WO2008112683A2 - Synthèse de gènes par amplification d'assemblages circulaires - Google Patents

Synthèse de gènes par amplification d'assemblages circulaires Download PDF

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WO2008112683A2
WO2008112683A2 PCT/US2008/056504 US2008056504W WO2008112683A2 WO 2008112683 A2 WO2008112683 A2 WO 2008112683A2 US 2008056504 W US2008056504 W US 2008056504W WO 2008112683 A2 WO2008112683 A2 WO 2008112683A2
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sample
dna
oligonucleotides
contacting
synthetic
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WO2008112683A3 (fr
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George M. Church
Duhee Bang
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • Embodiments of the present invention relate in general to methods for reducing and/or eliminating errors resulting from DNA assembly and/or DNA synthesis.
  • the present invention provides novel methods for synthesizing oligonucleotide (e.g., DNA) sequences (e.g., one or more genes, portions of a chromosome and/or the like) using a set of oligonucleotides (e.g., DNA oligonucleotides (e.g., synthetic DNA oligonucleotides, DNA fragments from natural sources and/or the like)).
  • oligonucleotide e.g., DNA sequences (e.g., one or more genes, portions of a chromosome and/or the like)
  • a set of oligonucleotides e.g., DNA oligonucleotides (e.g., synthetic DNA oligonucleotides, DNA fragments from natural sources and/or the like)).
  • a method for synthesizing a synthetic, single-stranded polynucleotide including the steps of providing a first sample having a plurality of plus strand oligonucleotides, providing a second sample having a plurality of minus strand oligonucleotides, contacting the first sample and the second sample to form a third sample, annealing plus strand oligonucleotides and minus strand oligonucleotides in the third sample, contacting the third sample with a ligase, and contacting the third sample with one or more exonucleases.
  • the method further includes amplifying the synthetic, single-stranded polynucleotide sequence such as by PCR, rolling circle amplification or hyper-branched rolling circle amplification.
  • a plus strand oligonucleotide overlaps a minus strand oligonucleotide by about 20 nucleotides.
  • the single-stranded polynucleotide structure is a circularized structure such as circularized, single- stranded DNA.
  • the plurality of plus strand oligonucleotides in the first sample are phosphorylated prior to annealing.
  • the one or more exonucleases are Exol and/or Exo III.
  • a synthetic, single-stranded polynucleotide structure having reduced errors when compared to a synthetic, single-stranded reference polynucleotide remains.
  • a method for synthesizing a synthetic, double- stranded polynucleotide including the steps of providing a first sample having a plurality of plus strand oligonucleotides, providing a second sample having a plurality of minus strand oligonucleotides, contacting the first sample and the second sample to form a third sample, annealing plus strand oligonucleotides and minus strand oligonucleotides in the third sample, contacting the third sample with a ligase, and contacting the third sample with one or more exonucleases and an endonuclease.
  • the method further includes amplifying the synthetic, double-stranded polynucleotide sequence, such as by PCR, rolling circle amplification or hyper- branched rolling circle amplification.
  • a plus strand oligonucleotide overlaps a minus strand oligonucleotide by about 20 nucleotides.
  • the double-stranded polynucleotide structure is free of gaps.
  • the double-stranded polynucleotide structure is a circularized structure such as circularized, double-stranded DNA.
  • the plurality of plus strand oligonucleotides in the first sample and the plurality of minus strand oligonucleotides in the second sample are phosphorylated prior to annealing.
  • the one or more exonucleases are Exol and/or Exo III and the endonuclease is Sl nuclease, endonuclease I or endonuclease V.
  • a synthetic, double-stranded polynucleotide structure having reduced errors when compared to a synthetic, double- stranded reference polynucleotide remains.
  • a method for correcting mismatches in a synthetic polynucleotide sequence including the steps of providing a first sample having a plurality of plus strand oligonucleotides, providing a second sample having a plurality of minus strand oligonucleotides, contacting the first sample and the second sample to form a third sample, annealing plus strand oligonucleotides and minus strand oligonucleotides in the third sample, contacting the third sample with a ligase, and contacting the third sample with a mismatch cleavage endonuclease and one or more exonucleases such that mismatches are corrected.
  • a method for selecting a correctly assembled, synthetic, single-stranded polynucleotide including providing a first sample having a plurality of plus strand oligonucleotides, providing a second sample having a plurality of minus strand oligonucleotides, contacting the first sample and the second sample to form a third sample, annealing plus strand oligonucleotides and minus strand oligonucleotides in the third sample, contacting the third sample with a ligase, and contacting the third sample with one or more exonucleases, such that a correctly assembled, synthetic, single-stranded polynucleotide structure remains is provided.
  • a method for synthesizing a correctly assembled, synthetic, double-stranded polynucleotide including the steps of providing a first sample having a plurality of plus strand oligonucleotides, providing a second sample having a plurality of minus strand oligonucleotides, contacting the first sample and the second sample to form a third sample, annealing plus strand oligonucleotides and minus strand oligonucleotides in the third sample, contacting the third sample with a ligase, and contacting the third sample with one or more exonucleases and an endonuclease such that a correctly assembled, synthetic, double-stranded polynucleotide structure remains.
  • a method for reducing errors during synthesis of a synthetic polynucleotide such that a synthetic polynucleotide structure is generated having reduced errors when compared to a synthetic reference polynucleotide using a method and/or composition described herein.
  • a synthetic polynucleotide is a synthetic, single-stranded polynucleotide.
  • a synthetic polynucleotide is a synthetic, double-stranded polynucleotide.
  • FIGS 1A-1B schematically depict the assembly of circular single-stranded DNA.
  • (A) depicts the assembly process.
  • (B) depicts an additional purification process.
  • Figures 2A-2B schematically depict the assembly of circular double-stranded DNA.
  • (A) depicts the assembly process.
  • (B) depicts the use of mismatch cleavage endonucleases together with exonucleases.
  • Figure 3 depicts an agarose gel of products obtained during assembly of circular double-stranded DNA.
  • Lane 1 shows a 2-log ladder (New England Biolabs, Beverly, MA).
  • Lane 2 shows a sample taken after the circular ligation reaction of 48 5'-phosphorylated oligonucleotides using AMPLIGASE ® .
  • Lane 3 shows a sample from an exonuclease cocktail-treated ligation mixture.
  • Lane 4 shows a sample from an exonuclease/endonuclease cocktail- and SURVEYORTM nuclease- treated ligation mixture.
  • Figure 4 depicts an agarose gel showing DNA amplification of an exonuclease cocktail- or exonuclease/endonuclease cocktail-treated ligation mixture.
  • Lane 1 shows a 2-log ladder (New England Biolabs).
  • Lane 2 shows a sample from a PCR amplification of an exonuclease cocktail-treated ligation mixture.
  • Lane 3 shows a sample from a PCR amplification of an exonuclease/endonuclease cocktail- and SURVEYORTM nuclease-treated ligation mixture.
  • Figure 5 depicts a schematic of circular assembly amplification showing the three-tier process in detail.
  • Figures 6A-6C depicts the Dpo4 gene (1.05kb) constructed by circular assembly amplification of 48 oligonucleotides.
  • (A) depicts an agarose gel of products obtained during the amplification. Lane 1 & 2; Constructs from PCR reactions on ligation mixtures with and without a guiding oligonucleotide. Lane 3 & 4; Constructs from PCR reactions on ligation mixtures treated with exonucleases. Lane 5 to 10; Constructs from PCR reactions on ligation mixtures treated with exonucleases followed by treatments with different concentrations of mismatch-cleaving endonuclease.
  • FIGS 7A-7C Depicts Circular assembly amplification for the synthesis of Pfu DNA polymerase.
  • A Schematic representation of the synthesis.
  • B PCR products resulting from the circular assembly amplification of the Pfu Polymerase gene fragments.
  • C PCR products resulting from the USER-mediated circular ligation of the three Pfu gene fragments assembled in (B).
  • FIGS 8A-8C Depicts Dpo4 gene (1.05kb) construction by various methods.
  • A circular assembly amplification of 48 oligonucleotides. Use of insufficient exonuclease led to incomplete exonuclease degradation as shown in (A) lane 3 and lane 9.
  • B & (C) PCA reaction performed at two different annealing temperatures (65 0 C for B and 7O 0 C for C); Lane 4 from both experiments was cloned.
  • FIGS 9A-B Fraction of clones with incorrect full length Dpo4 sequence made by various methods. Experiments are performed at annealing temperature (a) 65 0 C and (b) 7O 0 C, respectively. Error bars denote a standard deviation (s.d.).
  • Figure 10 Depicts Circular assembly amplification for the construction of genes of various sizes assessed by the synthesis of Pfu DNA polymerase gene fragments.
  • FIGS 1 IA-11C Synthesis of a human minisatellite repeat sequence by circular assembly amplification;
  • A Target DNA sequence (GenBank accession code: NTOl 1515).
  • B Lane 1 to 8; PCR products resulting from PCA reactions performed with different oligonucleotide concentrations. Lane 9 & 10; products resulting from PCR reactions on ligation mixtures. Lane 11 & 12; products resulting from PCR reactions on ligation mixtures treated with exonuc leases. Lane 12 (arrow) was used for further cloning and characterization of one clone shown in (C).
  • FIGs 12A-12B USER mediated-circular assembly amplification for the synthesis of tandem repeat Dpo4.
  • A Schematic representation of our strategy.
  • B Verification of the order of tandem repeats of the Dpo4 genes using PCR amplification of the regions shown by crescent lines in the diagram below. Lanes on the gel are labeled according to the diagram below the gel.
  • Figures 13A-13S DNA sequences of various oligonucleotides and polynucleotidesl
  • A Dpo4 sequence.
  • B 24 plus strand oligonucleotides for Dpo4.
  • C 24 minus oligonucleotides for Dpo4.
  • Q Amplification primers (containing deoxyU, dU) for PCR amplification of three segments.
  • Figures 14A-14B Comparison of sequence errors generated by various methods for the synthesis of Dpo4
  • A number of errors per sequencing. A breakdown of the types of errors detected by sequencing is shown in parenthesis (deletion: insertion: transition: transversion).
  • B Fraction of clones with incorrect full-length sequence.
  • the principles of the present invention may be applied with particular advantage for decreasing the error rate in multiplexing methods wherein polynucleotides are constructed by the assembly of oligonucleotides that have partially overlapping sequences, as well as for correcting errors that occur during polynucleotide synthesis.
  • polynucleotide sequences e.g., DNA
  • oligonucleotides e.g., DNA
  • various nucleases e.g., exonucleases, and/or endonucleases
  • a facile synthetic oligonucleotide error correction method using a mixture of endonucleases and exonucleases is also described herein.
  • polynucleotide e.g., DNA
  • polynucleotide e.g., DNA sequences
  • the terms “reduced errors,” “decreased error rate” and the like are intended to include, but are not limited to, a synthetic oligonucleotide and/or polynucleotide sequence which, when compared with a synthetic oligonucleotide and/or polynucleotide sequence synthesized by a method known in the art, has fewer incorrectly incorporated nucleic acids than the synthetic oligonucleotide and/or polynucleotide sequence synthesized by methods known in the art.
  • the number of errors in a synthetic oligonucleotide and/or polynucleotide sequence can be determined by comparing the oligonucleotide and/or polynucleotide sequence to a reference oligonucleotide and/or polynucleotide sequence (e.g., a template oligonucleotide and/or polynucleotide sequence or the oligonucleotide and/or polynucleotide sequence synthesized by methods known in the art) using methods known in the art (e.g., nucleic acid sequencing, nuclease digestion (e.g., endonuclease and/or exonuclease digestion) and the like).
  • a reference oligonucleotide and/or polynucleotide sequence e.g., a template oligonucleotide and/or polynucleotide sequence or the oligonucleotide and/or poly
  • a synthetic oligonucleotide and/or polynucleotide sequence will have less errors than the synthetic oligonucleotide and/or polynucleotide sequence synthesized by a method known in the art.
  • the error rate is reduced by 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%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 2000%, 3000%, 4000%, 5000%, 10,000%, 15,000%, 20,000% or more.
  • the error rate may be reduced by addition of the exonuclease alone or with endonuc lease.
  • the error rate may be reduced by 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%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 2000%, 3000%, 4000%, 5000%, 10,000%, 15,000%, 20,000% or more.
  • the error rate may be further reduced by addition of endonuclease.
  • the error rate may be reduced by a total of 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%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 2000%, 3000%, 4000%, 5000%, 10,000%, 15,000%, 20,000% or more.
  • mismatch binding enzymes can be included to further reduce error rates.
  • nucleic acid molecule As used herein, the terms “nucleic acid molecule,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Non- limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of a sequence, isolated RNA of a sequence, nucleic acid probes, and primers.
  • Polynucleotides useful in the methods of the invention may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.
  • a polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • T thymine
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • Polynucleotides may optionally include one or more non- standard nucleotide(s), nucleotide analog(s) and/or modified nucleo
  • modified nucleotides include, but are not limited to 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5 -methylaminomethyluracil, 5 -methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbox
  • the assembly methods described herein are not limited to circular structures.
  • the annealed oligonucleotides e.g., DNA
  • the annealed oligonucleotides are protected from exonuclease digestion using a variety of art-recognized methods such as, for example, assembly to other topological structures, or an introduction of exonuclease resistant chemical moieties to the DNA, combinations thereof and the like.
  • Correctly assembled circular polynucleotides (e.g., DNA) or correctly assembled polynucleotides otherwise protected from exonuclease digestion are stable during the nuclease treatments, but incorrectly assembled DNA, intermediate DNA fragments, and residual starting oligonucleotides are degraded.
  • a mixture of one or more mismatch cleavage endonucleases e.g., CEL I nuclease, T7 endonuclease or the like
  • exonucleases can be used (e.g., prior to an optional amplification step (e.g., a DNA amplification step)) to eliminate errors (e.g., mutations) incurred during oligonucleotide synthesis. This elimination of errors prior to amplification prevents a major source of errors during gene synthesis.
  • exonuclease refers to enzymes that cleave nucleotides one at a time from an end of a polynucleotide chain. These enzymes hydrolyze phosphodiester bonds from either the 3' or 5' terminus of polynucleotide molecules.
  • exonucleases include, but are not limited to, lambda Exo, T7 Exo, Exol, Exo III, RecJf, Exo T BAL-31 nuclease and the like.
  • a variety of exonucleases are known in the art and are available from commercial vendors (See New England Biolabs 2005 -06 Catalog and Technical Reference).
  • endonucleases refers to enzymes that cleave a phosphodiester bond within a polynucleotide chain.
  • endonucleases include, but are not limited to, mung bean nuclease, BAL-31 nuclease, and the like. Endonucleases also include mismatch cleavage endonucleases.
  • mismatch cleavage endonuclease refers to endonucleases that cleave duplex oligonucleotide mismatches including, but not limited to, enzymes such as Sl nuclease, CEL I nuclease, RNase, T7 endonuclease I, T4 endonuclease VII, Endo V, Mut S, Cleavase, Mut Y, thymine glycosylase and the like.
  • enzymes such as Sl nuclease, CEL I nuclease, RNase, T7 endonuclease I, T4 endonuclease VII, Endo V, Mut S, Cleavase, Mut Y, thymine glycosylase and the like.
  • endonucleases are known in the art and are available from commercial vendors (See New England Biolabs 2005 -06 Catalog and Technical Reference).
  • exonuclease-resistant and “exonuclease resistance” refer to one or more modifications of an oligonucleotide sequence that renders it resistant to degradation by exonucleases.
  • a variety of methods for rendering an oligonucleotide exonuclease-resistant include, but are not limited to, phosphoramidate internucleotide linkages, phosphormonothioate internucleotide linkages, phosphorodithioate internucleotide linkages, one or more terminal diols at the 3' and/or the 5' end, chromophore treatment, incorporation of 2'- alkoxy sugar modifications and the like.
  • Compositions and methods for rendering an oligonucleotide exonuclease-resistant are described in Povirk and Goldberg (1985) Biochemistry 24:4035; Monia et al. (1996) J. Biol. Chem. 271 :14533; Weis et al, U.S. Patent No. 5,245,022 and Froehler, U.S. Patent No. 5,256,775.
  • an oligonucleotide and/or a polynucleotide may be (e.g., temporarily) immobilized on a substrate to render it exonuclease-resistant, e.g., via a 5'-end and/or a 3'-end.
  • Substrates include, but are not limited to, plates, microarrays, slides (e.g., microscope slides), multi-well plates, Petri dishes, columns, beads, cells (e.g., S. aureus), agarose, particles, strands, gels, emulsions, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates and the like.
  • Substrate can refer to a matrix upon which oligonucleotides and/or polynucleotides are placed.
  • the support can be solid, semi-solid or a gel.
  • solid substrate includes, but is not limited to, materials such as glass silica, polymeric materials and the like.
  • solid support materials include, but are not limited to, glass, polacryloylmorpholide, silica, controlled pore glass, nitrocellulose, nylon, polystyrene, polystyrene/latex, carboxyl modified Teflon, polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO 2 , SiN 4 , modified silicon, or (poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene, polycarbonate, or combinations thereof.
  • Solid substrates include, but are not limited to, slides, plates, beads, particles, spheres, strands, sheets, tubing, containers (e.g., test tubes, micro fuge tubes, bowls, trays and the like), capillaries, films, polymeric chips and the like.
  • containers e.g., test tubes, micro fuge tubes, bowls, trays and the like
  • capillaries films, polymeric chips and the like.
  • at least one surface of the substrate is partially planar.
  • semi-solid includes, but is not limited to, a compressible matrix with both a solid and a liquid component, wherein the liquid occupies pores, spaces or other interstices between the solid matrix elements.
  • Semi-solid supports can be selected from polyacrylamide, cellulose, polyamide (nylon) and crossed linked agarose, dextran and polyethylene glycol.
  • a substrate can include a variety of different binding moieties to permit the coupling of one or more polynucleotides and/or concatemers to the support.
  • a suitable binding moiety includes, but is not limited to, a capture moiety such as a hydrophobic compound, an oligonucleotide, an antibody or fragment of an antibody, a protein, a peptide, a chemical cross-linker, an intercalator, a molecular cage (e.g., within a cage or other structure, e.g., protein cages, fullerene cages, zeolite cages, photon cages, and the like), or one or more elements of a capture pair, e.g., biotin- avidin, biotin-streptavidin, NHS-ester and the like, a thioether linkage, static charge interactions, van der Waals forces and the like.
  • a capture moiety such as a hydrophobic compound, an oligonucleotide,
  • a support can be functionalized with any of a variety of functional groups known in the art. Commonly used chemical functional groups include, but are not limited to, carboxyl, amino, hydroxyl, hydrazide, amide, chloromethyl, epoxy, aldehyde and the like.
  • polynucleotide structures described herein are amplified using methods including, but are not limited to, polymerase chain reaction (PCR), bridge PCR, emulsion PCR (ePCR), thermophilic helicase-dependent amplification (tHDA), linear polymerase reactions, strand displacement amplification (e.g., multiple displacement amplification), RCA (e.g., hyperbranched RCA, padlock probe RCA, linear RCA and the like) (Hutchison (2005) Proc. Natl. Acad. Sci. USA 102:17332), nucleic acid sequence-based amplification (NASBA) and the like, which are disclosed in the following references: Schweitzer et al. (2002) Nat. Biotech.
  • PCR polymerase chain reaction
  • ePCR emulsion PCR
  • tHDA thermophilic helicase-dependent amplification
  • linear polymerase reactions e.g., multiple displacement amplification
  • RCA e.g., hyperbranched RCA, padlock
  • JP 4-262799 rolling circle amplification
  • Church U.S. Patent Nos. 6,432,360, 6,511,803 and US 6,485,944 (replica amplification (e.g., polony amplification”); and the like.
  • PCR methods are provided.
  • the term "PCR” encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, emulsion PCR (ePCR) and the like. Reaction volumes range from a few hundred to a few hundred microliters.
  • Reverse transcription PCR or “RT-PCR,” refers to PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, e.g., Tecott et al, U.S. Patent No. 5,168,038.
  • Real-time PCR refers to PCR for which the amount of reaction product is monitored as the reaction proceeds.
  • Nested PCR refers to a two-stage PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon.
  • initial primers in reference to a nested amplification reaction refer to the primers used to generate a first amplicon
  • secondary primers refer to the one or more primers used to generate a second, or nested, amplicon.
  • Multiplexed PCR refers to PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture (See, e.g., Bernard et al (1999) Anal. Biochem., 273:221-228 (two-color real-time PCR)). Typically, distinct sets of primers are employed for each sequence being amplified.
  • Quantitative PCR refers to PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen. Quantitative PCR includes both absolute quantitation and relative quantitation of target sequences. Quantitative measurements are made using one or more reference sequences that may be assayed separately or together with a target sequence.
  • the reference sequence may be endogenous or exogenous to a sample or specimen, and in the latter case, may comprise one or more competitor templates.
  • Typical endogenous reference sequences include segments of transcripts of the following genes: ⁇ -actin, GAPDH, ⁇ 2 - microglobulin, ribosomal RNA, and the like. Techniques for quantitative PCR are well-known to those of ordinary skill in the art (See, e.g., Freeman et al. (1999) Biotechniques 26:112; Becker-Andre et al. (1989) Nucleic Acids Res. 17:9437; Zimmerman et al. (1996) Biotechniques 21 :268; Diviacco et al. (1992) Gene 122:3013; Becker-Andre et al. (1989) Nucleic Acids Res. 17:9437).
  • the assembly of polynucleotide (e.g., DNA) sequences is accomplished by assembly of circular and/or linear single-stranded polynucleotides (e.g., DNA).
  • the assembly of polynucleotide (e.g., DNA) sequences is accomplished by assembly of circular and/or linear double-stranded polynucleotides (e.g., DNA).
  • plus-strand oligonucleotide sequences and minus-strand oligonucleotide sequences are hybridized.
  • selective hybridization occurs when two nucleic acid sequences are substantially complementary, i.e., at least about 65% 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% complementary over a stretch of at least 14 to 25 nucleotides. See Kanehisa (1984) Nucleic Acids Res. 12: 203.
  • the terms “complementary” or “substantially complementary” refer to the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid.
  • Complementary nucleotides are, generally, A and T (or A and U), or C and G.
  • Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, or from about 98 to 100%.
  • substantial complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement.
  • selective hybridization will occur when there is at least about 65% complementarity over a stretch of at least 14 to 25 nucleotides, at least about 75% complementarity, or at least about 90% complementarity. See Kanehisa (1984) Nucl. Acids Res. 12:203.
  • Hybridization temperature varies inversely with primer annealing efficiency, as does the concentration of organic solvents, e.g., formamide, that might be included in a hybridization mixture, while increases in salt concentration facilitate binding. Under stringent hybridization conditions, longer probes hybridize more efficiently than do shorter ones, which are sufficient under more permissive conditions. Stringent hybridization conditions typically include salt concentrations of less than about 1 M, less than about 500 mM, or less than about 200 mM. Hybridization temperatures range from as low as 0 0 C to greater than 22 0 C, greater than about 30 0 C, and (most often) in excess of about 37 0 C.
  • the hybridization temperature can be about: 6O 0 C, 65 0 C, 7O 0 C, 75 0 C, and 80°c. Higher temperatures may be used to promote more stringent hybridization. For example, more specific hybridization may be achieved with higher temperatures when longer fragments are used. As several factors affect the stringency of hybridization, the combination of parameters is more important than the absolute measure of any one alone. Hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • T m is used in reference to "melting temperature.” Melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • T m is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • one or more oligonucleotide sequences are ligated together, e.g., after hybridization of plus-strands and minus-strands.
  • ligation is intended to include the formation of a covalent bond or linkage between the termini of two or more nucleic acids, e.g., oligonucleotides and/or polynucleotides. The nature of the bond or linkage may vary widely and the ligation may be carried out enzymatically or chemically.
  • Ligations are typically carried out enzymatically to form a phosphodiester linkage between a 5' carbon of a terminal nucleotide of one oligonucleotide with 3' carbon of another oligonucleotide.
  • a variety of ligation reactions are described in the following references: Whitely et al., U.S. Patent No. 4,883,750; Letsinger et al., U.S. Patent No. 5,476,930; Fung et al., U.S. Patent No. 5,593,826; Kool, U.S. Patent No. 5,426,180; Landegren et al., U.S. Patent No. 5,871,921; Xu and Kool (1999) Nucl.
  • methods of determining the nucleic acid sequence of one or more polynucleotides are provided. Determination of the nucleic acid sequence of a clonally amplified concatemer can be performed using variety of sequencing methods known in the art including, but not limited to, sequencing by hybridization (SBH), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan reporter probe digestion, pyrosequencing, fluorescent in situ sequencing (FISSEQ), allele-specif ⁇ c oligonucleotide ligation assays (e.g., oligonucleotide ligation assay (OLA), single template molecule OLA using a ligated linear probe and a rolling circle amplification (RCA) readout, ligated padlock probes, and/or single template molecule OLA using a ligated circular padlock probe and a rolling circle amplification
  • SBH sequencing by hybridization
  • detectable markers include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers and the like.
  • fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin and the like.
  • bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, aequorin and the like.
  • enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucorinidases, phosphatases, peroxidases, cholinesterases and the like.
  • Identifiable markers also include radioactive compounds such as 1251, 35S, 14C, or 3H. Identifiable markers are commercially available from a variety of sources.
  • the circular assembly method offers multiple advantages.
  • the exonucleases can be used in a single reaction vessel, i.e. a one-pot reaction, in a highly robust manner, and hence can be applied with automated gene synthesis processes.
  • one or more endonucleases may also be included in the same reaction vessel, or added to the reaction vessel after incubation with the exonucleases.
  • the assembly of ⁇ lkb gene an average length of a gene
  • the methods of the invention provide significant cost benefits by decreasing the expense of gene synthesis.
  • embodiments of the invention can be used to readily construct highly repetitive DNA sequences, which have hitherto been challenging to synthesize because the low-complexity of the sequences permits annealing events among homologous nucleotides leading to errors in gene synthesis.
  • cloning of multiple genes into the same plasmid provides an effective way of increasing copy-number for expression while minimizing the overhead of the plasmid backbone.
  • oligos DNA oligonucleotides
  • plus and minus strand DNA oligonucleotides
  • the plus strand and minus strand are designed in such a way that, upon annealing, complementary oligos will overlap by approximately 20 nucleotides and the annealed oligos form a circular structure. There will be no gap to fill for the plus strand, but there can be gaps to fill for the minus strand.
  • Plus strand oligos will be 5'-phosphorylated.
  • the minus strand and the 5'- phosphorylated plus strand will be subjected to annealing at approximately 60 0 C.
  • Thermostable ligase e.g., Taq ligase
  • Exonucleases e.g., Exol, ExoIII and the like
  • oligonucleotides were 5'-phosphorylated.
  • the 5'-phosphorylated oligonucleotides were annealed, and thermostable ligase was used to complete a circular double stranded-DNA structure.
  • Exonucleases and endonucleases were added for degradation of all but a correctly circularized DNA.
  • the use of mismatch cleavage endonucleases (e.g., CEL I nuclease, T7 endonuclease or the like) together with exonucleases eliminated errors incurred during oligonucleotide synthesis ( Figure 2B).
  • Outside primers were annealed, and the circular double stranded-DNA was amplified by PCR and/or RCA.
  • Assembly process #1 was accomplished as follows:
  • DNA sequence (1052 base pairs) from Dpo4 DNA-polymerase was chosen as a target.
  • 24 plus strand oligos and 24 minus strand oligos were designed in such a way that, upon annealing (at approximately 60 0 C), complementary oligos would overlap as a circular structure, leaving no gap to fill.
  • the length of each oligo was approximately 40 to 50 base pairs.
  • Each oligo was synthesized by a commercial oligo synthesis company (Integrated DNA Technology), and desalted. No further purification step was carried out. Each oligo was individually dissolved in water to 200 ⁇ M concentration.
  • This ligation mixture was incubated at 94 0 C for 5 minutes for melting, and was ramped to 60 0 C at 0.1 °C/sec for annealing, incubated at 70 0 C for two hours for ligation to for circular structures, and was stored at 4 0 C.
  • This thermo-reaction was carried out using a thermocycler. Samples were visualized on an agarose gel ( Figure 3).
  • Exonuclease and mismatch cleavage endonucleases were used for the degradation of all but correctly circularized DNA, and for the elimination of errors incurred during oligonucleotide synthesis.
  • a typical exonuclease cocktail was prepared by mixing 8 ⁇ l of water, 1 ⁇ l of NEB Buffer 1, 2 ⁇ l of Exol (20 units/ ⁇ l, from NEB), 0.4 ⁇ l of ExoIII (100 units/ ⁇ l, from NEB).
  • typical exonuclease/endonuclease cocktail was prepared by mixing 10 ⁇ l of exonuclease cocktail with 0.4 ⁇ l of endonuclease (e.g.
  • DNA oligos (plus and minus strands) will be prepared. There can be gaps to fill as long as complementary oligos overlap by approximately 20 nucleotides. dNTP and DNA polymerase will be added to fill any gaps. DNA ligase will be used to form circular, double stranded-DNA sequences by ligation of DNA strand junctions.
  • Exonucleases and/or endonucleases (e.g. Exol, ExoIII, Sl nuclease and the like) will be added for degradation of all but a correctly circularized DNA.
  • the use of mismatch cleavage endonucleases (e.g., CEL I nuclease, T7 endonuclease or the like) together with exonucleases will eliminate errors incurred during oligonucleotide synthesis.
  • Outside primers will be annealed, and the circular double stranded-DNA will be amplified by PCR and/or RCA.
  • This section describes a synthetic oligonucleotide error correction method using a mixture of endonucleases and exonucleases. Annealing and ligating synthetic oligonucleotides (both plus and minus strands) to 'circular double-stranded DNA' will be carried out, such that exonuclease and/or endonuclease digestion can be performed to remove all but error-free DNA sequences.
  • This error correction strategy can be applied to an individual oligo or a large pool of many different oligos in a same tube. This method is not limited to circular structures as long as an annealed DNA is protected from exonuclease digestion by the introduction of other topological structures or chemical moieties or both to the DNA. An exemplary procedure is described below.
  • oligonucleotides both plus and minus strands
  • Proper overhangs will be introduced to facilitate ligation into a circular form.
  • Generic primer sequences from both ends of the oligonucleotides will optionally be introduced, and the generic sequences can be removed at the end of the error correction process (Tian et al. (2004) Nature 432:1050-4).
  • the plus and minus strands will be phosphorylated in separate tubes. Annealing will be performed in the same tube.
  • Ligation will be performed using an appropriate DNA ligase. Dimeric or trimeric circles may be formed. However, as long as circular structures are formed, the DNA is protected from exonuclease digestion.
  • Mismatch cleavage endonucleases e.g., CEL I, T4 endonucleases or the like
  • exonucleases e.g., Exol, ExoIII or the like
  • the error-removed DNA can then be used with or without a DNA amplification step.
  • Generic primer sequences can be removed using enzymatic and/or chemical digestion methods (e.g. type-IIS restriction enzymes (Tian et al, Supra)).
  • the one-cycle gene synthesis approach utilizes three different tiers of selection ( Figure 5).
  • tier one By subsequently subjecting the ligation mixture to exonuclease treatment, circular molecules can be selected with the desired sequence (tier two).
  • tier two By subsequently subjecting the ligation mixture to exonuclease treatment, circular molecules can be selected with the desired sequence (tier two).
  • tier two By subsequently subjecting the ligation mixture to exonuclease treatment, circular molecules can be selected with the desired sequence (tier two).
  • tier two By utilizing a mismatch- cleaving endonuclease, circular DNA containing residual errors can be converted to a linear form that is degraded by exonucleases still present in the solution (
  • the 1056bp Dpo4 gene (Fig. 13 A; Sulfolobus solfataricus P2 DNA polymerase IV; (Ling, H., Boudsocq, F. & Woodgate, R., Yang, W. Cell. 107, 91-102. (2001)) was synthesized using the three-tier approach as outlined in Figure 5.
  • Dpo 4 is one of the smallest polymerases compatible with thermal cycling PCR (352 codons rather than 834 for Taq Polymerase)
  • Codon optimized Dpo4 DNA sequences were designed using the Gene Design program. (Richardson, S.M., Wheelan, S. J., Yarrington, R.M. & Boeke, J.D. Genome Res. 16 550-556. (2006)). 24 plus strand oligonucleotides and 23 minus strand oligonucleotides, each -40-50 base pair long (See Figures 13B and 13C), were designed to have a melting temperature of 6O 0 C using the nearest-neighbor method. (SantaLucia, J. Jr. Proc. Natl. Acad. Sci. USA 95:1460-1465. (1998)).
  • one more guiding oligo (24th minus strand) was designed to bridge, and hence, join the 5' and 3' ends of the Dpo4 sequence.
  • These oligonucleotides were synthesized by a commercial oligonucleotide synthesis company (Integrated DNA Technology). No purification step other than desalting was carried out.
  • Each oligonucleotide was individually dissolved in water to 200 ⁇ M concentration. Equal volumes of the 24 plus strand oligo solutions were pooled together, and then the oligonucleotides are 5'-phosphorylated by following procedures: 12 ⁇ l of the plus strand mixture was mixed with 120 ⁇ l of water, 12 ⁇ l of 1OX T4 ligase buffer, and 6 ⁇ l of T4 polynucleotide kinase (10 U/ ⁇ l, from New England Biolabs (NEB), Beverly, MA). The final concentration of each oligonucleotide was approximately 0.67 ⁇ M. This reaction mixture was incubated at 37 0 C overnight, and stored at -20 0 C. The pool of 23 minus strand oligos and 24 th minus strand oligo were 5'-phosphorylated the same way.
  • the 5'-phosphorylated oligos were annealed, and thermostable ligase was used to complete a circular double stranded-DNA structure; 2.4 ⁇ l of Ampligase (100 units/ ⁇ l, from Epicentre, Madison, WI) and 4.8 ⁇ l of 1OX Ampligase buffer were mixed with 24 ⁇ l of a pool of 5'-phosphorylated 24 plus strand oligos and 23 ⁇ l of a pool of 5'-phosphorylated 23 minus strand oligos. This mixture was split to two batches, and 0.5 ⁇ l of a 5 'phosphorylated 24 th minus strand oligo was added to the second batch.
  • the concentration of each oligonucleotide in the ligation reaction was approximately 0.3 ⁇ M.
  • the two batches of ligation mixture (with 47 oligonucleotides (as a control) & 48 oligonucleotides) were incubated at 95 0 C for 3 minutes for melting, and were ramped to 70 0 C at 0.1 °C/sec for annealing.
  • the reaction mixture was incubated at 70 0 C for two hours for ligation, and was stored at 4 0 C. This thermo-reaction was carried out using a thermocycler.
  • Exonucleases were used to degrade all but circularized DNA for the elimination of errors incurred during oligonucleotide synthesis.
  • a typical exonuclease cocktail was prepared by mixing 36 ⁇ l of water, 5 ⁇ l of NEB Buffer 1, 6 ⁇ l of exonuclease I (source from E. coli, 20 units/ ⁇ l, NEB), 3 ⁇ l of exonuclease III (100 units/ ⁇ l, NEB), and 3 ⁇ l of lambda exonuclease (100 units/ ⁇ l, NEB). Then, typically 0.5 ⁇ l aliquot from the circular assembly ligation reaction was mixed with 20 ⁇ l of exonuclease cocktail, and incubated at 37 0 C.
  • each reaction mixture was split to four batches (6 ⁇ l + 4 ⁇ l + 4 ⁇ l + 4 ⁇ l).
  • the first batch (6 ⁇ l) was incubated at 37 0 C overnight without any treatment.
  • the second batch (4 ⁇ l) was mixed with 0.5 ⁇ l of NEB buffer 4 and 1 ⁇ l of endonuclease V (100 units/ ⁇ l, NEB).
  • the third batch (4 ⁇ l) was mixed with 1.5 ⁇ l of aliquot from a cocktail made of lO ⁇ l of water +6 ⁇ l of NEB buffer4+ 2.4 ⁇ l of endonuclease V.
  • the fourth batch was mixed with 1.5 ⁇ l of aliquot of a cocktail made of lO ⁇ l of water +6 ⁇ l of NEB buffer4+ 1.2 ⁇ l of endonuclease V. All these batches were incubated overnight at 37 0 C.
  • a final extension at 72 0 C was carried out for 10 min, and stored at 4 0 C.
  • Product band was excised, and extracted using QIAquick gel extraction column (Qiagen, Valencia, CA).
  • the gel-purified Dpo4 gene products were cloned into pUC19 vector (NEB), and transformed into T7 express competent E. coli cells (NEB). Individual colonies were picked and grown in Luria-Bertani broth containing carbenicillin antibiotics. Plasmids from grown colonies were purified, and sequenced using four different sequencing primers (Fig. 13E). Sequencing data was analyzed by using a DNA sequence analysis program, Lasergene (DNAstar, Madison, WI).
  • a mismatch-cleaving endonuclease i.e. Endonuclease V from E. coli selected based on the comparison of the mismatch cleavage efficiencies of different endonucleases (See Fuhrmann, M., Oertel, W., Berthold, P. & Hegemann, P. Nucleic Acids Res. 33, e58. (2005)) was introduced.
  • the intensities of the bands resulting from PCR amplification of the endonuclease treated mixture were highly dependent on the quantity of the mismatch-cleaving enzyme (Fig. 6A, lanes 5 to 10) due to a non-specific activity of the enzyme.
  • PCA Polymerase Cycling Assembly
  • oligonucleotide pool concentrations ranging from 0.4 ⁇ M to 0.0125 ⁇ M per each oligonucleotide. Aliquots of the dilution series were used for PCA reactions. Using PAGE gel purified outside primers, PCA was carried out. A first primer (with an Xbal restriction site; see Fig. 13D), a second primer (with a Pstl restriction site; see Fig.
  • Circular assembly amplification method can also be used to synthesize large genes using the USER enzyme strategy ' (Geu-Flores, F., Nour-Eldin, H. H., Nielsen M. T. & Halkier B.A. Nucleic Acids Res. 35, e55. (2007)).
  • USER strategy takes advantage of USERTM (a mixture of uracil DNA glycosidase and DNA glycosylase-lyase endo VIII from New England Biolabs), where a deoxyuridine-excision reaction by the enzyme mix generates 3 ' overhangs on PCR amplified DNA prepared by the use of primers containing deoxyuridines (U) in the place of deoxythymidines.
  • Protein sequence (775 codons) from Pfu DNA Polymerase (Fig. 13L; GenBank accession code for protein sequence: P61875) was chosen as a target. Codon optimized 2325bp sequence was designed by using the Gene Design computer program. First, we prepared three ds-DNA fragments of a Pfu DNA polymerase (PfU(I -811), Pfu(812-1554), and Pfu(1555-2325)) via circular assembly amplification ( Figure 7A-7C).
  • 3'-overhangs were generated on the Pfu polymerase gene fragments using USERTM enzyme and constructed full-length circular structures by ligation of the three gene fragments, and by treating with exonuclease.
  • the large 3 '-overhangs (20 bp or more) made by incorporating two deoxyUridines into PCR overhangs resulted in higher stringency (melting temperature of 7O 0 C) during a circular ligation of ds-DNA ( Figure 7).
  • PCR was subsequently performed on the full-length circular ligation product, PfU(I- 2325) and the resulting product was cloned.
  • PfuTurboCx Hotstart DNA polymerase (Stratagene, CA) was used to amplify DNA sequence by PCR. 5% DMSO was added. PCR reaction was initiated by heating first at 95 0 C for 3 min, followed by 28 cycles of the subsequent program: 95 0 C for 30 s, 65 0 C for 30 s, and 72 0 C for 60s. A final extension at 72 0 C was carried out for 10 min, and stored at 4 0 C.
  • the PCR amplified DNA product was cloned into pUC19 vector (NEB), and transformed into NEB 5- alpha Competent E. coli cells (NEB). Sequencing of a clone illustrated this USER- mediated circular assembly amplification approach can be used to synthesize large (>2 kbp) genes without errors
  • Circular ligation reaction was performed as described above as an exception in the ligation temperature gradient to provide higher level of stringency for an annealing step; ligation mixtures were incubated at 95 0 C for 3 minutes for melting, and were ramped to 80 0 C at 0.1 °C/sec for annealing. The reaction mixture was incubated at 80 0C for one hour, 75 0 C for one hour, and 7O 0 C for one hour, and was stored at 4 0 C.
  • PCR amplification of Dpo4 and pUC19 were performed using primers containing two deoxyU (four primer sets for Dpo4 amplification and one primer set for pUC19 amplification, see Fig. 13 J for sequence information).
  • PCR amplified pUC19 DNA was introduced as a fifth segment for the circular assembly process.
  • PfuTurboCx Hotstart DNA polymerase (Stratagen, CA) was used to amplify DNA sequence by PCR. 5% DMSO was added.
  • PCR reaction was initiated by heating first at 95 0 C for 3 min, followed by cycles (30 for Dpo4, or 25 cycles for pUC19) of the subsequent program: 95 0 C for 30 s, 65 0 C for 30 s, and 72 0 C for 60s (for Dpo4), or 3min (for pUC19).
  • a final extension at 72 0 C was carried out for 10 min, and stored at 4 0 C.
  • 1 ⁇ l of aliquots from each PCR reaction was mixed with l ⁇ l of USERTM (NEB), l ⁇ l of 1OX Thermopol buffer (NEB) and 9 ⁇ l of water. Each reaction mixture was incubated at 37 0 C for 60 minutes.

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Abstract

L'invention porte sur des compositions et des méthodes de synthèse de polynucléotides, de réduction des erreurs lors de la synthèse des polynucléotides, de correction des disparités dans les séquences de polynucléotides de synthèse, et de sélection de séquences de polynucléotides correctement assemblées.
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US9670517B1 (en) 2012-01-16 2017-06-06 Integrated Dna Technologies, Inc. Synthesis of long nucleic acid sequences
WO2020001783A1 (fr) 2018-06-29 2020-01-02 Thermo Fisher Scientific Geneart Gmbh Assemblage à haut débit de molécules d'acides nucléiques
WO2020212391A1 (fr) 2019-04-15 2020-10-22 Thermo Fisher Scientific Geneart Gmbh Assemblage multiplex de molécules d'acides nucléiques
WO2021178809A1 (fr) 2020-03-06 2021-09-10 Life Technologies Corporation Synthèse et assemblage d'acide nucléique à fidélité de séquence élevée

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Cited By (17)

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US9670517B1 (en) 2012-01-16 2017-06-06 Integrated Dna Technologies, Inc. Synthesis of long nucleic acid sequences
US10704041B2 (en) 2012-02-01 2020-07-07 Codex Dna, Inc. Materials and methods for the synthesis of error-minimized nucleic acid molecules
CN104220602B (zh) * 2012-02-01 2018-10-30 合成基因组股份有限公司 用于合成错误最小化核酸分子的材料和方法
JP2015509005A (ja) * 2012-02-01 2015-03-26 シンセティック ジェノミクス インコーポレーテッド エラーを最少限に抑える核酸分子の合成のための材料及び方法
US20130225451A1 (en) * 2012-02-01 2013-08-29 Synthetic Genomics, Inc. Materials and methods for the synthesis of error-minimized nucleic acid molecules
AU2017272206B2 (en) * 2012-02-01 2020-01-16 Synthetic Genomics, Inc. Materials and methods for the synthesis of error-minimized nucleic acid molecules
US9771576B2 (en) 2012-02-01 2017-09-26 Synthetic Genomics, Inc. Materials and methods for the synthesis of error-minimized nucleic acid molecules
CN104220602A (zh) * 2012-02-01 2014-12-17 合成基因组股份有限公司 用于合成错误最小化核酸分子的材料和方法
US11884916B2 (en) 2012-02-01 2024-01-30 Telesis Bio Inc. Materials and methods for the synthesis of error-minimized nucleic acid molecules
AU2013214771B2 (en) * 2012-02-01 2017-09-07 Synthetic Genomics, Inc. Materials and methods for the synthesis of error-minimized nucleic acid molecules
EP3597764A3 (fr) * 2012-02-01 2020-05-06 SGI-DNA, Inc. Matériau et procédés de synthèse de molécules d'acide nucléique à réduction d'erreurs
JP2020078330A (ja) * 2012-02-01 2020-05-28 シンセティック ジェノミクス インコーポレーテッド エラーを最少限に抑える核酸分子の合成のための材料及び方法
WO2013116771A1 (fr) 2012-02-01 2013-08-08 Synthetic Genomics, Inc. Matériaux et procédés pour la synthèse de molécules d'acide nucléique présentant un minimum d'erreurs
JP7175290B2 (ja) 2012-02-01 2022-11-18 シンセティック ジェノミクス インコーポレーテッド エラーを最少限に抑える核酸分子の合成のための材料及び方法
WO2020001783A1 (fr) 2018-06-29 2020-01-02 Thermo Fisher Scientific Geneart Gmbh Assemblage à haut débit de molécules d'acides nucléiques
WO2020212391A1 (fr) 2019-04-15 2020-10-22 Thermo Fisher Scientific Geneart Gmbh Assemblage multiplex de molécules d'acides nucléiques
WO2021178809A1 (fr) 2020-03-06 2021-09-10 Life Technologies Corporation Synthèse et assemblage d'acide nucléique à fidélité de séquence élevée

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