WO2008112683A2 - Gene synthesis by circular assembly amplification - Google Patents

Abstract

Compositions and methods for synthesizing polynucleotides, for reducing errors during polynucleotide synthesis, for correcting mismatches in synthetic polynucleotide sequences, and for selecting correctly assembled polynucleotide sequences are provided.

Description

GENE SYNTHESIS BY CIRCULAR ASSEMBLY AMPLIFICATION

STATEMENT OF GOVERNMENT INTERESTS

[001] This application was funded in part by Department of Energy Grant No. DE-FG02- 02ER63445. The government has certain rights to the invention.

RELATED APPLICATIONS

[002] This application claims priority from U.S. provisional patent application number 60/989,517, filed November 21, 2007; and U.S. provisional patent application number 60/894,573, filed March 13, 2007, each of which is hereby incorporated herein by reference in its entirety for all purposes.

BACKGROUND Field of the Invention

[003] Embodiments of the present invention relate in general to methods for reducing and/or eliminating errors resulting from DNA assembly and/or DNA synthesis.

Description of Related Art

[004] Current methods of assembling DNA sequences from a pool of synthetic oligonucleotides include the use of various assembly PCR protocols, and the combination of DNA ligation and assembly PCR protocols. Cycling with thermostable DNA polymerase using a number of oligonucleotides results in the formation of increasingly larger size DNA fragments until the full-length gene is obtained (Stemmer (1995) Gene 164:49). Typically, these assembly processes are carried out by annealing of DNA oligonucleotides to form linear, double stranded DNA. These linear DNA are gel-purified, cut by restriction enzymes, and either inserted into circular vectors, or circularized for subsequent transformation into a cell (Smith (2003) Proc. Natl. Acad. Sci. USA 100:15440). SUMMARY

[005] 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)).

[006] In one embodiment, a method is provided 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. In certain aspects, the method further includes amplifying the synthetic, single-stranded polynucleotide sequence such as by PCR, rolling circle amplification or hyper-branched rolling circle amplification. In other aspects, a plus strand oligonucleotide overlaps a minus strand oligonucleotide by about 20 nucleotides. In certain aspects, the single-stranded polynucleotide structure is a circularized structure such as circularized, single- stranded DNA. In other aspects, the plurality of plus strand oligonucleotides in the first sample are phosphorylated prior to annealing. In still other aspects, the one or more exonucleases are Exol and/or Exo III. In other aspects, after contacting the third sample with an exonuclease, a synthetic, single-stranded polynucleotide structure having reduced errors when compared to a synthetic, single-stranded reference polynucleotide remains.

[007] In another embodiment, a method is provided 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. In certain aspects, the method further includes amplifying the synthetic, double-stranded polynucleotide sequence, such as by PCR, rolling circle amplification or hyper- branched rolling circle amplification. In certain aspects, a plus strand oligonucleotide overlaps a minus strand oligonucleotide by about 20 nucleotides. In other aspects, the double-stranded polynucleotide structure is free of gaps. In still other aspects, the double-stranded polynucleotide structure is a circularized structure such as circularized, double-stranded DNA. In certain aspects, 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. In other aspects, the one or more exonucleases are Exol and/or Exo III and the endonuclease is Sl nuclease, endonuclease I or endonuclease V. In other aspects, after contacting the third sample with an exonuclease and an endonuclease, a synthetic, double-stranded polynucleotide structure having reduced errors when compared to a synthetic, double- stranded reference polynucleotide remains.

[008] In another embodiment, a method is provided 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.

[009] In yet another embodiment, 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. [010] In still another embodiment, a method is provided 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.

[Oil] In another embodiment, a method is provided 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. In certain aspects, a synthetic polynucleotide is a synthetic, single-stranded polynucleotide. In other aspects, a synthetic polynucleotide is a synthetic, double-stranded polynucleotide.

[012] Further features and advantages of certain embodiments of the present invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[013] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

[014] Figures 1A-1B schematically depict the assembly of circular single-stranded DNA. (A) depicts the assembly process. (B) depicts an additional purification process.

[015] 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. [016] Figure 3 depicts an agarose gel of products obtained during assembly of circular double-stranded DNA. Lane 1 (left side) 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 SURVEYOR™ nuclease- treated ligation mixture.

[017] Figure 4 depicts an agarose gel showing DNA amplification of an exonuclease cocktail- or exonuclease/endonuclease cocktail-treated ligation mixture. Lane 1 (left side) 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 SURVEYOR™ nuclease-treated ligation mixture.

[018] Figure 5 depicts a schematic of circular assembly amplification showing the three-tier process in detail.

[019] 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. (B) & (C) Depict the number of errors per 10,000bp resulted from synthetic Dpo4 made by various methods. Experiments are performed at annealing temperature 65⁰C and 7O⁰C, respectively. Error bars denote a standard deviation (s.d.).

[020] Figures 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).

[021] Figures 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 (65⁰C for B and 7O⁰C for C); Lane 4 from both experiments was cloned.

[022] Figures 9A-B. Fraction of clones with incorrect full length Dpo4 sequence made by various methods. Experiments are performed at annealing temperature (a) 65⁰C and (b) 7O⁰C, respectively. Error bars denote a standard deviation (s.d.).

[023] Figure 10. Depicts Circular assembly amplification for the construction of genes of various sizes assessed by the synthesis of Pfu DNA polymerase gene fragments.

[024] Figures 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).

[025] Figures 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. [026] 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. (D) PCR amplification Primers for Dpo4. (E) Four Sequencing Primers for Dpo4. (F) Target Human Minisatellite Sequence (GenBank accession code: NTOl 1515). (G) 8 plus strand oligonucleotides for the human minisatellite sequence. (H) 8 minus strand oligonucleotides for the human minisatellite sequence. (I) PCR amplification Primers for the human minisatellite sequence. (J) Amplification primers (containing deoxyU, dU) for tandem Dpo4 repeat synthesis. (K) Dpo4 tandem repeat connectivity verification primers. (L) Pfu DNA polymerase sequence. (M) 55 plus strand oligonucleotides for Pfu DNA polymerase . (N) 55 minus oligonucleotides for Pfu DNA polymerase. (O) Guiding minus oligonucleotides for the circular ligations of different size Pfu DNA polymerase fragments. (P) Guiding minus oligonucleotides for the circular ligations of Pfu DNA polymerase fragments for making Pfu DNA polymerase by USER mediated circular assembly amplification of three DNA fragments. (Q) Amplification primers (containing deoxyU, dU) for PCR amplification of three segments. (R) Pfu DNA polymerase PCR amplification Primers. (S) Eight Sequencing Primers for Pfu plasmid.

[027] 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.

DETAILED DESCRIPTION

[028] 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.

[029] The assembly of polynucleotide sequences (e.g., DNA) from oligonucleotides has applications in a variety of synthetic biology projects including codon optimized DNA synthesis. In certain embodiments, an assembly of oligonucleotides (e.g., DNA) as a circular form is provided for gene synthesis, and various nucleases (e.g., exonucleases, and/or endonucleases) are provided to simplify selection of correctly assembled polynucleotide (e.g., DNA) sequences and for the easy elimination of errors incurred during oligonucleotide synthesis. A facile synthetic oligonucleotide error correction method using a mixture of endonucleases and exonucleases is also described herein.

[030] In certain embodiments, methods for constructing polynucleotide (e.g., DNA) sequences by annealing/assembly of circular single-stranded polynucleotide sequences or circular double-stranded polynucleotide sequences from a pool of oligonucleotides such that exonuclease and/or endonuclease digestion can be carried out to remove all but correctly assembled polynucleotide (e.g., DNA) sequences (e.g., those sequences having reduced or no sequence errors) are provided.

[031] As used herein 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).

[032] If the error rate is reduced or decreased, 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. In certain aspects, 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.

[033] For example, the error rate may be reduced by addition of the exonuclease alone or with endonuc lease. With the exonuclease addition included in the amplification, 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. With the endonuclease addition included in the amplification, 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. Optionally, mismatch binding enzymes can be included to further reduce error rates.

[034] 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.

[035] 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). Thus, the term "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 nucleotides.

[036] Examples of 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'-methoxycarboxymethyluracil, 5-methoxyuracil, 2- methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, A- thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6- diaminopurine and the like. Nucleic acid molecules may also be modified at the base moiety, sugar moiety or phosphate backbone.

[037] The assembly methods described herein are not limited to circular structures. In certain embodiments, the annealed oligonucleotides (e.g., DNA) 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. Furthermore, a mixture of one or more mismatch cleavage endonucleases (e.g., CEL I nuclease, T7 endonuclease or the like) and 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.

[038] As used herein, the term "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. Examples of 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).

[039] As used herein, the term "endonuclease" refers to enzymes that cleave a phosphodiester bond within a polynucleotide chain. Examples of endonucleases include, but are not limited to, mung bean nuclease, BAL-31 nuclease, and the like. Endonucleases also include mismatch cleavage endonucleases. As used herein, the term "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. A variety of endonucleases are known in the art and are available from commercial vendors (See New England Biolabs 2005 -06 Catalog and Technical Reference).

[040] As used herein, the terms "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 are known in the art and 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.

[041] In certain embodiments, 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. Methods of nucleic acids to substrates are described in U.S. Patent No. 6,432,360.

[042] "Substrate" can refer to a matrix upon which oligonucleotides and/or polynucleotides are placed. The support can be solid, semi-solid or a gel. As used herein, the term "solid substrate" includes, but is not limited to, materials such as glass silica, polymeric materials and the like. In certain embodiments, 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, SiO₂, SiN₄, modified silicon, or (poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene, polycarbonate, or combinations thereof.

[043] 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. In some embodiments, at least one surface of the substrate is partially planar. In other embodiments it is desirable to physically separate regions of the substrate to delineate synthetic regions, for example with trenches, grooves, wells or the like.

[044] As used herein, the term "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.

[045] 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. In certain aspects, 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 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.

[046] In certain embodiments, 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. 20:359; Demidov (2002) Expert Rev. MoL Diagn. 2(6):89 (RCA); Mullis et al, U.S. Patent Nos. 4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al., U.S. Patent No. 5,210,015 (real-time PCR with "Taqman" probes); Wittwer et al, U.S. Patent No. 6,174,670; Kacian et al., U.S. Patent No. 5,399,491 (NASBA); Lizardi, U.S. Patent No. 5,854,033; Aono et al., Japanese Patent Pub. 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.

[047] In certain exemplary embodiments, 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.

[048] "Real-time PCR" refers to PCR for which the amount of reaction product is monitored as the reaction proceeds. There are many forms of real-time PCR that differ primarily in the detection chemistries used for monitoring the reaction product, e.g., Gelfand et al., U.S. Patent No. 5,210,015 ("Taqman"); Wittwer et al., U.S. Patent Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al., U.S. Patent No. 5,925,517 (molecular beacons). Detection chemistries for real-time PCR are reviewed in Mackay et al. (2002) Nucleic Acids Res. 30:1292. "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. As used herein, "initial primers" in reference to a nested amplification reaction refer to the primers used to generate a first amplicon, and "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. [049] "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: β-actin, GAPDH, β₂- 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).

[050] In certain embodiments, the assembly of polynucleotide (e.g., DNA) sequences is accomplished by assembly of circular and/or linear single-stranded polynucleotides (e.g., DNA). In other embodiments, the assembly of polynucleotide (e.g., DNA) sequences is accomplished by assembly of circular and/or linear double-stranded polynucleotides (e.g., DNA).

[051] In certain embodiments, plus-strand oligonucleotide sequences and minus-strand oligonucleotide sequences are hybridized. Typically, 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.

[052] As used herein, 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%. Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, 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.

[053] Overall, five factors influence the efficiency and selectivity of hybridization of the primer to a second nucleic acid molecule. These factors, which are (i) primer length, (ii) the nucleotide sequence and/or composition, (iii) hybridization temperature, (iv) buffer chemistry and (v) the potential for steric hindrance in the region to which the primer is required to hybridize, are important considerations when non-random priming sequences are designed.

[054] There is a positive correlation between primer length and both the efficiency and accuracy with which a primer will anneal to a target sequence. Longer sequences have a higher T_m than do shorter ones, and are less likely to be repeated within a given target sequence, thereby cutting down on promiscuous hybridization. Primer sequences with a high G-C content or that comprise palindromic sequences tend to self-hybridize, as do their intended target sites, since unimolecular, rather than bimolecular, hybridization kinetics are generally favored in solution. At the same time, it is important to design a primer containing sufficient numbers of G-C nucleotide pairings to bind the target sequence tightly, since each such pair is bound by three hydrogen bonds, rather than the two that are found when A and T bases pair.

[055] 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 ⁰C to greater than 22 ⁰C, greater than about 30 ⁰C, and (most often) in excess of about 37 ⁰C. For example, the hybridization temperature can be about: 6O⁰C, 65⁰C, 7O⁰C, 75⁰C, 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.

[056] "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. Several equations for calculating the T_m of nucleic acids are well known in the art. As indicated by standard references, a simple estimate of the T_m value may be calculated by the equation. T_m =81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, "Quantitative Filter Hybridization," in Nucleic Acid Hybridization (1985). Other references (e.g., Allawi, H. T. & Santa Lucia, J., Jr., Biochemistry 36, 10581-94 (1997)) include alternative methods of computation which take structural and environmental, as well as sequence characteristics into account for the calculation of T_m.

[057] In certain embodiments, one or more oligonucleotide sequences are ligated together, e.g., after hybridization of plus-strands and minus-strands. As used herein, the term "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. Acids Res. 27:875; Higgins et al., Meth. in Enzymol (1979) 68:50; Engler et al. (1982) The Enzymes, 15:3 (1982); and Namsaraev, U.S. Patent Pub. No. 2004/0110213.

[058] In certain embodiments, 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-specifϊc 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 (RCA) readout) and the like. A variety of light-based sequencing technologies are known in the art (Landegren et al. (1998) Genome Res. 8:769-76; Kwok (2000) Pharmocogenomics 1 :95-100; and Shi (2001) Clin. Chem. 47:164-172).

[059] Examples of detectable markers include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers and the like. Examples of 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. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, aequorin and the like. Examples of 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.

[060] It is to be understood that the embodiments of the present invention which have been described are merely illustrative of some of the applications of the principles of the present invention. Numerous modifications may be made by those skilled in the art based upon the teachings presented herein without departing from the true spirit and scope of the invention. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety for all purposes.

[061] 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. Optionally, one or more endonucleases may also be included in the same reaction vessel, or added to the reaction vessel after incubation with the exonucleases. In addition, because the assembly of ~ lkb gene (an average length of a gene) can be done in single cycle with a smaller amount of sequencing required to find a perfect construct, the methods of the invention provide significant cost benefits by decreasing the expense of gene synthesis. Further, 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. In another aspect of the invention, 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. [062] The following examples are set forth as being representative of the present invention. These examples are not to be construed as limiting the scope of the invention as these and other equivalent embodiments will be apparent in view of the present disclosure, figures, and accompanying claims.

EXAMPLE I Assembly of Circular Single-Stranded DNA

[063] The assembly process is depicted in Figure IA. Two batches of DNA oligonucleotides ("oligos") (plus and minus strand) will be prepared. 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.

[064] Plus strand oligos will be 5'-phosphorylated. The minus strand and the 5'- phosphorylated plus strand will be subjected to annealing at approximately 60 ⁰C. Thermostable ligase (e.g., Taq ligase) will be used for the ligation of the 5'- phosphorylated plus strand. Exonucleases (e.g., Exol, ExoIII and the like) will be added for the degradation of all but a circularized single stranded-DNA.

[065] Outside primers will be annealed, and the circular single stranded-DNA will be amplified by PCR, RCA or HRCA.

[066] The additional purification process is depicted in Figure IB. Plus-strand and minus- strand circular ss-DNA will be prepared in two different batches using the method shown above (the steps prior to annealing) for each batch. Plus-strand and minus- strand circular ss-DNA will be annealed using topoisomerase to allow appropriate twisting. Endonucleases and/or exonucleases (e.g., Sl -nuclease, Exol, ExoIII and the like) will be used to digest the DNA. All but a correctly assembled DNA (i.e., DNA with a correct sequential assembly order) are subjected to degradation. EXAMPLE II Assembly of Circular Double-Stranded DNA (Process #1)

[067] This process is schematically depicted in Figure 2A. DNA oligos (plus and minus strand) were prepared. The plus strand and minus strand were designed in such a way that, upon annealing, complementary oligonucleotides overlapped as a circular structure and there was no gap to fill.

[068] All oligonucleotides were 5'-phosphorylated. The 5'-phosphorylated oligonucleotides were annealed, and thermostable ligase was used to complete a circular double stranded-DNA structure.

[069] Exonucleases and endonucleases (e.g., Exol, ExoIII, Sl nuclease and the like) 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 [070] Assembly process #1 was accomplished as follows:

[071] 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 ⁰C), 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 200μM concentration.

[072] 24 plus strand oligos were pooled together, and 5'-phosphorylated. 10 μl of the plus strand mixture was mixed with 100 μl of water, 10 μl of 1OX T4 ligase buffer, and 5 μl of T4 polynucleotide kinase (PNK: 10 U/μl, from New England Bio labs, Beverly, MA). The final concentration of each oligo was approximately 0.8 μM. This reaction mixture was incubated at 37 ⁰C overnight, and stored at -20 ⁰C. 24 minus strand oligos were 5'-phosphorylated the same way.

[073] 5'-phosphorylated oligos were annealed, and thermostable ligase was used to complete a circular double stranded-DNA structure. 1 μl of AMPLIGASE^® (100 units/μl, from EPICENTRE^®, Madison, WI) and 2 μl of AMPLIGASE^® buffer was mixed with 10 μl of a mixture of 5'-phosphorylated 24 plus strand oligos and lOμl of a mixture of 5'-phosphorylated 24 minus strand oligos. The final ligation reaction concentration of each oligonucleotide was approximately 0.4 μM. This ligation mixture was incubated at 94 ⁰C for 5 minutes for melting, and was ramped to 60 ⁰C at 0.1 °C/sec for annealing, incubated at 70 ⁰C for two hours for ligation to for circular structures, and was stored at 4 ⁰C. This thermo-reaction was carried out using a thermocycler. Samples were visualized on an agarose gel (Figure 3).

[074] 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 μl of water, 1 μl of NEB Buffer 1, 2μl of Exol (20 units/μl, from NEB), 0.4μl of ExoIII (100 units/μl, from NEB). Also, typical exonuclease/endonuclease cocktail was prepared by mixing 10 μl of exonuclease cocktail with 0.4 μl of endonuclease (e.g. SURVEYOR™ nuclease S from Transgenomic, Omaha, NE), T7 endonuclease I (10 units/μl, from NEB), or endonuclease V (10 units/μl, from NEB)). 5μl of circular ligation mixture (from step iii) was mixed with 10 μl of exonuclease cocktail or exonuclease/endonuclease cocktail. The reaction was incubated at 37 ⁰C for one hour to allow the enzymatic reaction to proceed, followed by incubation at 94 ⁰C for 20 minutes to allow denaturation of the nucleases. The reactions were stored at 4⁰C. Samples were visualized on an agarose gel (Figure 4).

[075] The amount of exonucleases and endonucleases, and reaction time for the nuclease treatment, and the order of adding nucleases will be varied to achieve additional optimal conditions for the reduction of errors. Also, a combination of exonucleases and/or endonucleases will be varied to determine additional optimal experimental parameters.

[076] Outside primers were annealed, and the double stranded-DNA was amplified by PCR for cloning into a desired vector. A first primer (with an Xbal restriction site), a second primer (with a Pstl restriction site), and high-fidelity DNA polymerase (IPROOF™ DNA polymerase from BioRad, Hercules, CA) were used to amplify DNA sequence by PCR. Conventional cloning procedures were carried out for the insertion of the amplified DNA to a desired vector including, but not limited to pUC19, pUC18, pTWINl, and/or TOPO^® (Invitrogen, Carlsbad, CA) vectors.

EXAMPLE III Assembly of Circular Double-Stranded DNA (Process #2)

[077] Assembly process #1 (Figure 2B) was accomplished as follows:

[078] 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.

[079] 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. EXAMPLE IV Synthetic Oligonucleotide Error Correction Method

[080] 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.

[081] Complementary copy of oligonucleotides (both plus and minus strands) will be synthesized. 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.

[082] 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.

[083] Mismatch cleavage endonucleases (e.g., CEL I, T4 endonucleases or the like) and exonucleases (e.g., Exol, ExoIII or the like) will be added into the circular DNA mixture. Once the endonucleases recognize and cleave any mismatches (e.g., due to errors incurred during oligonucleotide synthesis) in the circular double stranded- DNA, the cleaved DNA will be completely degraded by exonucleases. [084] 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)).

EXAMPLE V Circular Assembly Amplification for the Construction of Dpo4

[085] The one-cycle gene synthesis approach utilizes three different tiers of selection (Figure 5). First, single stranded oligonucleotides are assembled into circular molecules under a highly stringent annealing condition (7O⁰C), such that most error containing oligonucleotides would fail to anneal (tier one). By subsequently subjecting the ligation mixture to exonuclease treatment, circular molecules can be selected with the desired sequence (tier two). Finally, 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 (tier three). A final PCR reaction amplifies the remaining undegraded DNA from the reaction mixture.

[086] 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)

Target DNA Sequence and Oligonucleotide Sequence Design

[087] 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 6O⁰C using the nearest-neighbor method. (SantaLucia, J. Jr. Proc. Natl. Acad. Sci. USA 95:1460-1465. (1998)). In addition, 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.

5' Phosphorylation of Oligonucleotides

[088] Each oligonucleotide was individually dissolved in water to 200 μM concentration. Equal volumes of the 24 plus strand oligo solutions were pooled together, and then the oligonucleotides are 5'-phosphorylated by following procedures: 12 μl of the plus strand mixture was mixed with 120 μl of water, 12 μl of 1OX T4 ligase buffer, and 6 μl of T4 polynucleotide kinase (10 U/μl, from New England Biolabs (NEB), Beverly, MA). The final concentration of each oligonucleotide was approximately 0.67 μM. This reaction mixture was incubated at 37 ⁰C overnight, and stored at -20 ⁰C. The pool of 23 minus strand oligos and 24^th minus strand oligo were 5'-phosphorylated the same way.

Annealing of S'-Phosphorylated Oligos, and Circular Ligation Using Thermostable Ligase at 7O⁰C

[089] The 5'-phosphorylated oligos were annealed, and thermostable ligase was used to complete a circular double stranded-DNA structure; 2.4 μl of Ampligase (100 units/μl, from Epicentre, Madison, WI) and 4.8 μl of 1OX Ampligase buffer were mixed with 24 μl of a pool of 5'-phosphorylated 24 plus strand oligos and 23 μl of a pool of 5'-phosphorylated 23 minus strand oligos. This mixture was split to two batches, and 0.5 μl 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 μM. The two batches of ligation mixture (with 47 oligonucleotides (as a control) & 48 oligonucleotides) were incubated at 95 ⁰C for 3 minutes for melting, and were ramped to 70 ⁰C at 0.1 °C/sec for annealing. The reaction mixture was incubated at 70 ⁰C for two hours for ligation, and was stored at 4 ⁰C. This thermo-reaction was carried out using a thermocycler.

Exonuclease Degradation of Linear DNA

[090] 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 μl of water, 5 μl of NEB Buffer 1, 6μl of exonuclease I (source from E. coli, 20 units/μl, NEB), 3μl of exonuclease III (100 units/μl, NEB), and 3μl of lambda exonuclease (100 units/μl, NEB). Then, typically 0.5μl aliquot from the circular assembly ligation reaction was mixed with 20μl of exonuclease cocktail, and incubated at 37⁰C.

Use of Mismatch-Cleaving Endonuclease for the Conversion of Mismatch Containing Circular DNA to Linear DNA

[091] After four hours of exonuclease incubation, each reaction mixture was split to four batches (6 μl + 4 μl + 4 μl + 4 μl). The first batch (6μl) was incubated at 37⁰C overnight without any treatment. The second batch (4 μl) was mixed with 0.5 μl of NEB buffer 4 and 1 μl of endonuclease V (100 units/μl, NEB). The third batch (4μl) was mixed with 1.5μl of aliquot from a cocktail made of lOμl of water +6 μl of NEB buffer4+ 2.4 μl of endonuclease V. The fourth batch was mixed with 1.5μl of aliquot of a cocktail made of lOμl of water +6μl of NEB buffer4+ 1.2 μl of endonuclease V. All these batches were incubated overnight at 37⁰C.

PCR Amplification, Cloning and Sequencing

[092] Outside primers were synthesized and purified using PAGE gel (Integrated DNA Technology). Using these primers, the double stranded-DNA was amplified by PCR for cloning into a pUC19 vector. A first primer (with an Xbal restriction site; Fig. 13D), a second primer (with a Pstl restriction site; Fig. 13D), and iProof™ DNA polymerase (Bio-Rad, Hercules, CA) were used to amplify DNA sequence by PCR. PCR reaction was initiated by heating first at 95⁰C for 3 min, followed by 38 cycles of the subsequent program: 95⁰C for 30 s, 65⁰C for 30 s, and 72⁰C for 60 s. A final extension at 72⁰C was carried out for 10 min, and stored at 4⁰C. 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).

[093] Ligation of 5'phosphorylated oligonucleotides was carried out using a thermostable ligase at 7O⁰C with or without the bridging oligo, and PCR was subsequently performed on the ligation mixture. Both ligation mixtures led to desired products (lane 1 & 2 in Fig. 6A). However, upon introduction of a mixture of exonuclease I for single strand DNA degradation, and exonuclease III and lambda exonucleases for double strand DNA degradation, only the oligonucleotide pool containing the guide oligo yielded product. (Fig. 6A, lane 3 and 4). Use of insufficient exonuclease treatment lead to incomplete exonuclease degradation (Fig. 8A, lane 3 online). One of skill in the art can determine the correct amount of exonucleases to ensure that sequence from error-containing oligonucleotides is reduced or eliminated. Exonuclease treatment significantly reduced yield as would be expected if it is selecting for oligonucleotides of full length and proper sequence.

[094] In additional experiments, 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.

[095] To further characterize the assembly method of the present invention, products were cloned and sequenced. For points of reference, circular assembly amplification was carried out at a less stringent annealing temperature (65 ⁰C).

[096] Conventional Polymerase Cycling Assembly (PCA) was also carried out to synthesize Dpo4 (Fig. 8C and 8C) as follows. Each oligo was individually dissolved in water to 200 μM concentration. 24 plus strand oligos (oligo labeling from Fl to F24; Fig. 13B) and 23 minus strand oligos (oligo labeling from Rl to R23; Fig. 13C) were pooled together. 4 μl aliquot from the oligonucleotide pool was mixed with 32μl of water, and a series of six two-fold dilutions was carried out for the preparation of oligonucleotide pool concentrations ranging from 0.4μM to 0.0125μM 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. 13D), and high-fidelity DNA polymerase (iProof™ DNA polymerase from BioRad, Hercules, CA) were used to amplify DNA sequence. PCA reaction was initiated by heating first at 95⁰C for 3 min, followed by 35 cycles of the subsequent program: 95⁰C for 30 s, 65⁰C (or 7O⁰C) for 30 s, and 72⁰C for 60 s. A final extension at 72⁰C was carried out for 10 min, and stored at 4⁰C. (see Fig. 8 for gel analysis of Dpo4 constructs from this PCA reaction). Product band was excised, and extracted using microcentrifuge column (Qiagen, Valencia, CA) for cloning into a pUC19 vector. Transformation, sequencing (see primers at Fig. 13E), and sequence analysis were carried out as same procedures as Circular Assembly Amplification.

For each of the experiments we sequenced 10-12 clones and repeated each experiment two to four times. The sequencing results are summarized in Figures 6B, 6C, and 8, and Figures 14A and 14B. Exonuclease-degradation significantly improved the error rates. On average, an error rate of 0.036% was achieved after overnight exonuclease treatment following circularization performed at 7O⁰C. Adding endonuclease treatment to exonuclease treatment further reduced synthesis error rate, achieving the low error rate of 0.025%. In contrast, without exo- & endo-nuclease treatment, an error rate of 0.082% was observed from experiments at 7O⁰C ligation, her temperatures for annealing and ligation also contributed to error reduction to a moderate degree (Figure 6B & 6C). In contrast, gene synthesis by conventional PCA method resulted in error rate of 0.183% with annealing at 7O⁰C, suggesting the importance of the introduction of error correction for gene synthesis. Thus, using three tiers of selection reduced gene synthesis error rates by at least a factor of seven compared to conventional PCA method. Circular assembly amplification can be used to synthesize genes of various sizes (Fig. 10). EXAMPLE VI

Pfu DNA Polymerase Synthesis by USER-Mediated Circular Assembly

Amplification

[098] 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 USER™ (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.

[099] 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).

[0100] For each fragment, plus and minus strand oligonucleotides were designed in such a way that, upon annealing, complementary oligos would overlap as a circular structure, leaving no gap to fill. For the construction of each fragment, a protocol was followed for the synthesis of Dpo4 shown above. Guiding minus oligonucleotides for different- sized Pfu fragments and for the whole Pfu are shown in Figures 130 and 13P, respectively. In this assembly, however, the PCR amplification step required primers containing deoxyUridines (three primer sets, see Fig. 13Q). 3'-overhangs were generated on the Pfu polymerase gene fragments using USER™ 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 7O⁰C) during a circular ligation of ds-DNA (Figure 7).

[0101] 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 95⁰C for 3 min, followed by 28 cycles of the subsequent program: 95⁰C for 30 s, 65⁰C for 30 s, and 72⁰C for 60s. A final extension at 72⁰C was carried out for 10 min, and stored at 4⁰C. Without a further purification step, 1 μl of aliquots from each PCR reaction was mixed with lμl of USER™ (NEB), lμl of 1OX Thermopol buffer (NEB) and 9 μl of water. Each reaction mixture was incubated at 37 ⁰C for 60 minutes. An aliqout (2 μl ) from the USER™ treated reaction was mixed, and added 0.5 μl of Ampligase and 1 μl of 1OX Ampligase buffer, then incubated at 70 ⁰C for two hours, and stored at 4 ⁰C. Exonuclease degradation of linear DNA was performed as described above. Using outside primers, full-length double stranded-DNA was amplified. 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

EXAMPLE VII

Synthesis of a Human Minisatellite Sequence by Circular Assembly

Amplification

[0102] Assembly of low-complexity DNA sequences using conventional gene synthesis methods is challenging because promiscuous annealing events among homologous oligonucleotides results in the heterogeneous products. Exonuclease degradation of the circular ligation mixture can overcome problems with these sequences by eliminating incorrectly annealed DNA sequences, thereby selecting a desired circular DNA molecule for the next amplification step. A -300 bp human minisatellite region (GenBank accession code: NTOl 1515) composed was assembled using five tandem repeats of 45 bp each with 97% homology (Figures HA and 13F for a target sequence). Plus strand oligonucleotides (Fig. 13G) and minus strand oligonucleotides (Fig. 13H) were designed, and 5' phosphorylated.

[0103] 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 ⁰C for 3 minutes for melting, and were ramped to 80 ⁰C at 0.1 °C/sec for annealing. The reaction mixture was incubated at 80 0C for one hour, 75⁰C for one hour, and 7O⁰C for one hour, and was stored at 4 ⁰C.

[0104] Exonuclease degradation of linear DNA, PCR amplification, cloning and sequencing were performed as described above. Except that DNA amplification was performed by 22 PCR cycles (Primers are shown in Fig. 131); PCR amplicon was purified using Qiagen PCR purification column (Qiagen, Valencia, CA); transformation was performed using 5-alpha Competent E. coli cells (NEB).

[0105] We successfully constructed the desired sequence by circular assembly of 16 oligonucleotides (Fig. HB, lane 12 and Fig. 1OC for sequencing data). In contrast, conventional methods including a PCA (Fig. HB, lane 1 to 8) and a ligation-PCR method (Fig. HB, lane 9 to 10) resulted in heterogeneous products. However, in one experiment, circular assembly amplification did not assemble 14 tandem repeats of the 45 bp fragment.

EXAMPLE VIII

Circular Assembly Amplification of Large Tandem Repeats

[0106] Large tandem repeats can be prepared via USER-mediated circular assembly amplification strategy (Fig. 12A). 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 95⁰C for 3 min, followed by cycles (30 for Dpo4, or 25 cycles for pUC19) of the subsequent program: 95⁰C for 30 s, 65⁰C for 30 s, and 72⁰C for 60s (for Dpo4), or 3min (for pUC19). A final extension at 72⁰C was carried out for 10 min, and stored at 4⁰C. Without a further purification step, 1 μl of aliquots from each PCR reaction was mixed with lμl of USER™ (NEB), lμl of 1OX Thermopol buffer (NEB) and 9 μl of water. Each reaction mixture was incubated at 37 ⁰C for 60 minutes. Five DNA molecules were assembled by 70 ⁰C ligation of the ds-DNA containing 3' overhangs. Transformation followed by characterization of clones showed that four Dpo4 tandem repeats (-4,400 bp) and a pUC19 fragment (-2,700 bp) were correctly assembled (Fig. 12B). Aliqouts from USER™ treated reaction (lμl each from four Dpo4 mixtures and lμl from pUC19 mixture) were mixed with 0.5 μl of Ampligase and 1 μl of 1OX Ampligase buffer, 5 μl of water, and incubated at 70 ⁰C for two hours for ligation. A resulting construct was transformed into NEB 5 -alpha Competent E. coli cells (NEB). A colony was picked and characterized by using a set of PCR primers listed below.

EXAMPLE IX

References

Binkowski, B.F., Richmond, K.E., Kaysen, J., Sussman, M. R. & Belshaw, P.J. Nucleic Acids Res., 33:e55. (2005)

Carr, P.A., Park, J.S., Lee, Y.J., Yu, T., Zhang, S. & Jacobson, J.M. Nucleic Acids Res., 32:el62. (2004)

Fuhrmann, M., Oertel, W., Berthold, P. & Hegemann, P. Nucleic Acids Res., 33:e58. (2005)

Geu-Flores, F., Nour-Eldin, H.H., Nielsen M. T. & Halkier B.A. Nucleic Acids Res., 35:e55. (2007)

Han, J.S. & Boeke, J.D. Nature, 429:314-318. (2004) Hutchinson et al. (2005) Proc. Natl. Acad. Sci. U.S.A., 102:17332.

Kodumal, S.J., Patel, K.G., Reid, R., Menzella, H.G., Welch, M. & Santi, D.V. Proc. Natl. Acad. Sci. USA, 101:15573-15578. (2004)

Ling, H., Boudsocq, F. & Woodgate, R., Yang, W. Cell. 107, 91-102. (2001) Menzella, H.G. et al. Nat. Biotechnol, 23:1171-1176. (2005).

Richardson, S. M., Wheelan, S. J., Yarrington, R.M. & Boeke, J.D. Genome Res., 16:550-556. (2006)

SantaLucia, J. Jr. Proc. Natl. Acad. Sci. USA, 95: 1460-1465. (1998)

Smith, H.O., Hutchison, CA. 3rd, Pfannkoch, C. & Venter, J.C. Proc. Natl. Acad. Sci. USA, 100:15440-15445. (2003)

Stemmer, W.P., Crameri, A., Ha, K.D., Brennan, T.M. & Heyneker, H.L. Gene, 164:49-53. (1995)

Tian, J., Gong, H., Sheng, N., Zhou, X., Gulari, E., Gao, X. & Church, G. M. Nature, 432:1050-1054. (2004)

Xiong, A.S., Yao, Q.H., Peng, R.H., Li, X., Fan, H.Q., Cheng, Z.M. & Li, Y. Nucleic Acids Res., 32:e98. (2004)

Young, L. & Dong, Q. Nucleic Acids Res., 32:e59. (2004)

Claims

What is claimed is:

1. A method for synthesizing a synthetic, single-stranded polynucleotide comprising: 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 an exonuclease.

2. The method of claim 1, further comprising amplifying the synthetic, single-stranded polynucleotide sequence.

3. The method of claim 2, wherein the amplifying is performed using a method selected from the group consisting of PCR, rolling circle amplification and hyper-branched rolling circle amplification.

4. The method of claim 1, wherein a plus strand oligonucleotide overlaps a minus strand oligonucleotide by about 20 nucleotides.

5. The method of claim 1, wherein the single-stranded polynucleotide structure is a circularized structure.

6. The method of claim 1, wherein the single-stranded polynucleotide structure is circularized, single-stranded DNA.

7. The method of claim 1, wherein the plurality of plus strand oligonucleotides in the first sample are phosphorylated prior to annealing.

8. The method of claim 1, wherein the exonuclease is selected from the group consisting of Exol and Exo III.

9. The method of claim 1, wherein after contacting the third sample with an exonuclease, a synthetic, single-stranded polynucleotide structure having reduced errors when compared to a synthetic, single-stranded reference polynucleotide remains.

10. A method for synthesizing a synthetic, double-stranded polynucleotide comprising: 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 an exonuclease and an endonuclease.

11. The method of claim 10, further comprising amplifying the synthetic, double-stranded polynucleotide sequence.

12. The method of claim 11 , wherein the amplifying is performed using a method selected from the group consisting of PCR, rolling circle amplification and hyper-branched rolling circle amplification.

13. The method of claim 10, wherein a plus strand oligonucleotide overlaps a minus strand oligonucleotide by about 20 nucleotides.

14. The method of claim 10, wherein the double-stranded polynucleotide structure is free of gaps.

15. The method of claim 10, wherein the double-stranded polynucleotide structure is a circularized structure.

16. The method of claim 10, wherein the double-stranded polynucleotide structure is circularized, double-stranded DNA.

17. The method of claim 10, wherein 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.

18. The method of claim 10, wherein the exonuclease is selected from the group consisting of Exol and Exo III.

19. The method of claim 10, wherein the endonuclease is selected from the group consisting of Sl nuclease, endonuclease I and endonuclease V.

20. The method of claim 10, wherein after contacting the third sample with an exonuclease and an endonuclease, a synthetic, double-stranded polynucleotide structure having reduced errors when compared to a synthetic, double-stranded reference polynucleotide remains.

21. A method for correcting mismatches in a synthetic polynucleotide sequence comprising: 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 an exonuclease such that mismatches are corrected.

22. A method for selecting a correctly assembled, synthetic, single-stranded polynucleotide comprising: 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 an exonuclease, such that a correctly assembled, synthetic, single-stranded polynucleotide structure remains.

23. A method for synthesizing a correctly assembled, synthetic, double-stranded polynucleotide comprising: 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 an exonuclease and an endonuclease such that a correctly assembled, synthetic, double-stranded polynucleotide structure remains.