate.
  • features include, without limitation, wells in a substrate, beads (or other particles) in or on a substrate, projections from a substrate, ridges on a substrate or channels in a substrate.
  • the sites of an array can be separate substrates each bearing a different molecule. Different molecules attached to separate substrates can be identified according to the locations of the substrates on a surface to which the substrates are associated or according to the locations of the substrates in a liquid or gel.
  • arrays in which separate substrates are located on a surface include, without limitation, those having beads in wells
  • clonal population generally refers to a population of nucleic acids homogeneous with respect to a particular nucleotide sequence.
  • the homogenous sequence may be at least 10 nucleotides long but can be longer including for example but not limited to, at least 50, 100, 250, 500 or 1000 nucleotides long.
  • a clonal population can be derived from a single target nucleic acid or template nucleic acid.
  • Nucleic acids in a clonal population may have the same nucleotide sequence, however the nucleic acids may have a small number of mutations, such as but not limited to those due to amplification artifacts, can occur in a clonal population without departing from clonality.
  • primer generally refers to a short, single stranded nucleic acid utilized in the initiation of DNA synthesis.
  • Primers may be either RNA primers or DNA primers.
  • a pair of primers may be used to hybridize with sample DNA and define the region of SNA that will be amplified.
  • a primer may also target a specific locus for further amplification and analysis.
  • a primer is complementary to the existing DNA.
  • capture agent generally refers to a material, chemical, molecule or moiety thereof that is capable of attaching, retaining or binding to a target molecule, such as but not limited to, a target nucleic acid.
  • Capture agents include, but are not limited to, a capture nucleic acid that is complementary to at least a portion of a target nucleic acid, a member of a receptor-ligand binding pair capable of binding to a target nucleic acid, or linking moiety attached thereto, or a chemical reagent capable of forming a covalent bond with a target nucleic acid, or linking moiety attached thereto.
  • Poisson distribution generally refers to a discrete probability distribution that expresses the probability of a given number of events occurring in a fixed interval of time and/or space if these events occur with an average rate and independently of the time since the previous event.
  • the target material is spread across a large number of partitions where the average number of molecules per partition is estimated using Poisson distribution statistics. This distribution can be mathematically converted into molecular concentrations.
  • the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values
  • the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.
  • each when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection unless the context clearly dictates otherwise.
  • Nucleic acid sequencing is an important tool in a variety of genetic analysis.
  • DNA sequencing may involve the determination of the order of bases for an individual nucleic acid strand which may be important in understanding and utilizing genetic information.
  • One way of increasing efficiency is to perform many processes in parallel with simultaneous amplification of cloned fragments in high density arrays.
  • Fragmented DNA and the small amounts of DNA common in many genetic applications may be amplified prior to analysis.
  • This amplification may be clonal amplification of template DNA where DNA is amplified generating several millions of copies of a specific segment of DNA from a small amount of starting material, the template. Its specificity may rely on sequence hybridization and its sensitivity may rely on enzyme-based amplification.
  • One method of amplification, PCR may comprise a series of temperature cycles repeated. Each cycle denatures DNA duplexes, hybridizes two DNA oligonucleotides (primers) flanking the target sequence, and elongates those primers by a DNA polymerase. Each cycle results in a doubling of the number of target DNA molecules and thus exponential amplification.
  • Methods of amplification in commercial aspects may require a high throughput to efficiently analyze large and complex nucleic acids, and analyze multiple target sequences simultaneously.
  • Nucleic acids partitioned to individual reaction sites on an array may be targeted to the sites through dilution and statistical probability predicted by a Poisson distribution, which may describe patterns of low particle numbers in a volume.
  • a sample may be divided into multiple independent partitions such that each partition contains small amounts or no target sequences. These partitions may act as an individual PCR microreactor containing amplified target sequences able to be detected in them and thus determining the concentration of the target in the sample. In the amplification of large libraries, statistical variance may not follow a Poisson distribution.
  • the fraction of sites in an array that are clonal can exceed the fraction predicted by the Poisson distribution resulting in a super-Poisson distribution where there is more variance with the same mean as a Poisson distribution.
  • Clonal sites may have a super- Poisson distribution when the number of different target nucleic acids in the solution exceeds the number of amplification sites in the array which may enable amplification and library formation through kinetic exclusion.
  • Kinetic exclusion can occur when a process proceeds at a sufficiently rapid rate to effectively exclude another event or process from occurring.
  • the seeding of an array with target nucleic acids may form a solution where generation of copies of the target nucleic acid to fill each seeded site can proceed simultaneously under conditions where the amplification rate exceeds the seeding rate.
  • the relatively rapid rate at which copies are made at a site that has been seeded by a first target nucleic acid may effectively exclude a second nucleic acid from seeding the site for amplification improving amplification efficiency in large libraries.
  • the present disclosure provides a method for nucleic acid amplification, comprising contacting an array of reaction sites with a solution comprising a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules different from target nucleic acid molecules of the plurality of target nucleic acid molecules, such that a reaction site of the array of reaction sites comprises a target nucleic acid molecule of the plurality of target nucleic acid molecules and a non-target nucleic acid molecule of the plurality of non-target nucleic acid molecules.
  • the target nucleic acid molecule and the non-target nucleic acid molecule may be immobilized at the reaction site.
  • the target nucleic acid molecules and non-target nucleic acid molecules may be present in the solution at a first ratio of target nucleic acid molecules to non target nucleic acid molecules of at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100.
  • the target nucleic acid molecules and non-target nucleic acid molecules may be present in the solution at a first ratio of target nucleic acid molecules to non-target nucleic acid molecules of at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
  • the target and non-target nucleic acid molecules provided in solution may be immobilized to a reaction site to generate amplicons of the target and non-target nucleic acid molecules immobilized at the reaction site.
  • the reaction site may be a particle, planar, a sensor configured to detect a signal indicative a reaction associated with the target nucleic acid molecules or their amplicons, or an electronic sensor among others.
  • the target or non-target nucleic acid molecules may be amplified at a reaction site in the plurality of reaction sites. Additional target or non-target nucleic acid molecules in a plurality of target or non-target nucleic acid molecules may be immobilized at the array of reaction sites.
  • This amplification may occur at a rate sufficient to generate amplicons of the target and non-target nucleic acid molecules without amplification of other target or non-target nucleic acid molecules in the plurality of target or non-target nucleic acid molecules present at the reaction site.
  • a second ratio of amplicons of these target and non-target nucleic acid molecules may be immobilized at the reaction site to a copy or complement of the target nucleic acid molecule, non-target nucleic acid molecule, or any combination thereof may be at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100.
  • a second ratio of amplicons of these target and non-target nucleic acid molecules, non-target nucleic acid molecule, or any combination thereof may be at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:l
  • the reaction site may be one, two, or a plurality of oligonucleotides immobilized to them.
  • a portion of the target or non-target nucleic acid molecules may be hybridized with a sequence of one of the oligonucleotides in the plurality of oligonucleotides immobilized at the reaction site.
  • Amplification of the nucleic acid molecules may comprise conducting a nucleic acid extension reaction using one or more of these oligonucleotides and a target nucleic acid molecule. Amplification may occur with the aid of an enzyme such as but not limited to a recombinase or a polymerase. The amplicons generated of such a target nucleic acid molecule may be partially complementary or clonal to the target nucleic acid molecule.
  • This amplification process may occur contemporaneous with the flow of the solution of target and non-target nucleic acid molecules to the array of reaction sites. This amplification process may occur absent the flow of the solution of target and non-target nucleic acid molecules to the array of reaction sites.
  • amplification sequencing may be performed of the generated target nucleic acid molecules with sequencing-by synthesis, an electronic sensor, or other methods. Sequencing may occur by detecting a signal indicative of an impedance, an impedance change, a conductivity, a conductivity change, a charge, or a charge change, or another method. The process of amplification and sequencing may be performed for additional target nucleic acid molecules.
  • the nucleic acid molecules described herein may be target or non-target sequences.
  • a target nucleic acid(s) is one that is targeted to the array, immobilized, and copies generated. These amplicons are then amplified.
  • a non-target nucleic acid(s) is one that is targeted to the array, immobilized, and copies generated alongside the target nucleic acid(s). Amplicons of the target sequence may be immobilized to a copy of the non-target nucleic acid at the reaction site. Ultimately these poisoned amplification products may be blocked through an adapter sequence from amplifying alongside the target sequence.
  • the target and non-target sequences may exist in a ratio.
  • the ratio may include 100%, 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less of the target nucleic acids.
  • the ratio may include 100%, 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less of the non-target sequence.
  • the ratio may be any combination of the above or may be a combination not listed above.
  • the ratio of the target to non target sequence may change throughout the amplification process.
  • the nucleic acid molecules described herein may be RNA.
  • the methods and systems as described elsewhere herein may comprise methods to exclude a plurality of nucleic acid molecules from other array reaction sites. The excluding may generate arrays with less than about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less contamination from other arrays.
  • the excluding may generate arrays with more than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more contamination from other arrays.
  • the immobilization of RNA may be for a time. For example, the RNA can be immobilized while the RNA is being generated, and then the excess RNA can be washed away.
  • the nucleic acid molecules described herein may be DNA.
  • the methods and systems as described elsewhere herein may comprise methods and mechanisms configured to exclude a plurality of nucleic acid molecules from other arrays of reaction sites. The excluding may generate arrays with less than about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less contamination from other arrays.
  • the excluding may generate arrays with more than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more contamination from other arrays.
  • the immobilization of DNA may be for a time. For example, the DNA can be immobilized while the DNA is being generated, and then the excess DNA can be washed away.
  • a method for nucleic acid amplification may comprise bringing a template nucleic acid molecule for both or either a target and non-target sequences in contact with a reaction site such as an array of probes.
  • the template nucleic acid molecule may be immobilized at the reaction site such as by binding to a probe of the array of probes.
  • the template nucleic acid molecule may be used to synthesize a plurality of nucleic acid molecules at least partially complementary to sequences of other probes of the array of probes.
  • the nucleic acid molecules of the plurality of nucleic acid molecules may bind to the other probes of the array of probes, thereby generating occupied probes.
  • At least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules may be removed from the occupied probes, thereby generating active probes.
  • the template nucleic acid molecule and the active probes may be used to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the active probes.
  • the present disclosure provides a method for nucleic acid amplification which may comprise bringing a template nucleic acid molecule for both or either a target and non target sequence in contact with a reaction site such as an array of oligonucleotides.
  • the template nucleic acid molecule may bind to a probe of the array of oligonucleotides.
  • the template nucleic acid molecule may be used to synthesize a plurality of nucleic acid molecules at least partially complementary to sequences of other oligonucleotides of the array of oligonucleotides.
  • the nucleic acid molecules of the plurality of nucleic acid molecules may bind to the other oligonucleotides of the array of oligonucleotides, thereby generating occupied oligonucleotides. At least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules may be removed from the occupied oligonucleotides, thereby generating active oligonucleotides.
  • the template nucleic acid molecule and the active oligonucleotides may be used to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the active oligonucleotides.
  • An array of reaction sites may be contacted with a solution with many target or non-target sequences.
  • the array of reaction sites may be contacted with a solution with many nucleic acids where there are at least two populations of nucleic acids, such as a target and non-target nucleic acids. These target and non-target sequences exist in a ratio.
  • the array binds at least one nucleic acid of each population in the solution at a ratio.
  • the systems can bind a single sequence of interest at the reaction site or a plurality.
  • the array may be a plurality or electrically or magnetically immobilized probes.
  • sequences are immobilized to a reaction site where the nucleic acid molecules may be amplified to generate amplicons of target and non-target nucleic acid molecules immobilized at a reaction site.
  • Multiple nucleic acid molecules may be immobilized to the reaction site or just one molecule can be immobilized to the reaction site.
  • a plurality of either multiple or single molecules may be targeted to a plurality of reaction sites.
  • the target and non-target nucleic acid molecules may be immobilized at a reaction site at a ratio of at most about 1:1, 1:2, 1:3, 1 :4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1 : 100.
  • the target and non-target nucleic acid molecules may be immobilized at a reaction site at a ratio of at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
  • the ratio may be greater than, less than, or some value in between those stated above.
  • the amplifying may occur at a rate sufficient to generate the amplicons of the target and/or non-target nucleic acid molecules without amplification of other target and/or non-target nucleic acid molecules of the plurality of target and/or non-target nucleic acid molecules at a reaction site using kinetic exclusion.
  • This amplification may occur at a rate sufficient to generate amplicons of target and/or non-target nucleic acid molecules of a plurality of target and non-target molecules at a reaction site.
  • a target and/or non-target nucleic acid molecule(s) may be amplified to generate a copy or complement, or a plurality of copies or complements, of a target or non-target nucleic acid molecule immobilized at a reaction site.
  • Amplicons of either the target or non-target nucleic acids may be a clonal population, complementary, or partially complementary.
  • a reaction site may have a plurality of oligonucleotides immobilized to it.
  • the target nucleic acid molecule(s) may be hybridized with a oligonucleotide sequence(s) bound to the reaction site.
  • Multiple targets can be immobilized to many reaction sites on the array.
  • the reaction site can be a particle, plane, or a sensor configured to detect a signal indicative or a reaction associate with target or amplicons of target and the sensor can be but is not necessarily electronic.
  • An electrical sensor at the reaction site can detect a signal indicative of a target reaction or amplicon of a target reaction.
  • Amplification may occur by doing a nucleic acid extension reaction with the oligonucleotides and the target nucleic acid molecule(s) thus generating amplicons of the target nucleic acid molecule(s).
  • amplification may be performed with a nucleic acid extension reaction, such as, for example, PCR, blunt end cloning, or shotgun cloning among other methods.
  • Amplification may be performed with the aid of an enzyme such as a recombinase or a polymerase.
  • Contact with reaction sites can be done with or without fluid flow.
  • An array of reaction sites may be contacted with a solution with many target and non-target sequences using fluid flow. This contact of an array of reaction sites with solution may occur contemporaneous to fluid flow of the solution.
  • the array may comprise a microfluidic device.
  • the solution may be viscous or non-viscous.
  • the substrate can include a plurality of microfluidic modules integrally arranged with each other so as to be in fluid communication.
  • the substrate can include, for example, at least one inlet module having at least one inlet channel adapted to carry at least one dispersed phase fluid, at least one main channel adapted to carry at least one continuous phase fluid.
  • the inlet channel may be in fluid communication with the main channel at a junction.
  • the junction may include a fluidic nozzle designed for flow focusing such that the dispersed phase fluid is immiscible with the continuous phase fluid and forms a plurality of highly uniform, monodisperse droplets in the continuous phase fluid.
  • the flow of the dispersed phase and continuous phase can be pressure driven, for example.
  • the dispersed phase e.g. droplets
  • the microfluidic substrate can include one or more additional modules, including but not limited to, coalescence module, detection module, sorting module, collection module, waste module, delay module, droplet spacing module, or a mixing module. There may be zero, one, or more of each of the modules. Contact between the array of reaction sites with the solution containing nucleic acid molecules may happen at the same time as amplification through the flow of fluid.
  • An open channel may include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation), physical or chemical characteristics (hydrophobicity vs. hydrophilicity), other characteristics that can exert a force (e.g., a containing force) on a fluid, or any combination thereof.
  • the fluid within the channel may partially or completely fill the channel.
  • the fluid may be held or confined within the channel or a portion of the channel in some fashion, for example, using surface tension (e.g., such that the fluid is held within the channel within a meniscus, such as a concave or convex meniscus).
  • the substrate may include one or plurality of channels.
  • a channel may have various sizes.
  • the channel may have a largest dimension perpendicular to the direction of fluid flow along the channel of less than about 5 millimeters (mm), less than about 2 mm, less than about 1 mm, less than about 500 micrometers (mih), less than about 200 mih, less than about 100 mih, less than about 60 mih, less than about 50 mih, less than about 40 mih, less than about 30 mih, less than about 25 mih, less than about 10 mih, less than about 3 mih, less than about 1 micron, less than about 300 nanometers (nm), less than about 100 nm, less than about 30 nm, or less than about 10 nm or less in some cases.
  • larger channels, tubes, etc. can be used to store fluids in bulk and/or deliver a fluid to the channel.
  • the dimensions of the channel may be chosen such that fluid is able to freely flow through the channel. In some cases, more than one channel may be used.
  • an electric field may be applied to fluidic droplets to cause the droplets to experience an electric force.
  • the electric force exerted on the fluidic droplets may be, in some cases, at least about 10 16 Newtons (N)/pm 3 .
  • the electric force exerted on the fluidic droplets may be greater, e.g., at least about 10 15 N/pm 3 , at least about 10 14 N/pm 3 , at least about 10 13 N/pm 3 , at least about 10 12 N/pm 3 , at least about 10 u N/pm 3 , at least about 10 10 N/pm 3 , at least about 10 9 N/pm 3 , at least about 10 8 N/pm 3 , or at least about 10 7 N/pm 3 or more.
  • the electric force exerted on the fluidic droplets, relative to the surface area of the fluid may be at least about 10 15 N/pm 2 , and in some cases, at least about 10 14 N/pm 2 , at least about 10 13 N/pm 2 , at least about 10 12 N/pm 2 , at least about 10 11 N/pm 2 , at least about 10 10 N/pm 2 , at least about 10 9 N/pm 2 , at least about 10 8 N/pm 2 , at least about 10 7 N/pm 2 , or at least about 10 6 N/pm 2 or more.
  • the electric force exerted on the fluidic droplets may be at least about 10 9 Newtons (N), at least about 10 8 N, at least about 10 7 N, at least about 10 6 N, at least about 10 5 N, or at least about 10 4 N or more in some cases.
  • Fluid may flow through the microfluidic channels at about 15 microliters (pL)/minute (min.)
  • Target and non-target sequences may be present in a solution at such a density as to allow single strand binding of a target or non-target sequence at a reaction site. This can be done using Poisson statistics.
  • the density of a target or non-target sequence in a solution may be a high density, low density, average density, or any combination thereof.
  • the density may follow a Poisson distribution, a super-Poisson distribution, or a non-Poisson distribution.
  • the density may be constant, or it may vary.
  • the solution comprising the target nucleic acid molecules may be flowed at a flow rate about 1 microliter (pL)/minute (min) to about 12 pL/min.
  • the solution comprising the target nucleic acid molecules may be flowed at a flow rate about 1 pL/min to about 2 pL/min, about 1 pL/min to about 3 pL/min, about 1 pL/min to about 4 pL/min, about 1 pL/min to about 5 pL/min, about 1 pL/min to about 6 pL/min, about 1 pL/min to about 7 pL/min, about 1 pL/min to about 8 pL/min, about 1 pL/min to about 9 pL/min, about 1 pL/min to about 10 pL/min, about 1 pL/min to about 11 pL/min, about 1 pL/min to about 12 pL/min, about 2 pL/min to about 3
  • the solution comprising the target nucleic acid molecules may be flowed at about 1 gL/min, about 2 gL/min, about 3 gL/min, about 4 gL/min, about 5 gL/min, about 6 gL/min, about 7 gL/min, about 8 gL/min, about 9 gL/min, about 10 gL/min, about 11 gL/min, or about 12 gL/min.
  • the solution comprising the target nucleic acid molecules may be flowed at least about 1 gL/min, about 2 gL/min, about 3 gL/min, about 4 gL/min, about 5 gL/min, about 6 gL/min, about 7 gL/min, about 8 gL/min, about 9 gL/min, about 10 gL/min, or about 11 gL/min.
  • the solution comprising the target nucleic acid molecules may be flowed at most about 2 gL/min, about 3 gL/min, about 4 gL/min, about 5 gL/min, about 6 gL/min, about 7 gL/min, about 8 gL/min, about
  • the solution comprising the target nucleic acid molecules may be flowed at about 13 microliters (gL)/minute (min) to about 24 gL/min.
  • the solution comprising the target nucleic acid molecules may be flowed at about 13 gL/min to about 14 gL/min, about 13 gL/min to about 15 gL/min, about 13 gL/min to about 16 gL/min, about 13 gL/min to about 17 gL/min, about 13 gL/min to about 18 gL/min, about 13 gL/min to about 19 gL/min, about 13 gL/min to about 20 gL/min, about 13 gL/min to about 21 gL/min, about 13 gL/min to about 22 gL/min, about 13 gL/min to about 23 gL/min, about 13 gL/min to about 24 gL/min, about 14 gL/min to about 15 gL/min, about 14
  • the solution comprising the target nucleic acid molecules may be flowed at about 13 gL/min, about 14 gL/min, about 15 gL/min, about 16 gL/min, about 17 gL/min, about 18 gL/min, about 19 gL/min, about 20 gL/min, about 21 gL/min, about 22 gL/min, about 23 gL/min, or about 24 gL/min.
  • the solution comprising the target nucleic acid molecules may be flowed at least about 13 gL/min, about 14 gL/min, about 15 gL/min, about 16 gL/min, about 17 gL/min, about 18 gL/min, about 19 gL/min, about 20 gL/min, about 21 gL/min, about 22 gL/min, or about 23 gL/min.
  • the solution comprising the target nucleic acid molecules may be flowed at most about 14 gL/min, about 15 gL/min, about 16 gL/min, about 17 gL/min, about 18 gL/min, about 19 gL/min, about 20 gL/min, about 21 gL/min, about 22 gL/min, about 23 gL/min, or about 24 gL/min.
  • the solution comprising the target nucleic acid molecules may be flowed at about 25 microliters (gL)/minute (min) to about 36 gL/min.
  • the solution comprising the target nucleic acid molecules may be flowed at about 25 gL/min to about 26 gL/min, about 25 gL/min to about 27 gL/min, about 25 gL/min to about 28 gL/min, about 25 gL/min to about 29 gL/min, about 25 gL/min to about 30 gL/min, about 25 gL/min to about 31 gL/min, about 25 gL/min to about 32 gL/min, about 25 gL/min to about 33 gL/min, about 25 gL/min to about 34 gL/min, about 25 gL/min to about 35 gL/min, about 25 gL/min to about 36 gL/min, about 26 gL/min to about 27 gL/min, about 26
  • the solution comprising the target nucleic acid molecules may be flowed at about 25 gL/min, about 26 gL/min, about 27 gL/min, about 28 gL/min, about 29 gL/min, about 30 gL/min, about 31 gL/min, about 32 gL/min, about 33 gL/min, about 34 gL/min, about 35 gL/min, or about 36 gL/min.
  • the solution comprising the target nucleic acid molecules may be flowed at least about 25 gL/min, about 26 gL/min, about 27 gL/min, about 28 gL/min, about 29 gL/min, about 30 gL/min, about 31 gL/min, about 32 gL/min, about 33 gL/min, about 34 gL/min, or about 35 gL/min.
  • the solution comprising the target nucleic acid molecules may be flowed at most about 26 gL/min, about 27 gL/min, about 28 gL/min, about 29 gL/min, about 30 gL/min, about 31 gL/min, about 32 gL/min, about 33 gL/min, about 34 gL/min, about 35 gL/min, or about 36 gL/min.
  • a microfluidic channel may utilize a consistent flow design or an oscillatory flow design.
  • nucleic acids, droplets, or solution are in continuous-flow.
  • Nucleic acids, droplets, or solution may be stationary or semi-stationary.
  • Nucleic acids, droplets, or solution may be in motion.
  • a microfluidic device may utilize oscillating or bidirectional flow.
  • a microfluidic device may combine the cycling flexibility of a stationary chamber-based system and the fast dynamics of a continuous flow system.
  • Nucleic acids, droplets, or solutions may be transported back and forth through a single channel or may be transported in multiple channels or capillaries. The channel(s) may span various temperature zones.
  • a microfluidic device or array may be attached to a pumping system such as but not limited to external pumps and integrated micropumps. There may be on board power or an external power source. Centrifugal force and/or capillary forces may be used to control the fluid flow.
  • a compact disc format may be used to house the reaction chambers or other components.
  • a droplet may serve as a reactor environment allowing for fast reagent mixing and minimum surface adsorption.
  • Interfacial chemistry may be used to create such a reactor droplet (e.g., an oil-water plug may be flowed through a fluid capillary to create a water-in-oil droplet).
  • Nucleic acids, droplets, or solutions in a microfluidic device, in a well, attached to a support, or in an array may be incubated, split, and merged in a microfluidic device.
  • Droplets may vary in size. Droplets may be at least about 0.5 micrometers (pm), 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80, pm, 90 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1000 pm or more in diameter.
  • Nucleic acid, droplet, or solution formation frequency may be at least about 0.5 Hertz (Hz), 1 Hz, 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz, 10 Hz, 20 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 100 Hz, 200 Hz, 300 Hz, 400 Hz, 500 Hz, 600 Hz, 700 Hz, 800 Hz, 900 Hz, 1,000 Hz, 2,000 Hz, 3,000 Hz, 4,000 Hz, 5,000 Hz, 6,000 Hz, 7,000 Hz, 8,000 Hz, 9,000 Hz, 10,000 Hz or more.
  • the frequency may be less than or greater than those listed here or any value in between.
  • a target or non-target nucleic acid sequence may be immobilized at a reaction site which is or is coupled to an electronic sensor where the target and non-target nucleic acid molecules may be amplified to generate specific amplicons without amplifying other targets at the reaction site.
  • target and non-target nucleic acids captured at the amplification sites can be amplified at an average amplification rate.
  • the average amplification rate can exceed the average capture rate. This amplification using kinetic exclusion occurs at a rate sufficient to generate amplicons of a target nucleic acid molecule without amplification of other target nucleic acid molecules of the plurality of target nucleic acid molecules at a reaction site.
  • Amplicons can be partly complementary or clonal to the target sequence.
  • Amplification of non-target sequences may make complements of the non-target sequences immobilized at the reaction site.
  • Amplification of nucleic acid molecules may occur through a nucleic acid extension reaction of oligonucleotides and the target sequence.
  • Amplification of the target to generate amplicons of the target immobilized at the reaction site may happen with an enzyme like a recombinase or polymerase.
  • Amplification may occur through bridged/locked nucleic acid extension reactions.
  • Amplification of the target or non-target nucleic acid molecule(s) may generate amplicons of a target or non-target nucleic acid immobilized at the reaction at such a rate sufficient to generate amplicons without amplification of other targets on the array.
  • At least one nucleic acid of the first population and at least one nucleic acid of the second population are amplified to make a population of amplification products at the reaction site where the population of amplification products corresponds to at least one of the first set of nucleic acids and one of the second set of nucleic acids.
  • These two populations of nucleic acids may be the target and non-target nucleic acids.
  • the non-target nucleic acid molecule(s) may be copied and mixed together in solution with the target nucleic acid molecule(s).
  • a second ratio of amplicons of the target nucleic acids may be immobilized at a reaction site to the non-target nucleic acid at a reaction site.
  • a subset of the target amplicons may be used to identify a sequence of target nucleic acids. All reverse strands may be washed off the flow cell leaving forward strands.
  • One nucleic acid of the plurality of nucleic acids may be an adapter sequence and one nucleic acid may be a primer. At least one nucleic acid of each party may be a primer thus using two different primers for target and non-target nucleic acid sequences.
  • Primers may attach to the forward strands where a fluorescently tagged polymerase may be added to tag the nucleic acid strand.
  • An electronic sensor at the reaction site comprising one, two, or more electrodes may sense target amplicons or a signifier of amplicons of the target nucleic acid sequence.
  • the two populations may exist in a ratio in a plurality of amplification products.
  • the amplification products may correspond to at least one of each population.
  • the systems provide an adapter sequence to amplify the nucleic acid molecule(s) immobilized to the reaction site.
  • a blocking sequence is used to block the non-target nucleic acid amplification products from amplifying with target nucleic acid products. This blocking sequence inhibits nucleic acid extension of the non-target amplification products, the poisoned sequence, in the colony of interest.
  • the amplicons of the target nucleic acid(s) may be sequenced through either sequencing by synthesis or through an electronic sensor.
  • an enzyme such as a DNA polymerase or ligase enzyme may be used to extend many DNA strands in parallel.
  • Nucleotides or short oligonucleotides are provided either one at a time or may be modified with identifying tags to the base type of the incorporated nucleotide or oligonucleotide can be determined as extension proceeds.
  • the systems may sequence electronically using a sensor and one or more electrodes.
  • An electronic sensor may detect a change in electrical charge, current, impedance, or conductivity of one or more electrodes and use these electric differences to identify specific nucleic acids during sequencing.
  • the systems may be coupled to a colorimetric assay after amplification to sequence.
  • a four-color chemistry panel may be used to specifically and independently tag nucleotides with a unique emission spectrum.
  • a two-color sequencing chemistry may also be used where one of two colors, no colors, or both colors mixed are used to identify specific nucleotides.
  • Different colorimetric dyed can be used to detect the existence of amplicons.
  • a colorimetric detection may rely on production of magnesium pyrophosphate or as a by-product of the reaction between deoxynucleotide triphosphate and magnesium sulfate.
  • a colorimetric assay may utilize the emission of metal catalysts during enzymatic labeling.
  • Single color fluorescence optics may be used or coupled-enzyme chemiluminescence assays may be used.
  • Dyes may be used such as but not limited to calcein, hydroxynaphthol blue, propodium iodine, SYBR GREEN, SYTO-81, or Picogreen.
  • Gold nanoparticles may also be used.
  • Amplicons may be measured using a spectrophotometer, optical photomultiplier, a charge-coupled device, fluorescence microscope, or another modality. Single or multiplex detection or some combination thereof may be utilized.
  • the sequence of the target nucleic acid molecule may be identified at an accuracy of at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more. Such accuracy may be achieved without resequencing.
  • the sequence of the target nucleic acid molecule may be identified at a single-pass accuracy of at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.
  • a diffusion barrier may be used to contain nucleic acid molecules in a well.
  • the high viscosity buffer may be a hydrocarbon (e.g., an oil, squalene), a chemical compound (e.g., 1-Butyl- 3-methylimidazolium hexafluorophosphate or glycerol), a gel buffer, a viscoelastic polymer, or the like.
  • the buffer may have a viscosity of at least about 1 x 10 3 Pascal-seconds (IE-3 Pa s), 5E- 3 Pa s, IE-2 Pa s, 5E-2 Pa s, IE-1 Pa s, 5E-1 Pa s, 1 Pa s, 5 Pa s, 10 Pa s, 50 Pa s, 100 Pa s, 500 Pa s, 1,000 Pa s, or more.
  • the buffer may have a viscosity of at most about 1,000 Pa s, 500 Pa s, 100 Pa s, 50 Pa s, 10 Pa s, 5 Pa s, 1 Pa s, 5E-1 Pa s, IE-1 Pa s, 5E-2 Pa s, IE-2 Pa s, 5E-3 Pa s, IE-3 Pa s, or less.
  • a subset of nucleic acid molecules may be degraded to exclude the nucleic acid molecules for other arrays.
  • the nucleic acid molecules may be contained within well by the use of degrading elements.
  • Degrading elements may comprise enzymes, chemical degrading elements, light induced degrading elements, or any combination thereof.
  • the enzymes may be an RNase as described elsewhere herein, a DNase as described elsewhere herein, or a combination thereof.
  • the chemical degrading elements may be an acid (e.g., />-toluene sulfonic acid, nitric acid, ascorbic acid), abase (e.g., an amine, a hydroxide salt), a reductant (e.g., sodium hydride), an oxidizer (e.g., chromate, hydrogen peroxide), or any combination thereof.
  • the light induced degrading element may be a radical generator (e.g., N- bromosuccinimide (NBS), a cadmium selenide nanoparticle with an attached ferrocene molecule).
  • NBS N- bromosuccinimide
  • a light source can be configured to illuminate NBS, generating bromine radicals that degrade RNA.
  • the degrading element 506 may be coupled to a support.
  • the support may be a particle (e.g., a bead, a microparticle, a nanoparticle), a textured surface (e.g., pillars), or a combination thereof.
  • An electric field may be applied to the support.
  • a generator may generate the electric field.
  • the electric field may have a potential of at least about 0.001 volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V,
  • V 0.001 volts
  • the electric field may have a potential of at most about 10,000V, 5,000V, 1,000V, 240V, 120V, 50V, 20V, 15V, 12V, 10V, 9V, 8V, 7V, 6V, 5V, 4V, 3V, 2V, IV, 0.9V, 0.8V, 0.7V, 0.6V, 0.5V, 0.4V, 0.3V, 0.2V, 0.1V, 0.05V, 0.01 V, 0.005V, 0.001 V or less volts.
  • the electric field may be applied via electrodes that are electronically coupled to a generator.
  • the support may be placed on the electrodes. For example, a series of beads can be cast onto an electrode.
  • the electrode may be a metal electrode, a semiconductor electrode, a polymer electrode, or any combination thereof.
  • electrode materials may be metals, semiconductors, or conductive polymers.
  • the metals may be gold, silver, platinum, nickel, copper, iron, other transition metals, or alloys thereof.
  • the semiconductors may be organic semiconductors (e.g., C 6 o, phenyl-C61 -butyric acid methyl ester), inorganic semiconductors (e.g., silicon, cadmium telluride, indium tin oxide, gallium arsenide), or a combination thereof.
  • the conductive polymers may be polyfluorenes, polyacetylenes, poly(p-phenylene vinylene)s, polypyrroles, polyanilines, polythiophenes, poly(3,4-ethylenedioxythiophene), poly(p-phenylene sulfide), or any combination thereof.
  • the nucleic acids may be confined by applying an electric field.
  • the nucleic acid molecules may be within a well.
  • a generator may be electronically coupled to electrodes which apply an electric field between the electrodes.
  • the electric field may interact with labels attached to one or more of nucleic acid molecules.
  • the interacting may draw the nucleic acid molecules away from the top of the well and thus contain the nucleic acid molecules.
  • the labels may be a particle.
  • the particle may be a di electrophoretic particle.
  • the particle may be a metal particle (e.g., gold, aluminum, silver, platinum), a semiconductor particle (e.g., silicon, carbon, zinc sulfide), or a molecular unit (e.g., Ru(bpy) 3 2+ , ferrocene).
  • the particle may be attached to the 3’ end, the 5’ end, or both ends of the nucleic acid molecule.
  • a different particle may be attached to each end of the nucleic acid molecule.
  • the nucleic acids may be confined by applying a magnetic field.
  • the magnetic field may be applied to a plurality of nucleic acid molecules confined in a well using a magnet.
  • the magnet may be a permanent magnet (e.g., a rare-earth magnet, an iron-based magnet) or an electromagnet (e.g., a solenoid, a superconducting magnet).
  • At least one nucleic acid molecule of the nucleic acid molecules may comprise a label that interacts with the magnetic field.
  • the label may be a particle (e.g., an iron nanoparticle), a molecular species (e.g., a single molecule magnet, an iron containing molecule), or a combination thereof.
  • a nucleic acid can be attached to the surface of an iron nanoparticle cluster.
  • the label may be attached to the 3’ end, the 5’ end, or both ends of the nucleic acid molecule.
  • a different label may be attached to each end of the nucleic acid molecule.
  • the nucleic acids may be confined using an electrophoretic force.
  • Nucleic acid molecules may be generated in well.
  • an electric field can be applied between electrodes.
  • the electric field may generate an electrophoretic force that attracts the nucleic acid molecules down into the well.
  • a generator may generate the electric field.
  • the generator may generate a potential of at least about 0.001 volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more.
  • V 0.001 volts
  • the generator may generate a potential of at most about 10,000V, 5,000V, 1,000V, 240V, 120V, 50V, 20V, 15V, 12V, 10V, 9V, 8V, 7V, 6V, 5V, 4V, 3V, 2V, IV, 0.9V, 0.8V, 0.7V, 0.6V, 0.5V, 0.4V, 0.3V, 0.2V, 0.1V, 0.05V, 0.01 V, 0.005V, or less volts.
  • the electrodes may be separated by at least about 1 micrometers (pm), 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 50 pm, 75 pm, 100 pm, 125 pm, 150 pm, 175 pm, 200 pm, 225 pm, 250 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm or more micrometers.
  • the electrodes may be separated by at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm, 150 pm, 140 pm, 130 pm, 120 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 5 pm, 1 pm, 0.1 pm, or less micrometers.
  • a method for nucleic acid amplification may comprise brining a plurality of target nucleic acid molecules in contact with an array of probes.
  • the plurality of target nucleic molecules may be present at a concentration such that most a target nucleic acid molecule of the plurality of target nucleic acid molecules hybridizes to a probe of the array of probes.
  • the array of probes may be subject to conditions sufficient to synthesize a first plurality of nucleic acid molecules from the target nucleic acid molecule hybridized to the probe.
  • the first plurality of nucleic acid molecules may be hybridized to other probes of the array of probes.
  • the array of probes may be subject to conditions sufficient to remove or degrade at least a subset of the first plurality of nucleic acid molecules.
  • the array of probes may be subject to conditions sufficient to amplify the target nucleic acid molecule to yield a second plurality of nucleic acid molecules hybridized to the array of probes.
  • the appropriately configured system may be a system configured to perform a nucleic acid sequencing.
  • the system may be configured to amplify one or more nucleic acids.
  • the system can bring a template nucleic molecule in contact with an array of nucleotides using fluid flow in a microfluidic device.
  • the template nucleic acid molecule may bind to a probe of the array of probes.
  • the template nucleic molecule may comprise a nucleic molecule of interest (e.g., a DNA molecule to be sequenced) or a non-target molecule (e.g., a DNA molecule undesirable to sequence).
  • the template nucleic molecule may further comprise one or more moieties configured to bind to a probe.
  • the template nucleic molecule can be a fragment of a DNA sample with a probe attached to the 3’ end.
  • the template nucleic molecule can be a fragment of a DNA sample with a probe attached to the 5’ end.
  • the moiety configured to bind to the probe may be configured to bind with a portion of the probe.
  • the probe can be a 36-base probe, and the moiety can be 15 bases complimentary to the free end of the probe.
  • the binding of the template nucleic acid molecule with the probe may be a hybridization of complimentary bases.
  • the template nucleic acid molecule may have a concentration of at least about 0.001 nanograms (ng)/microliter (pL), 0.005 ng/pL, 0.01 ng/pL, 0.05 ng/pL, 0.1 ng/pL, 0.2 ng/pL, 0.3 ng/pL, 0.4 ng/pL, 0.5 ng/pL, 0.6 ng/pL, 0.7 ng/pL, 0.8 ng/pL, 0.9 ng/pL, 1 ng/pL, 2 ng/pL, 3 ng/pL, 4 ng/pL, 5 ng/pL, 6 ng/pL, 7 ng/pL, 8 ng/pL, 9(ng/pL), 10(ng/pL), 11 (ng/pL), 12(ng/pL), 13 (ng/pL), 14(ng/pL), 15 (ng/pL), 16 ng/pL, 17 ng/pL, 18 ng/p
  • the template nucleic acid molecule may have a concentration of at most about 1,000 ng/pL, 900 ng/pL, 800 ng/pL, 700 ng/pL, 600 ng/pL, 550 ng/pL, 500 ng/pL, 450 ng/pL, 400 ng/pL, 350 ng/pL, 300 ng/pL, 275 ng/pL, 250 ng/pL, 225 ng/pL, 200 ng/pL, 190 ng/pL, 180 ng/pL, 170 ng/pL, 160 ng/pL, 150 ng/gL, 140 ng/gL, 130 ng/gL, 120 ng/gL, 110 (ng/gL, 100 ng/gL, 95 ng/gL, 90 ng/gL, 85 ng/gL, 80 ng/gL, 75 ng/gL, 70 ng/gL, 65 ng/gL, 60 ng/gL
  • the template nucleic acid molecule may have a concentration range as defined by any two of the previous values.
  • the template nucleic acid molecule may have a concentration from 0.4 nanograms per microliter to 4 nanograms per microliter.
  • the system can use the template nucleic acid molecule to synthesize a plurality of nucleic acid molecules at least partially complementary to sequences of other oligonucleotides of the array of oligonucleotides.
  • the synthesizing a plurality of nucleic acid molecules may be a polymerase chain reaction.
  • the plurality of nucleic acid molecules may be RNA molecules, DNA molecules, or oligonucleotides.
  • the RNA molecules may be synthesized from the template nucleic acid molecule with the aid of a reagent.
  • the reagent may be an enzyme.
  • the enzyme may be an RNA polymerase.
  • the RNA polymerase may be a T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), or phage T3 RNA polymerase.
  • the plurality of nucleic acid molecules may be at least partially complimentary to sequences of other oligonucleotides of the array of nucleotides.
  • the plurality of nucleic acids may be at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more complimentary to sequences of other oligonucleotides of the array of nucleotides.
  • the plurality of nucleic acid molecules may be at most about 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less complimentary to sequences of other oligonucleotides of the array of nucleotides.
  • the target nucleic acid molecule may have a concentration of at least about 0.001 nanograms (ng)/microliter (gL), 0.005 ng/gL, 0.01 ng/gL, 0.05 ng/gL, 0.1 ng/gL, 0.2 ng/gL, 0.3 ng/gL, 0.4 ng/gL, 0.5 ng/gL, 0.6 ng/gL, 0.7 ng/gL, 0.8 ng/gL, 0.9 ng/gL, 1 ng/gL, 2 ng/gL, 3 ng/gL, 4 ng/gL, 5 ng/gL, 6 ng/gL, 7 ng/gL, 8 ng/gL, 9 ng/gL, 10 ng/gL, 11 ng/gL, 12 ng/gL, 13 ng/gL, 14 ng/gL, 15 ng/gL, 16 ng/gL, 17 ng/gL, 18
  • the target nucleic acid molecule may have a concentration of at most about 1,000 ng/gL, 900 ng/gL, 800 ng/gL, 700 ng/gL, 600 ng/gL, 550 ng/gL, 500 ng/mL, 450 ng/mL, 400 ng/gL, 350 ng/mL, 300 ng/gL, 275 ng/mL, 250 ng/mL, 225 ng/gL, 200 ng/mL, 190 ng/gL, 180 ng/mL, 170 ng/mL, 160 ng/gL, 150 ng/mL, 140 ng/gL, 130 ng/gL, 120 ng/gL, 110 ng/gL, 100 ng/gL, 95 ng/gL, 90 ng/gL, 85 ng/gL, 80 ng/gL, 75 ng/gL, 70 ng/gL, 65ng/gL, 60 ng/gL,
  • the other probes of the array of probes may comprise a common sequence.
  • the probes of the array of probes may be identical.
  • the plurality of nucleic acid molecules may be at least partially complementary to the common sequence.
  • an RNA molecule can be complimentary to all probes within a 15 micrometer square (gm 2 ) area, but not to probes outside that area.
  • the other probes of the array of probes may have a common sequence with other probes in an area of at least about 0.01 gm 2 , 0.05 gm 2 , 0.1 gm 2 , 0.5 gm 2 , 1 gm 2 , 5 gm 2 , 10 gm 2 , 15 gm 2 , 20 gm 2 , 25 gm 2 , 50 gm 2 , 75 gm 2 , 100 gm 2 , 150 gm 2 , 200 gm 2 , 250 gm 2 , 500 gm 2 , 750 gm 2 , 1,000 gm 2 , 5,000 gm 2 , 10,000 gm 2 , 50,000 gm 2 , 100,000 gm 2 , or more square micrometers.
  • the other probes of the array of probes may have a common sequence with other probes in an area of at most about 100,000 gm 2 , 50,000 gm 2 , 10,000 gm 2 , 5,000 gm 2 , 1,000 gm 2 , 750 gm 2 , 500 gm 2 , 250 gm 2 , 200 gm 2 , 150 gm 2 , 100 gm 2 , 75 gm 2 , 50 gm 2 , 25 gm 2 , 20 gm 2 , 15 gm 2 , 10 gm 2 , 5 gm 2 , 1 gm 2 , 0.5 gm 2 , 0.1 gm 2 , 0.05 gm 2 , 0.01 gm 2 , or less square micrometers.
  • Template nucleic acid molecules can be used to synthesize a plurality of nucleic acid molecules that are at least partially complementary to sequences of other probes of the array of probes. This operation may be performed with binding of the template nucleic acid molecule to at least two probes of the array of probes. The binding of the template nucleic acid molecule to at least two probes may impart a bridge geometry to the template nucleic acid molecule.
  • the array of probes may be among a plurality of arrays of probes.
  • the array of probes may comprise probes having sequences different from probes of at least one other array of the plurality of arrays of probes. For example, the probes coupled to the sensors of a 3x3 grid of sensors can each have a different sequence, leading to 9 different probe sequences.
  • the different probe sequences may result in less cross contamination of nucleic acids between sensors.
  • the lack of cross contamination may be particularly relevant in sensing arrays that do not comprise wells.
  • each bead of an array of beads having different probe sequences can prevent the RNA produced at each bead from diffusing to and binding onto another bead.
  • the arrays of the plurality of arrays of probes can be selectively activated for nucleic acid amplification reactions and sequencing by synthesis reactions. Select areas of the arrays of probes can be selectively activated for nucleic acid amplification reactions and sequencing by synthesis reactions.
  • a subset of the arrays of probes may be blocked from binding to nucleic acid molecules.
  • the subset of the arrays of probes can be blocked by a nucleic acid molecule of a first plurality of nucleic acid molecules.
  • the first plurality of nucleic acid molecules may comprise RNA molecules.
  • the template nucleic acid molecule can be among a plurality of template nucleic acid molecules. Individual template nucleic acid molecules can comprise different sequences.
  • Distinct template nucleic acid molecules can be bound to distinct select areas of the arrays of probes. These distinct template nucleic acid molecules can be selectively amplified or sequenced at corresponding, distinct, or select areas of the arrays of probes. Selective amplification of the distinct template nucleic acid molecules may generate a second plurality of nucleic acid molecules.
  • the second plurality of nucleic acid molecules may comprise DNA molecules.
  • the nucleic acid molecules of the plurality of nucleic acid molecules may be transported from the probe to the other probes of the array of probes.
  • the transportation may be via diffusion.
  • the transportation may be assisted diffusion.
  • the transportation may be an active transportation.
  • the active transportation may comprise cellular transportation methods (e.g., primary active transport, secondary active transport), optical methods (e.g., optical tweezers moving nucleic acid molecules), directed flow (e.g., flowing a liquid carrier in the direction of transport), or any combination thereof.
  • the transportation may be limited. For example, walls of a well can be placed around the nucleic acid molecules to limit the distance of diffusion.
  • the system can bind nucleic acid molecules of the plurality of nucleic acid molecules to the other probes of the array of probes, thereby generating occupied probes.
  • the binding of the nucleic acid to the probe may be configured to prevent additional nucleic acids or other template nucleic acid molecules from binding to the probe.
  • the binding of the nucleic acid to the probe may allow for one template nucleic acid to bind to a given area. For example, a target nucleic acid binds to a probe and produces a plurality of nucleic acids that block the surrounding probes from other target nucleic acids binding.
  • Synthesizing a plurality of nucleic acid molecules that are at least partially complementary to sequences of other probes of the array of probes and binding nucleic acid molecules of the plurality of nucleic acid molecules to the other probes of the array of probes may occur contemporaneously.
  • an RNA molecule generated by the template nucleic acid molecule can bind to a nearby probe immediately after being generated.
  • Synthesizing a plurality of nucleic acid molecules that are at least partially complementary to sequences of other probes of the array of probes and binding nucleic acid molecules of the plurality of nucleic acid molecules to the other probes of the array of probes may occur consecutively.
  • an RNA molecule generated by the template nucleic acid molecule can float in solution for a time before binding to a nearby probe.
  • the time between generation of a nucleic acid of the plurality of nucleic acids and the binding of the nucleic acid to the other probe may be at least about 0.1 seconds (s), 1 s, 2 s, 3 s, 4 s, 5 s, 10 s, 30 s, 60 s, 120 s, 180 s, 240 s, 300 s, 360 s, 600 s, 1200 s, 2400 s, 3600 s, or more.
  • the time between generation of a nucleic acid of the plurality of nucleic acids and the binding of the nucleic acid to the other probe may be at most about 3600 s, 2400 s, 1200 s, 600 s, 360 s, 300 s, 240 s, 180 s, 120 s, 60 s, 30 s, 10 s, 5 s, 4 s, 3 s, 2 s, 1 s, 0.1 s, or less.
  • the system can remove at least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules from the occupied probes, thereby generating active probes.
  • Removing at least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules from the occupied probes may comprise removing at least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules from the occupied probes with a reagent.
  • the removing at least a portion of the nucleic acid molecules may be removing substantially all nucleic acid molecules within an area. For example, all of the probes in a well of a sensing array can have the bound nucleic acid molecules removed.
  • the nucleic acids bound to probes on the surface of a bead can be removed.
  • the removing at least a portion of the nucleic acid molecules may be removing nucleotides of a given sequence.
  • nucleotides with the sequence ATACG can be removed, but nucleotides with the sequence TTAAG can remain.
  • the reagent may be an enzyme.
  • the enzyme may be an RNase.
  • the RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl, T2, U2, or V.
  • the reagent may be a chemical compound.
  • the chemical compound may be formamide, guanidine, sodium hydroxide, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, or urea.
  • the system can use the template nucleic acid molecule and the active probes to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the active probes.
  • Using template nucleic acid molecules and the active probes to amplify the template nucleic acid molecule may comprise conducting a reaction with aid of at least one recombinase, polymerase, or a combination thereof.
  • the recombinase may be a Tre recombinase, a Cre recombinase, a Hin recombinase, aDmcl recombinase, aRad51 recombinase, or a FLP recombinase.
  • the polymerase may be a DNA polymerase or an RNA polymerase.
  • the RNA polymerase may be a T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), or phage T3 RNA polymerase.
  • the DNA polymerase may be a DNA polymerase of family A, B, C, X, or Y.
  • the amplicons coupled to the active probes may be a clonal population of nucleic acids.
  • the clonal population of nucleic acids may be clones of the template nucleic acid.
  • the amplicons may be a partially clonal population.
  • the amplicons may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more clonal.
  • the amplicons may be at least about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less clonal.
  • the template nucleic acid molecules and the active probes can be used to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the active probes. This operation may further comprise sequencing at least a subset of the amplicons coupled to the active probes or derivatives thereof.
  • the derivatives may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more similar in sequence to the template nucleic acid.
  • the derivatives may be at least about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less similar in sequence to the template nucleic acid.
  • the subset may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the amplicons.
  • the subset may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the amplicons.
  • the sequencing may be sequencing-by-synthesis, Sanger sequencing, hydrogen ion detection sequencing, Polony sequencing, nanopore sequencing, rolling circle sequencing, or the like.
  • the nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, change in charge, or any combination thereof.
  • the nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of fluorescence, wavelength of fluorescence, intensity of fluorescence, time resolved fluorescence, or any combination thereof.
  • the sequencing may be performed by methods and systems as described elsewhere herein.
  • the array of probes may be attached to a solid support.
  • the solid support may be a bead, planar, a surface of a well, or any combination thereof.
  • a bead functionalized with probes can rest on a planar surface.
  • the bead may be a functionalized bead comprising a tosylated surface.
  • the bead may have a diameter of at least about 1 micrometer (pm), 5 pm, 10 pm, 25 pm, 50 pm, 75 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 750 pm, 1,000 pm, or more micrometers.
  • the bead may have a diameter of at most about 1,000 pm, 750 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 150 pm, 100 pm, 75 pm, 50 pm, 25 pm, 10 pm, 5 pm, 1 pm, or less micrometers.
  • the bead may be a component of a well-less sensing array.
  • the bead may be a polymer bead (e.g., latex, polystyrene), a glass bead, a metal bead, or the like.
  • the planar solid support may be a well-less sensing array.
  • the planar solid support may comprise one or more electrodes.
  • the electrodes may be dielectric stacks, metals, or a combination thereof.
  • the electrodes may be nanoneedles.
  • the well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1 pm, 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more micrometers.
  • the well may have a dimension of x by y by z, where x, y, and z are each independently at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm, 150 pm, 140 pm, 130 pm, 120 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 5 pm, 1 pm, 0.1 pm, or less micrometers.
  • the well can have an x dimension of 434 micrometers, a y dimension of 30 pm and a z dimension of 510 pm. In another example, the well can have an x and w dimension of 15 pm and a z dimension of 1 pm.
  • the system may comprise mechanisms configured to reduce or eliminate movement of RNA between sensors of an array of sensors.
  • the array of probes may be in sensory communication with a sensor.
  • the sensor may be an optical sensor, an electrical sensor, an ion sensor (e.g., a pH sensor), or any combination thereof.
  • the sensor may comprise an electrode.
  • the electrode may be a metal electrode (e.g., gold, copper, an alloy), a semiconductor electrode (e.g., silicon, gallium arsenide, an organic semiconductor), or a combination thereof.
  • the sensor may comprise a plurality of electrodes.
  • the plurality of electrodes may comprise at least about 1, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 250,000, 500,000, 750,000, 1,000,000, or more electrodes.
  • the plurality of electrodes may comprise at most about 1,000,000, 750,000, 500,000, 250,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 100, 50, 10, 5, 1, or less electrodes.
  • the sensor may be among an array of sensors.
  • the array of sensor may comprise sensors of one or more types.
  • an array of sensor may comprise an optical sensor and an electrical sensor.
  • the sensors of the array of sensors may be individually addressable. For example, each electrode of an array of 1,000,000 can be measured independently of each other electrode.
  • the array of probes may be among a plurality of arrays of probes.
  • the plurality of arrays of probes may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more arrays of probes.
  • the plurality of arrays of probes may be at most about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less arrays of probes.
  • the above operations may be repeated at another array of the plurality of arrays of probes.
  • the operations may be repeated for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more other arrays.
  • the operations may be repeated for at least about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less other arrays.
  • the operations may be repeated at each other array of the plurality of arrays of probe [0095]
  • the present disclosure provides methods for processing a template nucleic acid molecule.
  • a method for processing a template nucleic acid molecule may comprise providing a template nucleic molecule coupled to a probe of an array of probes.
  • the other probes of the array of probes may be blocked such that other template nucleic acid molecules are incapable of stably coupling to the other probes. At least a subset of the other probes may be blocked.
  • the template nucleic acid molecule and the deblocked or active probes may be used to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the deblocked or active probes.
  • the conditions sufficient to remove or degrade at least a subset of the first plurality of nucleic acid molecules may comprise removing or degrading the subset of the nucleic acid molecules with a reagent.
  • the reagent may be an enzyme.
  • the enzyme may be an RNase.
  • the RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl, T2, U2, or V.
  • the enzyme a may be a DNase.
  • the enzymes may be an RNase as described elsewhere herein, a DNase as described elsewhere herein, or a combination thereof.
  • the reagent may be a chemical compound.
  • the chemical compound may be formamide, guanidine, sodium hydroxide, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, or urea.
  • the chemical degrading elements may be an acid (e.g., p-toluene sulfonic acid, nitric acid, ascorbic acid), a base (e.g., an amine, a hydroxide salt), a reductant (e.g., sodium hydride), an oxidizer (e.g., chromate, hydrogen peroxide), or any combination thereof.
  • the light induced degrading element may be a radical generator (e.g., N- bromosuccinimide (NBS)), a cadmium selenide nanoparticle with an attached ferrocene molecule.
  • NBS N- bromosuccinimide
  • a light source can be configured to illuminate NBS, generating bromine radicals that degrade RNA.
  • the degrading elements may be coupled to a support.
  • the support may be a particle (e.g., a bead, a microparticle, a nanoparticle), a textured surface (e.g., pillars), or a combination thereof.
  • a plurality of RNase enzymes can be coupled to a plurality of support beads, and the support beads can be placed above the wells.
  • An electric field may be applied to the support.
  • a generator may generate the electric field.
  • the electric field may have a potential of at least about 0.001 volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120
  • the electric field may be at most about 10,000 V, 5,000 V, 1,000 V, 240 V, 120 V, 50 V, 20 V, 15 V, 12 V, 10 V, 9 V, 8 V, 7 V, 6 V, 5 V, 4 V, 3 V, 2 V, 1 V, 0.9 V, 0.8 V, 0.7 V, 0.6 V, 0.5 V, 0.4 V, 0.3 V, 0.2 V, 0.1 V, 0.05 V, 0.01 V, 0.005 V, or less volts.
  • the electric field may be applied via electrodes that are electronically coupled to the generator.
  • the support may be placed on the electrodes. For example, a series of beads can be cast onto an electrode.
  • the electrode may be a metal electrode, a semiconductor electrode, a polymer electrode, or any combination thereof.
  • the target nucleic acid molecule hybridized to the probe may comprise a promoter sequence.
  • the promoter sequence may be a T7 RNA polymerase promoter sequence.
  • the probes of the array of probes may comprise a complementary promoter sequence.
  • the complementary promoter sequence may be complimentary to the promoter sequence of the target nucleic acid molecule.
  • the target nucleic acid molecule may be able to hybridize with a probe via interaction of the promoter sequence with the complementary promoter sequence.
  • An array of amplification sites used in a method set forth herein can be present as one or more substrates.
  • types of substrate materials that can be used for an array include glass, modified glass, functionalized glass, inorganic glasses, microspheres, plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, an optical fiber or optical fiber bundles, polymers and multiwell plates.
  • Plastics may include but are not limited to acrylics, polystyrene, polypropylene, polyethylene, polybutylene, polyurethanes and TeflonTM.
  • silica-based materials include silicon and various forms of modified silicon.
  • a substrate can be within or part of a vessel such as a well, tube, channel, cuvette, Petri plate, or bottle
  • the sites of an array can be configured as features on a surface.
  • the features can be present in any of a variety of predetermined formats.
  • the sites can be wells, pits, channels, ridges, raised regions, pegs, posts or the like.
  • the sites can contain beads. However, the sites may not contain a bead or particle.
  • the sites of an array can be metal features on a non-metallic surface such as glass, plastic or other materials described above.
  • a metal layer can be deposited on a surface using methods such as wet plasma etching, dry plasma etching, atomic layer deposition, ion beam etching, chemical vapor deposition, vacuum sputtering or the like.
  • a metal layer can also be deposited by e-beam evaporation or sputtering.
  • Metal layer deposition techniques such as those described above, can be combined with photolithography techniques to create metal regions or patches on a surface.
  • An array of features can appear as a grid of spots or patches.
  • the features can be located in a repeating pattern or in an irregular non-repeating pattern.
  • Particularly useful patterns include but are not limited to hexagonal patterns, rectilinear patterns, grid patterns, asymmetric patterns, patterns having reflective symmetry, or patterns having rotational symmetry.
  • the pitch can be the same between different pairs of nearest neighbor features or the pitch can vary between different pairs of nearest neighbor features.
  • Features of an array can each have an area that is larger than about 100 nanometers squared (nm 2 ), 250 nm 2 , 500 nm 2 , 1 micrometers squared (pm 2 ), 2.5 pm 2 , 5 mih 2 , 10 mih 2 , 100 mih 2 , or 500 mih 2 .
  • features of an array can each have an area that is smaller than about 1 mm 2 , 500 mih 2 ,100 mih 2 , 25 mih 2 , 10 mih 2 , 5 mih 2 , 1 mih 2 , 500 nm 2 , or 100 nm 2 .
  • a region can have a size that is in a range between an upper and lower limit selected from those provided above.
  • the system may provide a template nucleic acid molecule coupled to a probe of an array of probes.
  • the template nucleic acid molecule may have a concentration of at least about 0.001 nanograms/microliter (ng/pL), 0.005 ng/pL, 0.01 ng/pL, 0.05 ng/pL, 0.1 ng/pL, 0.2 ng/pL, 0.3 ng/pL, 0.4 ng/pL, 0.5 ng/pL, 0.6 ng/pL, 0.7 ng/pL, 0.8 ng/pL, 0.9 ng/pL, 1 ng/pL, 2 ng/pL, 3 ng/pL, 4 ng/pL, 5 ng/pL, 6 ng/pL, 7 ng/pL, 8 ng/pL, 9 ng/pL, 10 ng/pL, 11 ng/pL, 12 ng/pL, 13 ng/pL, 14 ng/pL
  • the template nucleic acid molecule may have a concentration of at most about 1,000 ng/pL, 900 ng/pL, 800 ng/pL, 700 ng/pL, 600 ng/pL, 550 ng/pL, 500 ng/pL, 450 ng/pL, 400 ng/pL, 350 ng/pL, 300 ng/pL, 275 ng/pL, 250 ng/pL, 225 ng/pL, 200 ng/pL, 190 ng/pL, 180 ng/pL, 170 ng/pL, 160 ng/pL, 150 ng/pL, 140 ng/pL, 130 ng/pL, 120 ng/pL, 110 ng/pL, 100 ng/pL, 95 ng/pL, 90 ng/pL, 85 ng/pL, 80 ng/pL, 75 ng/pL, 70 ng/pL, 65 ng/pL, 60 ng/pL
  • the template nucleic acid molecule may have a concentration range as defined by any two of the previous values. For example, the template nucleic acid molecule may have a concentration from 0.4 nanograms per microliter to 4 nanograms per microliter.
  • the other probes of the array of probes may be blocked such that other template nucleic acid molecules may be incapable of stably coupling to the other probes.
  • the other probes of the array of probes may be blocked with nucleic acid molecules bound to the other probes of the array of probes.
  • the nucleic acid molecules may be DNA molecules or RNA molecules.
  • the other probes can be blocked with RNA molecules that bind to enough of the probe to prevent stable binding.
  • the amount the RNA molecules are configured to be bound to prevent stable binding can be a function of temperature and the ionic strength of the buffer solution around the probes.
  • the stability of the binding can be modulated by factors such as the length of the blocking nucleic acid, the sequence of the probe, the ionic strength of the solution (e.g., the salt concentration), the temperature, the presence of solvents (e.g., formamide, DMSO), the presence of ligands, the presence of metal ions, the pH of the solution, or any combination thereof.
  • the nucleic acids blocking the other probes may isolate the template nucleic acid.
  • the probes of the array of probes may be coupled to a support.
  • the support may be the interior of a well.
  • the support may be planer.
  • the support may be a bead.
  • the bead may be a component of a well-less sensing array or any combination of the aforementioned.
  • the probes may be coupled to a functional unit on the surface of the bead.
  • the support may be the interior of a well.
  • the support may be an electrode.
  • a bead functionalized with probes can rest on a planar surface.
  • the bead may be a functionalized bead comprising a tosylated surface.
  • the bead may have a diameter of at least about 1 micrometer (pm), 5 pm, 10 pm, 25 pm, 50 pm, 75 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 750 pm, 1,000 pm, or more micrometers.
  • the bead may have a diameter of at most about 1,000 pm, 750 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 150 pm, 100 pm, 75 pm, 50 pm, 25 pm, 10 pm, 5 pm, 1 pm, or less micrometers.
  • the bead may be a component of a well-less sensing array.
  • the bead may be a polymer bead (e.g., latex, polystyrene, a glass bead, a metal bead, or the like.
  • the planar solid support may be a well-less sensing array.
  • the planar solid support may comprise one or more electrodes.
  • the electrodes may be dielectric stacks, metals, or a combination thereof.
  • the electrodes may be nanoneedles.
  • the well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1 (pm), 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more micrometers.
  • 0.1 (pm) 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm
  • the well may have a dimension of x by y by z, where x, y, and z are each independently at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm, 150 pm, 140 pm, 130 pm, 120 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 5 pm, 1 pm, 0.1 pm, or less micrometers.
  • the well can have an x dimension of 434 micrometers, a y dimension of 30 micrometers, and a z dimension of 510 micrometers. In another example, the well can have an x and y dimension of 16 micrometers and a z dimension of 1 micrometer.
  • the system may comprise mechanisms configured to reduce or eliminate movement of RNA between sensors of an array of sensors.
  • the probes of the array of probes may be coupled to the support by a linking unit.
  • the linking unit may be a polymer, a thiol group, a silane group, or the like.
  • An electric field may be applied to the array of probes.
  • the electric field may be at least about 0.001 volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more.
  • V 0.001 volts
  • the electric field may be at most about 10,000 V, 5,000 V, 1,000 V, 240 V, 120 V, 50 V, 20 V, 15 V, 12 V, 10 V, 9 V, 8 V, 7 V, 6 V, 5 V, 4 V, 3 V, 2 V, 1 V, 0.9 V, 0.8 V, 0.7 V, 0.6 V, 0.5 V, 0.4 V, 0.3 V, 0.2 V, 0.1 V, 0.05 V, 0.01 V, 0.005 V, 0.001 V or less volts.
  • the electric field may be applied through a metal electrode (e.g., gold, platinum, copper, silver), a semiconductor electrode (e.g., silicon, gallium arsenide), an organic semiconductor electrode (e.g., poly(3,4-ethylenedioxythiophene)-poly styrene sulfonate (PDOT:PSS), fullerene doped polymers), or any combination thereof.
  • a metal electrode e.g., gold, platinum, copper, silver
  • a semiconductor electrode e.g., silicon, gallium arsenide
  • an organic semiconductor electrode e.g., poly(3,4-ethylenedioxythiophene)-poly styrene sulfonate (PDOT:PSS), fullerene doped polymers
  • the electric field may be applied over a distance of at least about 0.1 micrometer (pm), 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more micrometers.
  • the electric field may be applied over a distance of at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm, 150 pm, 140 pm, 130 pm, 120 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 5 pm, 1 pm, 0.1 pm, or less micrometers.
  • a pair of gold electrodes 100 micrometers apart can be used to apply a 0.5 V potential to the array of probes.
  • a magnetic field may be applied to the array of probes.
  • the magnetic field may be at least about 1 x 10 6 tesla (IE-6 T), IE-5 T, IE-4 T, IE-3 T, IE-2 T, IE-1 T, 1E0 T, 1E1 T, or more.
  • the magnetic field may be at most about 1E1 T, 1E0 T, IE-1 T, IE-2 T, IE-3 T, IE-4, IE-5 T, IE-6 T, or less.
  • the magnetic field may be applied using a permanent magnet (e.g., a Samarium Cobalt magnet, a Neodymium Iron Boron magnet) or an electromagnet (e.g., a solenoid).
  • the magnetic field may be applied over a distance of at least about 0.1 pm, 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more micrometers.
  • the magnetic field may be applied over a distance of at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm, 150 pm, 140 pm, 130 pm, 120 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 5 pm, 1 pm, 0.1 pm, or less micrometers.
  • a solenoid coil can be placed 500 micrometers behind the array of probes and used to apply a 0.3 tesla magnetic field.
  • the array of probes may be in sensory communication with a sensor.
  • the sensor may be an optical sensor, an electrical sensor, an ion sensor (e.g., a pH sensor), or any combination thereof.
  • the sensor may comprise an electrode.
  • the electrode may be a metal electrode (e.g., gold, copper, an alloy), a semiconductor electrode (e.g., silicon, gallium arsenide, an organic semiconductor), or a combination thereof.
  • the sensor may comprise a plurality of electrodes.
  • the plurality of electrodes may comprise at least about 1, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 250,000, 500,000, 750,000, 1,000,000, or more electrodes.
  • the plurality of electrodes may comprise at most about 1,000,000, 750,000, 500,000, 250,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 100, 50, 10, 5, 1, or less electrodes.
  • the sensor may be among an array of sensors.
  • the array of sensors may comprise sensors of one or more types.
  • an array of sensor may comprise an optical sensor and an electrical sensor.
  • the sensors of the array of sensors may be individually addressable.
  • each electrode of an array of 1,000,000 electrodes can be measured independently of each other electrode.
  • the system may deblock at least a subset of the other probes.
  • the deblocking may be performed with the aid of a reagent.
  • the reagent may be a chemical reagent, a physical process, an enzyme, or any combination thereof.
  • the chemical reagent may be a solvent (e.g., methanol, formamide), a ligand, a metal ion source, a proton source (e.g., an acid), a base (e.g., sodium hydroxide), a radical source, or any combination thereof.
  • the physical process may be applying energy (e.g., heating, sonication), applying light (e.g., an ultraviolet laser), or a combination thereof.
  • the enzyme may be an RNase or a DNase.
  • the RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl, T2, U2, or V.
  • the DNase may be DNase I, II, or micrococcal
  • the system may use the template nucleic acid molecule and the deblocked or active probes to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the deblocked or active probes.
  • Deblocking of a potential subset of probes and using the template nucleic acid molecules and the deblocked or active probes to amplify the template nucleic acid molecules may occur in a well.
  • the well may be a well of a plurality of wells of a sensing array.
  • the well may comprise one or more beads. For example, a single bead may be at least partially contained by the well.
  • the well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, or more micrometers.
  • the well may have a dimension of x by y by z, where x, y, and z are each independently at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.1, or less micrometers.
  • the well can have a width of 150 micrometers, a depth of 105 micrometers, and a height of 437 micrometers.
  • the well can have a length and width of 15 micrometers and a depth of 3 micrometers.
  • the amplicons coupled to the active probes may be a clonal population of nucleic acids.
  • a template nucleic acid molecule can be coupled to a probe surrounded by an array of nucleotides that were recently deblocked.
  • the template nucleic acid molecule can be amplified such that clones of the template nucleic acid molecule occupy the recently deblocked or active probes.
  • the amplicons may be a partially clonal population.
  • the amplicons may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more clonal.
  • the amplicons may be at most 100%, 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less clonal.
  • a template nucleic acid can be coupled to a probe in a well, where all of the other probes in the well are blocked.
  • the other probes after deblocking the other probes and generating amplicons of the template nucleotide, the other probes can have a 100% clonal population, as all of the amplicons are derived from the template nucleic acid.
  • Using the template nucleic acid molecule and the deblocked or active probes to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the deblocked or active probes may further comprise sequencing at least a subset of the amplicons coupled to the active probes or derivatives thereof.
  • the derivatives may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more similar in sequence to the template nucleic acid.
  • the derivatives may be at least about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less similar in sequence to the template nucleic acid.
  • the subset may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the amplicons.
  • the subset may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the amplicons.
  • the sequencing may be sequencing-by synthesis, Sanger sequencing, hydrogen ion detection sequencing, Polony sequencing, nanopore sequencing, rolling circle sequencing, or the like.
  • the nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, change in charge, or any combination thereof.
  • the nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of fluorescence, wavelength of fluorescence, intensity of fluorescence, time resolved fluorescence, or any combination thereof.
  • the sequencing may be performed by methods and systems as described elsewhere herein.
  • the method may be repeated at another array of the plurality of arrays of probes.
  • the method may be repeated for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more other arrays.
  • the method may be repeated for at least about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less other arrays.
  • the method may be repeated at each other array of the plurality of arrays of probes.
  • the repeating the method may further comprise sequencing at least a subset of the substantially clonal populations at the another array of the plurality of arrays of probes.
  • the subjecting the array of probes to conditions sufficient to amplify the target nucleic acid molecule to yield a second plurality of nucleic acid molecules hybridized to the array of probes may comprise conducing a reaction with aid of a recombinase, a polymerase, or any combination thereof.
  • the recombinase may be a Tre recombinase, a Cre recombinase, a Hin recombinase, a Dmcl recombinase, a Rad51 recombinase, or a FLP recombinase.
  • the polymerase may be a DNA polymerase or an RNA polymerase.
  • the RNA polymerase may be an RNA polymerase as described elsewhere herein.
  • the DNA polymerase may be a DNA polymerase of family A, B, C, X, or Y.
  • the method may further comprise sequencing at least a subset of the second plurality of nucleic acid molecules hybridized to the array of probes.
  • the subset may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the second plurality of nucleic acid molecules.
  • the subset may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the second plurality of nucleic acid molecules.
  • the sequencing may be sequencing-by-synthesis, Sanger sequencing, hydrogen ion detection sequencing, Polony sequencing, nanopore sequencing, rolling circle sequencing, or the like.
  • the sequencing may be performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, change in charge, or any combination thereof.
  • the sequencing may be performed with measurement of signals indicative of fluorescence, wavelength of fluorescence, intensity of fluorescence, time resolved fluorescence, or any combination thereof.
  • the sequencing may be performed by methods and systems as described elsewhere herein.
  • An array of probes may be used instead of the array of oligonucleotides in the methods and systems described herein.
  • antibodies can be used instead of oligonucleotides.
  • An array of probes may be intermixed with the array of oligonucleotides.
  • the nucleic acid amplification process can be implemented on an appropriately configured system as described elsewhere herein.
  • the system can bring a template nucleic molecule in contact with an array of nucleotides.
  • the template nucleic acid molecule may bind to an oligonucleotide of the array of oligonucleotides.
  • the template nucleic molecule may comprise a nucleic molecule of interest (e.g., a DNA molecule to be sequenced).
  • the template nucleic molecule may further comprise one or more moieties configured to bind to a probe.
  • the template nucleic molecule can be a fragment of a DNA sample with an oligonucleotide attached to the 3’ end.
  • the template nucleic molecule can be a fragment of a DNA sample with an oligonucleotide attached to the 5’ end.
  • the moiety configured to bind to the probe may be configured to bind with a portion of the probe.
  • the probe can be a 36-base oligonucleotide, and the moiety can be 15 bases complimentary to the free end of the oligonucleotide.
  • the binding of the template nucleic acid molecule with the oligonucleotide may be a hybridization of complimentary bases.
  • the template nucleic acid molecule may have a concentration of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,
  • the template nucleic acid molecule may have a concentration of at most about 1,000, 900, 800, 700, 600, 550, 500, 450, 400, 350, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, or less nanograms per microliter.
  • the template nucleic acid molecule may have a concentration range as defined by any two of the previous values. For example, the template nucleic acid molecule may have a concentration from 0.4 to 4 nanograms per microliter.
  • the system can use the template nucleic acid molecule to synthesize a plurality of nucleic acid molecules at least partially complementary to sequences of other oligonucleotides of the array of oligonucleotides.
  • the synthesizing a plurality of nucleic acid molecules may be a polymerase chain reaction.
  • the plurality of nucleic acid molecules may be RNA molecules, DNA molecules, or oligonucleotides.
  • the RNA molecules may be synthesized from the template nucleic acid molecule with the aid of a reagent.
  • the reagent may be an enzyme.
  • the enzyme may be a RNA polymerase.
  • the RNA polymerase may be a T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), or phage T3 RNA polymerase.
  • the plurality of nucleic acid molecules may be at least partially complimentary to sequences of other oligonucleotides of the array of nucleotides.
  • the plurality of nucleic acids may be at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more complimentary to sequences of other oligonucleotides of the array of nucleotides.
  • the plurality of nucleic acid molecules may be at most about 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less complimentary to sequences of other oligonucleotides of the array of nucleotides.
  • the other oligonucleotides of the array of oligonucleotides may comprise a common sequence.
  • the oligonucleotides of the array of oligonucleotides may be identical.
  • the plurality of nucleic acid molecules may be at least partially complementary to the common sequence.
  • an RNA molecule can be complimentary to all oligonucleotides within a 15 micrometer square area, but not to oligonucleotides outside that area.
  • the other oligonucleotides of the array of oligonucleotides may have a common sequence with other oligonucleotides in an area of at least about 0.01 micrometers 2 (pm 2 ), 0.05 pm 2 , 0.1 pm 2 , 0.5 pm 2 , 1 pm 2 , 5 pm 2 , 10 pm 2 , 15 pm 2 , 20 pm 2 , 25 pm 2 , 50 pm 2 , 75 pm 2 , 100 pm 2 , 150 pm 2 , 200 pm 2 , 250 pm 2 , 500 pm 2 , 750 pm 2 , 1,000 pm 2 , 5,000 pm 2 , 10,000 pm 2 , 50,000 pm 2 , 100,000 pm 2 , or more square micrometers.
  • the other oligonucleotides of the array of oligonucleotides may have a common sequence with other oligonucleotides in an area of at most about 100,000 pm 2 , 50,000 pm 2 , 10,000 pm 2 , 5,000 pm 2 , 1,000 pm 2 , 750 pm 2 , 500 pm 2 , 250 pm 2 , 200 pm 2 , 150 pm 2 , 100 pm 2 , 75 pm 2 , 50 pm 2 , 25 pm 2 , 20 pm 2 , 15 pm 2 , 10 pm 2 , 5 pm 2 , 1 pm 2 , 0.5 pm 2 , 0.1 pm 2 , 0.05 pm 2 , 0.01 pm 2 , or less square micrometers.
  • the synthesizing may be performed with binding of the template nucleic acid molecule to at least two oligonucleotides of the array of oligonucleotides.
  • the binding of the template nucleic acid molecule to at least two oligonucleotides may impart a bridge geometry to the template nucleic acid molecule.
  • the array of oligonucleotides may be among a plurality of arrays of oligonucleotides.
  • the array of oligonucleotides may comprise oligonucleotides having sequences different from oligonucleotides of at least one other array of the plurality of arrays of oligonucleotides.
  • the oligonucleotides coupled to the sensors of a 3x3 grid of sensors can each have a different sequence, leading to 9 different oligonucleotide sequences.
  • the different oligonucleotide sequences may result in less cross contamination of nucleic acids between sensors.
  • the lack of cross contamination may be particularly relevant in sensing arrays that do not comprise wells.
  • each bead of an array of beads having different oligonucleotide sequences can prevent the RNA produced at each bead from diffusing to and binding onto another bead.
  • the arrays of the plurality of arrays of oligonucleotides can be selectively activated for nucleic acid amplification reactions and sequencing by synthesis reactions. Select areas of the arrays of oligonucleotides can be selectively activated for nucleic acid amplification reactions and sequencing by synthesis reactions. A subset of the arrays of oligonucleotides may be blocked from binding to nucleic acid molecules.
  • the template nucleic acid molecule can be among a plurality of template nucleic acid molecules. Individual template nucleic acid molecules can comprise different sequences and distinct template nucleic acid molecules can be bound to distinct select areas of the arrays of oligonucleotides. These distinct template nucleic acid molecules can be selectively amplified or sequenced at corresponding, distinct, or select areas of the arrays of oligonucleotides.
  • Transporting the plurality of nucleic acid molecules produced from the template nucleic acid molecule may be performed when the template nucleic acid is bound to the oligonucleotide.
  • the nucleic acid molecules of the plurality of nucleic acid molecules may be transported from the oligonucleotide to the other oligonucleotides of the array of oligonucleotides.
  • the transportation may be via diffusion.
  • the transportation may be assisted diffusion.
  • the transportation may be an active transportation.
  • the active transportation may comprise cellular transportation methods (e.g., primary active transport, secondary active transport), optical methods (e.g., optical tweezers moving nucleic acid molecules), directed flow (e.g., flowing a liquid carrier in the direction of transport), or any combination thereof.
  • the transportation may be limited. For example, walls of a well can be placed around the nucleic acid molecules to limit the distance of diffusion.
  • the system can bind nucleic acid molecules of the plurality of nucleic acid molecules to the other oligonucleotides of the array of oligonucleotides, thereby generating occupied oligonucleotides.
  • the binding of the nucleic acid to the oligonucleotide may be configured to prevent additional nucleic acids or other template nucleic acid molecules from binding to the oligonucleotide.
  • the binding of the nucleic acid to the oligonucleotide may allow for one template nucleic acid to bind to a given area. For example, a target nucleic acid binds to an oligonucleotide and produces a plurality of nucleic acids that block the surrounding oligonucleotides from other target nucleic acids binding.
  • the synthesis of the plurality of nucleic acid molecules and the transport of the plurality of nucleic acid molecules may occur contemporaneously.
  • an RNA molecule generated by the template nucleic acid molecule can bind to a nearby oligonucleotide immediately after being generated.
  • the synthesis of the plurality of nucleic acid molecules and the transport of the plurality of nucleic acid molecules may occur consecutively.
  • an RNA molecule generated by the template nucleic acid molecule can float in solution for a time before binding to a nearby oligonucleotide.
  • the time between generation of a nucleic acid of the plurality of nucleic acids and the binding of the nucleic acid to the other oligonucleotide may be at least about 0.1 seconds (s), 1 s, 2 s, 3 s, 4 s, 5 s, 10 s, 30 s, 60 s, 120 s, 180 s, 240 s, 300 s, 360 s, 600 s, 1200 s, 2400 s, 3600 s, or more.
  • the time between generation of a nucleic acid of the plurality of nucleic acids and the binding of the nucleic acid to the other oligonucleotide may be at most about 3600 s, 2400 s, 1200 s, 600 s, 360 s, 300 s, 240 s, 180 s, 120 s, 60 s, 30 s, 10 s, 5 s, 4 s, 3 s, 2 s, 1 s, 0.1 s, or less.
  • the system can remove at least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules from the occupied oligonucleotides, thereby generating active oligonucleotides.
  • Removal of the nucleic acid molecules may comprise removing at least a portion of the nucleic acid molecules of the plurality of nucleic acid molecules from the occupied oligonucleotides with a reagent.
  • the removing at least a portion of the nucleic acid molecules may be removing substantially all nucleic acid molecules within an area. For example, all of the oligonucleotides in a well of a sensing array can have the bound nucleic acid molecules removed.
  • the nucleic acids bound to oligonucleotides on the surface of a bead can be removed.
  • the removing at least a portion of the nucleic acid molecules may be removing nucleotides of a given sequence.
  • nucleotides with the sequence ATACG can be removed, but nucleotides with the sequence TTAAG can remain.
  • the reagent may be an enzyme.
  • the enzyme may be an RNase.
  • the RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl, T2, U2, or V.
  • the reagent may be a chemical compound.
  • the chemical compound may be formamide, guanidine, sodium hydroxide, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, or urea.
  • the system can use the template nucleic acid molecule and the active oligonucleotides to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the active oligonucleotides.
  • the amplification may comprise conducting a reaction with aid of at least one recombinase, polymerase, or a combination thereof.
  • the recombinase may be a Tre recombinase, a Cre recombinase, a Hin recombinase, a Dmcl recombinase, a Rad51 recombinase, or a FLP recombinase.
  • the polymerase may be a DNA polymerase or an RNA polymerase.
  • the RNA polymerase may be a T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), or phage T3 RNA polymerase.
  • the DNA polymerase may be a DNA polymerase of family A, B, C, X, or Y.
  • the amplicons coupled to the active oligonucleotides may be a clonal population of nucleic acids.
  • the clonal population of nucleic acids may be clones of the template nucleic acid.
  • the amplicons may be a partially clonal population.
  • the amplicons may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more clonal.
  • the amplicons may be at least about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less clonal.
  • the amplification may further comprise sequencing at least a subset of the amplicons coupled to the active oligonucleotides or derivatives thereof.
  • the derivatives may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more similar in sequence to the template nucleic acid.
  • the derivatives may be at least about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less similar in sequence to the template nucleic acid.
  • the subset may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the amplicons.
  • the subset may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the amplicons.
  • the sequencing may be sequencing-by-synthesis, Sanger sequencing, hydrogen ion detection sequencing, Polony sequencing, nanopore sequencing, rolling circle sequencing, or the like.
  • the nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, change in charge, or any combination thereof.
  • the nucleotide bases incorporated in the sequencing can be detected by a measurement of signals indicative of fluorescence, wavelength of fluorescence, intensity of fluorescence, time resolved fluorescence, or any combination thereof.
  • the sequencing may be performed by methods and systems as described elsewhere herein.
  • the array of oligonucleotides may be among a plurality of arrays of oligonucleotides.
  • the plurality of arrays of oligonucleotides may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more arrays of oligonucleotides.
  • the plurality of arrays of oligonucleotides may be at most about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less arrays of oligonucleotides.
  • the operations may be repeated at another array of the plurality of arrays of oligonucleotides.
  • the operations may be repeated for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more other arrays.
  • the operations may be repeated for at least about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less other arrays.
  • the operations may be repeated at each other array of the plurality of arrays of oligonucleotides.
  • the oligonucleotides of the array of nucleotides may comprise a common sequence.
  • the oligonucleotides of the array of oligonucleotides may be identical.
  • the plurality of nucleic acid molecules may be at least partially complementary to the common sequence.
  • the plurality of nucleic acids may be at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more complimentary to sequences of other oligonucleotides of the array of nucleotides.
  • the plurality of nucleic acid molecules may be at most about 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,
  • the first plurality of nucleic acid molecules may be a plurality of RNA molecules.
  • the synthesizing the plurality of nucleic acid molecules from the target nucleic acid molecule may be performed with the aid of an enzyme.
  • the enzyme may be an RNA polymerase.
  • the RNA polymerase may be a T7 RNA polymerase, a RNAP I, II, or III polymerase, chloroplastic ssRNAP, SP6 RNA polymerase, RNA replicase, mitochondrial RNA polymerase (POLRMT), or phage T3 RNA polymerase.
  • the synthesizing the plurality of nucleic acid molecules from the target nucleic acid molecule may involve transporting a subset of the first plurality of nucleic acid molecules to the other oligonucleotides of the array of oligonucleotides.
  • the transporting may be via diffusion.
  • the transporting may be assisted diffusion.
  • the transporting may be an active transporting.
  • the active transporting may comprise cellular transportation methods (e.g., primary active transport, secondary active transport), optical methods (e.g., optical tweezers moving nucleic acid molecules), directed flow (e.g., flowing a liquid carrier in the direction of transport), or any combination thereof.
  • the transporting may be limited. For example, walls of a well can be placed around the nucleic acid molecules to limit the distance of simple diffusion.
  • the array of oligonucleotides may be attached to a solid support.
  • the solid support may be a bead, planar, a surface of a well, or any combination thereof.
  • a bead functionalized with oligonucleotides can rest on a planar surface.
  • the bead may be a functionalized bead comprising a tosylated surface.
  • the bead may have a diameter of at least about 1 micrometer (pm), 5 pm, 10 pm, 25 pm, 50 pm, 75 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 750 pm, 1,000 pm, or more micrometers.
  • the bead may have a diameter of at most about 1,000 pm, 750 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 150 pm, 100 pm, 75 pm, 50 pm, 25 pm, 10 pm, 5 pm, 1 pm, or less micrometers.
  • the bead may be a component of a well-less sensing array.
  • the bead may be a polymer bead (e.g., latex, polystyrene), a glass bead, a metal bead, or the like.
  • the planar solid support may be a well-less sensing array.
  • the planar solid support may comprise one or more electrodes.
  • the electrodes may be dielectric stacks, metals, or a combination thereof.
  • the electrodes may be nanoneedles.
  • the well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1 pm, 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more micrometers.
  • the well may have a dimension of x by y by z, where x, y, and z are each independently at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm, 150 pm, 140 pm, 130 pm, 120 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 5 pm, 1 pm, 0.1 pm, or less micrometers.
  • the well can have an x dimension of 434 pm, a y dimension of 30 pm, and a z dimension of 510 pm. In another example, the well can have an x and y dimension of 16 pm and a z dimension of 1 pm.
  • the system may comprise mechanisms configured to reduce or eliminate movement of RNA between sensors of an array of sensors.
  • the array of oligonucleotides may be in sensory communication with a sensor.
  • the sensor may be an optical sensor, an electrical sensor, an ion sensor, or any combination thereof.
  • the sensor may comprise an electrode.
  • the electrode may be a metal electrode, a semiconductor electrode, or a combination thereof.
  • the sensor may comprise a plurality of electrodes.
  • the plurality of electrodes may comprise at least about 1, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 250,000, 500,000, 750,000, 1,000,000, or more electrodes.
  • the plurality of electrodes may comprise at most about 1,000,000, 750,000, 500,000, 250,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 100, 50, 10, 5, 1, or less electrodes.
  • the sensor may be among an array of sensors.
  • the array of sensors may comprise sensors of one or more types.
  • an array of sensor may comprise an optical sensor and an electrical sensor.
  • the sensors of the array of sensors may be individually addressable. For example, each electrode of an array of 1,000,000 electrodes can be measured independently of each other electrode.
  • the oligonucleotides of the array of oligonucleotides may be coupled to a support.
  • the support may be planer.
  • the support may be a bead.
  • the bead may be a component of a well-less sensing array.
  • the oligonucleotides may be coupled to a functional unit on the surface of the bead.
  • the support may be the interior of a well.
  • the support may be an electrode.
  • the oligonucleotides of the array of oligonucleotides may be coupled to the support by a linking unit.
  • the linking unit may be a polymer, a thiol group, a silane group, or the like.
  • An electric field may be applied to the array of oligonucleotides.
  • the electric field may be at least about 0.001 volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more.
  • V 0.001 volts
  • the electric field may be at most about 10,000 V, 5,000 V, 1,000 V, 240 V, 120 V, 50 V, 20 V, 15 V, 12 V, 10 V, 9 V, 8 V, 7 V, 6 V, 5 V, 4 V, 3 V, 2 V, 1 V, 0.9 V, 0.8 V, 0.7 V, 0.6 V, 0.5 V, 0.4 V, 0.3 V, 0.2 V, 0.1 V, 0.05 V, 0.01 V, 0.005 V, 0.001 V or less volts.
  • the electric field may be applied through a metal electrode (e.g., gold, platinum, copper, silver), a semiconductor electrode (e.g., silicon, gallium arsenide), an organic semiconductor electrode (e.g., poly(3,4- ethylenedioxythiophene)-polystyrene sulfonate (PDOT:PSS), fullerene doped polymers), or any combination thereof.
  • a metal electrode e.g., gold, platinum, copper, silver
  • a semiconductor electrode e.g., silicon, gallium arsenide
  • an organic semiconductor electrode e.g., poly(3,4- ethylenedioxythiophene)-polystyrene sulfonate (PDOT:PSS), fullerene doped polymers
  • the electric field may be applied over a distance of at least about 0.1 pm, 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more micrometers.
  • the electric field may be applied over a distance of at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm, 150 pm, 140 pm, 130 pm, 120 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 5 pm, 1 pm, 0.1 pm, or less micrometers.
  • a pair of gold electrodes 100 pm apart can be used to apply a 0.5 V potential to the array of oligonucleotides.
  • a magnetic field may be applied to the array of oligonucleotides.
  • the magnetic field may be at least about 1 x 10 6 tesla (IE-6 T, IE-5 T, IE-4 T, IE-3 T, IE-2 T, IE-1 T, 1E0 T, 1E1 T, or more.
  • the magnetic field may be at most about 1E1 T, 1E0 T, IE-1 T, IE-2 T, IE-3 T, IE-4, IE-5 T, IE-6 T, or less.
  • the magnetic field may be applied using a permanent magnet (e.g., a Samarium Cobalt magnet, a Neodymium Iron Boron magnet) or an electromagnet (e.g., a solenoid).
  • the magnetic field may be applied over a distance of at least about 0.1 pm, 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more micrometers.
  • the magnetic field may be applied over a distance of at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm, 150 pm, 140 pm, 130 pm, 120 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 5 pm, 1 pm, 0.1 pm, or less micrometers.
  • a solenoid coil can be placed 500 pm behind the array of oligonucleotides and used to apply a 0.3 tesla magnetic field.
  • Amplicons may be generated in a well.
  • the well may be a well of a plurality of wells of a sensing array.
  • the well may comprise one or more beads.
  • a single bead may be at least partially contained by the well.
  • the well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1 pm, 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more micrometers.
  • the well may have a dimension of x by y by z, where x, y, and z are each independently at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm, 150 pm, 140 pm, 130 pm, 120 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 5 pm, 1 pm, 0.1 pm, or less micrometers.
  • the well can have a width of 150 pm, a depth of 105 pm, and a height of 437 pm. In another example the well can have a length and width of 15 pm and a depth of 3 pm.
  • the amplicons coupled to the active oligonucleotides may be a clonal population of nucleic acids.
  • the clonal population of nucleic acids may be clones of the template nucleic acid.
  • the amplicons may be a partially clonal population.
  • the amplicons may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more clonal.
  • the amplicons may be at least about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less clonal.
  • the method may further comprise sequencing at least a subset of the amplicons coupled to the active oligonucleotides or derivatives thereof.
  • the subset may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the amplicons.
  • the subset may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the amplicons.
  • the sequencing may be sequencing-by-synthesis, Sanger sequencing, hydrogen ion detection sequencing, Polony sequencing, nanopore sequencing, rolling circle sequencing, or the like.
  • the sequencing may be performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, change in charge, or any combination thereof.
  • the sequencing may be performed with measurement of signals indicative of fluorescence, wavelength of fluorescence, intensity of fluorescence, time resolved fluorescence, or any combination thereof.
  • the sequencing may be performed by methods and systems as described elsewhere herein.
  • the conditions sufficient to remove or degrade at least a subset of the first plurality of nucleic acid molecules may comprise removing or degrading the subset of the nucleic acid molecules with a reagent.
  • the reagent may be an enzyme.
  • the enzyme may be an RNase.
  • the RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl, T2, U2, or V.
  • the reagent may be a chemical compound.
  • the chemical compound may be formamide, guanidine, sodium hydroxide, sodium salicylate, dimethyl sulfoxide (DMSO, propylene glycol, or urea.
  • the target nucleic acid molecule hybridized to the oligonucleotide may comprise a promoter sequence.
  • the promoter sequence may be a T7 RNA polymerase promoter sequence.
  • the oligonucleotides of the array of oligonucleotides may comprise a complementary promoter sequence.
  • the complementary promoter sequence may be complimentary to the promoter sequence of the target nucleic acid molecule.
  • the target nucleic acid molecule may be able to hybridize with an oligonucleotide via interaction of the promoter sequence with the complementary promoter sequence.
  • the excluding may be performed by degrading a subset of the plurality of nucleic acid molecules.
  • the degrading may be performed with degrading elements.
  • the degrading elements may be enzymes, chemical degrading elements, light induced degrading elements, or any combination thereof.
  • the enzymes may be an RNase as described elsewhere herein, a DNase as described elsewhere herein, or a combination thereof.
  • the chemical degrading elements may be an acid (e.g.,/ toluene sulfonic acid, nitric acid, ascorbic acid), a base (e.g., an amine, a hydroxide salt), a reductant (e.g., sodium hydride), an oxidizer (e.g., chromate, hydrogen peroxide), or any combination thereof.
  • the light induced degrading element may be a radical generator (e.g., N- bromosuccinimide (NBS), a cadmium selenide nanoparticle with an attached ferrocene molecule).
  • NBS N- bromosuccinimide
  • a light source can be configured to illuminate NBS, generating bromine radicals that degrade RNA.
  • the degrading elements may be coupled to a support.
  • the support may be a particle (e.g., a bead, a microparticle, a nanoparticle), a textured surface (e.g., pillars), or a combination thereof.
  • a plurality of RNase enzymes can be coupled to a plurality of support beads, and the support beads can be placed above the wells.
  • An electric field may be applied to the support.
  • a generator may generate the electric field.
  • the electric field may have a potential of at least about 0.001 volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more.
  • V 0.001 volts
  • the electric field may be at most about 10,000 V, 5,000 V, 1,000 V, 240 V, 120 V, 50 V, 20 V, 15 V, 12 V, 10 V, 9 V, 8 V, 7 V, 6 V, 5 V, 4 V, 3 V, 2 V, 1 V, 0.9 V, 0.8 V, 0.7 V, 0.6 V, 0.5 V, 0.4 V, 0.3 V, 0.2 V, 0.1 V, 0.05 V, 0.01 V, 0.005 V, or less volts.
  • the electric field may be applied via electrodes that are electronically coupled to the generator.
  • the support may be placed on the electrodes. For example, a series of beads can be cast onto an electrode.
  • the electrode may be a metal electrode, a semiconductor electrode, a polymer electrode, or any combination thereof.
  • the system may deblock at least a subset of the other oligonucleotides.
  • the deblocking may be performed with the aid of a reagent.
  • the reagent may be a chemical reagent, a physical process, an enzyme, or any combination thereof.
  • the chemical reagent may be a solvent (e.g., methanol, formamide), a ligand, a metal ion source, a proton source (e.g., an acid), a base (e.g., sodium hydroxide), a radical source, or any combination thereof.
  • the physical process may be applying energy (e.g., heating, sonication), applying light (e.g., an ultraviolet laser), or a combination thereof.
  • the enzyme may be an RNase or a DNase.
  • the RNase may be RNase A, D, H, III, L, P, PH, M, R, T, Tl, T2, U2, or V.
  • the DNase may be DNase I, II, or micrococcal nuclease.
  • the system may use the template nucleic acid molecule and the deblocked oligonucleotides to amplify the template nucleic acid molecule, thereby generating amplicons coupled to the active oligonucleotides
  • the method may further comprise next sequencing at least a subset of the second plurality of nucleic acid molecules hybridized to the array of oligonucleotides.
  • the subset may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or more of the second plurality of nucleic acid molecules.
  • the subset may be at most about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the second plurality of nucleic acid molecules.
  • the sequencing may be sequencing-by-synthesis, Sanger sequencing, hydrogen ion detection sequencing, Polony sequencing, nanopore sequencing, rolling circle sequencing, or the like.
  • the sequencing may be performed with measurement of signals indicative of impedance, change in impedance, conductivity, change in conductivity, charge, change in charge, or any combination thereof.
  • the sequencing may be performed with measurement of signals indicative of fluorescence, wavelength of fluorescence, intensity of fluorescence, time resolved fluorescence, or any combination thereof.
  • the sequencing may be performed by methods and systems as described elsewhere herein.
  • the method may be repeated at another array of the plurality of arrays of oligonucleotides.
  • the method may be repeated for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more other arrays.
  • the method may be repeated for at least about 1,000,000, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 250, 100, 50, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less other arrays.
  • the method may be repeated at each other array of the plurality of arrays of oligonucleotides.
  • the repeating the method may further comprise sequencing at least a subset of the substantially clonal populations at another array of the plurality of arrays of oligonucleotides.
  • FIG. 1 shows a computer system 101 that is programmed or otherwise configured to perform the methods described herein.
  • the computer system 101 can regulate various aspects of the present disclosure, such as, for example, determining the ratio of target nucleic acids to non-target nucleic acids.
  • the computer system 101 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device.
  • the electronic device can be a mobile electronic device.
  • the computer system 101 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 105, which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the computer system 101 also includes memory or memory location 110 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 115 (e.g., hard disk), communication interface 120 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 125, such as cache, other memory, data storage and/or electronic display adapters.
  • the memory 110, storage unit 115, interface 120 and peripheral devices 125 are in communication with the CPU 105 through a communication bus (solid lines), such as a motherboard.
  • the storage unit 115 can be a data storage unit (or data repository) for storing data.
  • the computer system 101 can be operatively coupled to a computer network (“network”) 130 with the aid of the communication interface 120.
  • the network 130 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
  • the network 130 in some cases is a telecommunication and/or data network.
  • the network 130 can include one or more computer servers, which can enable distributed computing, such as cloud computing.
  • the network 1130 in some cases with the aid of the computer system 101, can implement a peer-to-peer network, which may enable devices coupled to the computer system 101 to behave as a client or a server.
  • the CPU 105 can execute a sequence of machine-readable instructions, which can be embodied in a program or software.
  • the instructions may be stored in a memory location, such as the memory 110.
  • the instructions can be directed to the CPU 105, which can subsequently program or otherwise configure the CPU 105 to implement methods of the present disclosure. Examples of operations performed by the CPU 105 can include fetch, decode, execute, and writeback.
  • the CPU 105 can be part of a circuit, such as an integrated circuit.
  • a circuit such as an integrated circuit.
  • One or more other components of the system 101 can be included in the circuit.
  • the circuit is an application specific integrated circuit (ASIC).
  • ASIC application specific integrated circuit
  • the storage unit 115 can store files, such as drivers, libraries and saved programs.
  • the storage unit 115 can store user data, e.g., user preferences and user programs.
  • the computer system 101 in some cases can include one or more additional data storage units that are external to the computer system 101, such as located on a remote server that is in communication with the computer system 101 through an intranet or the Internet.
  • the computer system 101 can communicate with one or more remote computer systems through the network 130.
  • the computer system 101 can communicate with a remote computer system of a user (e.g., a cellular network).
  • remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants.
  • the user can access the computer system 101 via the network 130.
  • Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 101, such as, for example, on the memory 110 or electronic storage unit 115.
  • the machine executable or machine readable code can be provided in the form of software.
  • the code can be executed by the processor 105.
  • the code can be retrieved from the storage unit 115 and stored on the memory 110 for ready access by the processor 105.
  • the electronic storage unit 115 can be precluded, and machine-executable instructions are stored on memory 110.
  • the code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code or can be compiled during runtime.
  • the code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as- compiled fashion.
  • aspects of the systems and methods provided herein can be embodied in programming.
  • Various aspects of the technology may be thought of as “products” or “articles of manufacture” may be in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
  • Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read only memory, random-access memory, flash memory) or a hard disk.
  • “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • a machine readable medium such as computer-executable code
  • a tangible storage medium such as computer-executable code
  • Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings.
  • Volatile storage media include dynamic memory, such as main memory of such a computer platform.
  • Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system.
  • Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications.
  • RF radio frequency
  • IR infrared
  • Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data.
  • Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
  • the computer system 101 can include or be in communication with an electronic display 135 that comprises a user interface (UI) 140 for providing, for example, the ratio of target nucleic acid molecules to non-target nucleic acid molecules or the flow rate of the solution comprising the target nucleic acid molecules.
  • UI user interface
  • Examples of UTs include, without limitation, a graphical user interface (GUI) and web-based user interface.
  • Methods and systems of the present disclosure can be implemented by way of one or more algorithms.
  • An algorithm can be implemented by way of software upon execution by the central processing unit 105.
  • the algorithm can, for example, determine the ratio of target nucleic acid molecules to non-target nucleic acid molecules or the flow rate of the solution comprising the target nucleic acid molecules.
  • Target DNA molecules comprising a target sequence are mixed with non-target DNA molecules comprising a non-target sequence in a 1 -Butyl-3 -methylimidazolium solution at a ratio of 50: 1 target DNA molecule to non-target DNA molecule.
  • the target DNA molecules comprise 10 nucleotide bases.
  • the non-target DNA molecules comprise 50 nucleotide bases.
  • the 1-Butyl- 3-methylimidazolium solution is formulated to achieve 100 Pa s viscosity.
  • the solution comprising the target DNA molecules and non-target DNA molecules is eluted onto a substrate comprising 100,000 oligonucleotide probes at a flow rate of 7 pL/min.
  • the oligonucleotide probes are occupied by substantially target DNA molecules.
  • the bound target DNA molecules are clonally amplified to generate amplicons. These amplicons undergo multiple phase DNA synthesis reactions to generate synthesized complementary DNA molecules of a known sequence, thereby generating the nucleotide sequence that is complementary to the target sequence.
  • Target DNA molecules comprising a target sequence are mixed with non-target DNA molecules comprising a non-target sequence in a 1 -Butyl-3 -methylimidazolium solution at a ratio of 50: 1 target DNA molecule to non-target DNA molecule.
  • the target DNA molecules comprise 10 nucleotide bases.
  • the non-target DNA molecules comprise 50 nucleotide bases.
  • the 1-Butyl- 3 -methylimidazolium solution is formulated to achieve 100 Pa s viscosity.
  • the solution comprising the target DNA molecules and non-target DNA molecules is eluted onto a substrate comprising 100,000 oligonucleotide probes at a flow rate of 7 pL/min.
  • a population of amplification products is introduced comprising a blocking sequence complementary to the non target sequence.
  • the oligonucleotide probes are occupied by both target and non-target DNA molecules in a ratio of 50:1.
  • the blocking sequence inhibits nucleic acid extension of the non target amplification products, the poisoned sequence in the colony of interest, thus isolating the target sequence and allowing the target DNA molecules to amplify.
  • the bound target DNA molecules are clonally amplified to generate amplicons.
  • amplicons undergo multiple phases of DNA synthesis reactions to generate synthesized complementary DNA molecules of a known sequence, thereby generating a nucleotide molecule that is complementary to the target DNA molecule.
  • a washing solution is applied to remove the non-amplified non-target nucleic acid molecules leaving the amplified target sequence for further analysis.

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Abstract

L'invention concerne un procédé d'amplification d'acides nucléiques. Les systèmes et les procédés décrits dans la description permettent d'amplifier des oligonucléotides cibles et non cibles sur un réseau où il peut y avoir une multitude d'oligonucléotides cibles et non cibles. Le ciblage d'oligonucléotides par rapport à leurs brins complémentaires est effectué à l'aide d'un débit spécifique sur le réseau. Le procédé d'amplification peut bloquer l'amplification des oligonucléotides non cibles et peut générer des amplicons de la cible immobilisée au niveau du site de réaction à une vitesse suffisante pour générer des amplicons sans amplification d'autres cibles sur le réseau. Les procédés et les compositions décrits dans la description peuvent fournir des procédés et des mécanismes plus efficaces d'amplification d'acides nucléiques.
PCT/US2021/018558 2020-02-19 2021-02-18 Procédés et systèmes de traitement d'acides nucléiques Ceased WO2021168097A1 (fr)

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AU2017299803B2 (en) * 2016-07-22 2023-06-29 Illumina, Inc. Single cell whole genome libraries and combinatorial indexing methods of making thereof
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US20180187252A1 (en) * 2017-01-05 2018-07-05 Illumina, Inc. Kinetic exclusion amplification of nucleic acid libraries

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