WO2008054543A2 - Oligonucléotides pour l'assemblage mutiplexé d'acides nucléiques - Google Patents

Oligonucléotides pour l'assemblage mutiplexé d'acides nucléiques Download PDF

Info

Publication number
WO2008054543A2
WO2008054543A2 PCT/US2007/012095 US2007012095W WO2008054543A2 WO 2008054543 A2 WO2008054543 A2 WO 2008054543A2 US 2007012095 W US2007012095 W US 2007012095W WO 2008054543 A2 WO2008054543 A2 WO 2008054543A2
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
oligonucleotides
oligonucleotide
different
amplification
Prior art date
Application number
PCT/US2007/012095
Other languages
English (en)
Other versions
WO2008054543A3 (fr
Inventor
Brian M. Baynes
Original Assignee
Codon Devices, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Codon Devices, Inc. filed Critical Codon Devices, Inc.
Publication of WO2008054543A2 publication Critical patent/WO2008054543A2/fr
Publication of WO2008054543A3 publication Critical patent/WO2008054543A3/fr

Links

Classifications

    • 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
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Definitions

  • aspects of the invention relate to synthetic oligonucleotides and methods for obtaining correct oligonucleotides for nucleic acid assembly.
  • Recombinant and synthetic nucleic acids have many applications in research, industry, agriculture, and medicine.
  • Recombinant and synthetic nucleic acids can be used to express and obtain large amounts of polypeptides, including enzymes, antibodies, growth factors, receptors, and other polypeptides that may be used for a variety of medical, industrial, or agricultural purposes.
  • Recombinant and synthetic nucleic acids also can be used to produce genetically modified organisms including modified bacteria, yeast, mammals, plants, and other organisms.
  • Genetically modified organisms may be used in research (e.g., as animal models of disease, as tools for understanding biological processes, etc.), in industry (e.g., as host organisms for protein expression, as bioreactors for generating industrial products, as tools for environmental remediation, for isolating or modifying natural compounds with industrial applications, etc.), in agriculture (e.g., modified crops with increased yield or increased resistance to disease or environmental stress, etc.), and for other applications.
  • Recombinant and synthetic nucleic acids also may be used as therapeutic compositions (e.g., for modifying gene expression, for gene therapy, etc.) or as diagnostic tools (e.g., as probes for disease conditions, etc.).
  • nucleic acids e.g., naturally occurring nucleic acids
  • combinations of nucleic acid amplification, mutagenesis, nuclease digestion, ligation, cloning and other techniques may be used to produce many different recombinant nucleic acids.
  • Chemically synthesized polynucleotides are often used as primers or adaptors for nucleic acid amplification, mutagenesis, and cloning.
  • nucleic acids are made (e.g., chemically synthesized) and assembled to produce longer target nucleic acids of interest.
  • nucleic acids are made (e.g., chemically synthesized) and assembled to produce longer target nucleic acids of interest.
  • multiplex assembly techniques are being developed for assembling oligonucleotides into larger synthetic nucleic acids that can be used in research, industry, agriculture, and/or medicine.
  • the invention relates in general to methods and products aimed at facilitating the use of chemically-synthesized oligonucleotides in various applications, including multiplex nucleic acid assembly.
  • the invention provides a heterogeneous population of synthetic oligonucleotides each having a nucleotide sequence comprising a target sequence identical to a portion of a sequence of a target nucleic acid, an inner amplification sequence pair consisting of a 5' inner amplification sequence located 5' of the target sequence and a 3' inner amplification sequence located 3' of the target sequence, an outer amplification sequence pair consisting of a 5' outer amplification sequence located 5' of the 5' inner amplification sequence and a 3' outer amplification sequence located 3' of the 3' inner amplification sequence.
  • the invention provides a synthetic oligonucleotide having a nucleotide sequence comprising a target sequence identical to a portion of a sequence of a target nucleic acid, an inner amplification sequence pair consisting of a 5' inner amplification sequence located 5' of the target sequence and a 3' inner amplification sequence located 3 ' of the target sequence, an outer amplification sequence pair consisting of a 5' outer amplification sequence located 5' of the 5' inner amplification sequence and a 3' outer amplification sequence located 3' of the 3' inner amplification sequence.
  • the synthetic oligonucleotide further comprises at least one spacer sequence located between one member of the inner amplification sequence pair and the target sequence. In some embodiments, the synthetic oligonucleotide further comprises at least one spacer sequence located between one member of the outer amplification sequence pair and one member of the inner amplification sequence pair, provided both members are located on the same side of the target sequence.
  • a first spacer sequence is located between the 5' inner amplification sequence and the target sequence, and a second spacer sequence is located between the 3 ' inner amplification sequence and the target sequence.
  • the at least one spacer sequence is between 1 and 20 nucleotides long. In some embodiments, the at least one spacer sequence is about 5 nucleotides long. However, any suitable spacer sequence may be used as the invention is not limited in this respect.
  • the invention provides a plurality of oligonucleotide sets, wherein each oligonucleotide set is synthesized to have a nucleotide sequence comprising a target sequence identical to a portion of a sequence of a target nucleic acid, an inner amplification sequence pair consisting of a 5' inner amplification sequence located 5' of the target sequence and a 3' inner amplification sequence located 3' of the target sequence, an outer amplification sequence pair consisting of a 5' outer amplification sequence located 5' of the 5' inner amplification sequence and a 3' outer amplification sequence located 3' of the 3' inner amplification sequence, wherein each oligonucleotide set has a different target sequence, a different inner amplification sequence pair, and an identical outer amplification sequence pair.
  • At least one oligonucleotide set comprises at least one spacer sequence located between one member of the inner amplification sequence pair and the target sequence. In some embodiments, at least one oligonucleotide set comprises at least one spacer sequence located between one member of the outer amplification sequence pair and one member of the inner amplification sequence pair, provided both members are located on the same side of the target sequence.
  • the at least one spacer sequence is between 1 and 20 nucleotides long. In some embodiments, the at least one spacer sequence is about 5 nucleotides long. However, any suitable spacer sequence may be used as the invention is not limited in this respect.
  • each oligonucleotide set is synthesized to have a sequence of the same length. In certain embodiments, at least two oligonucleotide sets have target sequences of different lengths and spacer sequences of different lengths, but wherein the - A -
  • the oligonucleotides are synthesized on a solid support.
  • the solid support is an array such as a chip. In some embodiments, more than 100 different oligonucleotide sets are synthesized on a single array or chip.
  • aspects of the invention also relate to pools or sets of oligonucleotides containing different target sequences with or without one or more flanking universal, selection, and/or spacer sequences.
  • a service may be provided wherein a pool or set of oligonucleotides may be ordered and prepared according to techniques described herein in order to provide high yield of correct oligonucleotides (e.g., with no or very few deletions, for example with less than 10%, and preferably less than 5%, less than 1%, less than 0.1%, less than 0.01%, less than 0.001% or fewer oligonucleotides in a pool or set having unwanted deletions).
  • aspects of the invention also include providing one or more design strategies for a customer wanting to prepare pools or sets of oligonucleotides having different specified sequences.
  • compositions or kits containing one or more universal and/or selection primers along with sequence information and instructions for their use according to embodiments of the invention may be labeled with an affinity tag for purification or isolation and/or a detection tag to allow detection or monitoring of oligonucleotide preparation steps.
  • an affinity tag for purification or isolation and/or a detection tag to allow detection or monitoring of oligonucleotide preparation steps.
  • Certain tags such as biotin may be used for purification and/or detection. However, any suitable affinity and/or detection tags may be included.
  • FIG. 1 illustrates non-limiting aspects of an embodiment of a polymerase-based multiplex oligonucleotide assembly reaction
  • FIG. 2 illustrates non-limiting aspects of an embodiment of sequential assembly of a plurality of oligonucleotides in a polymerase-based multiplex assembly reaction
  • FIG. 3 illustrates a non-limiting embodiment of a ligase-based multiplex oligonucleotide assembly reaction
  • FIG. 4 illustrates several non-limiting embodiments of ligase-based multiplex oligonucleotide assembly reactions on supports;
  • FIG. 5 outlines a non-limiting embodiment of a nucleic acid assembly procedure;
  • FIG. 6 illustrates a non-limiting embodiment of an oligonucleotide and the acts performed on the oligonucleotide to arrive at the target sequence
  • FIG. 7 illustrates three non-limiting embodiments of oligonucleotides.
  • oligonucleotides on a solid substrate allows parallel synthesis of a plurality of oligonucleotides.
  • a solid substrate such as microchip or glass slides
  • Oligonucleotides synthesized in parallel are harvested from the chip or slide and combined, thereby usually resulting in a heterogeneous pool.
  • chip-based oligonucleotide synthesis commonly deletes one or more bases of the desired sequence from the product.
  • the probability of a chemically synthesized oligonucleotide having a deletion within the target sequence increases with length.
  • the coupling efficiency of nucleotides during chemical synthesis is substantially reduced as the oligonucleotide gets longer, particularly at lengths greater than 50 bases.
  • the end result is a heterogeneous population of oligonucleotides having some but not completely common sequence.
  • the yield of oligonucleotide from chip-based methods is also lower than with other methods.
  • the invention is therefore premised in part on methods and products for facilitating the use of chemically-synthesized oligonucleotides.
  • the invention contemplates increasing the quality and quantity of chemically synthesized oligonucleotides, particularly those produced on a solid substrate, such as a microchip.
  • the invention provides methods for enriching oligonucleotides having a desired sequence (and thereby excluding oligonucleotides comprising one or more deletions), and for increasing amounts of oligonucleotide generated from particular chemical synthesis methods. It is to be understood that the methods of the invention can be applied to any oligonucleotide chemical synthesis method having limitations as set forth herein, including a pronounced deletion rate.
  • chip-based methods and chip-based oligonucleotides as exemplary embodiments. It should be appreciated that methods and configurations described herein in the context of chip-based oligonucleotides can be applied to any oligonucleotide (or pools or subsets of oligonucleotides) regardless of their synthesis (e.g., for oligonucleotides from chip-based, synthesis, column-based synthesis, synthesis on any solid support, or synthesis on any porous material, gel, or in solution, etc., or any combination thereof).
  • the invention accomplishes increased quality and quantity of oligonucleotides by addition of specific sequences on one or both ends of particular chemically synthesized oligonucleotides. That is, the oligonucleotides are synthesized to have one and preferably more particular sequences that flank its target sequence.
  • the target sequence is the sequence that is identical (or complementary, depending on the strand comparison) to a region of a target nucleic acid desired to be synthesized using for example the assembly based methods described herein.
  • the length of the target sequence may vary, and the invention is not limited in this respect. Suitable ranges for oligonucleotide target sequences are provided herein.
  • the terms "target sequence” and "central assembly sequence” are used interchangeably herein.
  • flanking additional sequences described herein are referred to generally as non-target sequences.
  • the length of the non-target sequence may also vary and the invention is not limited in this respect, unless otherwise indicated.
  • the non-target sequence may add 5- 100, 10-50, or 15-30 bases to the target sequence length.
  • the non-target sequences may include, according to the invention, spacer sequences and amplification sequences. The function of these sequences will be discussed herein.
  • Amplification sequences on an oligonucleotide may be defined as sequences that are either identical to or complementary to an amplification primer. It should be appreciated that since an oligonucleotide is single-stranded, one of a pair of amplification primers is complementary to and hybridizes to an amplification sequence at the 3' end of the oligonucleotide.
  • the second amplification primer is identical to an amplification sequence at the 5' end of the oligonucleotide. Accordingly, the second primer hybridizes to the 3' end of the complement of the oligonucleotide, rather than the oligonucleotide itself.
  • This configuration applies to all amplification primer pairs that are used for exponential amplification (regardless of whether they are universal or selection primers as described herein). It should be appreciated that some embodiments of the invention may involve a single amplification that is used for linear amplification.
  • a primer is complementary to the 3' end of the oligonucleotide if it is designed to amplify the oligonucleotide, or complementary to the 3' end of the complement of the oligonucleotide if it is designed to amplify the complement in a linear fashion.
  • 3' refers to sequences that are 3' from the target sequence on an oligonucleotide (or 3' from the complement of the target sequence on a complement of the oligonucleotide).
  • 5' refers to sequences that are 5' to the target sequence on an oligonucleotide (or 5' to the complement of the target sequence on a complement of the oligonucleotide).
  • spacer sequences may be defined as sequences on an oligonucleotide of the invention that are neither target sequences nor amplification sequences (or complements of either thereof). Typically, spacer sequences may be located between any amplification sequence and the target sequence (or the complement of any thereof).
  • aspects of the invention may be used to prepare pools of oligonucleotides for multiplex nucleic acid assembly reactions that involve the assembly of a plurality of nucleic acids (e.g., polynucleotides, oligonucleotides, etc.) to form a longer nucleic acid product.
  • Methods and compositions of the invention may be used to remove error containing oligonucleotides from a pool of oligonucleotides prior to their use in a variety of nucleic acid assembly procedures.
  • Methods and composition also may be used to prepare separate subsets of oligonucleotides from an initial pool of oligonucleotides (e.g., from a pool of oligonucleotides that were synthesized on a chip).
  • Separate oligonucleotide subsets may be used to assemble different intermediate fragments that are assembled in parallel prior to being combined during a subsequent cycle of a multi- step multiplex assembly reaction.
  • separate subsets may be used to assemble different target nucleic acids (e.g., sequence variants of a target nucleic acid of interest or unrelated target nucleic acids) or fragments thereof (e.g., intermediate fragments in an assembly).
  • target nucleic acids e.g., sequence variants of a target nucleic acid of interest or unrelated target nucleic acids
  • fragments thereof e.g., intermediate fragments in an assembly
  • FIG. 6 illustrates a non-limiting embodiment of a method for preparing an oligonucleotide for an assembly reaction.
  • an oligonucleotide is shown having a central target sequence flanked by 5' and 3' selection amplification sequences, which in turn are flanked by spacer sequences, which are flanked by 5' and 3' universal amplification sequences.
  • the selection, spacer, and universal sequences are shown as the 5' and 3' non-target sequences.
  • a heterogeneous pool of oligonucleotides may include a plurality of different oligonucleotides, each having a different target sequence but the same 5' and 3' universal non-target sequences.
  • a first subset of oligonucleotides in this pool may share the same 5' and 3' selection sequences (referred to as first 5' and 3' selection sequences) that are not found on other subsets of oligonucleotides in the pool.
  • amplification using universal or outer amplification primers amplifies all of the plurality of different oligonucleotides in the starting pool.
  • All of these amplified oligonucleotides include the spacer sequence, if present. Accordingly, in some embodiments they can be size-selected to remove deletion (or insertion) containing oligonucleotides. Subsequently, in act 2) amplification with a first pair of selection primers amplifies only the first subset (or sub-pool) of oligonucleotides that share the first 5' and 3' selection sequences. In the configuration shown in FIG. 6, the first subset of oligonucleotides no longer contains either the outer universal amplification sequences or the spacer sequences.
  • act 3 the remaining inner amplification sequences are removed from the first subset of oligonucleotides to generate a preparation of different oligonucleotides having different target sequences, but containing no non-target sequences.
  • oligonucleotides can be used for assembly as described herein. It should be appreciated that these oligonucleotides may be double- stranded and in some embodiments may be processed to generate appropriate single strands. However, in some embodiments, double-stranded oligonucleotides may be used directly for assembly as described herein.
  • FIG. 6 is non-limiting and that different combinations of universal sequences, selection sequences, and spacer sequences may be used.
  • only a single universal sequence is used (e.g., a 3' sequence that can be used for linear amplification of a single-stranded complement of the initial pool of oligonucleotides.
  • only a single selection sequence may be used (e.g., 5' or 3') for either linear amplification (e.g., of either strand of an amplified double-stranded oligonucleotide pool) or for exponential amplification in combination with a primer for one of the universal sequences.
  • a spacer sequence is optional, and may be located in any suitable position (e.g., 5' and/or 3' to the target, between a universal sequence and a selection sequence, between a selection sequence and the target sequence, or any combination thereof since two or more spacer sequences may be used).
  • the invention contemplates the addition of spacer sequence on an oligonucleotide having a particular target sequence.
  • the length and sequence of the spacer sequence will vary depending on the application and on the length of the other oligonucleotides synthesized in parallel.
  • the spacer sequence is designed to increase the length of the oligonucleotide with the desired result that all oligonucleotides synthesized on, for example, a chip will be of the same length. As a result, when the oligonucleotides are harvested from the chip and pooled, the majority will have the same length.
  • Oligonucleotides that include a deletion however will be shorter than the majority of oligonucleotides, and it is possible to identify and thus remove those shorter oligonucleotides from the pool.
  • Methods for separating oligonucleotides based on length include but are not limited to HPLC, size exclusion chromatography/columns, and the like.
  • the use of spacer sequences is particularly important when oligonucleotides of differing lengths are generated and ultimately pooled together. In this latter embodiment, since the desired oligonucleotides will differ in length, it is impossible to distinguish between oligonucleotides having the desired sequence and those having a deletion.
  • Spacer sequence length and sequence will be governed by the particular oligonucleotide synthesis, but is otherwise not limited. Spacer sequence lengths may be 1-5 bases, 1-10 bases, or 1-20 bases, although they are not intended to be so limited. Spacer sequence can be 5' and/or 3' to the oligonucleotide target sequence.
  • FIG. 7 illustrates a non-limiting example of three oligonucleotide species A, B, and C, where all three oligonucleotides comprise target sequences of different length.
  • each comprises an outer pair of "universal” amplification sequences, which is common to all.
  • Each also comprises an inner “selection cassette, " comprising "selection” amplification sequences and optional "spacer” sequences.
  • a different specific "spacer” may be designed for each oligonucleotide.
  • a spacer sequence may be used to adjust the length of each oligonucleotide such that a species with a shorter target sequence is lengthened with an additional segment (the spacer sequence). This can be used to obtain a uniform oligonucleotide length across a heterogeneous mixture of oligonucleotides having target sequences of different lengths.
  • common universal amplification sequences are used so that a single universal amplification primer or a single pair of universal amplification primers may be used to amplify a pool of oligonucleotides having different central sequences (e.g., different target sequences).
  • common selection sequences may be included on subsets of oligonucleotides so that different sets of oligonucleotides may be amplified and isolated from the pool of oligonucleotides using a single subset-specific selection primer or a single subset-specific pair of selection primers.
  • sets and “subsets” may be used interchangeably herein to refer to smaller sub-pools of oligonucleotides relative to an initial heterogeneous pool of oligonucleotides.
  • a pool of oligonucleotides may be used to refer to a heterogeneous population of different synthetic oligonucleotides (e.g., synthesized on one or more columns, chips, or other solid supports or in other gel or soluble protocols).
  • Oligonucleotide A comprises a target sequence of 25 nucleotides in length
  • Oligonucleotide B comprises a target sequence of 30 nucleotides
  • Oligonucleotide C comprises a target sequence of 32 nucleotides.
  • 7 extra nucleotides in the form of spacer sequence are added onto A to compensate for the difference in size.
  • spacer sequence is situated in the 5' non-target sequences and in the 3' non-target sequences.
  • a spacer sequence of 2 nucleotides is incorporated into B to standardize length. Given that the remaining sequences (i.e., the universal and selective sequences) are identical in size, all three oligonucleotide species now have the same total number of nucleotides (and the same length), unless a deletion occurred during synthesis. Size selection (and exclusion), as described herein, can be performed before or after amplification, as described herein. The order will generally depend on the sensitivity or detection limit of the selection method used. Thus, while amplification prior to size selection may result in amplification of many deleted oligonucleotides, it may still be necessary if the deleted oligonucleotides are not even detected in the absence of amplification.
  • a pool or first set of oligonucleotides are designed to include at least one or two outer flanking primer binding sequences (e.g., a 5', a 3', or both 5' and 3' outer flanking sequences) that are common to all oligonucleotides. It should be appreciated that 5' and 3' outer flanking sequences may be the same, complements of each other, or unrelated.
  • a first subset of the pool of oligonucleotides also may include one or two first inner primer-binding flanking sequences (e.g., a 5', a 3', or both 5' and 3' first inner flanking sequences) that are different from the outer flanking sequences and are common only to the first subset of oligonucleotides.
  • first inner primer-binding flanking sequences e.g., a 5', a 3', or both 5' and 3' first inner flanking sequences
  • Additional subsets of the pool of oligonucleotides may include one or two different inner primer-binding flanking sequences (e.g., a 5', a 3', or both 5' and 3' inner flanking sequences) that are specific and common for each subset of oligonucleotides, but different from the outer flanking sequences and different from the inner flanking sequences of each other subset of oligonucleotides. It should be appreciated that subset-specific 5' and 3' inner flanking sequences may be the same, complements of each other, or unrelated.
  • one or more primers that are specific for the outer flanking sequences can be used to amplify the entire pool of oligonucleotides whereas one or two primers that are specific for each subset-specific inner flanking sequences may be used to specifically amplify each subset of oligonucleotides (e.g., first, second, third, fourth, fifth, etc., subset of oligonucleotides) .
  • a spacer sequence may be included at either or both ends (e.g., 5', 3', or both) of the target sequence of an oligonucleotide.
  • a spacer sequence is flanked by outer flanking sequences (e.g., 3' to a 5' outer flanking sequence, and 5' to a 3' outer flanking sequence), but is not always flanked by inner flanking sequences. Accordingly, in some embodiments, the spacer sequence(s) may be located between the outer and inner flanking sequences. However, in some embodiments, the spacer sequence(s) are located between the inner flanking sequence(s) and the target sequence (e.g., 3', 5', or both relative to the target sequence).
  • an oligonucleotide may be designed to include two or more spacer sequences and may include one or more spacer sequences between outer and inner flanking sequences and one or more spacer sequences between inner flanking sequences and the target sequence.
  • spacer sequences may be defined as those sequences that are not part of the target sequence and also are not complementary to a primer that is used for amplification of the pool or a subset of the pool of oligonucleotides. The spacer sequences ultimately may be removed along with the outer and inner flanking primer-binding sequences to produce a target sequence that is used for assembly.
  • spacer sequences are useful in a variety of applications and is not limited to applications drawn to removal of deletion-containing species from those having a correct size.
  • the present invention contemplates uses of spacer sequences for the purpose of differentiating a first set of oligonucleotides from a second set of oligonucleotides in a pool, when the oligonucleotides from both sets have target sequences of the same size.
  • a first set of oligonucleotides may be designed to contain a mutation, such as a single base substitution, while a second set does not (e.g., wild type sequence). Both of the species of target sequences contain the same number of nucleotides, and thus have identical sizes.
  • a spacer may be included in the first (or second) set of oligonucleotides, outside the target sequence as described above.
  • the first set of oligonucleotides and the second set of oligonucleotides can be separated by size.
  • each set can be separately processed (e.g., using other selection cassettes) for use.
  • the spacer described herein in combination with various other non-target sequences may offer utility for a number of applications where size differentiation amongst oligonucleotides in a mixture is deemed useful.
  • the size differentiation step for which the use of spacers is useful may be a final step (where target sequences of variable length which are synthesized and processed collectively by initial size adjustment using a spacer), initial step (where target sequences of identical size are 'tagged' by a spacer or an intermediate step to differentiate by size), or an intermediate step.
  • different combinations and configurations of spacers may be used to generate a plurality of different subsets of oligonucleotides each having a different common size.
  • each subset having a different common size may be designed for a different purpose and the different sizes may be used for selection after synthesis and amplification to further purify only the oligonucleotides of interest for a particular purpose.
  • oligonucleotides in a pool all may include spacers designed so that the size of all the oligonucleotides amplified by universal primers is the same. These can be size selected to remove deletions (and/or insertions). However, a first subset of the oligonucleotides may have a spacer between the 5 ' and/or 3 ' universal sequences and the inner selection sequences.
  • a second subset of the oligonucleotides may have a spacer between the 5' and/or 3' selection sequence and the target sequence. Therefore, amplification with first selection primers yields a first set of oligonucleotides without spacers, whereas amplification with the second selection primers yields a second set of oligonucleotides having spacers. Accordingly, the first and second sets of oligonucleotides can be separated by size. It should be appreciated that this configuration allows the first and second sets of oligonucleotides to be separated by size even if the first selection primers (and corresponding selection sequences) are the same as the second selection primer (and corresponding selection sequences).
  • This separation also may be used even if the target sequences vary in size, provided that the size differences between different target sequences is small relative to the length of the spacer so that the first and second heterogeneous populations of oligonucleotides could still be separated based on the presence or absence of spacer.
  • Amplification sequences and oligonucleotide quantity Generally, a defined region on a microchip contains a plurality of oligonucleotides of a given species at fmole levels. These levels are not optimal for subsequent applications including multiplex nucleic acid assembly.
  • the invention addresses this by providing methods and products for amplifying the oligonucleotides either universally or specifically following chemical synthesis. It should be appreciated that amplification reactions described herein may be exponential (e.g., using two primers that hybridize to opposite ends of complementary copies of an oligonucleotide) or linear (e.g., using a single primer that hybridizes to the 3' end of the oligonucleotide template or the complement thereof).
  • Different combinations of linear and exponential amplification may be used by using different combinations of one or two universal primers and one or two selection primers during amplification. It should be appreciated that single amplification primers may be used for linear amplification even if the oligonucleotide contains both 5' and 3' primer binding sequences.
  • the amplifications described herein are referred to as universal amplifications or selection amplifications.
  • Universal amplifications employ universal primers that are complementary to the universal sequences of the oligonucleotides.
  • Selection amplifications, as described herein, may employ a pair of selection primers that are complementary to the selection sequences of the oligonucleotides. Selection amplifications however may alternatively employ one universal primer and one selection primer.
  • the population of oligonucleotides synthesized on a solid substrate are amplified simultaneously using a pair of universal primers to produce sufficient quantities of the oligonucleotides, followed by amplification of a set of oligonucleotides within the population using a pair of selection primers specific to that set.
  • This approach provides an almost unlimited quantity of the starting population of oligonucleotides, without the need for dividing the starting population.
  • a pool may contain any number of different oligonucleotides, for example from about 100 to about 10 7 different oligonucleotides (e.g., about 100; about 100-1,000; about 1,000; about 1,000-10,000; about 10,000; about 10,000-100,000; about 100,000; about 100,000-1,000,000; about 10 6 , about 10 6 -10 7 , about 10 , or more different oligonucleotides).
  • a subset may contain any smaller number of different oligonucleotides (e.g., a subset may represent about 1%, about 5%, about 10%, about 25%, about 50%, or fewer or more of the oligonucleotides in the pool).
  • a subset may include about 10, about 25, about 50, about 75, about 100, about 500, about 1,000 or more different oligonucleotides (or any intermediate integer number of different oligonucleotides).
  • each different oligonucleotide may be represented by more than one molecule in an initial pool (e.g., about 10, about 100, about 10 3 , about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , or more copies of each different oligonucleotide may be present in an initial pool).
  • each oligonucleotide may be present in a pool in femptomolar, picomolar, nanomolar, micromolar, or higher or lower amounts.
  • universal amplification refers to amplification of all oligonucleotides within a pool (e.g., all oligonucleotides harvested from a chip).
  • Universal amplification can be accomplished through the use of universal sequences at the 5' and 3' ends of the oligonucleotide. These universal sequences may be common to all oligonucleotides generated on the chip. This allows amplification of all oligonucleotides within the pool using only a single pair of primers (i.e., the universal primer pair). In addition, since all the oligonucleotides being amplified will be of the same length, all oligonucleotides should be amplified to the same extent, barring any sequence-specific effects.
  • Universal sequence and universal amplification sequence are used interchangeably herein.
  • Universal sequences are also referred to herein as outer amplification sequence pairs which in turn are comprised of 5 ' and 3 ' outer amplification sequences.
  • the sequence and length of the universal sequence will vary depending on the application and various factors including but not limited to the length of the target sequence.
  • the amplification step may be carried out directly on the chip. Alternatively the amplification step may be carried out following oligonucleotide release from the chip.
  • oligonucleotides designed to have different target sequences
  • the chip-based methods may therefore be used to generate several sets of oligonucleotides, each of which is required for the assembly of a particular nucleic acid.
  • Members of each set will therefore additionally contain common selection sequences which can be used to amplify a set of oligonucleotides from the pool.
  • each set of oligonucleotides will be distinguished from other sets based at least in part on its selection sequence, and therefore amplification of one set will result in its enrichment (and the exclusion of other sets).
  • the selection sequences will be present 5' and 3' of the target sequence, and will be internal to the universal sequences (see FIGs. 6 and 7).
  • the term internal refers to a non-end position of a sequence within an oligonucleotide.
  • a 5' selection sequence that is internal to the universal sequences is situated between the 5' universal sequence and the target sequence.
  • a 3' selection sequence that is internal to the universal sequences is situated between the 3' universal sequence and the target sequence.
  • the non- target sequences are discrete and non-overlapping.
  • selection sequence and selection amplification sequence are used interchangeably herein.
  • Selection sequences are also referred to herein as inner amplification sequences (or pairs) which in turn are comprised of 5' and 3 inner amplification sequences.
  • the invention further contemplates amplification of a set of oligonucleotides within the population using one selection sequence and one universal sequence.
  • a set of oligonucleotides may comprise 5' and 3' universal sequences and either a 5' or a 3' selection sequence, but not both.
  • Selective amplification can then be accomplished using the primers which hybridize to the 5' universal sequence and the 3' selective sequence, or alternatively to the 5' selective sequence and the 3' universal sequence.
  • oligonucleotide may include one or two selection sequences.
  • the selection sequence should be at the 5' end of the oligonucleotide so that it can be recognized by a selection primer that hybridizes to the complement of the oligonucleotide that was generated by linear amplification using the single universal primer.
  • a single selection sequence may be at either the 5' or 3' end if a pair of universal primers are used for exponential amplification generating an amplified double stranded oligonucleotide pool. It should be appreciated that a primer binding sequence is defined as a universal sequence based on the fact that it is present on all different oligonucleotides in a pool and not based on any particular sequence of nucleotides.
  • a primer binding sequence is defined as a selection sequence based on the fact that it is present on only a subset of different oligonucleotides within a pool and not based on any particular sequence of nucleotides.
  • a spacer sequence (or portions thereof) may be present on different numbers of nucleotides within a pool or within a subset depending on the configuration.
  • the space is defined as a spacer based on the fact that it is not used for primer binding (and not based on any particular sequence of nucleotides).
  • a given sequence may be used as a universal amplification sequence, a selection amplification sequence, or a spacer sequence depending on the configuration of the pool of oligonucleotides and the primers that are used for amplification.
  • a particular sequence is used as a universal, selection, or spacer sequence, it should be appreciated that the universal, selection, and spacer sequences should be different from each other. Also, the amplification sequences should be different from the target sequences.
  • oligonucleotide configurations for preparing pools and sets of amplified oligonucleotides as described herein, care should be taken to select and use amplification sequences that are not present on the target sequences (or the complements thereof) of the oligonucleotides in the pool.
  • a sequence that is not identical or perfectly complementary to an oligonucleotide may still cause problems if the sequence similarity or complementarity is sufficient to cause levels of mis-priming and/or incorrect amplification that would interfere with the intended oligonucleotide amplification procedure.
  • primer binding sequences and primers as used herein are typically perfectly complementary (100% complementary - meaning that each G, A, T, and C on the primer will hybridize to a C, T, A, and G, respectively on the template when the primer binds to the template without any mismatched or unpaired nucleotides on the primer or on the template within the region of hybridization).
  • sequences that are not perfectly complementary may be used as primers and primer binding sequences if the degree of complementarity is sufficient for selective amplification of the desired oligonucleotide regions as described herein.
  • Oligonucleotides designed according to the invention therefore may consist of various combinations of target, universal, selection and spacer sequences as described herein.
  • the oligonucleotides generally consist of, in the 5' ⁇ 3' direction, 5' non-target sequence, target sequence, and 3' non-target sequence.
  • the oligonucleotides should preferably include a target sequence and 5' and 3' universal sequences. In some embodiments the oligonucleotides will further comprise at least one selection sequence.
  • oligonucleotides may consist of 5' universal sequence, optional spacer sequence, 5' selection sequence, target sequence, 3' selection sequence, optional spacer sequence, and 3' universal sequence.
  • An example of an oligonucleotide is shown in FIG. 7.
  • a spacer sequence, if present is located between the 5' universal sequence and the 5' selection sequence, if present, or between the 3' selection sequence, if present, and the 3' universal sequence.
  • a spacer sequence is located between a selection sequence (i.e., an inner amplification sequence) and the corresponding end of the target sequence (i.e., 5' or 3').
  • a spacer sequence is present on both sides (5' and 3') of the target sequence.
  • use of spacer sequences may be strategically designed in any combination as described herein. For example, if the difference in the total length of a plurality of oligonucleotides in a synthesis pool is relatively small (e.g., a few nucleotides longer or shorter), use of one spacer may suffice to adjust the size difference. However, if the size of a subset of oligonucleotides to be synthesized in a pool is significantly different from that of other species in the pool, two or more spacers may be designed into the oligonucleotides to accommodate the size difference. Accordingly, as a skilled artisan will appreciate, the present invention is not intended to limit the use of spacer sequences, in combination with universal and/or selection amplification sequences, to a particular configuration.
  • the total length of the oligonucleotides generally will not vary, except for those oligonucleotides comprising target sequences with deletions.
  • the total length may be 50-1000, 50-500, 50-100, 50-75, 50-60, bases or any integer therebetween, although the invention is not intended to be so limited.
  • the invention embraces specific oligonucleotides as well as a heterogeneous plurality of oligonucleotides having these elements.
  • heterogeneous means that a pool (or population or plurality) of oligonucleotides contains different species of oligonucleotides. These species may differ based on target sequence and/or presence, sequence and/or length of selective sequences and spacer sequences. All species in the heterogeneous pool however will generally share common universal sequences.
  • a pool of heterogeneous oligonucleotides may be synthesized on a chip, then amplified using a primer pair against universal amplification sequences contained in each of the species in the pool.
  • a second set of primers may be used to selectively amplify only those oligonucleotides containing a corresponding selection amplification sequence (e.g., inner amplification sequence).
  • a set of oligonucleotides is a mixture of heterogeneous species of oligonucleotides that may be selectively amplified from a greater pool of oligonucleotides.
  • a set of oligonucleotides may represent a mixture of oligonucleotides to be used for a given purpose.
  • a set of oligonucleotides may represent fragments of a gene which can be assembled.
  • outer amplification, inner amplification, spacer, and target sequences may be used for a pool of oligonucleotides regardless of whether they are synthesized on a chip or other solid support or are provided as a solution or dried preparation of oligonucleotides that are not attached to a chip or other solid support.
  • Non-target sequences such as universal, spacer and other selection sequences can be achieved in a number of ways.
  • the universal and spacer sequences are removed by virtue of a second amplification that employs the pair of selection sequences.
  • the selection sequences (or other non-target sequences situated adjacent to the target sequence) can be removed in a number of ways as described in greater detail herein.
  • a general strategy for obtaining a target sequence by removal of non-target sequences from an oligonucleotide is exemplified in FIG. 6. As illustrated, one method for removing non-target sequence, particularly sequence situated immediately adjacent to the target sequence is by enzymatic cleavage of the sequence from the target sequence.
  • Type Hs restriction enzymes are enzymes that bind to a double stranded nucleic acid at one site, referred to as the recognition site, and make a single double stranded cut outside of the recognition site.
  • the double stranded cut referred to as the cleavage site, is generally situated 0-20 bases away from the recognition site.
  • the recognition site is generally about 4-7 bp long. All type Hs restriction enzymes exhibit at least partial asymmetric recognition. Asymmetric recognition means that 5' ⁇ 3' recognition sequences are different for each strand of the nucleic acid.
  • the enzyme activity also shows polarity meaning that the cleavage sites are located on only one side of the recognition site.
  • Examples include but are not limited to BstF5 I, BtsC I, BsrD I, Bts I, AIw I, Bcc I, BsmA I, Ear I, MIy I (blunt), PIe I, Bmr I, Bsa I, BsmB I, Fau I, MnI I, Sap I, Bbs I, BciV I, Hph I, Mbo II, BfuA I, BspCN I, BspM I, SfaN I, Hga I, BseR I, Bbv I, Eci I, Fok I, BceA I, BsmF I, BtgZ I, BpuE I, Bsg I, Mme I, BseG I, Bse3D I, BseM I, AcIW I, Alw26 I, Bst6 1, BstMA I, Eaml 104 I, Ksp632 I, Pps I, Sch I (blu
  • the location of the recognition site within the non-target sequence will vary depending upon the enzyme used and the distance between its recognition and spacer sites.
  • the invention contemplates that the double stranded cut may be situated within the target sequence or at an end of the target sequence so that no non-target sequence is retained.
  • enzymes with greater distances between their recognition and cleavage sites can accommodate a spacer sequence that is immediately adjacent to the target sequence and thus between the selection sequence and the target sequence.
  • Another method for removing non-target sequence from an oligonucleotide employs uracil DNA glycosylase. This enzyme hydrolyzes uracil-glycosidic bonds in nucleic acids thereby removing uracil and creating alkali-sensitive abasic sites in the DNA which can be subsequently hydrolyzed by endonuclease, heat or alkali treatment.
  • This approach requires the deliberate incorporation of one or more uracil bases in the non-target sequence, preferably immediately adjacent to the target sequence.
  • the uracil residues may in some instances also be situated within the target sequence itself, preferably within bases from either or both ends.
  • Uracil DNA glycosylase is commercially available from suppliers such as Roche Applied Science.
  • T4 DNA polymerase possesses 3' ⁇ 5' exonuclease activity. While this activity favors single-stranded regions, it can function, albeit somewhat less efficiently, on blunt ends. Accordingly, it is expected that amplified and blunt ended double stranded oligonucleotides contacted with T4 DNA polymerase will be cleaved at their 3' ends.
  • T4 The 5' ⁇ 3' polymerase activity of T4 will then attempt to replace the excised nucleotide.
  • Excision into the target sequence can be avoided by providing the nucleotide complementary to the ultimate 5' nucleotide in the target sequence. In doing so, an equilibrium will be reached between the excision of this nucleotide and its re-incorporation by T4.
  • this approach dictates the sequence adjacent to the target sequence as well as the ultimate nucleotide of the target sequence.
  • the remaining 5' single stranded overhang can be removed using a single stranded specific nuclease such as but not limited to S 1 nuclease.
  • aspects of the invention may include automating one or more acts described herein. For example, amplification, size selection and sequence removal acts may be automated in order to generate the desired product automatically. Acts of the invention may be automated using, for example, a computer system.
  • aspects of the invention may be used in conjunction with any suitable multiplex nucleic acid assembly procedure.
  • enrichment techniques of the invention may be use in connection with or more of the multiplex nucleic acid assembly procedures described below.
  • a nucleic acid assembly reaction may involve the assembly of a plurality of nucleic acids (e.g., polynucleotides, oligonucleotides, etc.) to form a longer nucleic acid product.
  • Methods and compositions of the invention may be used to remove error containing oligonucleotides from a pool of oligonucleotides prior to their use in a variety of nucleic acid assembly procedures.
  • Non-limiting examples of assembly reactions are described herein and illustrated in FIGs. 1-4.
  • FIG. 5 illustrates a method for assembling a nucleic acid in accordance with one embodiment of the invention.
  • sequence information is obtained.
  • the sequence information may be the sequence of a predetermined target nucleic acid that is to be assembled.
  • the sequence may be received in the form of an order from a customer. The order may be received electronically or on a paper copy.
  • the sequence may be received as a nucleic acid sequence (e.g., DNA or RNA).
  • the sequence may be received as a protein sequence.
  • the sequence may be converted into a DNA sequence. For example, if the sequence obtained in act 500 is an RNA sequence, the Us may be replaced with Ts to obtain the corresponding DNA sequence. If the sequence obtained in act 500 is a protein sequence, it may be converted into a DNA sequence using appropriate codons for the amino acids.
  • codons for each amino acid consideration may be given to one or more of the following factors: i) using codons that correspond to the codon bias in the organism in which the target nucleic acid may be expressed, ii) avoiding excessively high or low GC or AT contents in the target nucleic acid (for example, above 60% or below 40%; e.g., greater than 65%, 70%, 75%, 80%, 85%, or 90%; or less than 35%, 30%, 25%, 20%, 15%, or 10%), and iii) avoiding sequence features that may interfere with the assembly procedure (e.g., the presence of repeat sequences or stem loop structures). However, these factors may be ignored in some embodiments as the invention is not limited in this respect.
  • a DNA sequence determination may omit one or more steps relating to the analysis of the GC or AT content of the target nucleic acid sequence (e.g., the GC or AT content may be ignored in some embodiments) or one or more steps relating to the analysis of certain sequence features (e.g., sequence repeats, inverted repeats, etc.) that could interfere with an assembly reaction performed under standard conditions but may not interfere with an assembly reaction including one or more concerted assembly steps.
  • sequence features e.g., sequence repeats, inverted repeats, etc.
  • the sequence information may be analyzed to determine an assembly strategy. This may involve determining whether the target nucleic acid will be assembled as a single fragment or if several intermediate fragments will be assembled separately and then combined in one or more additional rounds of assembly to generate the target nucleic acid.
  • a sequence analysis may involve scanning for the presence of one or more interfering sequence features that are known or predicted to interfere with a polymerase- based assembly.
  • An interfering sequence feature may be the presence of high GC content (e.g., a GC content of greater than 50%, greater than 60%, greater than 70%, greater than 80% or greater than 90% over a length of 10 or more bases, for example 10- 20, 20-50, 50-100 or more than 100 bases).
  • a region of a target nucleic acid with a high GC content should be assembled in a ligase-based reaction.
  • an interfering sequence structure may be the presence of two or more identical or homologous sequences that are repeated (e.g., direct or inverted repeats) within a fragment (e.g., a fragment between about 200 and about 1,000 bases long, or a longer or shorter fragment) that is to be assembled in a single reaction.
  • the length of interfering repeats may be as short as 6 or 7 bases. However, repeats that are 8, 9, 10, 11, 12 or more bases long may be more interfering.
  • a fragment containing interfering repeats is assembled using a ligase-based assembly. Alternatively, the design of the fragments being assembled in a single step may be changed to avoid the presence of two identical or homologous sequences within the same assembly fragment.
  • input nucleic acids e.g., oligonucleotides
  • the sizes and numbers of the input nucleic acids may be based in part on the type of assembly reaction (e.g., the type of polymerase-based assembly, ligase-based assembly, chemical assembly, or combination thereof) that is being used for each fragment.
  • the input nucleic acids also may be designed to avoid 5' and/or 3' regions that may cross-react incorrectly and be assembled to produce undesired nucleic acid fragments. Other structural and/or sequence factors also may be considered when designing the input nucleic acids.
  • some of the input nucleic acids may be designed to incorporate one or more specific sequences (e.g., primer binding sequences, restriction enzyme sites, etc.) at one or both ends of the assembled nucleic acid fragment.
  • the input nucleic acids are obtained. These may be synthetic oligonucleotides that are synthesized on-site or obtained from a different site (e.g., from a commercial supplier).
  • one or more input nucleic acids may be amplification products (e.g., PCR products), restriction fragments, or other suitable nucleic acid molecules. Synthetic oligonucleotides may be synthesized using any appropriate technique as described in more detail herein.
  • oligonucleotide preparations may be selected or screened to remove error-containing molecules as described in more detail herein.
  • an assembly reaction may be performed for each nucleic acid fragment.
  • the input nucleic acids may be assembled using any appropriate assembly technique (e.g., a polymerase-based assembly, a ligase-based assembly, a chemical assembly, or any other multiplex nucleic acid assembly technique, or any combination thereof).
  • An assembly reaction may result in the assembly of a number of different nucleic acid products in addition to the predetermined nucleic acid fragment.
  • an assembly reaction may be processed to remove incorrectly assembled nucleic acids (e.g., by size fractionation) and/or to enrich correctly assembled nucleic acids (e.g., by amplification, optionally followed by size fractionation).
  • correctly assembled nucleic acids may be amplified (e.g., in a PCR reaction) using primers that bind to the ends of the predetermined nucleic acid fragment.
  • act 530 may be repeated one or more times. For example, in a first round of assembly a first plurality of input nucleic acids (e.g., oligonucleotides) may be assembled to generate a first nucleic acid fragment.
  • the first nucleic acid fragment may be combined with one or more additional nucleic acid fragments and used as starting material for the assembly of a larger nucleic acid fragment.
  • this larger fragment may be combined with yet further nucleic acids and used as starting material for the assembly of yet a larger nucleic acid. This procedure may be repeated as many times as needed for the synthesis of a target nucleic acid. Accordingly, progressively larger nucleic acids may be assembled. At each stage, nucleic acids of different sizes may be combined. At each stage, the nucleic acids being combined may have been previously assembled in a multiplex assembly reaction.
  • nucleic acids being combined may have been obtained from different sources (e.g., PCR amplification of genomic DNA or cDNA, restriction digestion of a plasmid or genomic DNA, or any other suitable source).
  • sources e.g., PCR amplification of genomic DNA or cDNA, restriction digestion of a plasmid or genomic DNA, or any other suitable source.
  • nucleic acids generated in each cycle of assembly may contain sequence errors if they incorporated one or more input nucleic acids with sequence error(s).
  • a fidelity optimization procedure may be performed after a cycle of assembly in order to remove or correct sequence errors. It should be appreciated that fidelity optimization may be performed after each assembly reaction when several successive cycles of assembly are performed. However, in certain embodiments fidelity optimization may be performed only after a subset (e.g., 2 or more) of successive assembly reactions are complete. In some embodiments, no fidelity optimization is performed.
  • act 540 is an optional fidelity optimization procedure.
  • Act 540 may be used in some embodiments to remove nucleic acid fragments that seem to be correctly assembled (e.g., based on their size or restriction enzyme digestion pattern) but that may have incorporated input nucleic acids containing sequence errors as described herein. For example, since synthetic oligonucleotides may contain incorrect sequences due to errors introduced during oligonucleotide synthesis, it may be useful to remove nucleic acid fragments that have incorporated one or more error-containing oligonucleotides during assembly. In some embodiments, one or more assembled nucleic acid fragments may be sequenced to determine whether they contain the predetermined sequence or not. This procedure allows fragments with the correct sequence to be identified.
  • error containing-nucleic acids may be double-stranded homoduplexes having the error on both strands (i.e., incorrect complementary nucleotide(s), deletion(s), or addition(s) on both strands), because the assembly procedure may involve one or more rounds of polymerase extension (e.g., during assembly or after assembly to amplify the assembled product) during which an input nucleic acid containing an error may serve as a template thereby producing a complementary strand with the complementary error.
  • polymerase extension e.g., during assembly or after assembly to amplify the assembled product
  • a preparation of double-stranded nucleic acid fragments may be suspected to contain a mixture of nucleic acids that have the correct sequence and nucleic acids that incorporated one or more sequence errors during assembly.
  • sequence errors may be removed using a technique that involves denaturing and reannealing the double-stranded nucleic acids.
  • single strands of nucleic acids that contain complementary errors may be unlikely to reanneal together if nucleic acids containing each individual error are present in the nucleic acid preparation at a lower frequency than nucleic acids having the correct sequence at the same position. Rather, error containing single strands may reanneal with a complementary strand that contains no errors or that contains one or more different errors.
  • error- containing strands may end up in the form of heteroduplex molecules in the reannealed reaction product.
  • Nucleic acid strands that are error-free may reanneal with error- containing strands or with other error-free strands.
  • Reannealed error-free strands form homoduplexes in the reannealed sample.
  • Any suitable method for removing heteroduplex molecules may be used, including chromatography, electrophoresis, selective binding of heteroduplex molecules, etc.
  • mismatch binding proteins that selectively (e.g., specifically) bind to heteroduplex nucleic acid molecules may be used.
  • One example includes using MutS, a MutS homolog, or a combination thereof to bind to heteroduplex molecules.
  • the MutS protein which appears to function as a homodimer, serves as a mismatch recognition factor.
  • MSH MutS Homolog
  • the MSH2-MSH6 complex (also known as MutS ⁇ ) recognizes base mismatches and single nucleotide insertion/deletion loops
  • the MSH2-MSH3 complex (also known as MutS ⁇ ) recognizes insertions/deletions of up to 12-16 nucleotides, although they exert substantially redundant functions.
  • a mismatch binding protein may be obtained from recombinant or natural sources.
  • a mismatch binding protein may be heat-stable.
  • a thermostable mismatch binding protein from a thermophilic organism may be used.
  • thermostable DNA mismatch binding proteins include, but are not limited to: Tth MutS (from Thermus thermophilics); Taq MutS (from Thermits aquaticus); Apy MutS (from Aquifex pyrophilus); Tma MutS (from Thermotoga maritimd); any other suitable MutS; or any combination of two or more thereof.
  • protein-bound heteroduplex molecules e.g., heteroduplex molecules bound to one or more MutS proteins
  • may be removed from a sample using any suitable technique binding to a column, a filter, a nitrocellulose filter, etc., or any combination thereof. It should be appreciated that this procedure may not be 100% efficient. Some errors may remain for at least one of the following reasons.
  • the fidelity optimization act 540 may be repeated one or more times after each assembly reaction. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles of fidelity optimization may be performed after each assembly reaction.
  • the nucleic acid is amplified after each fidelity optimization procedure. It should be appreciated that each cycle of fidelity optimization will remove additional error-containing nucleic acid molecules. However, the proportion of correct sequences is expected to reach a saturation level after a few cycles of this procedure.
  • the size of an assembled nucleic acid that is fidelity optimized may be determined by the expected number of sequence errors that are suspected to be incorporated into the nucleic acid during assembly.
  • an assembled nucleic acid product should include error free nucleic acids prior to fidelity optimization in order to be able to enrich for the error free nucleic acids. Accordingly, error screening (e.g., using MutS or a MutS homolog) should be performed on shorter nucleic acid fragments when input nucleic acids have higher error rates.
  • one or more nucleic acid fragments of between about 200 and about 800 nucleotides are assembled prior to fidelity optimization. After assembly, the one or more fragments may be exposed to one or more rounds of fidelity optimization as described herein. In some embodiments, several assembled fragments may be ligated together (e.g., to produce a larger nucleic acid fragment of between about 1,000 and about 5,000 bases in length, or larger), and optionally cloned into a vector, prior to fidelity optimization as described herein. At act 550, an output nucleic acid is obtained.
  • an output nucleic acid may be cloned with one or more other nucleic acids (e.g., other output nucleic acids) for subsequent applications.
  • Subsequent applications may include one or more research, diagnostic, medical, clinical, industrial, therapeutic, environmental, agricultural, or other uses.
  • aspects of the invention may include automating one or more acts described herein. For example, sequence analysis, the identification of interfering sequence features, assembly strategy selection (including fragment design and selection, the choice of a particular combination of extension-based and ligation-based assembly reactions, etc.), fragment production, single-stranded overhang production, and/or concerted assembly may be automated in order to generate the desired product automatically. Acts of the invention may be automated using, for example, a computer system.
  • aspects of the invention may be used in conjunction with any suitable multiplex nucleic acid assembly procedure.
  • concerted assembly steps may be used in connection with or more of the multiplex nucleic acid assembly procedures described below.
  • multiplex nucleic acid assembly relates to the assembly of a plurality of nucleic acids to generate a longer nucleic acid product.
  • multiplex oligonucleotide assembly relates to the assembly of a plurality of oligonucleotides to generate a longer nucleic acid molecule.
  • nucleic acids e.g., single or double-stranded nucleic acid degradation products, restriction fragments, amplification products, naturally occurring small nucleic acids, other polynucleotides, etc.
  • a multiplex assembly reaction e.g., along with one or more oligonucleotides
  • an assembled nucleic acid molecule that is longer than any of the single starting nucleic acids (e.g., oligonucleotides) that were added to the assembly reaction.
  • one or more nucleic acid fragments that each were assembled in separate multiplex assembly reactions may be combined and assembled to form a further nucleic acid that is longer than any of the input nucleic acid fragments.
  • one or more nucleic acid fragments that each were assembled in separate multiplex assembly reactions may be combined with one or more additional nucleic acids (e.g., single or double-stranded nucleic acid degradation products, restriction fragments, amplification products, naturally occurring small nucleic acids, other polynucleotides, etc.) and assembled to form a further nucleic acid that is longer than any of the input nucleic acids.
  • one or more multiplex assembly reactions may be used to generate target nucleic acids having predetermined sequences.
  • a target nucleic acid may have a sequence of a naturally occurring gene and/or other naturally occurring nucleic acid (e.g., a naturally occurring coding sequence, regulatory sequence, non-coding sequence, chromosomal structural sequence such as a telomere or centromere sequence, etc., any fragment thereof or any combination of two or more thereof).
  • a target nucleic acid may have a sequence that is not naturally-occurring.
  • a target nucleic acid may be designed to have a sequence that differs from a natural sequence at one or more positions.
  • a target nucleic acid may be designed to have an entirely novel sequence.
  • target nucleic acids may include one or more naturally occurring sequences, non-naturally occurring sequences, or combinations thereof.
  • multiplex assembly may be used to generate libraries of nucleic acids having different sequences.
  • a library may contain nucleic acids having random sequences.
  • a predetermined target nucleic acid may be designed and assembled to include one or more random sequences at one or more predetermined positions.
  • a target nucleic acid may include a functional sequence (e.g., a protein binding sequence, a regulatory sequence, a sequence encoding a functional protein, etc., or any combination thereof).
  • a target nucleic acid may lack a specific functional sequence (e.g., a target nucleic acid may include only non-functional fragments or variants of a protein binding sequence, regulatory sequence, or protein encoding sequence, or any other non-functional naturally-occurring or synthetic sequence, or any non-functional combination thereof).
  • Certain target nucleic acids may include both functional and non-functional sequences.
  • a target nucleic acid may be assembled in a single multiplex assembly reaction (e.g., a single oligonucleotide assembly reaction). However, a target nucleic acid also may be assembled from a plurality of nucleic acid fragments, each of which may have been generated in a separate multiplex oligonucleotide assembly reaction. It should be appreciated that one or more nucleic acid fragments generated via multiplex oligonucleotide assembly also may be combined with one or more nucleic acid molecules obtained from another source (e.g., a restriction fragment, a nucleic acid amplification product, etc.) to form a target nucleic acid. In some embodiments, a target nucleic acid that is assembled in a first reaction may be used as an input nucleic acid fragment for a subsequent assembly reaction to produce a larger target nucleic acid.
  • a target nucleic acid may be assembled in a single multiplex assembly reaction (e.g., a single oligonucleotide assembly reaction).
  • different strategies may be used to produce a target nucleic acid having a predetermined sequence.
  • different starting nucleic acids e.g., different sets of predetermined nucleic acids
  • predetermined nucleic acid fragments may be assembled using one or more different in vitro and/or in vivo techniques.
  • nucleic acids e.g., overlapping nucleic acid fragments
  • an enzyme e.g., a ligase and/or a polymerase
  • a chemical reaction e.g., a chemical ligation
  • in vivo e.g., assembled in a host cell after transfection into the host cell
  • each nucleic acid fragment that is used to make a target nucleic acid may be assembled from different sets of oligonucleotides.
  • a nucleic acid fragment may be assembled using an in vitro or an in vivo technique (e.g., an in vitro or in vivo polymerase, recombinase, and/or ligase based assembly process).
  • an in vitro assembly reaction may involve one or more polymerases, ligases, other suitable enzymes, chemical reactions, or any combination thereof.
  • a predetermined nucleic acid fragment may be assembled from a plurality of different starting nucleic acids (e.g., oligonucleotides) in a multiplex assembly reaction (e.g., a multiplex enzyme-mediated reaction, a multiplex chemical assembly reaction, or a combination thereof).
  • a multiplex assembly reaction e.g., a multiplex enzyme-mediated reaction, a multiplex chemical assembly reaction, or a combination thereof.
  • the assembly reactions described herein may be performed using starting nucleic acids obtained from one or more different sources (e.g., synthetic or natural polynucleotides, nucleic acid amplification products, nucleic acid degradation products, oligonucleotides, etc.).
  • the starting nucleic acids may be referred to as assembly nucleic acids (e.g., assembly oligonucleotides).
  • assembly nucleic acids e.g., assembly oligonucleotides
  • an assembly nucleic acid has a sequence that is designed to be incorporated into the nucleic acid product generated during the assembly process.
  • the description of the assembly reactions in the context of single-stranded nucleic acids is not intended to be limiting.
  • one or more of the starting nucleic acids illustrated in the figures and described herein may be provided as double stranded nucleic acids. Accordingly, it should be appreciated that where the figures and description illustrate the assembly of single-stranded nucleic acids, the presence of one or more complementary nucleic acids is contemplated. Accordingly, one or more double-stranded complementary nucleic acids may be included in a reaction that is described herein in the context of a single-stranded assembly nucleic acid. However, in some embodiments the presence of one or more complementary nucleic acids may interfere with an assembly reaction by competing for hybridization with one of the input assembly nucleic acids.
  • an assembly reaction may involve only single- stranded assembly nucleic acids (i.e., the assembly nucleic acids may be provided in a single-stranded form without their complementary strand) as described or illustrated herein.
  • the presence of one or more complementary nucleic acids may have no or little effect on the assembly reaction.
  • complementary nucleic acid(s) may be incorporated during one or more steps of an assembly.
  • assembly nucleic acids and their complementary strands may be assembled under the same assembly conditions via parallel assembly reactions in the same reaction mixture.
  • a nucleic acid product resulting from the assembly of a plurality of starting nucleic acids may be identical to the nucleic acid product that results from the assembly of nucleic acids that are complementary to the starting nucleic acids (e.g., in some embodiments where the assembly steps result in the production of a double-stranded nucleic acid product).
  • an oligonucleotide may be a nucleic acid molecule comprising at least two covalently bonded nucleotide residues. In some embodiments, an oligonucleotide may be between 10 and 1,000 nucleotides long.
  • an oligonucleotide may be between 10 and 500 nucleotides long, or between 500 and 1,000 nucleotides long. In some embodiments, an oligonucleotide may be between about 20 and about 100 nucleotides long (e.g., from about 30 to 90, 40 to 85, 50 to 80, 60 to 75, or about 65 or about 70 nucleotides long), between about 100 and about 200, between about 200 and about 300 nucleotides, between about 300 and about 400, or between about 400 and about 500 nucleotides long. However, shorter or longer oligonucleotides may be used. An oligonucleotide may be a single-stranded nucleic acid.
  • a double-stranded oligonucleotide may be used as described herein.
  • an oligonucleotide may be chemically synthesized as described in more detail below.
  • an input nucleic acid e.g., oligonucleotide
  • the resulting product may be double-stranded.
  • one of the strands of a double-stranded nucleic acid may be removed before use so that only a predetermined single strand is added to an assembly reaction.
  • each oligonucleotide may be designed to have a sequence that is identical to a different portion of the sequence of a predetermined target nucleic acid that is to be assembled. Accordingly, in some embodiments each oligonucleotide may have a sequence that is identical to a portion of one of the two strands of a double-stranded target nucleic acid.
  • the two complementary strands of a double stranded nucleic acid are referred to herein as the positive (P) and negative (N) strands. This designation is not intended to imply that the strands are sense and anti-sense strands of a coding sequence.
  • a P strand may be a sense strand of a coding sequence
  • a P strand may be an anti-sense strand of a coding sequence
  • a target nucleic acid may be either the P strand, the N strand, or a double-stranded nucleic acid comprising both the P and N strands.
  • oligonucleotides may be designed to have different lengths.
  • one or more different oligonucleotides may have overlapping sequence regions (e.g., overlapping 5' regions or overlapping 3' regions). Overlapping sequence regions may be identical (i.e., corresponding to the same strand of the nucleic acid fragment) or complementary (i.e., corresponding to complementary strands of the nucleic acid fragment).
  • the plurality of oligonucleotides may include one or more oligonucleotide pairs with overlapping identical sequence regions, one or more oligonucleotide pairs with overlapping complementary sequence regions, or a combination thereof. Overlapping sequences may be of any suitable length.
  • overlapping sequences may encompass the entire length of one or more nucleic acids used in an assembly reaction.
  • Overlapping sequences may be between about 5 and about 500 nucleotides long (e.g., between about 10 and 100, between about 10 and 75, between about 10 and 50, about 20, about 25, about 30, about 35, about 40, about 45, about 50, etc.) However, shorter, longer or intermediate overlapping lengths may be used. It should be appreciated that overlaps between different input nucleic acids used in an assembly reaction may have different lengths.
  • the combined sequences of the different oligonucleotides in the reaction may span the sequence of the entire nucleic acid fragment on either the positive strand, the negative strand, both strands, or a combination of portions of the positive strand and portions of the negative strand.
  • the plurality of different oligonucleotides may provide either positive sequences, negative sequences, or a combination of both positive and negative sequences corresponding to the entire sequence of the nucleic acid fragment to be assembled.
  • the plurality of oligonucleotides may include one or more oligonucleotides having sequences identical to one or more portions of the positive sequence, and one or more oligonucleotides having sequences that are identical to one or more portions of the negative sequence of the nucleic acid fragment.
  • One or more pairs of different oligonucleotides may include sequences that are identical to overlapping portions of the predetermined nucleic acid fragment sequence as described herein (e.g., overlapping sequence portions from the same or from complementary strands of the nucleic acid fragment).
  • the plurality of oligonucleotides includes a set of oligonucleotides having sequences that combine to span the entire positive sequence and a set oligonucleotides having sequences that combine to span the entire negative sequence of the predetermined nucleic acid fragment.
  • the plurality of oligonucleotides may include one or more oligonucleotides with sequences that are identical to sequence portions on one strand (either the positive or negative strand) of the nucleic acid fragment, but no oligonucleotides with sequences that are complementary to those sequence portions.
  • a plurality of oligonucleotides includes only oligonucleotides having sequences identical to portions of the positive sequence of the predetermined nucleic acid fragment. In one embodiment, a plurality of oligonucleotides includes only oligonucleotides having sequences identical to portions of the negative sequence of the predetermined nucleic acid fragment. These oligonucleotides may be assembled by sequential ligation or in an extension-based reaction (e.g., if an oligonucleotide having a 3' region that is complementary to one of the plurality of oligonucleotides is added to the reaction).
  • a nucleic acid fragment may be assembled in a polymerase- mediated assembly reaction from a plurality of oligonucleotides that are combined and extended in one or more rounds of polymerase-mediated extensions.
  • a nucleic acid fragment may be assembled in a ligase-mediated reaction from a plurality of oligonucleotides that are combined and ligated in one or more rounds of ligase-mediated ligations.
  • a nucleic acid fragment may be assembled in a non- enzymatic reaction (e.g., a chemical reaction) from a plurality of oligonucleotides that are combined and assembled in one or more rounds of non-enzymatic reactions.
  • a nucleic acid fragment may be assembled using a combination of polymerase, ligase, and/or non-enzymatic reactions.
  • polymerase(s) and ligase(s) may be included in an assembly reaction mixture.
  • a nucleic acid may be assembled via coupled amplification and ligation or ligation during amplification.
  • the resulting nucleic acid fragment from each assembly technique may have a sequence that includes the sequences of each of the plurality of assembly oligonucleotides that were used as described herein.
  • primerless assemblies since the target nucleic acid is generated by assembling the input oligonucleotides rather than being generated in an amplification reaction where the oligonucleotides act as amplification primers to amplify a pre-existing template nucleic acid molecule corresponding to the target nucleic acid.
  • Polymerase-based assembly techniques may involve one or more suitable polymerase enzymes that can catalyze a template-based extension of a nucleic acid in a 5' to 3' direction in the presence of suitable nucleotides and an annealed template.
  • a polymerase may be thermostable.
  • a polymerase may be obtained from recombinant or natural sources.
  • a thermostable polymerase from a thermophilic organism may be used.
  • a polymerase may include a 3 ' ⁇ 5 ' exonuclease/proofreading activity.
  • a polymerase may have no, or little, proofreading activity (e.g., a polymerase may be a recombinant variant of a natural polymerase that has been modified to reduce its proofreading activity).
  • thermostable DNA polymerases include, but are not limited to: Taq (a heat-stable DNA polymerase from the bacterium Thermus aquaticus); Pfu (a thermophilic DNA polymerase with a 3' ⁇ 5' exonuclease/proofreading activity from Pyrococcus furiosus, available from for example Promega); VentR® DNA Polymerase and VentR® (exo-) DNA Polymerase (thermophilic DNA polymerases with or without a 3 '— ⁇ 5 ' exonuclease/proofreading activity from Thermococcus litoralis; also known as TIi polymerase); Deep VentR® DNA Polymerase and Deep VentR® (exo-) DNA Polymerase (thermophilic DNA polymerases
  • coli DNA Polymerase I which retains polymerase activity, but has lost the 5 '—* ⁇ 3 ' exonuclease activity, available from, for example, Promega and NEB); SequenaseTM (T7 DNA polymerase deficient in 3'-5' exonuclease activity); Phi29 (bacteriophage 29 DNA polymerase, may be used for rolling circle amplification, for example, in a TempliPhiTM DNA Sequencing Template Amplification Kit, available from Amersham Biosciences); TopoTaqTM (a hybrid polymerase that combines hyperstable DNA binding domains and the DNA unlinking activity of Methanopyrus topoisomerase, with no exonuclease activity, available from Fidelity Systems); TopoTaq HiFi which incorporates a proofreading domain with exonuclease activity; PhusionTM (a Pyrococcus-Wko, enzyme with a processivity-enhancing domain, available from New England Biolabs); any
  • Ligase-based assembly techniques may involve one or more suitable ligase enzymes that can catalyze the covalent linking of adjacent 3' and 5' nucleic acid termini (e.g., a 5' phosphate and a 3' hydroxyl of nucleic acid(s) annealed on a complementary template nucleic acid such that the 3' terminus is immediately adjacent to the 5' terminus).
  • a ligase may catalyze a ligation reaction between the 5' phosphate of a first nucleic acid to the 3' hydroxyl of a second nucleic acid if the first and second nucleic acids are annealed next to each other on a template nucleic acid).
  • a ligase may be obtained from recombinant or natural sources.
  • a ligase may be a heat- stable ligase.
  • a thermostable ligase from a thermophilic organism may be used.
  • thermostable DNA ligases include, but are not limited to: Tth DNA ligase (from Thermus thermophilus, available from, for example, Eurogentec and GeneCraft); Pfu DNA ligase (a hyperthermophilic ligase from Pyrococcus furiosus); Taq ligase (from Thermus aquaticus), any other suitable heat-stable ligase, or any combination thereof.
  • one or more lower temperature ligases may be used (e.g., T4 DNA ligase).
  • a lower temperature ligase may be useful for shorter overhangs (e.g., about 3, about 4, about 5, or about 6 base overhangs) that may not be stable at higher temperatures.
  • Non-enzymatic techniques can be used to ligate nucleic acids. For example, a 5'- end (e.g., the 5' phosphate group) and a 3'-end (e.g., the 3' hydroxyl) of one or more nucleic acids may be covalently linked together without using enzymes (e.g., without using a ligase). In some embodiments, non-enzymatic techniques may offer certain advantages over enzyme-based ligations.
  • non-enzymatic techniques may have a high tolerance of non-natural nucleotide analogues in nucleic acid substrates, may be used to ligate short nucleic acid substrates, may be used to ligate RNA substrates, and/or may be cheaper and/or more suited to certain automated (e.g., high throughput) applications.
  • Non-enzymatic ligation may involve a chemical ligation.
  • nucleic acid termini of two or more different nucleic acids may be chemically ligated.
  • nucleic acid termini of a single nucleic acid may be chemically ligated (e.g., to circularize the nucleic acid). It should be appreciated that both strands at a first double-stranded nucleic acid terminus may be chemically ligated to both strands at a second double-stranded nucleic acid terminus. However, in some embodiments only one strand of a first nucleic acid terminus may be chemically ligated to a single strand of a second nucleic acid terminus. For example, the 5' end of one strand of a first nucleic acid terminus may be ligated to the 3' end of one strand of a second nucleic acid terminus without the ends of the complementary strands being chemically ligated.
  • a chemical ligation may be used to form a covalent linkage between a 5' terminus of a first nucleic acid end and a 3' terminus of a second nucleic acid end, wherein the first and second nucleic acid ends may be ends of a single nucleic acid or ends of separate nucleic acids.
  • chemical ligation may involve at least one nucleic acid substrate having a modified end (e.g., a modified 5' and/or 3' terminus) including one or more chemically reactive moieties that facilitate or promote linkage formation.
  • chemical ligation occurs when one or more nucleic acid termini are brought together in close proximity (e.g., when the termini are brought together due to annealing between complementary nucleic acid sequences). Accordingly, annealing between complementary 3' or 5' overhangs (e.g., overhangs generated by restriction enzyme cleavage of a double-stranded nucleic acid) or between any combination of complementary nucleic acids that results in a 3' terminus being brought into close proximity with a 5' terminus (e.g., the 3' and 5' termini are adjacent to each other when the nucleic acids are annealed to a complementary template nucleic acid) may promote a template-directed chemical ligation.
  • complementary 3' or 5' overhangs e.g., overhangs generated by restriction enzyme cleavage of a double-stranded nucleic acid
  • any combination of complementary nucleic acids that results in a 3' terminus being brought into close proximity with a 5' terminus e.g
  • Examples of chemical reactions may include, but are not limited to, condensation, reduction, and/or photochemical ligation reactions. It should be appreciated that in some embodiments chemical ligation can be used to produce naturally-occurring phosphodiester interaucleotide linkages, non-naturally-occurring phosphamide pyrophosphate internucleotide linkages, and/or other non-naturally-occurring internucleotide linkages. In some embodiments, the process of chemical ligation may involve one or more coupling agents to catalyze the ligation reaction.
  • a coupling agent may promote a ligation reaction between reactive groups in adjacent nucleic acids (e.g., between a 5'- reactive moiety and a 3 '-reactive moiety at adjacent sites along a complementary template).
  • a coupling agent may be a reducing reagent (e.g., ferricyanide), a condensing reagent such (e.g., cyanoimidazole, cyanogen bromide, carbodiimide, etc.), or irradiation (e.g., UV irradiation for photo-ligation).
  • a chemical ligation may be an autoligation reaction that does not involve a separate coupling agent.
  • autoligation the presence of a reactive group on one or more nucleic acids may be sufficient to catalyze a chemical ligation between nucleic acid termini without the addition of a coupling agent (see, for example, Xu Y & Kool ET, 1997, Tetrahedron Lett. 38:5595-8).
  • Non-limiting examples of these reagent-free ligation reactions may involve nucleophilic displacements of sulfur on bromoacetyl, tosyl, or iodo-nucleoside groups (see, for example, Xu Y et al., 2001, Nat Biotech 19:148-52).
  • Nucleic acids containing reactive groups suitable for autoligation can be prepared directly on automated synthesizers (see, for example, Xu Y & Kool ET, 1999, Nuc. Acids Res. 27:875-81).
  • a phosphorothioate at a 3' terminus may react with a leaving group (such as tosylate or iodide) on a thymidine at an adjacent 5' terminus.
  • two nucleic acid strands bound at adjacent sites on a complementary target strand may undergo auto-ligation by displacement of a 5 '-end iodide moiety (or tosylate) with a 3 '-end sulfur moiety.
  • the product of an autoligation may include a non-naturally-occurring internucleotide linkage (e.g., a single oxygen atom may be replaced with a sulfur atom in the ligated product).
  • a synthetic nucleic acid duplex can be assembled via chemical ligation in a one step reaction involving simultaneous chemical ligation of nucleic acids on both strands of the duplex.
  • a mixture of 5'- phosphorylated oligonucleotides corresponding to both strands of a target nucleic acid may be chemically ligated by a) exposure to heat (e.g., to 97 0 C) and slow cooling to form a complex of annealed oligonucleotides, and b) exposure to cyanogen bromide or any other suitable coupling agent under conditions sufficient to chemically ligate adjacent 3' and 5' ends in the nucleic acid complex.
  • a synthetic nucleic acid duplex can be assembled via chemical ligation in a two step reaction involving separate chemical ligations for the complementary strands of the duplex.
  • each strand of a target nucleic acid may be ligated in a separate reaction containing phosphorylated oligonucleotides corresponding to the strand that is to be ligated and non-phosphorylated oligonucleotides corresponding to the complementary strand.
  • the non-phosphorylated oligonucleotides may serve as a template for the phosphorylated oligonucleotides during a chemical ligation (e.g. using cyanogen bromide).
  • the resulting single-stranded ligated nucleic acid may be purified and annealed to a complementary ligated single-stranded nucleic acid to form the target duplex nucleic acid (see, for example, Shabarova ZA et al., 1991, Nuc. Acids Res. 19:4247-51).
  • aspects of the invention may be used to enhance different types of nucleic acid assembly reactions (e.g., multiplex nucleic acid assembly reactions). Aspects of the invention may be used in combination with one or more assembly reactions described in, for example, Carr et al., 2004, Nucleic Acids Research, Vol. 32, No 20, el 62 (9 pages); Richmond et al., 2004, Nucleic Acids Research, Vol. 32, No 17, pp. 5011-5018; Caruthers et al., 1972, J. MoI. Biol. 72, 475-492; Hecker et al., 1998, Biotechniques 24:256-260; Kodumal et al., 2004, PNAS Vol. 101, No. 44, pp.
  • synthesis and assembly methods described herein may be performed in any suitable format, including in a reaction tube, in a multi-well plate, on a surface, on a column, in a microfiuidic device (e.g., a microfluidic tube), a capillary tube, etc.
  • FIG. 1 shows one embodiment of a plurality of oligonucleotides that may be assembled in a polymerase-based multiplex oligonucleotide assembly reaction.
  • FIG. IA shows two groups of oligonucleotides (Group P and Group N) that have sequences of portions of the two complementary strands of a nucleic acid fragment to be assembled.
  • Group P includes oligonucleotides with positive strand sequences (Pi, P 2 , ...
  • Group N includes oligonucleotides with negative strand sequences (N T , ..., N n+ i, N n , N n-1 , ..., N 2 , N 1 , shown from 5' ⁇ 3' on the negative strand).
  • N T oligonucleotides with negative strand sequences
  • none of the P group oligonucleotides overlap with each other and none of the N group oligonucleotides overlap with each other.
  • one or more of the oligonucleotides within the S or N group may overlap.
  • FIG. IA shows gaps between consecutive oligonucleotides in Group P and gaps between consecutive oligonucleotides in Group N.
  • each P group oligonucleotide (except for Pi) and each N group oligonucleotide (except for N ⁇ ) overlaps with complementary regions of two oligonucleotides from the complementary group of oligonucleotides.
  • Pj and N T overlap with a complementary region of only one oligonucleotide from the other group (the complementary 3 '-most oligonucleotides Ni and P T , respectively).
  • FIG. IB shows a structure of an embodiment of a Group P or Group N oligonucleotide represented in FIG. IA.
  • This oligonucleotide includes a 5' region that is complementary to a 5' region of a first oligonucleotide from the other group, a 3' region that is complementary to a 3' region of a second oligonucleotide from the other group, and a core or central region that is not complementary to any oligonucleotide sequence from the other group (or its own group).
  • This central region is illustrated as the B region in FIG. IB.
  • the sequence of the B region may be different for each different oligonucleotide.
  • the B region of an oligonucleotide in one group corresponds to a gap between two consecutive oligonucleotides in the complementary group of oligonucleotides.
  • the 5 '-most oligonucleotide in each group does not have a 5' region that is complementary to the 5' region of any other oligonucleotide in either group. Accordingly, the 5 '-most oligonucleotides (P 1 and N T ) that are illustrated in FIG. IA each have a 3' complementary region and a 5' non-complementary region (the B region of FIG. IB), but no 5' complementary region.
  • any one or more of the oligonucleotides in Group P and/or Group N can be designed to have no B region.
  • a 5 '-most oligonucleotide has only the 3' complementary region (meaning that the entire oligonucleotide is complementary to the 3' region of the 3'-most oligonucleotide from the other group (e.g., the 3' region of Ni or P T shown in FIG. IA).
  • one of the other oligonucleotides in either Group P or Group N has only a 5' complementary region and a 3' complementary region (meaning that the entire oligonucleotide is complementary to the 5' and 3' sequence regions of the two overlapping oligonucleotides from the complementary group).
  • only a subset of oligonucleotides in an assembly reaction may include B regions. It should be appreciated that the length of the 5', 3', and B regions may be different for each oligonucleotide.
  • the length of the 5' region is the same as the length of the complementary 5' region in the 5' overlapping oligonucleotide from the other group.
  • the length of the 3' region is the same as the length of the complementary 3' region in the 3' overlapping oligonucleotide from the other group.
  • a 3 '-most oligonucleotide may be designed with a 3' region that extends beyond the 5' region of the 5 '-most oligonucleotide.
  • an assembled product may include the 5' end of the 5 '-most oligonucleotide, but not the 3' end of the 3 '-most oligonucleotide that extends beyond it.
  • FIG. 1C illustrates a subset of the oligonucleotides from FIG. IA, each oligonucleotide having a 5', a 3', and an optional B region. Oligonucleotide P n is shown with a 5' region that is complementary to (and can anneal to) the 5' region of oligonucleotide N n - I .
  • Oligonucleotide P n also has a 3' region that is complementary to (and can anneal to) the 3' region of oligonucleotide N n .
  • N n is also shown with a 5' region that is complementary (and can anneal to) the 5' region of oligonucleotide P n+ i. This pattern could be repeated for all of oligonucleotides P 2 to PT and N 1 to NT- i (with the 5 '-most oligonucleotides only having 3' complementary regions as discussed herein).
  • oligonucleotides from Group P and Group N may anneal to form a long chain such as the oligonucleotide complex illustrated in FIG. IA.
  • subsets of the oligonucleotides may form shorter chains and even oligonucleotide dimers with annealed 5' or 3' regions. It should be appreciated that many copies of each oligonucleotide are included in a typical reaction mixture. Accordingly, the resulting hybridized reaction mixture may contain a distribution of different oligonucleotide dimers and complexes.
  • Polymerase-mediated extension of the hybridized oligonucleotides results in a template- based extension of the 3' ends of oligonucleotides that have annealed 3' regions. Accordingly, polymerase-mediated extension of the oligonucleotides shown in FIG. 1C would result in extension of the 3' ends only of oligonucleotides P n and N n generating extended oligonucleotides containing sequences that are complementary to all the regions of N n and P n , respectively.
  • Extended oligonucleotide products with sequences complementary to all of N n- 1 and P n + 1 would not be generated unless oligonucleotides P n- i and N n+1 were included in the reaction mixture. Accordingly, if all of the oligonucleotide sequences in a plurality of oligonucleotides are to be incorporated into an assembled nucleic acid fragment using a polymerase, the plurality of oligonucleotides should include 5 '-most oligonucleotides that are at least complementary to the entire 3' regions of the 3'-most oligonucleotides.
  • the 5'-most oligonucleotides also may have 5' regions that extend beyond the 3' ends of the 3 '-most oligonucleotides as illustrated in FIG. IA.
  • a ligase also may be added to ligate adjacent 5' and 3' ends that may be formed upon 3' extension of annealed oligonucleotides in an oligonucleotide complex such as the one illustrated in FIG. IA.
  • a single cycle of polymerase extension extends oligonucleotide pairs with annealed 3' regions.
  • a single cycle of polymerase extension would result in the extension of the 3' ends of the Pi/Ni, P 2 ZN 2 , ..., P n-1 /N n-1 , P n /N n , Pn+i/N n+1 , ..., P ⁇ /N ⁇ oligonucleotide pairs.
  • a single molecule could be generated by ligating the extended oligonucleotide dimers.
  • a single molecule incorporating all of the oligonucleotide sequences may be generated by performing several polymerase extension cycles.
  • FIG. ID illustrates two cycles of polymerase extension (separated by a denaturing step and an annealing step) and the resulting nucleic acid products. It should be appreciated that several cycles of polymerase extension may be required to assemble a single nucleic acid fragment containing all the sequences of an initial plurality of oligonucleotides. In one embodiment, a minimal number of extension cycles for assembling a nucleic acid may be calculated as Iog 2 n, where n is the number of oligonucleotides being assembled. In some embodiments, progressive assembly of the nucleic acid may be achieved without using temperature cycles.
  • an enzyme capable of rolling circle amplification may be used (e.g., phi 29 polymerase) when a circularized nucleic acid (e.g., oligonucleotide) complex is used as a template to produce a large amount of circular product for subsequent processing using MutS or a MutS homolog as described herein.
  • a circularized nucleic acid e.g., oligonucleotide
  • annealed oligonucleotide pairs P n /N n and P n+ i/N n+1 are extended to form oligonucleotide dimer products incorporating the sequences covered by the respective oligonucleotide pairs.
  • N n is extended to incorporate sequences that are complementary to the B and 5' regions of N n (indicated as N' n in FIG. ID).
  • N n+ i is extended to incorporate sequences that are complementary to the 5' and B regions of P n+ i (indicated as P' n+ i in FIG. ID).
  • These dimer products may be denatured and reannealed to form the starting material of step 2 where the 3' end of the extended P n oligonucleotide is annealed to the 3' end of the extended N n+ i oligonucleotide.
  • This product may be extended in a polymerase-mediated reaction to form a product that incorporates the sequences of the four oligonucleotides (P n , N n , P n +1, N n+1 ).
  • One strand of this extended product has a sequence that includes (in 5' to 3' order) the 5', B, and 3' regions of P n , the complement of the B region of N n , the 5', B, and 3' regions of P n+1 , and the complements of the B and 5' regions of N n+ 1 .
  • the other strand of this extended product has the complementary sequence.
  • reaction products shown in FIG. ID are a subset of the reaction products that would be obtained using all of the oligonucleotides of Group P and Group N.
  • a first polymerase extension reaction using all of the oligonucleotides would result in a plurality of overlapping oligonucleotide dimers from Pj/Nj to P ⁇ /N ⁇ .
  • Each of these may be denatured and at least one of the strands could then anneal to an overlapping complementary strand from an adjacent (either 3' or 5') oligonucleotide dimer and be extended in a second cycle of polymerase extension as shown in FIG. ID.
  • Subsequent cycles of denaturing, annealing, and extension produce progressively larger products including a nucleic acid fragment that includes the sequences of all of the initial oligonucleotides. It should be appreciated that these subsequent rounds of extension also produce many nucleic acid products of intermediate length.
  • the reaction product may be complex since not all of the 3' regions may be extended in each cycle.
  • unextended oligonucleotides may be available in each cycle to anneal to other unextended oligonucleotides or to previously extended oligonucleotides.
  • extended products of different sizes may anneal to each other in each cycle.
  • FIG. 2 shows an embodiment of a plurality of oligonucleotides that may be assembled in a directional polymerase-based multiplex oligonucleotide assembly reaction. In this embodiment, only the 5 '-most oligonucleotide of Group P may be provided. In contrast to the example shown in FIG. 1, the remainder of the sequence of the predetermined nucleic acid fragment is provided by oligonucleotides of Group N.
  • the 3 ' -most oligonucleotide of Group N (N 1 ) has a 3 ' region that is complementary to the 3' region Of P 1 as shown in FIG. 2B. However, the remainder of the oligonucleotides in Group N have overlapping (but non-complementary) 3' and 5' regions as illustrated in FIG. 2B for oligonucleotides N1-N3.
  • Each Group N oligonucleotide (e.g., N n ) overlaps with two adjacent oligonucleotides: one overlaps with the 3' region (N n-1 ) and one with the 5' region (N n+1 ), except for Ni that overlaps with the 3' regions OfP 1 (complementary overlap) and N2 (non-complementary overlap), and NT that overlaps only with N ⁇ -i. It should be appreciated that all of the overlaps shown in FIG.
  • each oligonucleotide may have 3', B, and 5 'regions of different lengths (including no B region in some embodiments). In some embodiments, none of the oligonucleotides may have B regions, meaning that the entire sequence of each oligonucleotide may overlap with the combined 5' and 3' region sequences of its two adjacent oligonucleotides.
  • Assembly of a predetermined nucleic acid fragment from the plurality of oligonucleotides shown in FIG. 2 A may involve multiple cycles of polymerase-mediated extension. Each extension cycle may be separated by a denaturing and an annealing step.
  • FIG. 2C illustrates the first two steps in this assembly process.
  • step 1 annealed oligonucleotides P 1 and N 1 are extended to form an oligonucleotide dimer.
  • P 1 is shown with a 5' region that is non-complementary to the 3' region OfN 1 and extends beyond the 3' region of N] when the oligonucleotides are annealed.
  • P 1 may lack the 5' non-complementary region and include only sequences that overlap with the 3' region OfN 1 .
  • the product OfP 1 extension is shown after step 1 containing an extended region that is complementary to the 5' end OfN 1 .
  • the single strand illustrated in FIG. 2C may be obtained by denaturing the oligonucleotide dimer that results from the extension Of P 1 ZN 1 in step 1.
  • the product of Pi extension is shown annealed to the 3' region of N 2 . This annealed complex may be extended in step 2 to generate an extended product that now includes sequences complementary to the B and 5' regions of N 2 .
  • cycles of extension may be obtained by denaturing the oligonucleotide dimer that results from the extension reaction of step 2. Additional cycles of extension may be performed to further assemble a predetermined nucleic acid fragment. In each cycle, extension results in the addition of sequences complementary to the B and 5' regions of the next Group N oligonucleotide. Each cycle may include a denaturing and annealing step. However, the extension may occur under the annealing conditions. Accordingly, in one embodiment, cycles of extension may be obtained by alternating between denaturing conditions (e.g., a denaturing temperature) and annealing/extension conditions (e.g., an annealing/extension temperature).
  • denaturing conditions e.g., a denaturing temperature
  • annealing/extension conditions e.g., an annealing/extension temperature
  • T (the number of group N oligonucleotides) may determine the minimal number of temperature cycles used to assemble the oligonucleotides.
  • progressive extension may be achieved without temperature cycling.
  • an enzyme capable promoting rolling circle amplification may be used (e.g., TempliPhi).
  • TempliPhi an enzyme capable promoting rolling circle amplification
  • a reaction mixture containing an assembled predetermined nucleic acid fragment also may contain a distribution of shorter extension products that may result from incomplete extension during one or more of the cycles or may be the result of an Pi/Nj extension that was initiated after the first cycle.
  • 2D illustrates an example of a sequential extension reaction where the 5'- most P 1 oligonucleotide is bound to a support and the Group N oligonucleotides are unbound.
  • the reaction steps are similar to those described for FIG. 2C.
  • an extended predetermined nucleic acid fragment will be bound to the support via the 5'- most P 1 oligonucleotide.
  • the complementary strand (the negative strand) may readily be obtained by denaturing the bound fragment and releasing the negative strand.
  • the attachment to the support may be labile or readily reversed (e.g., using light, a chemical reagent, a pH change, etc.) and the positive strand also may be released.
  • FIG. 2E illustrates an example of a sequential reaction where P 1 is unbound and the Group N oligonucleotides are bound to a support. The reaction steps are similar to those described for FIG. 2C. However, an extended predetermined nucleic acid fragment will be bound to the support via the 5 '-most N ⁇ oligonucleotide. Accordingly, the complementary strand (the positive strand) may readily be obtained by denaturing the bound fragment and releasing the positive strand.
  • the attachment to the support may be labile or readily reversed (e.g., using light, a chemical reagent, a pH change, etc.) and the negative strand also may be released. Accordingly, either the positive strand, the negative strand, or the double- stranded product may be obtained.
  • oligonucleotides may be used to assemble a nucleic acid via two or more cycles of polymerase-based extension.
  • at least one pair of oligonucleotides have complementary 3' end regions.
  • FIG. 2F illustrates an example where an oligonucleotide pair with complementary 3' end regions is flanked on either side by a series of oligonucleotides with overlapping non-complementary sequences.
  • the oligonucleotides illustrated to the right of the complementary pair have overlapping 3' and 5' regions (with the 3' region of one oligonucleotide being identical to the 5' region of the adjacent oligonucleotide) that corresponding to a sequence of one strand of the target nucleic acid to be assembled.
  • the oligonucleotides illustrated to the left of the complementary pair have overlapping 3' and 5' regions (with the 3' region of one oligonucleotide being identical to the 5' region of the adjacent oligonucleotide) that correspond to a sequence of the complementary strand of the target nucleic acid.
  • oligonucleotides may be assembled via sequential polymerase-based extension reactions as described herein (see also, for example, Xiong et al., 2004, Nucleic Acids Research, Vol. 32, No. 12, e98, 10 pages, the disclosure of which is incorporated by reference herein). It should be appreciated that different numbers and/or lengths of oligonucleotides may be used on either side of the complementary pair. Accordingly, the illustration of the complementary pair as the central pair in FIG. 2F is not intended to be limiting as other configuration of a complementary oligonucleotide pair flanked by a different number of non-complementary pairs on either side may be used according to methods of the invention.
  • FIG. 3 shows an embodiment of a plurality of oligonucleotides that may be assembled in a ligase reaction.
  • FIG. 3 A illustrates the alignment of the oligonucleotides showing that they do not contain gaps (i.e., no B region as described herein). Accordingly, the oligonucleotides may anneal to form a complex with no nucleotide gaps between the 3' and 5' ends of the annealed oligonucleotides in either Group P or Group N. These oligonucleotides provide a suitable template for assembly using a ligase under appropriate reaction conditions.
  • FIG. 3 B shows two individual ligation reactions. These reactions are illustrated in two steps. However, it should be appreciated that these ligation reactions may occur simultaneously or sequentially in any order and may occur as such in a reaction maintained under constant reaction conditions (e.g., with no temperature cycling) or in a reaction exposed to several temperature cycles. For example, the reaction illustrated in step 2 may occur before the reaction illustrated in step 1. In each ligation reaction illustrated in FIG.
  • a Group N oligonucleotide is annealed to two adjacent Group P oligonucleotides (due to the complementary 5' and 3' regions between the P and N oligonucleotides), providing a template for ligation of the adjacent P oligonucleotides.
  • ligation of the N group oligonucleotides also may proceed in similar manner to assemble adjacent N oligonucleotides that are annealed to their complementary P oligonucleotide. Assembly of the predetermined nucleic acid fragment may be obtained through ligation of all of the oligonucleotides to generate a double stranded product.
  • a single stranded product of either the positive or negative strand may be obtained.
  • a plurality of oligonucleotides may be designed to generate only single-stranded reaction products in a ligation reaction.
  • a first group of oligonucleotides (of either Group P or Group N) may be provided to cover the entire sequence on one strand of the predetermined nucleic acid fragment (on either the positive or negative strand).
  • a second group of oligonucleotides may be designed to be long enough to anneal to complementary regions in the first group but not long enough to provide adjacent 5' and 3' ends between oligonucleotides in the second group.
  • This provides substrates that are suitable for ligation of oligonucleotides from the first group but not the second group.
  • the result is a single-stranded product having a sequence corresponding to the oligonucleotides in the first group.
  • a ligase reaction mixture that contains an assembled predetermined nucleic acid fragment also may contain a distribution of smaller fragments resulting from the assembly of a subset of the oligonucleotides.
  • FIG. 4 shows an embodiment of a ligase-based assembly where one or more of the plurality of oligonucleotides is bound to a support.
  • the 5' most oligonucleotide of the P group oligonucleotides is bound to a support.
  • Ligation of adjacent oligonucleotides in the 5' to 3' direction results in the assembly of a predetermined nucleic acid fragment.
  • FIG. 4A illustrates an example where adjacent oligonucleotides P 2 and P 3 are added sequentially. However, the ligation of any two adjacent oligonucleotides from Group P may occur independently and in any order in a ligation reaction mixture.
  • N 2 when Pi is ligated to the 5' end of N 2 , N 2 may be in the form of a single oligonucleotide or it already may be ligated to one or more downstream oligonucleotides (N 3 , N 4 , etc.).
  • N 3 , N 4 downstream oligonucleotides
  • the 5 '-most (e.g., P 1 for Group P, or NT for Group N) or the 3'-most (e.g., Pj for Group P, or Ni for Group N) oligonucleotide may be bound to a support since the reaction can proceed in any direction.
  • a predetermined nucleic acid fragment may be assembled with a central oligonucleotide (i.e., neither the 5 '-most or the 3 '-most) that is bound to a support provided that the attachment to the support does not interfere with ligation.
  • a central oligonucleotide i.e., neither the 5 '-most or the 3 '-most
  • FIG. 4B illustrates an example where a plurality of N group oligonucleotides are bound to a support and a predetermined nucleic acid fragment is assembled from P group oligonucleotides that anneal to their complementary support-bound N group oligonucleotides.
  • FIG. 4B illustrates a sequential addition.
  • adjacent P group oligonucleotides may be ligated in any order.
  • the bound oligonucleotides may be attached at their 5' end, 3' end, or at any other position provided that the attachment does not interfere with their ability to bind to complementary 5' and 3' regions on the oligonucleotides that are being assembled.
  • This reaction may involve one or more reaction condition changes (e.g., temperature cycles) so that ligated oligonucleotides bound to one immobilized N group oligonucleotide can be dissociated from the support and bind to a different immobilized N group oligonucleotide to provide a substrate for ligation to another P group oligonucleotide.
  • reaction condition changes e.g., temperature cycles
  • support-bound ligase reactions that generate a full length predetermined nucleic acid fragment also may generate a distribution of smaller fragments resulting from the assembly of subsets of the oligonucleotides.
  • a support used in any of the assembly reactions described herein may include any suitable support medium.
  • a support may be solid, porous, a matrix, a gel, beads, beads in a gel, etc.
  • a support may be of any suitable size.
  • a solid support may be provided in any suitable configuration or shape (e.g., a chip, a bead, a gel, a microfluidic channel, a planar surface, a spherical shape, a column, etc.).
  • oligonucleotide assembly reactions may be used to assemble a plurality of overlapping oligonucleotides (with overlaps that are either 575', 373', 573', complementary, non-complementary, or a combination thereof).
  • Many of these reactions include at least one pair of oligonucleotides (the pair including one oligonucleotide from a first group or P group of oligonucleotides and one oligonucleotide from a second group or N group of oligonucleotides) have overlapping complementary 3' regions.
  • a predetermined nucleic acid may be assembled from non-overlapping oligonucleotides using blunt-ended ligation reactions.
  • the order of assembly of the non-overlapping oligonucleotides may be biased by selective phosphorylation of different 5' ends.
  • size purification may be used to select for the correct order of assembly.
  • the correct order of assembly may be promoted by sequentially adding appropriate oligonucleotide substrates into the reaction (e.g., the ligation reaction) .
  • a purification step may be used to remove starting oligonucleotides and/or incompletely assembled fragments.
  • a purification step may involve chromatography, electrophoresis, or other physical size separation technique.
  • a purification step may involve amplifying the full length product. For example, a pair of amplification primers (e.g., PCR primers) that correspond to the predetermined 5' and 3' ends of the nucleic acid fragment being assembled will preferentially amplify full length product in an exponential fashion.
  • a pair of amplification primers e.g., PCR primers
  • the sequence of the predetermined fragment will be provided by the oligonucleotides as described herein.
  • the oligonucleotides may contain additional sequence information that may be removed during assembly or may be provided to assist in subsequent manipulations of the assembled nucleic acid fragment. Examples of additional sequences include, but are not limited to, primer recognition sequences for amplification (e.g., PCR primer recognition sequences), restriction enzyme recognition sequences, recombination sequences, other binding or recognition sequences, labeled sequences, etc.
  • one or more of the 5'-most oligonucleotides, one or more of the 3 '-most oligonucleotides, or any combination thereof may contain one or more additional sequences.
  • the additional sequence information may be contained in two or more adjacent oligonucleotides on either strand of the predetermined nucleic acid sequence.
  • an assembled nucleic acid fragment may contain additional sequences that may be used to connect the assembled fragment to one or more additional nucleic acid fragments (e.g., one or more other assembled fragments, fragments obtained from other sources, vectors, etc.) via ligation, recombination, polymerase-mediated assembly, etc.
  • purification may involve cloning one or more assembled nucleic acid fragments.
  • the cloned product may be screened (e.g., sequenced, analyzed for an insert of the expected size, etc.).
  • a nucleic acid fragment assembled from a plurality of oligonucleotides may be combined with one or more additional nucleic acid fragments using a polymerase-based and/or a ligase-based extension reaction similar to those described herein for oligonucleotide assembly. Accordingly, one or more overlapping nucleic acid fragments may be combined and assembled to produce a larger nucleic acid fragment as described herein.
  • double-stranded overlapping oligonucleotide fragments may be combined.
  • single-stranded fragments, or combinations of single-stranded and double-stranded fragments may be combined as described herein.
  • a nucleic acid fragment assembled from a plurality of oligonucleotides may be of any length depending on the number and length of the oligonucleotides used in the assembly reaction.
  • a nucleic acid fragment (either single-stranded or double-stranded) assembled from a plurality of oligonucleotides may be between 50 and 1,000 nucleotides long (for example, about 70 nucleotides long, between 100 and 500 nucleotides long, between 200 and 400 nucleotides long, about 200 nucleotides long, about 300 nucleotides long, about 400 nucleotides long, etc.).
  • One or more such nucleic acid fragments e.g., with overlapping 3' and/or 5' ends
  • a full length product assembled from smaller nucleic acid fragments also may be isolated or purified as described herein (e.g., using a size selection, cloning, selective binding or other suitable purification procedure).
  • any assembled nucleic acid fragment (e.g., full-length nucleic acid fragment) described herein may be amplified (prior to, as part of, or after, a purification procedure) using appropriate 5' and 3' amplification primers.
  • P Group and N Group oligonucleotides are used herein for clarity purposes only, and to illustrate several embodiments of multiplex oligonucleotide assembly.
  • the Group P and Group N oligonucleotides described herein are interchangeable, and may be referred to as first and second groups of oligonucleotides corresponding to sequences on complementary strands of a target nucleic acid fragment.
  • Oligonucleotides may be synthesized using any suitable technique. For example, oligonucleotides may be synthesized on a column or other support (e.g., a chip).
  • a synthetic oligonucleotide may be of any suitable size, for example between 10 and 1 ,000 nucleotides long (e.g., between 10 and 200, 200 and 500, 500 and 1 ,000 nucleotides long, or any combination thereof).
  • An assembly reaction may include a plurality of oligonucleotides, each of which independently may be between 10 and 200 nucleotides in length (e.g., between 20 and 150, between 30 and 100, 30 to 90, 30-80, 30-70, 30-60, 35-55, 40-50, or any intermediate number of nucleotides).
  • oligonucleotides may be provided as single stranded synthetic products. However, in some embodiments, oligonucleotides may be provided as double-stranded preparations including an annealed complementary strand. Oligonucleotides may be molecules of DNA, RNA, PNA, or any combination thereof. A double-stranded oligonucleotide may be produced by amplifying a single-stranded synthetic oligonucleotide or other suitable template (e.g., a sequence in a nucleic acid preparation such as a nucleic acid vector or genomic nucleic acid).
  • a plurality of oligonucleotides designed to have the sequence features described herein may be provided as a plurality of single-stranded oligonucleotides having those feature, or also may be provided along with complementary oligonucleotides.
  • an oligonucleotide may be phosphorylated (e.g., with a 5' phosphate).
  • an oligonucleotide may be non-phosphorylated.
  • an oligonucleotide may be amplified using an appropriate primer pair with one primer corresponding to each end of the oligonucleotide (e.g., one that is complementary to the 3' end of the oligonucleotide and one that is identical to the 5' end of the oligonucleotide).
  • an oligonucleotide may be designed to contain a central assembly sequence (designed to be incorporated into the target nucleic acid) flanked by a 5' amplification sequence (e.g., a 5' universal sequence) and a 3' amplification sequence (e.g., a 3' universal sequence).
  • Amplification primers corresponding to the flanking amplification sequences may be used to amplify the oligonucleotide (e.g., one primer may be complementary to the 3' amplification sequence and one primer may have the same sequence as the 5' amplification sequence).
  • the amplification sequences then may be removed from the amplified oligonucleotide using any suitable technique to produce an oligonucleotide that contains only the assembly sequence.
  • a plurality of different oligonucleotides e.g., about 5, 10,
  • a preparation of an oligonucleotide designed to have a certain sequence may include oligonucleotide molecules having the designed sequence in addition to oligonucleotide molecules that contain errors (e.g., that differ from the designed sequence at least at one position).
  • a sequence error may include one or more nucleotide deletions, additions, substitutions (e.g., transversion or transition), inversions, duplications, or any combination of two or more thereof.
  • Oligonucleotide errors may be generated during oligonucleotide synthesis. Different synthetic techniques may be prone to different error profiles and frequencies. In some embodiments, error rates may vary from 1/10 to 1/200 errors per base depending on the synthesis protocol that is used. However, in some embodiments lower error rates may be achieved. Also, the types of errors may depend on the synthetic techniques that are used. For example, in some embodiments chip-based oligonucleotide synthesis may result in relatively more deletions than column-based synthetic techniques.
  • one or more oligonucleotide preparations may be processed to remove (or reduce the frequency of) error-containing oligonucleotides.
  • a hybridization technique may be used wherein an oligonucleotide preparation is hybridized under stringent conditions one or more times to an immobilized oligonucleotide preparation designed to have a complementary sequence. Oligonucleotides that do not bind may be removed in order to selectively or specifically remove oligonucleotides that contain errors that would destabilize hybridization under the conditions used.
  • this processing may not remove all error-containing oligonucleotides since many have only one or two sequence errors and may still bind to the immobilized oligonucleotides with sufficient affinity for a fraction of them to remain bound through this selection processing procedure.
  • a nucleic acid binding protein or recombinase may be included in one or more of the oligonucleotide processing steps to improve the selection of error free oligonucleotides. For example, by preferentially promoting the hybridization of oligonucleotides that are completely complementary with the immobilized oligonucleotides, the amount of error containing oligonucleotides that are bound may be reduced.
  • this oligonucleotide processing procedure may remove more error-containing oligonucleotides and generate an oligonucleotide preparation that has a lower error frequency (e.g., with an error rate of less than 1/50, less than 1/100, less than 1/200, less than 1/300, less than 1/400, less than 1/500, less than 1/1,000, or less than 1/2,000 errors per base.
  • a lower error frequency e.g., with an error rate of less than 1/50, less than 1/100, less than 1/200, less than 1/300, less than 1/400, less than 1/500, less than 1/1,000, or less than 1/2,000 errors per base.
  • a plurality of oligonucleotides used in an assembly reaction may contain preparations of synthetic oligonucleotides, single-stranded oligonucleotides, double- stranded oligonucleotides, amplification products, oligonucleotides that are processed to remove (or reduce the frequency of) error-containing variants, etc., or any combination of two or more thereof.
  • synthetic oligonucleotides synthesized on an array are not amplified prior to assembly.
  • a polymerase-based or ligase-based assembly using non-amplified oligonucleotides may be performed in a microfluidic device.
  • a synthetic oligonucleotide may be amplified prior to use. Either strand of a double-stranded amplification product may be used as an assembly oligonucleotide and added to an assembly reaction as described herein.
  • a synthetic oligonucleotide may be amplified using a pair of amplification primers (e.g., a first primer that hybridizes to the 3' region of the oligonucleotide and a second primer that hybridizes to the 3' region of the complement of the oligonucleotide).
  • the oligonucleotide may be synthesized on a support such as a chip (e.g., using an ink-jet- based synthesis technology).
  • the oligonucleotide may be amplified while it is still attached to the support. In some embodiments, the oligonucleotide may be removed or cleaved from the support prior to amplification.
  • the two strands of a double-stranded amplification product may be separated and isolated using any suitable technique. In some embodiments, the two strands may be differentially labeled (e.g., using one or more different molecular weight, affinity, fluorescent, electrostatic, magnetic, and/or other suitable tags). The different labels may be used to purify and/or isolate one or both strands. In some embodiments, biotin may be used as a purification tag.
  • the strand that is to be used for assembly may be directly purified (e.g., using an affinity or other suitable tag).
  • the complementary strand is removed (e.g., using an affinity or other suitable tag) and the remaining strand is used for assembly.
  • a synthetic oligonucleotide may include a central assembly sequence flanked by 5' and 3' amplification sequences.
  • the central assembly sequence is designed for incorporation into an assembled nucleic acid.
  • the flanking sequences are designed for amplification and are not intended to be incorporated into the assembled nucleic acid.
  • the flanking amplification sequences may be used as universal primer sequences to amplify a plurality of different assembly oligonucleotides that share the same amplification sequences but have different central assembly sequences.
  • the flanking sequences are removed after amplification to produce an oligonucleotide that contains only the assembly sequence.
  • one of the two amplification primers may be biotinylated.
  • the nucleic acid strand that incorporates this biotinylated primer during amplification can be affinity purified using streptavidin (e.g., bound to a bead, column, or other surface).
  • streptavidin e.g., bound to a bead, column, or other surface.
  • the amplification primers also may be designed to include certain sequence features that can be used to remove the primer regions after amplification in order to produce a single-stranded assembly oligonucleotide that includes the assembly sequence without the flanking amplification sequences.
  • the non-biotinylated strand may be used for assembly.
  • the assembly oligonucleotide may be purified by removing the biotinylated complementary strand.
  • the amplification sequences may be removed if the non-biotinylated primer includes a dU at its 3' end, and if the amplification sequence recognized by (i.e., complementary to) the biotinylated primer includes at most three of the four nucleotides and the fourth nucleotide is present in the assembly sequence at (or adjacent to) the junction between the amplification sequence and the assembly sequence.
  • the double-stranded product is incubated with T4 DNA polymerase (or other polymerase having a suitable editing activity) in the presence of the fourth nucleotide (without any of the nucleotides that are present in the amplification sequence recognized by the biotinylated primer) under appropriate reaction conditions. Under these conditions, the 3' nucleotides are progressively removed through to the nucleotide that is not present in the amplification sequence (referred to as the fourth nucleotide above). As a result, the amplification sequence that is recognized by the biotinylated primer is removed. The biotinylated strand is then removed.
  • T4 DNA polymerase or other polymerase having a suitable editing activity
  • UDG uracil-DNA glycosylase
  • This technique generates a single-stranded assembly oligonucleotide without the flanking amplification sequences. It should be appreciated that this technique may be used to process a single amplified oligonucleotide preparation or a plurality of different amplified oligonucleotides in a single reaction if they share the same amplification sequence features described above.
  • the biotinylated strand may be used for assembly.
  • the assembly oligonucleotide may be obtained directly by isolating the biotinylated strand.
  • the amplification sequences may be removed if the biotinylated primer includes a dU at its 3' end, and if the amplification sequence recognized by (i.e., complementary to) the non-biotinylated primer includes at most three of the four nucleotides and the fourth nucleotide is present in the assembly sequence at (or adjacent to) the junction between the amplification sequence and the assembly sequence.
  • the double-stranded product is incubated with T4 DNA polymerase (or other polymerase having a suitable editing activity) in the presence of the fourth nucleotide (without any of the nucleotides that are present in the amplification sequence recognized by the non-biotinylated primer) under appropriate reaction conditions. Under these conditions, the 3' nucleotides are progressively removed through to the nucleotide that is not present in the amplification sequence (referred to as the fourth nucleotide above). As a result, the amplification sequence that is recognized by the non- biotinylated primer is removed. The biotinylated strand is then isolated (and the non- biotinylated strand is removed).
  • T4 DNA polymerase or other polymerase having a suitable editing activity
  • the isolated biotinylated strand is then treated with UDG to remove the biotinylated primer sequence.
  • This technique generates a single- stranded assembly oligonucleotide without the flanking amplification sequences. It should be appreciated that this technique may be used to process a single amplified oligonucleotide preparation or a plurality of different amplified oligonucleotides in a single reaction if they share the same amplification sequence features described above.
  • biotinylated primer may be designed to anneal to either the synthetic oligonucleotide or to its complement for the amplification and purification reactions described above.
  • non-biotinylated primer may be designed to anneal to either strand provided it anneals to the strand that is complementary to the strand recognized by the biotinylated primer.
  • an oligonucleotide may be modified by incorporating a modified-base (e.g., a nucleotide analog) during synthesis, by modifying the oligonucleotide after synthesis, or any combination thereof.
  • a modified-base e.g., a nucleotide analog
  • modifications include, but are not limited to, one or more of the following: universal bases such as nitroindoles, dP and dK, inosine, uracil; halogenated bases such as BrdU; fluorescent labeled bases; non-radioactive labels such as biotin (as a derivative of dT) and digoxigenin (DIG); 2,4-Dinitrophenyl (DNP); radioactive nucleotides; post-coupling modification such as dR-NH 2 (deoxyribose-NH 2 ); Acridine (6-chloro-2- methoxiacridine); and spacer phosphoramides which are used during synthesis to add a spacer 'arm' into the sequence, such as C3, C8 (octanediol), C9, C12, HEG (hexaethlene glycol) and C 18.
  • universal bases such as nitroindoles, dP and dK, inosine, uracil
  • nucleic acid binding proteins or recombinases are preferably not included in a post-assembly fidelity optimization technique (e.g., a screening technique using a MutS or MutS homolog), because the optimization procedure involves removing error-containing nucleic acids via the production and removal of heteroduplexes. Accordingly, any nucleic acid binding proteins or recombinases (e.g., RecA) that were included in the assembly steps are preferably removed (e.g., by inactivation, column purification or other suitable technique) after assembly and prior to fidelity optimization.
  • a post-assembly fidelity optimization technique e.g., a screening technique using a MutS or MutS homolog
  • the invention provides methods for assembling synthetic nucleic acids with increased efficiency.
  • the resulting assembled nucleic acids may be amplified in vitro (e.g., using PCR, LCR, or any suitable amplification technique), amplified in vivo (e.g., via cloning into a suitable vector), isolated and/or purified.
  • An assembled nucleic acid (alone or cloned into a vector) may be transformed into a host cell (e.g., a prokaryotic, eukaryotic, insect, mammalian, or other host cell).
  • the host cell may be used to propagate the nucleic acid.
  • the nucleic acid may be integrated into the genome of the host cell.
  • the nucleic acid may replace a corresponding nucleic acid region on the genome of the cell (e.g., via homologous recombination). Accordingly, nucleic acids may be used to produce recombinant organisms.
  • a target nucleic acid may be an entire genome or large fragments of a genome that are used to replace all or part of the genome of a host organism. Recombinant organisms also may be used for a variety of research, industrial, agricultural, and/or medical applications.
  • a host cell may be prokaryotic (e.g., bacterial such as E. coli or B. subtilis) or eukaryotic (e.g., a yeast, mammal or insect cell).
  • host cells may be bacterial cells (e.g., Escherichia coli, Bacillus subtilis, Mycobacterium spp., M. tuberculosis, or other suitable bacterial cells), yeast cells (for example, Saccharomyces spp., Picchia spp., Candida spp., or other suitable yeast species, e.g., S. cerevisiae, C. albicans, S.
  • pombe, etc. Xenopus cells, mouse cells, monkey cells, human cells, insect cells (e.g., SF9 cells and Drosophila cells), worm cells (e.g., Caenorhabditis spp.), plant cells, or other suitable cells, including for example, transgenic or other recombinant cell lines.
  • insect cells e.g., SF9 cells and Drosophila cells
  • worm cells e.g., Caenorhabditis spp.
  • plant cells or other suitable cells, including for example, transgenic or other recombinant cell lines.
  • a number of heterologous cell lines may be used, such as Chinese Hamster Ovary cells (CHO).
  • nucleic acid when integrating a nucleic acid into a eukaryotic genome (e.g., a mammalian genome) care should be taken to select sites that will allow sufficient expression (e.g., silenced regions of the genome should be avoided, whereas a site comprising an enhancer may be appropriate).
  • a eukaryotic genome e.g., a mammalian genome
  • Many of the techniques described herein can be used together, applying combinations of one or more extension-based and/or ligation-based assembly techniques at one or more points to produce long nucleic acid molecules.
  • concerted assembly may be used to assemble oligonucleotide duplexes and nucleic acid fragments of less than 100 to more than 10,000 base pairs in length (e.g., 100 mers to 500 mers, 500 mers to 1,000 mers, 1,000 mers to 5,000 mers, 5, 000 mers to 10,000 mers, 25,000 mers, 50,000 mers, 75,000 mers, 100,000 mers, etc.).
  • methods described herein may be used during the assembly of an entire genome (or a large fragment thereof, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) of an organism (e.g., of a viral, bacterial, yeast, or other prokaryotic or eukaryotic organism), optionally incorporating specific modifications into the sequence at one or more desired locations.
  • an organism e.g., of a viral, bacterial, yeast, or other prokaryotic or eukaryotic organism
  • nucleic acid products e.g., including nucleic acids that are amplified, cloned, purified, isolated, etc.
  • any of the nucleic acid products may be packaged in any suitable format (e.g., in a stable buffer, lyophilized, etc.) for storage and/or shipping (e.g., for shipping to a distribution center or to a customer).
  • any of the host cells e.g., cells transformed with a vector or having a modified genome
  • cells may be prepared in a suitable buffer for storage and or transport (e.g., for distribution to a customer).
  • cells may be frozen.
  • other stable cell preparations also may be used.
  • Host cells may be grown and expanded in culture.
  • Host cells may be used for expressing one or more RNAs or polypeptides of interest (e.g., therapeutic, industrial, agricultural, and/or medical proteins).
  • the expressed polypeptides may be natural polypeptides or non-natural polypeptides.
  • the polypeptides may be isolated or purified for subsequent use. Accordingly, nucleic acid molecules generated using methods of the invention can be incorporated into a vector.
  • the vector may be a cloning vector or an expression vector.
  • a vector may comprise an origin of replication and one or more selectable markers (e.g., antibiotic resistant markers, auxotrophic markers, etc.).
  • the vector may be a viral vector.
  • a viral vector may comprise nucleic acid sequences capable of infecting target cells.
  • a prokaryotic expression vector operably linked to an appropriate promoter system can be used to transform target cells.
  • a eukaryotic vector operably linked to an appropriate promoter system can be used to transfect target cells or tissues.
  • RNAs or polypeptides may be isolated or purified.
  • Nucleic acids of the invention also may be used to add detection and/or purification tags to expressed polypeptides or fragments thereof.
  • polypeptide-based fusion/tag include, but are not limited to, hexa- histidine (His 6 ) Myc and HA, and other polypeptides with utility, such as GFP, GST, MBP, chitin and the like.
  • polypeptides may comprise one or more unnatural amino acid residue(s).
  • antibodies can be made against polypeptides or fragment(s) thereof encoded by one or more synthetic nucleic acids.
  • synthetic nucleic acids may be provided as libraries for screening in research and development (e.g., to identify potential therapeutic proteins or peptides, to identify potential protein targets for drug development, etc.).
  • a synthetic nucleic acid may be used as a therapeutic (e.g., for gene therapy, or for gene regulation).
  • a synthetic nucleic acid may be administered to a patient in an amount sufficient to express a therapeutic amount of a protein.
  • a synthetic nucleic acid may be administered to a patient in an amount sufficient to regulate (e.g., down-regulate) the expression of a gene.
  • an assembly procedure may involve a combination of acts that are performed at one site (in the United States or outside the United States) and acts that
  • aspects of the invention may include automating one or more acts described herein.
  • a sequence analysis may be automated in order to generate a synthesis strategy automatically.
  • the synthesis strategy may include i) the design of the starting nucleic acids that are to be assembled into the target nucleic acid, ii) the choice of the assembly technique(s) to be used, iii) the number of rounds of assembly and error screening or sequencing steps to include, and/or decisions relating to subsequent processing of an assembled target nucleic acid.
  • one or more steps of an assembly reaction may be automated using one or more automated sample handling devices (e.g., one or more automated liquid or fluid handling devices).
  • reaction reagents including one or more of the following: starting nucleic acids, buffers, enzymes (e.g., one or more ligases and/or polymerases), nucleotides, nucleic acid binding proteins or recombinases, salts, and any other suitable agents such as stabilizing agents.
  • reaction reagents may include one or more reagents or reaction conditions suitable for extension-based assembly, ligation-based assembly, or combinations thereof.
  • Automated devices and procedures also may be used to control the reaction conditions.
  • an automated thermal cycler may be used to control reaction temperatures and any temperature cycles that may be used.
  • a thermal cycler may be automated to provide one or more reaction temperatures or temperature cycles suitable for incubating nucleic acid fragments prior to transformation.
  • subsequent purification and analysis of assembled nucleic acid products may be automated.
  • fidelity optimization steps e.g., a MutS error screening procedure
  • Sequencing also may be automated using a sequencing device and automated sequencing protocols. Additional steps (e.g., amplification, cloning, etc.) also may be automated using one or more appropriate devices and related protocols. It should be appreciated that one or more of the device or device components described herein may be combined in a system (e.g., a robotic system). Assembly reaction mixtures (e.g., liquid reaction samples) may be transferred from one component of the system to another using automated devices and procedures (e.g., robotic manipulation and/or transfer of samples and/or sample containers, including automated pipetting devices, etc.). The system and any components thereof may be controlled by a control system.
  • Assembly reaction mixtures e.g., liquid reaction samples
  • automated devices and procedures e.g., robotic manipulation and/or transfer of samples and/or sample containers, including automated pipetting devices, etc.
  • the system and any components thereof may be controlled by a control system.
  • acts of the invention may be automated using, for example, a computer system (e.g., a computer controlled system).
  • a computer system on which aspects of the invention can be implemented may include a computer for any type of processing (e.g., sequence analysis and/or automated device control as described herein).
  • processing steps may be provided by one or more of the automated devices that are part of the assembly system.
  • a computer system may include two or more computers.
  • one computer may be coupled, via a network, to a second computer.
  • One computer may perform sequence analysis.
  • the second computer may control one or more of the automated synthesis and assembly devices in the system.
  • additional computers may be included in the network to control one or more of the analysis or processing acts.
  • Each computer may include a memory and processor.
  • the computers can take any form, as the aspects of the present invention are not limited to being implemented on any particular computer platform.
  • the network can take any form, including a private network or a public network (e.g., the Internet).
  • Display devices can be associated with one or more of the devices and computers.
  • a display device may be located at a remote site and connected for displaying the output of an analysis in accordance with the invention. Connections between the different components of the system may be via wire, wireless transmission, satellite transmission, any other suitable transmission, or any combination of two or more of the above.
  • sequence information e.g., a target sequence, a processed analysis of the target sequence, etc.
  • a public network such as the Internet
  • a remote location to be processed by computer to produce any of the various types of outputs discussed herein (e.g., in connection with oligonucleotide design).
  • a public network such as the Internet
  • outputs discussed herein (e.g., in connection with oligonucleotide design).
  • the aspects of the present invention described herein are not limited in that respect, and that numerous other configurations are possible.
  • all of the analysis and processing described herein can alternatively be implemented on a computer that is attached locally to a device, an assembly system, or one or more components of an assembly system.
  • sequence information e.g., a target sequence, a processed analysis of the target sequence, etc.
  • a communication medium e.g., the network
  • the information can be loaded onto a computer readable medium that can then be physically transported to another computer for processing in the manners described herein.
  • a combination of two or more transmission/delivery techniques may be used.
  • computer implementable programs for performing a sequence analysis or controlling one or more of the devices, systems, or system components described herein also may be transmitted via a network or loaded onto a computer readable medium as described herein. Accordingly, aspects of the invention may involve performing one or more steps within the United States and additional steps outside the United States.
  • sequence information (e.g., a customer order) may be received at one location (e.g., in one country) and sent to a remote location for processing (e.g., in the same country or in a different country), for example, for sequence analysis to determine a synthesis strategy and/or design oligonucleotides.
  • a portion of the sequence analysis may be performed at one site (e.g., in one country) and another portion at another site (e.g., in the same country or in another country).
  • different steps in the sequence analysis may be performed at multiple sites (e.g., all in one country or in several different countries). The results of a sequence analysis then may be sent to a further site for synthesis.
  • different synthesis and quality control steps may be performed at more than one site (e.g., within one county or in two or more countries).
  • An assembled nucleic acid then may be shipped to a further site (e.g., either to a central shipping center or directly to a client).
  • each of the different aspects, embodiments, or acts of the present invention described herein can be independently automated and implemented in any of numerous ways.
  • each aspect, embodiment, or act can be independently implemented using hardware, software or a combination thereof.
  • the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers.
  • any component or collection of components that perform the functions described above can be generically considered as one or more controllers that control the above-discussed functions.
  • the one or more controllers can be implemented in numerous ways, such as with dedicated hardware, or with general purpose hardware (e.g., one or more processors) that is programmed using microcode or software to perform the functions recited above.
  • one implementation of the embodiments of the present invention comprises at least one computer-readable medium (e.g., a computer memory, a floppy disk, a compact disk, a tape, etc.) encoded with a computer program (i.e., a plurality of instructions), which, when executed on a processor, performs one or more of the above-discussed functions of the present invention.
  • the computer-readable medium can be transportable such that the program stored thereon can be loaded onto any computer system resource to implement one or more functions of the present invention discussed herein.
  • the reference to a computer program which, when executed, performs the above-discussed functions is not limited to an application program running on a host computer.
  • computer program is used herein in S generic sense to reference any type of computer code (e.g., software or microcode) that can be employed to program a processor to implement the above-discussed aspects of the present invention. It should be appreciated that in accordance with several embodiments of the present invention wherein processes are implemented in a computer readable medium, the computer implemented processes may, during the course of their execution, receive input manually (e.g., from a user).
  • a system controller which may provide control signals to the associated nucleic acid synthesizers, liquid handling devices, thermal cyclers, sequencing devices, associated robotic components, as well as other suitable systems for performing the desired input/output or other control functions.
  • the system controller along with any device controllers together form a controller that controls the operation of a nucleic acid assembly system.
  • the controller may include a general purpose data processing system, which can be a general purpose computer, or network of general purpose computers, and other associated devices, including communications devices, modems, and/or other circuitry or components necessary to perform the desired input/output or other functions.
  • the controller can also be implemented, at least in part, as a single special purpose integrated circuit (e.g., ASIC) or an array of ASICs, each having a main or central processor section for overall, system- level control, and separate sections dedicated to performing various different specific computations, functions and other processes under the control of the central processor section.
  • the controller can also be implemented using a plurality of separate dedicated programmable integrated or other electronic circuits or devices, e.g., hard wired electronic or logic circuits such as discrete element circuits or programmable logic devices.
  • the controller can also include any other components or devices, such as user input/output devices (monitors, displays, printers, a keyboard, a user pointing device, touch screen, or other user interface, etc.), data storage devices, drive motors, linkages, valve controllers, robotic devices, vacuum and other pumps, pressure sensors, detectors, power supplies, pulse sources, communication devices or other electronic circuitry or components, and so on.
  • the controller also may control operation of other portions of a system, such as automated client order processing, quality control, packaging, shipping, billing, etc., to perform other suitable functions known in the art but not described in detail herein.
  • aspects of the invention may be useful to streamline nucleic acid assembly reactions. Accordingly, aspects of the invention relate to marketing methods, compositions, kits, devices, and systems for increasing nucleic acid assembly throughput involving combinations of one or more extension-based and/or ligation-based assembly techniques described herein.
  • aspects of the invention may be useful for reducing the time and/or cost of production, commercialization, and/or development of synthetic nucleic acids, and/or related compositions. Accordingly, aspects of the invention relate to business methods that involve collaboratively (e.g., with a partner) or independently marketing one or more methods, kits, compositions, devices, or systems for analyzing and/or assembling synthetic nucleic acids as described herein. For example, certain embodiments of the invention may involve marketing a procedure and/or associated devices or systems involving nucleic acid assembly techniques described herein. In some embodiments, synthetic nucleic acids, libraries of synthetic nucleic acids, host cells containing synthetic nucleic acids, expressed polypeptides or proteins, etc., also may be marketed.
  • Marketing may involve providing information and/or samples relating to methods, kits, compositions, devices, and/or systems described herein.
  • Potential customers or partners may be, for example, companies in the pharmaceutical, biotechnology and agricultural industries, as well as academic centers and government research organizations or institutes.
  • Business applications also may involve generating revenue through sales and/or licenses of methods, kits, compositions, devices, and/or systems of the invention.
  • kits comprising at least one universal amplification primer or primer pair (e.g., 1-10, or more different ones) and at least one or more different selection primers or primer pairs (e.g., 1-10, 10-50, about 50, 50-100, 100-500 or more different ones).
  • the primers may be of any suitable length, for example about 10-50 nucleotides long (e.g., about 15, about 20, about 25, about 30, about 35, about 40, or about 45 nucleotides long) or longer or shorter.
  • the length of the primer may depend one or more factors known to one of skill in the art including the length of the synthetic oligonucleotides being amplified, the complexity of the pools or sets of oligonucleotides, the AT and/or GC content of the primers, and/or other factors known to one of skill in the art.
  • Primers (and/or spacers) may have certain sequence characteristics that allow them to be removed from target sequences after amplification as described herein.
  • the primers may be provided with instructions for amplification and removal of non-target sequences as described herein.
  • primers may be provided with one or more affinity and/or detection tags.
  • step (1) a primerless assembly of oligonucleotides is performed and in step (2) an assembled nucleic acid fragment is amplified in a primer-based amplification.
  • a 993 base-long promoter>EGFP construct was assembled from 50-mer abutting oligonucleotides using a 2-step PCR assembly.
  • oligonucleotide pools were prepared as follows: 36 overlapping 50-mer oligonucleotides and two 5' terminal 59-mers were separated into 4 pools, each corresponding to overlapping 200-300 nucleotide segments of the final construct. The total oligonucleotide concentration in each pool was 5 ⁇ M.
  • a primerless PCR extension reaction was used to stitch (assemble) overlapping oligonucleotides in each pool.
  • the PCR extension reaction mixture was as follows: oligonucleotide pool (5 ⁇ M total) 1.0 ⁇ l ( ⁇ 25 nM final each) dNTP (10 mM each) 0.5 ⁇ l (250 ⁇ M final each)
  • primerless PCR product 1.0 ⁇ l primer 5' (1.2 ⁇ M) 5 ⁇ l (300 nM final) primer 3' (1.2 ⁇ M) 5 ⁇ l (300 nM final) dNTP (10 mM each) 0.5 ⁇ l (250 ⁇ M final each)
  • the amplified sub-segments were assembled using another round of primerless PCR as follows.
  • a diluted amplification product was prepared for each sub-segment by diluting each amplified sub-segment PCR product 1 : 10 (4 ⁇ l mix + 36 ⁇ l dH 2 O).
  • This diluted mix was used as follows: diluted sub-segment mix 1.0 ⁇ l dNTP (1 OmM each) 0.5 ⁇ l (250 ⁇ M final each) Pfu buffer (1 Ox) 2.0 ⁇ l
  • Pfu polymerase (2.5 U/ ⁇ l) 0.5 ⁇ l dH 2 O to 20 ⁇ l
  • the following PCR cycle conditions were used: start 2 min. 95 0 C 30 cycles of 95 0 C 30 sec, 65 0 C 30 sec, 72 0 C 1 min. final 72 0 C 2 min. extension step
  • the full-length 993 nucleotide-long promoter>EGFP was amplified in the following PCR mix: assembled sub-segments 1.0 ⁇ l primer 5' (1.2 ⁇ M) 5 ⁇ l (300 nM final) primer 3 ' (1.2 ⁇ M) 5 ⁇ l (300 nM final) dNTP (10 mM each) 0.5 ⁇ l (250 ⁇ M final each)
  • the present invention provides among other things methods for assembling large polynucleotide constructs and organisms having increased genomic stability. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne des procédés d'augmentation de la qualité et de la quantité d'oligonucléotides synthétisés en particulier chimiquement, notamment ceux synthétisés en utilisant des procédés basés sur puce électronique, et les produits ainsi obtenus.
PCT/US2007/012095 2006-05-20 2007-05-20 Oligonucléotides pour l'assemblage mutiplexé d'acides nucléiques WO2008054543A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US80176006P 2006-05-20 2006-05-20
US60/801,760 2006-05-20

Publications (2)

Publication Number Publication Date
WO2008054543A2 true WO2008054543A2 (fr) 2008-05-08
WO2008054543A3 WO2008054543A3 (fr) 2008-08-14

Family

ID=39344817

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/012095 WO2008054543A2 (fr) 2006-05-20 2007-05-20 Oligonucléotides pour l'assemblage mutiplexé d'acides nucléiques

Country Status (1)

Country Link
WO (1) WO2008054543A2 (fr)

Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2630264A1 (fr) * 2010-10-22 2013-08-28 President and Fellows of Harvard College Amplification orthogonale et assemblage de séquences d'acide nucléique
EP2684964A1 (fr) * 2012-07-09 2014-01-15 OakLabs GmbH Procédés à base de micro-réseau d'identification de nucléotides uniques avec des positions spécifiques dans des acides nucléiques
US9023601B2 (en) 2002-09-12 2015-05-05 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
WO2015021080A3 (fr) * 2013-08-05 2015-05-28 Twist Bioscience Corporation Banques de gènes synthétisés de novo
WO2015175832A1 (fr) * 2014-05-16 2015-11-19 Illumina, Inc. Techniques de synthèse d'acide nucléique
US9217144B2 (en) 2010-01-07 2015-12-22 Gen9, Inc. Assembly of high fidelity polynucleotides
US9216414B2 (en) 2009-11-25 2015-12-22 Gen9, Inc. Microfluidic devices and methods for gene synthesis
WO2015184016A3 (fr) * 2014-05-27 2016-03-10 The Broad Institute, Inc. Assemblage à haut rendement d'éléments génétiques
US9677067B2 (en) 2015-02-04 2017-06-13 Twist Bioscience Corporation Compositions and methods for synthetic gene assembly
US9895673B2 (en) 2015-12-01 2018-02-20 Twist Bioscience Corporation Functionalized surfaces and preparation thereof
EP3155146A4 (fr) * 2014-06-16 2018-02-28 Agilent Technologies, Inc. Assemblage de gènes à haut débit dans des gouttelettes
US9981239B2 (en) 2015-04-21 2018-05-29 Twist Bioscience Corporation Devices and methods for oligonucleic acid library synthesis
US10053688B2 (en) 2016-08-22 2018-08-21 Twist Bioscience Corporation De novo synthesized nucleic acid libraries
US10081807B2 (en) 2012-04-24 2018-09-25 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
US10202608B2 (en) 2006-08-31 2019-02-12 Gen9, Inc. Iterative nucleic acid assembly using activation of vector-encoded traits
US10207240B2 (en) 2009-11-03 2019-02-19 Gen9, Inc. Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly
US10308931B2 (en) 2012-03-21 2019-06-04 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
US10417457B2 (en) 2016-09-21 2019-09-17 Twist Bioscience Corporation Nucleic acid based data storage
US10457935B2 (en) 2010-11-12 2019-10-29 Gen9, Inc. Protein arrays and methods of using and making the same
US10669304B2 (en) 2015-02-04 2020-06-02 Twist Bioscience Corporation Methods and devices for de novo oligonucleic acid assembly
US10696965B2 (en) 2017-06-12 2020-06-30 Twist Bioscience Corporation Methods for seamless nucleic acid assembly
WO2020212391A1 (fr) * 2019-04-15 2020-10-22 Thermo Fisher Scientific Geneart Gmbh Assemblage multiplex de molécules d'acides nucléiques
US10844373B2 (en) 2015-09-18 2020-11-24 Twist Bioscience Corporation Oligonucleic acid variant libraries and synthesis thereof
US10894242B2 (en) 2017-10-20 2021-01-19 Twist Bioscience Corporation Heated nanowells for polynucleotide synthesis
US10894959B2 (en) 2017-03-15 2021-01-19 Twist Bioscience Corporation Variant libraries of the immunological synapse and synthesis thereof
US10907274B2 (en) 2016-12-16 2021-02-02 Twist Bioscience Corporation Variant libraries of the immunological synapse and synthesis thereof
US10936953B2 (en) 2018-01-04 2021-03-02 Twist Bioscience Corporation DNA-based digital information storage with sidewall electrodes
US11060137B2 (en) * 2016-12-14 2021-07-13 Codex Dna, Inc. Methods for assembling DNA molecules
US11072789B2 (en) 2012-06-25 2021-07-27 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing
US11084014B2 (en) 2010-11-12 2021-08-10 Gen9, Inc. Methods and devices for nucleic acids synthesis
US11332738B2 (en) 2019-06-21 2022-05-17 Twist Bioscience Corporation Barcode-based nucleic acid sequence assembly
US11377676B2 (en) 2017-06-12 2022-07-05 Twist Bioscience Corporation Methods for seamless nucleic acid assembly
US11407837B2 (en) 2017-09-11 2022-08-09 Twist Bioscience Corporation GPCR binding proteins and synthesis thereof
US11434262B2 (en) * 2011-01-26 2022-09-06 President And Fellows Of Harvard College Transcription activator-like effectors
US11492665B2 (en) 2018-05-18 2022-11-08 Twist Bioscience Corporation Polynucleotides, reagents, and methods for nucleic acid hybridization
US11492727B2 (en) 2019-02-26 2022-11-08 Twist Bioscience Corporation Variant nucleic acid libraries for GLP1 receptor
US11492728B2 (en) 2019-02-26 2022-11-08 Twist Bioscience Corporation Variant nucleic acid libraries for antibody optimization
US11512347B2 (en) 2015-09-22 2022-11-29 Twist Bioscience Corporation Flexible substrates for nucleic acid synthesis
US11550939B2 (en) 2017-02-22 2023-01-10 Twist Bioscience Corporation Nucleic acid based data storage using enzymatic bioencryption
US11629377B2 (en) 2017-09-29 2023-04-18 Evonetix Ltd Error detection during hybridisation of target double-stranded nucleic acid
US11702662B2 (en) 2011-08-26 2023-07-18 Gen9, Inc. Compositions and methods for high fidelity assembly of nucleic acids
US11970697B2 (en) 2020-10-19 2024-04-30 Twist Bioscience Corporation Methods of synthesizing oligonucleotides using tethered nucleotides
US12018065B2 (en) 2021-04-27 2024-06-25 Twist Bioscience Corporation Variant nucleic acid libraries for coronavirus

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047643A1 (fr) * 1998-03-18 1999-09-23 Quark Biotech, Inc. Methode de selection-soustraction utilisee dans l'identification de genes
WO2006044956A1 (fr) * 2004-10-18 2006-04-27 Codon Devices, Inc. Procedes d'assemblage de polynucleotides synthetiques de haute fidelite

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047643A1 (fr) * 1998-03-18 1999-09-23 Quark Biotech, Inc. Methode de selection-soustraction utilisee dans l'identification de genes
WO2006044956A1 (fr) * 2004-10-18 2006-04-27 Codon Devices, Inc. Procedes d'assemblage de polynucleotides synthetiques de haute fidelite

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RICHMOND K E ET AL: "Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis" NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 32, no. 17, 1 January 2004 (2004-01-01), pages 5011-5018, XP002344586 ISSN: 0305-1048 *

Cited By (93)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10640764B2 (en) 2002-09-12 2020-05-05 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
US10774325B2 (en) 2002-09-12 2020-09-15 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
US10450560B2 (en) 2002-09-12 2019-10-22 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
US9023601B2 (en) 2002-09-12 2015-05-05 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
US9051666B2 (en) 2002-09-12 2015-06-09 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
US10202608B2 (en) 2006-08-31 2019-02-12 Gen9, Inc. Iterative nucleic acid assembly using activation of vector-encoded traits
US10207240B2 (en) 2009-11-03 2019-02-19 Gen9, Inc. Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly
US9216414B2 (en) 2009-11-25 2015-12-22 Gen9, Inc. Microfluidic devices and methods for gene synthesis
US9968902B2 (en) 2009-11-25 2018-05-15 Gen9, Inc. Microfluidic devices and methods for gene synthesis
US9217144B2 (en) 2010-01-07 2015-12-22 Gen9, Inc. Assembly of high fidelity polynucleotides
US11071963B2 (en) 2010-01-07 2021-07-27 Gen9, Inc. Assembly of high fidelity polynucleotides
US9925510B2 (en) 2010-01-07 2018-03-27 Gen9, Inc. Assembly of high fidelity polynucleotides
EP2630264A1 (fr) * 2010-10-22 2013-08-28 President and Fellows of Harvard College Amplification orthogonale et assemblage de séquences d'acide nucléique
EP2630264A4 (fr) * 2010-10-22 2014-04-02 Harvard College Amplification orthogonale et assemblage de séquences d'acide nucléique
US20140045728A1 (en) * 2010-10-22 2014-02-13 President And Fellows Of Harvard College Orthogonal Amplification and Assembly of Nucleic Acid Sequences
US10457935B2 (en) 2010-11-12 2019-10-29 Gen9, Inc. Protein arrays and methods of using and making the same
US10982208B2 (en) 2010-11-12 2021-04-20 Gen9, Inc. Protein arrays and methods of using and making the same
US11084014B2 (en) 2010-11-12 2021-08-10 Gen9, Inc. Methods and devices for nucleic acids synthesis
US11845054B2 (en) 2010-11-12 2023-12-19 Gen9, Inc. Methods and devices for nucleic acids synthesis
US11434262B2 (en) * 2011-01-26 2022-09-06 President And Fellows Of Harvard College Transcription activator-like effectors
US11702662B2 (en) 2011-08-26 2023-07-18 Gen9, Inc. Compositions and methods for high fidelity assembly of nucleic acids
US10308931B2 (en) 2012-03-21 2019-06-04 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
US10927369B2 (en) 2012-04-24 2021-02-23 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
US10081807B2 (en) 2012-04-24 2018-09-25 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
US11072789B2 (en) 2012-06-25 2021-07-27 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing
WO2014009007A1 (fr) * 2012-07-09 2014-01-16 Oaklabs Gmbh Méthodes à base de micropuces repérant des nucléotides uniques dans les sites spécifiques d'acides nucléiques
EP2684964A1 (fr) * 2012-07-09 2014-01-15 OakLabs GmbH Procédés à base de micro-réseau d'identification de nucléotides uniques avec des positions spécifiques dans des acides nucléiques
US9409139B2 (en) 2013-08-05 2016-08-09 Twist Bioscience Corporation De novo synthesized gene libraries
US10632445B2 (en) 2013-08-05 2020-04-28 Twist Bioscience Corporation De novo synthesized gene libraries
WO2015021080A3 (fr) * 2013-08-05 2015-05-28 Twist Bioscience Corporation Banques de gènes synthétisés de novo
US9889423B2 (en) 2013-08-05 2018-02-13 Twist Bioscience Corporation De novo synthesized gene libraries
US10272410B2 (en) 2013-08-05 2019-04-30 Twist Bioscience Corporation De novo synthesized gene libraries
US9839894B2 (en) 2013-08-05 2017-12-12 Twist Bioscience Corporation De novo synthesized gene libraries
GB2533173A (en) * 2013-08-05 2016-06-15 Twist Bioscience Corp De Novo synthesized gene libraries
US10384188B2 (en) 2013-08-05 2019-08-20 Twist Bioscience Corporation De novo synthesized gene libraries
US11559778B2 (en) 2013-08-05 2023-01-24 Twist Bioscience Corporation De novo synthesized gene libraries
US9833761B2 (en) 2013-08-05 2017-12-05 Twist Bioscience Corporation De novo synthesized gene libraries
US11185837B2 (en) 2013-08-05 2021-11-30 Twist Bioscience Corporation De novo synthesized gene libraries
EA034459B1 (ru) * 2013-08-05 2020-02-11 Твист Байосайенс Корпорейшн Синтезированные de novo библиотеки генов
US9403141B2 (en) 2013-08-05 2016-08-02 Twist Bioscience Corporation De novo synthesized gene libraries
US10583415B2 (en) 2013-08-05 2020-03-10 Twist Bioscience Corporation De novo synthesized gene libraries
US10618024B2 (en) 2013-08-05 2020-04-14 Twist Bioscience Corporation De novo synthesized gene libraries
US10773232B2 (en) 2013-08-05 2020-09-15 Twist Bioscience Corporation De novo synthesized gene libraries
US9555388B2 (en) 2013-08-05 2017-01-31 Twist Bioscience Corporation De novo synthesized gene libraries
US10639609B2 (en) 2013-08-05 2020-05-05 Twist Bioscience Corporation De novo synthesized gene libraries
US11452980B2 (en) 2013-08-05 2022-09-27 Twist Bioscience Corporation De novo synthesized gene libraries
CN106536734A (zh) * 2014-05-16 2017-03-22 Illumina公司 核酸合成技术
US10570447B2 (en) 2014-05-16 2020-02-25 Illumina, Inc. Nucleic acid synthesis techniques
US20170191126A1 (en) * 2014-05-16 2017-07-06 Illumina, Inc. Nucleic acid synthesis techniques
CN106536734B (zh) * 2014-05-16 2020-12-22 Illumina公司 核酸合成技术
WO2015175832A1 (fr) * 2014-05-16 2015-11-19 Illumina, Inc. Techniques de synthèse d'acide nucléique
AU2015259024B2 (en) * 2014-05-16 2021-07-01 Illumina, Inc. Nucleic acid synthesis techniques
WO2015184016A3 (fr) * 2014-05-27 2016-03-10 The Broad Institute, Inc. Assemblage à haut rendement d'éléments génétiques
US11898141B2 (en) 2014-05-27 2024-02-13 The Broad Institute, Inc. High-throughput assembly of genetic elements
EP3155146A4 (fr) * 2014-06-16 2018-02-28 Agilent Technologies, Inc. Assemblage de gènes à haut débit dans des gouttelettes
US10669304B2 (en) 2015-02-04 2020-06-02 Twist Bioscience Corporation Methods and devices for de novo oligonucleic acid assembly
US9677067B2 (en) 2015-02-04 2017-06-13 Twist Bioscience Corporation Compositions and methods for synthetic gene assembly
US11697668B2 (en) 2015-02-04 2023-07-11 Twist Bioscience Corporation Methods and devices for de novo oligonucleic acid assembly
US10744477B2 (en) 2015-04-21 2020-08-18 Twist Bioscience Corporation Devices and methods for oligonucleic acid library synthesis
US9981239B2 (en) 2015-04-21 2018-05-29 Twist Bioscience Corporation Devices and methods for oligonucleic acid library synthesis
US11691118B2 (en) 2015-04-21 2023-07-04 Twist Bioscience Corporation Devices and methods for oligonucleic acid library synthesis
US11807956B2 (en) 2015-09-18 2023-11-07 Twist Bioscience Corporation Oligonucleic acid variant libraries and synthesis thereof
US10844373B2 (en) 2015-09-18 2020-11-24 Twist Bioscience Corporation Oligonucleic acid variant libraries and synthesis thereof
US11512347B2 (en) 2015-09-22 2022-11-29 Twist Bioscience Corporation Flexible substrates for nucleic acid synthesis
US10384189B2 (en) 2015-12-01 2019-08-20 Twist Bioscience Corporation Functionalized surfaces and preparation thereof
US10987648B2 (en) 2015-12-01 2021-04-27 Twist Bioscience Corporation Functionalized surfaces and preparation thereof
US9895673B2 (en) 2015-12-01 2018-02-20 Twist Bioscience Corporation Functionalized surfaces and preparation thereof
US10975372B2 (en) 2016-08-22 2021-04-13 Twist Bioscience Corporation De novo synthesized nucleic acid libraries
US10053688B2 (en) 2016-08-22 2018-08-21 Twist Bioscience Corporation De novo synthesized nucleic acid libraries
US10417457B2 (en) 2016-09-21 2019-09-17 Twist Bioscience Corporation Nucleic acid based data storage
US11562103B2 (en) 2016-09-21 2023-01-24 Twist Bioscience Corporation Nucleic acid based data storage
US11263354B2 (en) 2016-09-21 2022-03-01 Twist Bioscience Corporation Nucleic acid based data storage
US10754994B2 (en) 2016-09-21 2020-08-25 Twist Bioscience Corporation Nucleic acid based data storage
US11060137B2 (en) * 2016-12-14 2021-07-13 Codex Dna, Inc. Methods for assembling DNA molecules
US10907274B2 (en) 2016-12-16 2021-02-02 Twist Bioscience Corporation Variant libraries of the immunological synapse and synthesis thereof
US11550939B2 (en) 2017-02-22 2023-01-10 Twist Bioscience Corporation Nucleic acid based data storage using enzymatic bioencryption
US10894959B2 (en) 2017-03-15 2021-01-19 Twist Bioscience Corporation Variant libraries of the immunological synapse and synthesis thereof
US11332740B2 (en) 2017-06-12 2022-05-17 Twist Bioscience Corporation Methods for seamless nucleic acid assembly
US10696965B2 (en) 2017-06-12 2020-06-30 Twist Bioscience Corporation Methods for seamless nucleic acid assembly
US11377676B2 (en) 2017-06-12 2022-07-05 Twist Bioscience Corporation Methods for seamless nucleic acid assembly
US11407837B2 (en) 2017-09-11 2022-08-09 Twist Bioscience Corporation GPCR binding proteins and synthesis thereof
US11629377B2 (en) 2017-09-29 2023-04-18 Evonetix Ltd Error detection during hybridisation of target double-stranded nucleic acid
US10894242B2 (en) 2017-10-20 2021-01-19 Twist Bioscience Corporation Heated nanowells for polynucleotide synthesis
US11745159B2 (en) 2017-10-20 2023-09-05 Twist Bioscience Corporation Heated nanowells for polynucleotide synthesis
US10936953B2 (en) 2018-01-04 2021-03-02 Twist Bioscience Corporation DNA-based digital information storage with sidewall electrodes
US11492665B2 (en) 2018-05-18 2022-11-08 Twist Bioscience Corporation Polynucleotides, reagents, and methods for nucleic acid hybridization
US11732294B2 (en) 2018-05-18 2023-08-22 Twist Bioscience Corporation Polynucleotides, reagents, and methods for nucleic acid hybridization
US11492728B2 (en) 2019-02-26 2022-11-08 Twist Bioscience Corporation Variant nucleic acid libraries for antibody optimization
US11492727B2 (en) 2019-02-26 2022-11-08 Twist Bioscience Corporation Variant nucleic acid libraries for GLP1 receptor
WO2020212391A1 (fr) * 2019-04-15 2020-10-22 Thermo Fisher Scientific Geneart Gmbh Assemblage multiplex de molécules d'acides nucléiques
US11332738B2 (en) 2019-06-21 2022-05-17 Twist Bioscience Corporation Barcode-based nucleic acid sequence assembly
US11970697B2 (en) 2020-10-19 2024-04-30 Twist Bioscience Corporation Methods of synthesizing oligonucleotides using tethered nucleotides
US12018065B2 (en) 2021-04-27 2024-06-25 Twist Bioscience Corporation Variant nucleic acid libraries for coronavirus

Also Published As

Publication number Publication date
WO2008054543A3 (fr) 2008-08-14

Similar Documents

Publication Publication Date Title
WO2008054543A2 (fr) Oligonucléotides pour l'assemblage mutiplexé d'acides nucléiques
US20090087840A1 (en) Combined extension and ligation for nucleic acid assembly
US20200231976A1 (en) Iterative nucleic acid assembly using activation of vector-encoded traits
US20070231805A1 (en) Nucleic acid assembly optimization using clamped mismatch binding proteins
US11702662B2 (en) Compositions and methods for high fidelity assembly of nucleic acids
US20190010529A1 (en) Compositions and methods for synthesis of high fidelity oligonucleotides
WO2007120624A2 (fr) Réactions d'assemblage concerté d'acides nucléiques
AU2019283944A1 (en) Methods for Nucleic Acid Assembly and High Throughput Sequencing
US20150203839A1 (en) Compositions and Methods for High Fidelity Assembly of Nucleic Acids
WO2007123742A2 (fr) Méthodes et compositions améliorant la fidélité d'assemblage de plusieurs acides nucléiques
US10421993B2 (en) Methods and compositions for reducing non-specific amplification products
JP6755890B2 (ja) 非特異的増幅産物を減少させる為の方法及び組成物
WO2007136833A2 (fr) Procédés et compositions pour la production d'aptamères et utilisations de ces procédés et de ces compositions
US20090136986A1 (en) Methods and cells for creating functional diversity and uses thereof
US20100129873A1 (en) Highly Parallel Gel-Free Cloning Method

Legal Events

Date Code Title Description
NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07867129

Country of ref document: EP

Kind code of ref document: A2