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Gene Synthesis: Methods and Applications
Randall A. Hughes,*,† Aleksandr E. Miklos,*,† and Andrew D. Ellington*,† Contents
1. Introduction 2. Oligonucleotide Synthesis 2.1. Solid phase phosphoramidite synthesis 2.2. Microchip-based oligonucleotide synthesis 3. Gene Assembly Methodologies 3.1. Ligation-mediated assembly 3.2. PCR-mediated assembly 4. Gene Design Software 5. Synthesis Fidelity/Error Correction Methods and Considerations 5.1. Oligonucleotide purification 5.2. Reading frame selection 5.3. Mismatch binding and cleavage 5.4. Correcting errors in synthetic genes by site-directed mutagenesis 6. Applications of Gene Synthesis 6.1. Codon optimization 6.2. Synthetic biology 7. Example of High-Throughput Gene Synthesis Using Protein Fabrication Automation 8. Conclusions References 278 278 278 280 282 282 284 291 293 294 294 295 297 297 297 299 300 303 303

DNA synthesis techniques and technologies are quickly becoming a cornerstone of modern molecular biology and play a pivotal role in the field of synthetic biology. The ability to synthesize whole genes, novel genetic pathways, and even entire genomes is no longer the dream it was 30 years ago. Using little more than a thermocycler, commercially synthesized
* Applied Research Laboratories, The University of Texas at Austin, Austin, Texas, USA Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA


Methods in Enzymology, Volume 498 ISSN 0076-6879, DOI: 10.1016/B978-0-12-385120-8.00012-7

2011 Elsevier Inc. All rights reserved.



Randall A. Hughes et al.

oligonucleotides, and DNA polymerases, a standard molecular biology laboratory can synthesize several kilobase pairs of synthetic DNA in a week using existing techniques. Herein, we review the techniques used in the generation of synthetic DNA, from the chemical synthesis of oligonucleotides to their assembly into long, custom sequences. Software and websites to facilitate the execution of these approaches are explored, and applications of DNA synthesis techniques to gene expression and synthetic biology are discussed. Finally, an example of automated gene synthesis from our own laboratory is provided.

1. Introduction
The ability to design and synthesize custom DNA sequences is at the heart of many current transformative research efforts in biotechnology, and the emerging field of synthetic biology. Interlocking technological improvements and research efforts have driven the development of cost-effective, highthroughput, and high-fidelity methods for the synthesis of de novo DNA sequences of ever increasing length and complexity. While beginning to end chemical synthesis of very long (> 500 nt) DNA sequences remains elusive, techniques which combine chemical oligonucleotide synthesis and enzymatic assembly of oligonucleotides into longer DNA duplexes have undergone extensive optimization. Using existing methods, a standard molecular biology laboratory can routinely synthesize DNA sequences up to 2 kbp within a week using just a thermocycler, DNA polymerases, and commercially synthesized oligonucleotides. This is a far cry from the 5 years it took to synthesize the first synthetic gene (a 77 nt tRNA gene) some 40 years ago (Agarwal et al., 1970). In the past decade alone, researchers have gone beyond the mere synthesis of genes to the synthesis of multiple gene operons and even the synthesis of the genomes for entire organisms (Cello et al., 2002; Gibson et al., 2008a, 2010a; Kodumal et al., 2004; Smith et al., 2003). Herein, we review the relevant methodologies for gene synthesis from synthetic oligonucleotides and give an example of high-throughput gene synthesis from our own laboratory.

2. Oligonucleotide Synthesis
2.1. Solid phase phosphoramidite synthesis
All gene synthesis technologies rely on the chemical synthesis of oligonucleotides to supply the building blocks for enzymatic assembly. The most commonly used method for oligonucleotide synthesis is the cyclical fourstep phosphoramidite synthesis method developed in the 1980s (Caruthers et al., 1983, 1987; Fig. 12.1). DNA oligonucleotides are synthesized in a

Gene Synthesis: Methods and Applications


Cycle start

Next cycle
(or release) DMTO O Basen



Basen –1


HO O O P O O CN O Basen –1 DMTO O Basen O P CN O Basen –1 O O


Basen –1


DMTO O Basen


Oxidation and capping




Figure 12.1 Oligonucleotide synthesis from phosphoramidites. The initial nucleoside is tethered to controlled pore glass via its 30 hydroxyl. The synthetic cycle involves deprotection, coupling, oxidation, and capping. Synthesis proceeds in a 30 –50 direction.

30 –50 manner by coupling acid-activated deoxynucleoside phosphoramidites to an initial deoxynucleoside attached to a solid support (usually controlled pore glass (CPG) or polystyrene (PS) beads) through its 30 hydroxyl group. In most commercial DNA synthesizers, the solid support matrix is packed into a flow-through column and the reagents necessary for the phosphoramidite synthesis cycle flow by the solid support matrix. The addition of each nucleotide monomer to the growing oligonucleotide chain is carried out in four steps (Fig. 12.1): (1) deprotection: a weak acid is used to remove the dimethoxytrityl (DMT) ether group from the 50 -end of the growing oligonucleotide chain leaving a reactive 50 -hydroxyl (-OH) for the next coupling step; (2) coupling: the reactive 50 -OH reacts with a tetrazoleactivated monomer by simultaneous addition of the monomer and the activator solutions. (3) capping: any uncoupled 50 -OH groups are blocked by acylation to minimize the formation of deletion products; and finally (4) oxidation: the relatively unstable phosphite triester internucleotide linkages are oxidized into more stable phosphotriester linkages. This cyclic process is repeated until the oligonucleotide is complete. The oligonucleotide is then cleaved from the solid support, and the remaining protecting

Lashkari et al. DNA . Hughes et al. Rayner et al. 2003). This scale is compatible with traditional microliter scale gene synthesis reactions while simultaneously reducing reagent consumption over traditional techniques by 100-fold (Lee et al.. 2002. 1987.280 Randall A. Microchip-based oligonucleotide synthesis Emerging DNA synthesis technologies may eventually reduce the waste. In particular. 1996). groups are removed by treatment with a strong base such as ammonium hydroxide. Horvath et al. while further increasing throughput. Sindelar and Jaklevic... 1998. Sindelar and Jaklevic. 2. cost. 2009). 1998). Quake and coworkers have developed microfluidic reaction devices in which the distribution of minute amounts of synthesis reagents into gated microfluidic reaction vessels leads to the synthesis of oligonucleotides at a  100-pmol scale. Caruthers et al. However.. 1998.. Lashkari et al. 1995. The robustness of solid phase phosphoramidite synthesis makes it easily amenable to automation. In this method. 2002. An alternative two-step version of the phosphoramidite synthesis cycle has been published that could also potentially reduce reagent consumption (Sierzchala et al. the DMT protecting group on the 50 -OH of each phosphoramidite is replaced with a carbonate group. Further improvements in throughput and concomitant reductions in cost may be achieved by miniaturization. 1985. Rayner et al. 1983.. depurination side reactions limit the quality and yields of oligonucleotides above 100 nt (Hall et al. the synthesis of up to 1536Â 20-mer oligonucleotides can now be carried out in a matter of hours (Cheng et al. 2010). although for gene synthesis relatively small amounts (picomoles) are necessary. While the amount of time necessary to synthesize a given oligonucleotide will vary between automated synthesizer platforms. A peroxy anion then serves as a nucleophile to simultaneously remove the 50 -carbonate protecting group and oxidize the phosphite triester internucleotide linkage to the phosphotriester linkage. and this method is now used in almost all commercially available DNA synthesizers (Caruthers... This procedure should also reduce the mutations observed in synthetic DNA that arise from depurination upon acid deprotection (Septak.. during acid deprotection. 1987. Automated synthesizers have a parallel throughput of up to 1536 oligonucleotides (Cheng et al... 1995). and errors associated with current automated solid phase oligonucleotide synthesizers. 2010).. 1995).. Different machines can generate oligonucleotides in a range of synthesis scales.. A recent modification of the traditional reaction conditions using a novel detritylation process has been reported to remedy this problem and can lead to the improved synthesis of oligonucleotides up to 150 nt in length (LeProust et al. Horvath et al. Synthesis of oligonucleotides with lengths < 100 nt is common place using solid phase phosphoramidite chemistry with coupling efficiencies that can approach 99% (Rayner et al.2. 1987). 1995.

Singh-Gasson et al.. photo-generated acids can be produced by these methods which allows for deprotection using standard phosphoramidites (Gao et al. Richmond et al. reviewed in Tian et al. While the multiplex synthesis capabilities of chip-based devices (up to 105 different sequences per chip) are almost unparalleled. Zhou et al..4-(methylenedioxy)-6-nitrophenyl)ethyl chloroformate (MeNPOC) or 2-(2-nitrophenyl)propoxy-carbonyl (NPPOC) on the phosphoramidite monomers or linkers (Gao et al.. Fodor et al. Excess reagents are washed away. A relatively low-cost alternative to these methods is to direct light to particular positions via digital photolithography with a micromirror device such as the NimbleGen device available from Roche. 2001)..Gene Synthesis: Methods and Applications 281 synthesis on microarrays or microfluidic-devices is becoming increasingly commonplace (Barone et al. 2004). large-volume synthesis of DNA chips... 2004). These methods are therefore primarily practical for the large-scale.. Zhou et al. Alternatively. Many of the microarray synthesis methods rely on lightdirected synthesis. 2001. 2009) show substantial promise in eventually helping to reduce the cost of oligonucleotide synthesis and thus the cost of gene synthesis itself.S)-1-(3. 1999.. photoremoval of protecting groups. However. 1991). 2001. the total synthetic yield of any given oligonucleotide (107–108 molecules) is too low to use these oligonucleotides directly in many conventional molecular biology reactions. As with other solid phase synthetic methods. further refinement and commercialization will likely be necessary . 2004).. Digital photolithography methods are amenable to different photochemistries.. Tian et al. George Church and colleges may have found a possible solution to this problem by using postsynthesis PCR amplification to increase the quantities of synthesized oligonucleotides to the concentrations required for standard DNA assembly methods (Tian et al. Fodor et al. The extraordinary spatial control available through illumination allows the parallel synthesis of many thousands of individual strands on a single microchip (Barone et al. including cleaving photolabile protecting groups such as (R. 2001. which in turn activates that position for participation in the next round of nucleotide coupling. 2004. resulting in the removal of the photolabile (or acid labile) protecting group on the 50 -OH. and coupling is repeated until the oligonucleotide sequence is completed. The primary drawback of the photolithography-based methods is the need for large numbers of unique prefabricated photomasks for each synthesis step. photolithography masks combined with photolabile nucleotide monomers direct the light-mediated reactions at particular positions on a microchip.. Hughes et al.. and the process of masking. reagents for chain extension flow past the surface of the chip and react with the previously deprotected positions. Areas not covered by the mask are exposed to light. While both of these technologies (along with several others. 1991. 2004. In one implementation. 2001...

3.. 2008). 1991).1. the yields are exceedingly poor and the synthesis error rate increases as a function of oligonucleotide length (because of the multiple acid deprotection steps). After sub-fragments of a gene are individually assembled. the ligase chain reaction (LCR) (Au et al.” involves splitting the desired gene product into multiple fragments composed of overlapping.. Grundstrom et al.. before these technologies see enough market penetrance to displace traditional automated solid phase synthesizers. These methods can be roughly grouped into ligation-mediated assembly and PCR-mediated assembly methodologies. the first chemically synthesized gene for a protein.282 Randall A. The newly formed . 1979). In consequence. In this method.2). Each of the gene fragments are assembled by annealing the pooled oligonucleotides for each fragment together and then ligating them en masse. 3. The first chemically synthesized gene encoded a 30 bp fragment of the yeast alanine tRNA and was constructed in the late 1960s using rudimentary oligonucleotide synthesis techniques and DNA ligasemediated strand joining (Gupta et al. For example. Therefore. heated to denature the oligonucleotides. 50 -phosphorylated oligonucleotides with carefully designed overlap sequences that span both strands of a desired DNA duplex are mixed together. 1968). While the synthesis of oligomers of lengths up to 600 nt has been reported (Ciccarelli et al. human insulin A (63 bp). Ligation-mediated assembly The joining of shorter DNA sequences together into longer DNA sequences using DNA ligase represents the earliest example of synthetic gene synthesis. A more modern version of this ligation-mediated assembly method called “Shotgun Ligation. 1985) (Fig. and then cooled to promote both annealing of the oligonucleotides and ligation by the thermostable ligase.. the full-length gene is created by pooling and ligating the subfragments together.. was similarly assembled using DNA ligase (Hsiung et al. a number of methods have been developed in the past three decades to assemble relatively short synthetic oligonucleotides into longer gene sequences. The discovery of thermostable ligases has since resulted in the synthesis of even longer DNA sequences (reviewed in Xiong et al.. 1998) involves a temperature cycling reaction similar to PCR. Hughes et al. 1989. it is not generally recommended that very long pieces be used for gene synthesis. 12. phosphorylated oligonucleotides (Eren and Swenson. Over a decade later. Gene Assembly Methodologies Chemical synthesis is typically used to synthesize oligonucleotides of up to 120–150 nt in length.

The authors allowed the oligonucleotides to anneal based upon the sequence complementarity of the overlap regions.Gene Synthesis: Methods and Applications 283 Ligase Product Ligase Figure 12. products can serve as the templates for additional ligation events.2 Ligase-based gene synthesis methods. longer oligonucleotides that encode one strand of the desired duplex can be bridged by small complementary oligonucleotides that align ligation junctions (Adams et al. For example.. Mehta et al. Once the full-length. one of the first demonstrations of PCR-mediated assembly was with a 924-bp gene coding for horseradish peroxidise isozyme (Smith et al. ligated them together. the complementary strand can be generated by primer extension with a DNA polymerase by PCR. For example.. Top. single-stranded DNA sequence has been assembled by ligation. By first creating only a single strand of the eventual DNA duplex. 1988. ligase may also be used in a stepwise synthesis approach where the growing product is tethered to a solid support and oligonucleotide pairs are washed over and sequentially ligated to the tethered product. the authors broke the genes into double-stranded oligonucleotide pairs that were 30–60 nt in length and which contained 6–9 nt overlaps. and then amplified . 1997).. ligase may be used to ligateassembled (gap-free) overlapping oligonucleotides into a single strand. Depending upon the length of the desired gene product. One of the advantages of ligation-mediated assembly over PCRmediated methods is the potential cost savings from reductions in oligonucleotide synthesis. 1990).. Chen et al. 1998).. assembly of gene fragments by LCR is often accompanied by a final PCR step to amplify or stitch together the shorter gene fragments produced by the ligation reactions (Au et al. and the temperature cycling process is continued until the desired gene or gene fragment has been assembled. the cost of oligonucleotide synthesis can be roughly halved. In this study. Bottom. 1990.

4-kb genome of the bacteriaphage jX174 (Smith et al. 2007). This technology has been fully automated allowing for the efficient.1-kb gene encoding a beta-lactamase gene fragment (Stemmer et al. These methodologies can be characterized as “one-pot” or single-step assemblies where the desired gene product is assembled (often as a mixture of PCR products of varying lengths) in a single enzymatic reaction or as multiple step assemblies where the gene is first divided into separate subassembly reactions.. the new section of DNA duplex can be ligated together using DNA ligase. the assembled gene by PCR using flanking primers ( Jayaraman et al. 1996). PCR-mediated assembly The most commonly used gene synthesis techniques currently rely on the polymerase chain reaction (PCR) to mediate assembly of a desired DNA sequence from short oligonucleotides. 1995). (1995). leading to a mixture of elongated gene fragments that included the full-length desired product. Hughes et al. 12. A version of this protocol that relied on enzymatically phosphorylated oligonucleotides was used to assemble the entire 5. largely complementary reference sequence to guide the assembly of a mismatched DNA duplex and led to the assembly of a 1.. 2002. All of the oligonucleotides encoding the gene were pooled together and assembled in a onepot polymerase chain assembly (PCA) reaction.2. 3.2). one-pot PCR-mediated gene assembly from oligonucleotides has been reported by Stemmer et al. the various subassemblies are then mixed and “stitched” together in a series of hierarchical thermal cycling reactions to yield the fully assembled gene products. For example. a DNA polymerase was used to stitch and extend 56Â 40 nt oligonucleotides designed to cover both strands of a 1. In each round of synthesis. 1991). The Blue Heron technology assembles a DNA duplex sequence on a solid support by iterative annealing and ligation of oligonucleotide pairs. This process is repeated until the entire gene sequence is sequentially assembled. high-throughput synthesis of DNA sequences at a commercial scale. Blue Heron Biotechnology uses a solid support-based ligation-mediated oligonucleotide assembly process to synthesize customersupplied DNA sequences (Fig. In these latter methods. In this method.. Once annealed.. A ligase-independent. A slightly different version of this protocol used a known. the next section of the DNA duplex anneals to the previously assembled DNA through designed sequence overlaps..284 Randall A. The desired product was then amplified from the PCA reaction in a standard . Ligation-mediated assembly has found a home in some commercial gene synthesis operations due to its inherently low mutagenesis rate (no errors due to DNA polymerase) and its relative ease of use (Mulligan et al. 2003).9-kb Bacillus thuringiensis d-endotoxin gene that had been altered for optimized expression in transgenic alfalfa and tobacco (Strizhov et al.

reduce error rates. 1989) to develop a PCR assembly method that did not require optimization of the reaction conditions and could be performed without the need for Set of gapless. As the number of oligonucleotides needed to assemble increasingly complex DNA sequences increased. Variations of the onepot PCA synthesis of genes from oligonucleotides have also been employed to reduce the amount (and thus the cost) of oligonucleotide synthesis... Full-length product is amplified from the mixture using flanking primers. . up to and including the full-length gene. Along these lines. Most of these methods required multiple PCRs that built a gene in sections prior to splicing the sections together. The overlap extension process is frequently utilized to stitch multiple subsections (subassemblies) of genes together in an ordered fashion through designed overlap sequences. 1994.. the oligonucleotides encoding each strand of the DNA duplex are staggered. Young and Dong combined dual asymmetrical PCR (DA-PCR) (Sandhu et al. A gapless series of overlapping oligonucleotides extend one another sequentially to produce a mixture of products. 1995).3 The Stemmer method for gene assembly.. Instead. and increase throughput. 2004. 1989).3)..7-kb plasmid was assembled from overlapping oligonucleotides (Stemmer et al.. To demonstrate the utility of this method. 2004a... alternative methodologies for oligonucleotide design and PCR assembly were devised to increase the rates of successful assembly.Gene Synthesis: Methods and Applications 285 PCR using the outermost primers (Fig. with overlap sequences between adjacent oligonucleotides being filled in during polymerase elongation. Xiong et al. 2006.. 12. overlapping oligonucleotides is amplified forming a mixture of products. 1995). 2006a). and this method was also successfully used to synthesize a 2. a 2. a process known as overlap extension (Chen et al. These variations typically design the oligonucleotides such that the entire sequence of the DNA duplex to be synthesized is not represented in the synthesized oligonucleotides. Product Figure 12. This mixture is resolved by PCR with flanking primers to selectively amplify the desired full-length product.1-kb gene from Plasmodium falciparum (pfsub-1) (Stemmer et al. The one-pot PCR assembly methodology has since been used to synthesize a number of long gene sequences (Kodumal et al. Reisinger et al. 1992) and overlap extension PCR (OE-PCR) (Horton et al. Horton et al.

2 times the length of the gene versus the previously required two times the length (Stemmer et al. The products of each DA-PCR (primary subassemblies) could then be stitched together into the full-length gene product via an OE-PCR step. double-stranded gene product to 1. phosphorylated or gel-purified oligonucleotides (Young and Dong. this method led to the preferential extension and amplification of longer subassemblies over shorter products.. The product is constructed by first assembling 400–500 bp blocks from overlapping oligonucleotides. In this method. a 1230-bp Peniophora lycii phytase gene (Xiong et al. These blocks are then assembled into a full-length gene in a second PCR step. 2004a). This methodology also reduced the quantity of synthesized oligonucleotide bases necessary to synthesize the full-length. the oligonucleotides that encoded the gene of interest were designed such that the sequence overlaps between each subassembly (composed of four overlapping oligonucleotides) were longer than the overlaps between adjacent oligonucleotides. repetitive sequences. Hughes et al. a 1245-bp HBV PRS-S1S2S large Block 1 Block 2 1. The authors demonstrated that this method was a relatively high-fidelity (error rate 0.5 pmol each per reaction 30 pmol each per reaction Second step Product Figure 12. fragments of the gene of interest were synthesized by mixing 10–12 60-mer oligonucleotides with 20 bp overlaps in a single PCR to assemble each  400–500 bp gene fragment. two-step DNA synthesis (PTDS) method for the synthesis of long gene sequences based on the sequential OE-PCR assembly of genes from overlapping synthetic oligonucleotides (Fig.4 PCR-based two-step DNA synthesis (PTDS) strategy used by Xiong et al. When combined with a fivefold excess of the outermost primer pair. 2004).. 12.. Higher concentrations of the outermost flanking primers (example concentrations are at right) facilitate the assembly of individual blocks. the authors have synthesized the genes for a 657-bp rice transcription factor. The use of multiple subassembly reactions reduced the likelihood of nonspecific annealing and thereby increased the purity of the assembled product. 2006b). . Xiong et al. (2004a) described another PCR-based. In this method.12% average) and cost-effective way to assemble larger gene sequences regardless of high G/C content. Using this method. or complex secondary structures. 1995).4). The full-length gene was then assembled from the gene fragments in a secondary OE-PCR with the outermost primers (Xiong et al.286 Randall A.

2007). Primers are added in a concentration gradient with the innermost pair having the lowest concentration and the outermost pair having the highest concentration. Gao et al. 2004a). bidirectional PCRs that will yield Oligonucleotide Concentration Overlaps adjusted to match T ms across assemblies Figure 12. inside-out (TBIO) nucleation PCR-mediated gene synthesis (Fig. This is followed by the annealing and extension of the next pair of sense and antisense oligonucleotide primers to the previously created duplex DNA. This serial extension scheme builds the gene up in stages and aids in the assembly of genes which are otherwise difficult to assemble. (2003) introduced the concept of thermodynamically balanced. and 2382-bp vip3aI and 5367-bp CrtEBWY genes (Xiong et al. The sense-strand primers and the antisense primers meet at a point in the center of the gene (or gene fragment). An improved version of this methodology has also been described that includes additional gel purification steps for the oligonucleotide substrates. and an OE-PCR-based error correction step to improve the overall fidelity of the synthesized product (Xiong et al.Gene Synthesis: Methods and Applications 287 surface antigen (Lou et al... while antisense-strand primers encode the C-terminal half. 12.e. a nucleation event initially happens at the center of the gene sequence in which the first pair of overlapping sense and antisense primers is extended by PCR. inside-out (TBIO) assembly. Once the oligonucleotides necessary to assemble the gene fragments have been made. The TBIO method involves a five-step design and synthesis protocol and is especially notable for its unique primer design and assembly scheme. Overlaps between primers are balanced to obtain uniform annealing temperatures across the assembly. thermodynamically balanced). In the TBIO assembly scheme.5 Thermodynamically balanced. This results in selective pressure for amplification of ever longer products and ultimately favors the full-length gene. the second step in the assembly process is to use from four to six pairs of  60 nt TBIO primers for inside-out. During assembly. sense-strand primers encode the N-terminal half of a gene sequence..5). which anneal through overlap sequences designed to have identical annealing temperatures (i.. . 2006a).

rather than a mixture of four to six primer pairs at a time. Hoover and Lubkowski. The primer assembly of the gene fragments was almost identical to that of the TBIO method except that the authors initially used a one-pot PCR synthesis (although mechanistically assembly still occurred in an inside-out bidirectional manner). Using this methodology... making this one of the more high-fidelity gene synthesis methods available. Of the 15 synthetic genes sequenced. the authors report the successful synthesis of the 449 bp gene encoding for a PAZ (Piwi/Argonaute/Zwille) domain and the 1826 bp gene encoding for a polA DNA polymerase mutant (Marsic et al. 2008). Another strategy for gene assembly that is very similar to the TBIO assembly method is named successive extension PCR (Peng et al. 2003. The authors compared the TBIO method to “conventional” thermodynamically balanced (TBC) gene assembly methods for the synthesis of the human protein kinase genes PKB2 (1. which encourages the inside-out extension reaction.0 kb in length (Peng et al.. Xiong et al. the error rate for the TBIO PCR-based synthesis method ranged from 0% to 0. cryIA(c) from 26 oligonucleotides. After the first primer pair in the center of the desired gene is extended in a short seven cycle PCR extension. 2003). The first 13 primers encoded the N-terminal half of the gene and the last 13 primers encoded the C-terminal half of the gene. bidirectional PCRs with additional sets of sense and antisense primers.. S6K1 (1... Other TBC methods often have error rates between 0. Smith et al.5 kb). It was observed that equal concentrations of each oligonucleotide resulted in relatively low yields and extensive “smearing” of DNA fragments over 1. used this method to synthesize the gene encoding the B. The primers in a TBIO reaction are added in a gradient of concentrations with the innermost primers added at the lowest concentration and the outermost primers at the highest concentration. The assembly is much cleaner and eliminates the time-consuming need to purify products between reactions. 2003. the gene fragment is gel-purified to remove any spurious products and is further extended by inside-out.1% and 1% (Binkowski et al..7 kb) (Gao et al. thuringiensis d-endotoxin.6 kb).  400–500 bp gene fragments upon assembly. Xiong et al. and PDK1 (1. 2003). Next. starting with the centermost primer pair (Marsic et al. Peng et al.. 2004a....3% (Gao et al. However. has been published in which the authors performed the PCR assembly using small volumes (12 mL) and few cycles (7 cycles) with one TBIO primer pair at a time. 2003). 2002.b). The inside-out. Xiong et al. 2003. called sequential TBIO or seqTBIO. 2008). bidirectional assembly process is repeated with additional sets of primers until the fulllength gene is obtained. with 20 bp overlaps throughout (Peng et al. Hughes et al.. 2004b). 2005. 2004a). another seven cycle extension reaction is set up with the next pair of TBIO primers. these problems could be corrected by adjusting the amounts of the primers added to the one-pot . A modification of the original TBIO method.288 Randall A.

used to calculate oligonucleotide concentrations for the primary reactions. 2004a). 68 º. Oligonucleotide assembly scheme (top) for a three-pair per fragment by two fragments per gene assembly. (0. . Xiong et al. Primary reactions assemble oligonucleotides from inside-out due to a concentration gradient of material (top left) in 16 cycles of PCR (conditions right). are given in the lower left. The entire process can be automated on a liquid handling robot. 12. and the concentrations actually used for this sample “3 Â 2” reaction. Additionally.Gene Synthesis: Methods and Applications 289 assembly reaction (1. while simultaneously improving fidelity (Gordeeva et al. Secondary reaction 95 º. 2006a. 2 min 15 s 30 s 30 s Primary reaction 1 Primary reaction 2 16 cycles Dilute primary reactions 3:1. The recipe for the amplification master mix is given in the lower right.b).6 Gene synthesis as described by Cox et al. 15 s 60 º.5 pmol for the innermost primers and 30 pmol for the outermost primers) followed by amplifying the full-length gene product in a secondary PCR using the outermost primers (Xiong et al.. 60 º. 2004a.5 mL as template for secondary. and these gene fragments were then stitched together in a secondary OE-PCR into the full-length gene has been reported to improve the synthesis yield of the fully assembled product (Xiong et al. a variation of this procedure has been successfully automated for high-throughput assembly of genes encoding several hundred mutant proteins at a scale and cost savings heretofore unseen in laboratory-scale gene synthesis applications (Cox et al.. The formula from Cox et al.. 30 s 16 cycles Product [Pi] = [P ]T n–1 Σj = 0 cj c n–j [Pi ] = concentration of ith primer pair c = constant..1 nM 95.75) [P ]T = total [primer] (≈600 nM ) 67. 30 s 72º.. similar to that used in Reddy et al. The primary reactions are diluted and used as templates for a secondary (bottom) reaction that assembles and amplifies the final product via another 16 cycles of PCR (conditions right). 94 º. Young and Dong. 2003. 2004a. A version of this procedure in which a gene was first subdivided into multiple fragments for one-pot TBIO PCR assembly from oligonucleotides. 95 º. 2 min 94 º. 2010).65 ≤ c ≤ 0. add 1.9 nM 137 nM Each 50 mL reaction uses 13 mL of an amplification master mix comprised materials from the NovaGen KOD Hot-Start polymerase kit: 500 mL 500 mL 300 mL 100 mL 10ϫ KOD buffer dNTP mix 25 mM MgSO4 Hot-Start KOD Figure 12.. Recently an improved PCR-based gene synthesis (IPS) method was described that may lower cost of gene synthesis and eliminate the need for multiple gel purification steps associated with other assembly procedures (Gao et al. 2004).6). 2007) (Fig.

290 Randall A.7 The improved PCR synthesis (IPS) method. In the first two steps.7). which are in turn assembled into a final product. Hughes et al. the gene sequence is divided into 300–400 bp gene fragments and the oligonucleotides necessary to assemble each fragment are divided into multiple two-primer (one 60 nt primer and its complementary 30 nt “splint” primer) 5-cycle PCR-mediated extension reactions. 12. One strand of the desired DNA duplex sequence is synthesized in whole via end-to-end 60 nt oligonucleotide primers without overlaps. 2006a). 12. brief extension cycle each of the extended oligonucleotide pairs are pooled into assembly PCRs that lead to the synthesis of 300–400 bp gene fragments. The third step of the IPS method is the joining of these 300–400 bp gene fragments together into a full-length gene and amplification using the outmost primers. On the opposing strand. Errors discovered in the final product can be readily corrected using original primer pairs that comprise the region containing the error. The 15 bp extensions in turn form overlaps for adjacent fragments (now 75 nt long) to anneal to one another.7).. any errors in the assembled sequence can be corrected by an OE-PCR step without the need for the synthesis of additional oligonucleotides (Gordeeva et al. 5 cycles of PCR (make mini-fragments) 20 cycles of PCR (make main fragments) PCR for final product Figure 12. These pairs then build larger fragments.. This method simplifies the correction of synthetic errors by starting from mini-fragments that are formed by briefly extending primer pairs. The IPS method then synthesizes a gene using a three-step process. Due to the unique design of the oligonucleotide primers used in the IPS method. . 30 nt primers are designed that have 15 bp of complementary overlap with each of the abutted oligonucleotides and also a uniform melting temperature (Tm). This is a decided improvement over other OE-PCR-mediated error correction methodologies that require the synthesis of additional primers (Xiong et al. Following this first. These shorter oligonucleotides act like splints to orient the longer 60 nt oligonucleotides and provide overlap sequences for annealing and assembly of adjacent oligonucleotides. The primary cost savings afforded by the IPS method arises during the design of the oligonucleotide primers (Fig. This is because any 60 nt or longer fragments containing a mutation can be reamplified and reassembled using existing oligonucleotides and then stitched back together using sequence verified segments of a previously cloned (and sequenced) variant. 2010) (Fig.

Gene Design Software While the technical considerations accompanying gene synthesis are daunting. Wu et al. However. 4. There are software packages. While other sized DNA products are still produced by this method. and convenient (and other DNA products can still be removed by gel purification). results in the assembly of the desired sequence along with the many intermediate and spurious products.genocad. that will allow users to drag-and-drop genetic elements based on “parts databases” such as the Registry of Standard Biological Parts (http://www. and the mixture is assembled into the full-length gene using PCR. simple. and the other to either amplify the fully assembled gene from a population of assembly products or to stitch together smaller gene fragments. we are more concerned with the handful of packages that enable users to design overlapping oligonucleotides for gene synthesis. The desired gene is then amplified from the mixture by adding an excess of the outermost primers and carrying out a secondary PCR. (2006) simplified this procedure by simply initially adding at least a 10-fold excess of the outermost primers to the primary assembly PCR. such as GeneDesigner (https://www. most PCR assembly methods rely on at least two separate PCRs to generate Villalobos et al. 2009). In the traditional PCA methodology. while Taq and Pfu (Stratagene) DNA polymerases could not be used to assemble the genes under the same reaction conditions. . . However. if successful. the authors noted that this methodology was highly dependant on the DNA polymerase used for the PCR: KOD HiFi (Novagen) and KOD XL (Novagen) could be used to assemble a 777 bp Gene A fragment and a 936 bp Gene C fragment. 2006) and GenoCAD (http://www. This process. (1995).Gene Synthesis: Methods and Applications 291 As we have seen. However. (2006) have described a one-pot method that is at root a simplification of the traditional two-step method originally reported by Stemmer et al.partsregistry. 40-mer oligonucleotides with 18–20 bp of sequence overlap between adjacent oligonucleotides encode both strands of a DNA duplex with no sequence gaps between the oligonucleotides. Czar et al. it is nonetheless quick. full-length genes: one to assemble the oligonucleotides into gene fragments.. Wu et al. this type of software is beyond the scope of this review as it uses preassembled DNA parts which are not necessary derived from synthetic starting materials. the design phase is perhaps the most The oligonucleotides encoding for the entire DNA duplex are then mixed at equal molar concentrations.dna20. thereby encouraging the amplification of the desired full-length product from the outset.

. 2005) and Gene2Oligo (http://berry.8) The additional features include a “Codon Juggling” module that will produce not only optimized sequences (based on standard frequency tables) but also next-most optimized sequences and most different sequences (iso-coding sequences with as little DNA sequence identity as possible).html. Richardson et al. Also present are features to locate and remove restriction sites and user-specified arbitrary sequences from and the overlap regions are globally normalized to have similar Tm 12.a-star. This program has been under active development and includes a variety of tools beyond mere gene synthesis design. This program has the advantage that it has been under development for quite some time. Hoover and GeneDesign ( AssemblyPCRoligomaker... It is worth mentioning that this website is particularly self-explanatory and easy to use. Hughes et al.8 Gene synthesis as implemented by GeneDesign. Assembly PCR Oligo Maker (http://publish. it is noteworthy for carrying out a pairwise sequence alignment to identify and Overlaps adjusted to match Tms across assemblies Block 1 Restriction sites to facilitate product assembly Block 2 Figure 2009) also designs oligonucleotides for gapless PCR or LCR assembly and breaks large sequences into smaller segments for multipart assembly. It will reverse-translate an amino acid sequence with a variety of preexisting codon-usage tables as well as user-entered codon frequency data and permits a cutoff value for low-use codons to be specified. In its current version. Rouillard et al.ibn.umich.nih. TmPrime (http://prime. However. 2002). . Rydzanicz et al.. Bode et al. This hierarchical approach can facilitate the synthesis of very large stretches of DNA. 2004) both automate the design of oligonucleotides for use in Stemmer-style assembly methods. The synthesis design module will break a gene into  500 bp fragments joined by unique restriction sites and then further break each of those fragments into overlapping oligonucleotides (Fig. it is capable of setting up overlap extension PCR assembly reactions as well as TBIO assemblies with user-specified Tm windows and oligonucleotide lengths. 2010). One of the easiest to use design programs is DNAWorks (http://helixweb. The product is synthesized in  500 bp segments with unique restriction sites at each segment junction to facilitate final assembly by ligation. Each segment is assembled essentially by the Stemmer method but the overlaps contain gaps. 2006.292 Randall A.

or homodimers. Errors are introduced into the synthesis process during both chemical synthesis steps and enzymatic assembly steps.. 2010). 2004). this software package will construct entire families of variant genes while reusing as many oligonucleotides as possible. This is an extremely beneficial and thrifty feature for protein engineering projects that require the construction of numerous variants of a single parental gene. During chemical oligonucleotide synthesis. Insertions are typically caused by unwanted DMT cleavage by tetrazole and can occur at a frequency of up to 0.4% per position (Hall et al.. the coupling efficiency for the addition of each successive monomer is typically  98% (Hall et al.Gene Synthesis: Methods and Applications 293 remove hetero. 2010) and has also been adapted to efficiently construct ensembles of antibody genes (Reddy et al. Going beyond design and considering the use of laboratory automation for synthesizing genes. The oligonucleotide design modules attempt to balance Tms and permit user-specified minimum and maximum oligonucleotide lengths. GeneFab and FabMgr are programs that can be used to first design and implement a two-step gene assembly process (where smaller segments are assembled using inside-out nucleation PCR) and then join these fragments into a final product using conventional overlap extension PCR (Cox et al. GeneComposer (http://www. 2009. Tian et al. 5. This software can be used to create scripts that can be used by TECAN Evo liquid handling robotic workstations to set up gene assembly reactions.5% per position. 2009) is a software package with a mixture of high-level design features and the capability to break down a gene into oligonucleotide sequences for assembly. 2009) 2009. Insertions and deletions are the most common errors that arise. potential mishybridization events. This software has been used to explore the sequence space around protein-coding genes (Allert et al.. Zhou et al. .genecomposer.. Lorimer et al.. but the assembly strategy is limited to gapless overlaps.. and hairpins from the design. Synthesis Fidelity/Error Correction Methods and Considerations One of the primary problems with current chemical gene synthesis methods is the need to reduce or correct introduced sequence errors. a boon for assembling multiple genes. 2009). 2007). Deletions can occur as a result of incomplete capping or deprotection and can occur at 0... In addition to generating code to facilitate the use of liquid handling robots in gene construction. The accumulation of these deleterious by-products can lead to only about 30% of any synthesized oligonucleotide (100-mer) being the desired sequence (Hall et al.

removing synthesis by-products by PAGE is a costly. albeit at a much lower rate than those introduced by oligonucleotide synthesis. then functional selection can be used to prescreen and enrich mutation-deficient sequences. Xiong et al.. 2004). Consequentially. For synthetic protein-coding sequences whose function cannot be readily assayed. and labor involved in gene assembly. 5.. and this can indeed reduce error rates in the final assembled products by several fold (Xiong et al... 2002. Xiong et al. Young and Dong. 1997.. though errors still can and do occur (Andre et al. Again. truncated variants by denaturing polyacrylamide gel electrophoresis (PAGE) (Sambrook and Russell. labor-intensive process. Cline et al. 1995. several alternative. 2004a). 5. A premium is placed on the use of high-fidelity DNA polymerases in virtually all PCR-based assembly methods. Reading frame selection If the desired gene is a protein-coding sequence. Taken together.1. even though these are the predominant cause of synthesis-related errors in any gene assembly process (Temsamani et al. 1996). Hoover and Lubkowski. Tian et al. However.. To increase the purity of input oligonucleotides. 2005. 2006a). cost. 2004. Oligonucleotide purification The quality and purity of the oligonucleotides used in chemical gene synthesis is the large source of potential error. Kodumal et al. higher throughput methods have been devised to reduce or eliminate introduced synthetic errors. Hughes et al.. and multiple clones must be sequenced to obtain the desired DNA sequence. 2004). 2003. The assembly of relatively short oligonucleotides into longer DNA sequences requires enzymatic extension by DNA polymerases that can also introduce errors into synthetic genes.294 Randall A. many gene synthesis protocols rely on purification of full-length oligonucleotides from capped.2. all of these sources of potential error in synthetic gene synthesis lead to error rates of 1–10 mutations per kilobase of DNA (Binkowski et al. low-throughput. 2006a. It is also difficult to separate full-length oligonucleotides from single deletion variants. Purification of gene fragments by agarose gel electrophoresis steps is often used in multiple two-step assembly PCR protocols to reduce error and promote the successful assembly of a desired DNA product (Gao et al. These introduced errors must generally be detected by screening. several methods have been developed to eliminate errors associated with chemical DNA synthesis and to increase the throughput of the synthesis process itself. it may instead be possible to screen for clones that have the proper .. 2001).. gel purification steps add to the time. Therefore..

.. 2000). The cells expressing genes with deletions will be sensitive to the antibiotic and only cells with genes that lack these deletions will survive on selective media. 2004). 2004. 1997).. 12. Tian et al. binds to heteroduplex DNA sequences containing mismatches. G-G.. Many of these single nucleotide deletions will cause reading frame shifts within a protein-coding sequence that will in turn lead to premature translation termination (Cho et al. while others will not (generally correct) (Fig. new duplexes will form. bind. more general methods for error correction will have to be applied. By denaturing (heating) the synthesized DNA duplexes (some strands with mutations. 1991). and cleave mismatched duplexes. the MutS protein is immobilized on beads or other solid support. 12. the Escherichia coli mismatch repair MutHLS proteins locate.. 2004. the synthetic genes can be cloned into a frame-shift selection vector that will express a fusion with a reporter protein. 2002. Mismatch binding and cleavage If the gene to be synthesized does not encode a protein. To remove these variants. and foments their selective removal from the pool of sequences. This method of presequencing has been reported to enrich for correct sequences by four. 5.. The uncleaved (largely mutation free) duplexes that survive the cleavage can be purified by agarose gel electrophoresis based on their size and then subsequently cloned and sequenced. or A-A mismatches. some without) and then reannealing (cooling) the mixture. The Mut S and Mut L proteins are known to bind the mismatched position and then recruit the Mut H endonuclease to scan and cleave the DNA duplex at the first d(GATC) sequence 30 to the mutation site (Smith and Modrich.9). as many of the errors that accumulate are single nucleotide deletions from the n À 1 by-products of chemical oligonucleotide synthesis (Temsamani et al. 2007). The heteroduplexes that contain mismatches or bulges due to deletions or insertions can then be detected and/or cleaved by mismatch repair proteins. The authors demonstrated that this method can effectively enrich for sequences that lack G-T. some of which contain mismatches (generally due to errors). A-C. There was up to a 10-fold enrichment of correct sequences over nontreated control sequences (Smith and Modrich. A similar enrichment technique relies on a thermostable version of the MutS protein from Thermus aquaticus. 1995. as well as deletions or insertions. Many methods take advantage of the mismatch repair proteins responsible for maintaining sequence fidelity during replication of genomic DNA (Modrich. 2007. usually an antibiotic resistance element (Cox et al. This is especially useful. 1996. leaving mutation-free duplexes to be eluted from a column (Carr et al. In this instance. Gerth et al.9). 1992).. Fig. In one such method. Lutz et al. 1997). 1996.. This method has been shown to enrich for .3. Seehaus et al..Gene Synthesis: Methods and Applications 295 reading fivefold and virtually eliminates single and double deletion variants (Cox et al.

only one error per 3500 bp was observed. The authors demonstrated that two rounds of shuffling could significantly reduce the errors associated with chemical synthesis of a green fluorescent protein variant (GFPuv).9 Error correction schemes based on heteroduplex removal. 12.296 Randall A.. Shuffling has an efficiency advantage when error-free transcripts are relatively rare. a further 3. 2005.. Following this purification.5. which is frequently the case when synthesizing genes greater than 1 kb in length. have further modified this technique to include DNA shuffling. Hughes et al. .. and those mismatches that make it past the first screen will be further eliminated when the shuffled genes are again passed over the MutS column. and immobilized MutS is used to remove fragments containing 4. reassembly of the cleaved genes will enrich the correct consensus sequence (Binkowski et al. Any resultant mismatch-bearing heteroduplexes are removed by degradation with a mismatch-specific endonuclease (top right) or by binding to immobilized MutS (middle right and bottom right). Iterative cycles of removal and resynthesis can be accomplished by shuffling the fragments (bottom right). correct genes by as much as 15-fold and has an error rate of only 1 per 10 kb (Carr et al. By removing only short fragments containing errors. This is useful because the purification of mismatches by MutS is not fully efficient. Mismatch-specific endonuclease Heteroduplex Formation Gel purify correct (full-length) product Immobilized MutS Captures heteroduplexes Fragment Deplete heteroduplex fragments with immobilized MutS Reassemble Figure 12. 2004).3-fold decrease relative to no iterative binding and shuffling (Binkowski et al.9). Binkowski et al. Fig. the error-free fragments comprising the remainder of each synthetic gene can be used to reassemble error-free genes. Heteroduplexes are formed through melting and reannealing the synthetic products. Heteroduplex containing mixtures are digested with an endonuclease. 2005).

2002). Codon optimization Synonymous codon usage varies by organism. Other more specific mismatch detecting endonucleases including phage T4 endonuclease VII..Gene Synthesis: Methods and Applications 297 Several other methods for enriching mutation-free sequences from assembled DNAs use other mismatch endonucleases. 2004). may be preferable to others (Salerno et al. 2004). Gordeeva et al. Fig. However. Correcting errors in synthetic genes by site-directed mutagenesis Finally.. the direct correction of known errors can be used to generate desired DNA sequences following assembly (Gordeeva et al. 2005. 5. 1999. any oligonucleotide-based.. 2010.4. (2006a) design new oligonucleotide primers that contain the targeted correction and reassemble the gene via overlap extension PCR.. Xiong et al. 6.. coli endonucleases were shown to be superior in this regard and reduced errors by 400-fold over no endonuclease-treated controls (Fuhrmann et al.1.9). Young and Dong. Gordeeva et al.. 2010). 2002. T7 endonuclease I. Pincas et al. and the mutation is just a statistical anomaly. used Endo V endonuclease from Thermotoga maritima to cleave heteroduplexes one base 30 to the mismatch followed by thermostable ligase from Thermus species AK16D to proofread and repair any nonspecific nicks (Huang et al. Pincas et al.. coli endonuclease V have been shown to be useful for identifying and cleaving synthetic heteroduplex DNAs containing mutations (Fuhrmann et al. Xiong et al.. The use of these mismatch cleaving endonucleases increases the quality of the primary PCR assembly reactions to the point that mutation-free synthetic genes over 1 kb can be made with relative ease (Fuhrmann et al. this phenomenon has been reviewed elsewhere both in terms of evolutionary implications (Hershberg and Petrov. employ a similar methodology except that they do not employ new oligonucleotides (as the original oligonucleotides are presumably correct. In practice. 2006a). 2010). They demonstrate that one can simply rebuild the section of the gene which contained the error by using the original synthesis primers and then reassemble the gene again by overlap extension PCR (Gordeeva et al.. 2005). such as the widely used QuickChange method from Stratagene. 2005. those that can manage error correction without the need for subcloning. Applications of Gene Synthesis 6. 2005. Wang and Malcolm. site-specific mutagenesis protocol is likely to work for correcting errors in synthetic DNA sequences. and E. The T4 and E. 12.. 2008) and in terms of implications for heterologous expression .

transcription terminators.. the OPTIMIZER website (http://genomes. (2009) constructed 40 iso-coding variants for two genes using a Monte Carlo approach that should have explored a wide range of parameters thought to affect expression (secondary structure. There proved to be no correlation between CAI and GFP fluorescence. Hughes et al. GC content. More sophisticated decision trees regarding codon-use can also be implemented. 2009). 2005) uses codon adaptation index (CAI) values (Sharp and Li. but can also take into account these additional DNA sequence or secondary structure.. 2007) uses information from a database of genes that are predicted to be highly expressed (Puigbo et al. next-best codons may be introduced to avoid creating undesirable elements in the DNA sequence (restriction sites. AT content. and this has been commercialized through the company DNA2.. although they also noted some effect of 50 RNA structures. generated a synthetic library of 154 iso-coding GFP genes with random silent mutations. an amino acid sequence can be reverse-translated using highly utilized codons for an expression host. Several groups have subjected sequence-based expression optimization strategies to extensive experimental tests.jcat. codon frequency. 2009).. Indeed increasing the AT content at the extremes (particularly at the 50 end) was shown to increase expression levels of targeted protein sequences (Allert et al. Grote et al.. regardless of CAI values. Puigbo et al. At the most basic level. Similarly. 2004).urv. 2010). of proteins (Gustafsson et al. Based on the observed expression levels. They found that AT content at the 50 and 30 ends of genes was significantly higher than in the middle and used a Monte Carlo approach to explore whether this skewing impacted the expression of 285 synthetic variants of three genes. 1987) to optimize codon usage for a wide range of prokaryotic hosts and a handful of eukaryotes. Welch et al. (2010) examined 816 complete bacterial genomes for CAI values. AT content. These authors have developed a codon-usage model based on this empirical optimization. Kudla et al. the JCat Web site (www. for example. Taken together. Removing RNA structure (often by enhancing AT content) is also likely . Welch et al. these studies clearly indicate that merely recoding a gene using commonly used host codons will not maximize expression. 2008) to optimize codon usage. The process by which an amino acid sequence is rendered as a DNA sequence with codon usage suitable to a given organism is known as codon optimization. and RNA secondary structure at several positions within open reading frames. Allert et al.298 Randall A. and this is almost automatically implemented for E. but instead the greatest expression of fluorescence stemmed from the occurrence of weak RNA structures in the first 28 bases of the open reading frame (Kudla et al. inverted repeats) OPTIMIZER/.. coli in many DNA editing programs. For example. they concluded that the use of codons which corresponded to tRNAs which were well charged during amino acid depletion led to optimal expression. Finally.

2010. Young and Alper. it can be reasonably said that advances in synthesis and assembly techniques have led to the emergence and definition of the discipline. Next came the synthesis of the genome for the 5. 2010). regulatory elements.. Shetty et al.. To invoke the late Richard Feynman: “What I cannot create.2. . coli. 2002. and (most importantly) engineerable way.Gene Synthesis: Methods and Applications 299 to be important. and transplant entire bacterial genomes. and even entire pathways has been undertaken (Burbelo et al. 2002).5 kb poliovirus cDNA from synthetic oligonucleotides into subfragment assemblies followed by iterative cloning of assembled synthons into the full-length genome was both a technical and controversial advance when it was completed in 2002 (Cello et al. Craig Venter and colleagues at the Venter Institute have greatly expanded the limits of chemically synthesized genomes and have developed a number of techniques to synthesize..” Overall. the construction of synthetic reporters. Synthetic biology ˆ tre of synthetic biology is the desire to engineer and underThe raison d’e stand biological components in a modular. synthetic biology seeks to demonstrate an understanding of biology by rebuilding it (or at least parts of it). Recently. would be illuminating.. That said the derivation of rules for the expression of synthetic genes is still in its infancy. Most importantly. scalable. 2009. The viral genome for the 1918 “Spanish” Influenza A strain has even been assembled from synthetic oligonucleotides (Neumann et al.3 kb jX174 bacteriaphage by ligation of synthetic oligonucleotides into subassemblies followed by PCR assembly into the full-length chromosome (Cello et al. 6. Smith et al. Calculating and comparing the codon usages of the high-expressing variants from Allert et al.. 1999). 2010.. Most remarkably. J. assemble. Applications for gene synthesis within the burgeoning field of synthetic biology are largely up to the imagination of the people doing the experiments. the ability to make synthetic DNA means that researchers are no longer limited to natural sources or natural encodings. 2010). passage. though. at least for E.. Irrespective of school of thought. As a result. despite multiple competing definitions and sects. with the highexpressing codon usages from Welch et al. enzymes. I do not understand. Indeed. 2003). the technological frontier in DNA synthesis has been expanded beyond the modular assembly of biological components into the synthesis of entire genomes. These peregrinations have been abetted by attempting to modularize biology using standardized “parts” such as Biobricks (Kelly et al. The assembly of the 7.. Khalil and Collins. at the center of this discipline is the synthesis of synthetic DNA. The synthesis of complete genomes has become a possible (if not yet practical) reality. 2008) or BglBricks (Anderson et al.

2007). While novel techniques such as genome transplantation (Lartigue et al. this group applied these same techniques to synthesize the 1. Finally. In this regard. Another clever assembly method takes advantage of yeast’s ability to readily perform homologous recombination with DNA fragments (Gibson et al. 2009). thermostable Tag ligase seals up nicks in the assembled sequence. and Phusion DNA polymerase can be used to assemble large overlapping subassemblies of DNA (Gibson et al.. and the cloning of whole bacterial genomes using yeast vector systems (Benders et al. 2010b)..3-kb mouse mitochondrial genome from synthetic oligonucleotides (Gibson et al. 2009) and the complete 16. a combination of T5 exonuclease. Recently. 2010) have so far proved most relevant to manipulating genomic DNA. Hughes et al. Overlapping DNA duplexes are first chewed back at their 50 ends by T5 exonuclease to yield long single-stranded overlaps.300 Randall A. The first step in this (and most other) synthetic method is to break down the target sequence into a set of overlapping primers compatible with the general assembly strategy. Taq ligase. the authors have synthesized sections of the bacterial Mycoplasma genitalium genome (Gibson et al. some of the assembly methods are also relevant to laboratory-scale DNA synthesis. The adjacent complementary fragment is then used as a primer for a fill-in reaction with Phusion DNA polymerase. Example of High-Throughput Gene Synthesis Using Protein Fabrication Automation Our own version of high-throughput gene synthesis is called protein fabrication automation (PFA). For example.08-Mbp genome for the bacteria Mycoplasma mycoides (Gibson et al. 7.. passaging synthetic genomic DNA (Lartigue et al. 2008b). Using this method.b) showed that 25 overlapping DNA fragments could be transformed into yeast to produce the 592-kb synthetic genome for the bacterium Mycoplasma genitalium... 2009)... In the case of PFA. We are currently equipped with a Mermade 192 oligonucleotide synthesizer and a TECAN Evo 200 liquid handling robotic platform to synthesize genes from oligonucleotides in a high-throughput manner. 2010a). the enzymatic assembly of DNA fragments up to several hundred kilobases in vitro opens the way to the construction of novel operons and biosynthetic pathways. Gibson et al.. the strategy is to assemble primary fragments of approximately 200–600 bp using inside-out nucleation PCR and then to concatenate these fragments into a final product via . (2008a. The TECAN robot is equipped with integrated automated thermocyclers to perform assembly PCR with little operator intervention. derived from advances made by Homme Hellinga at Duke and largely implemented by Dr Colin Cox.

enabling the overall workflow to be entirely automated on a robotic liquid handling workstation. 2010). As an example. and a mispriming graph to aid in manually adjusting derived assembly schemes. the location of each primer.Gene Synthesis: Methods and Applications 301 overlap extension PCR (Fig. As the antibody sequences were destined for protein expression in E. this is a turnkey operation that goes from oligonucleotide synthesis to full-length genes. The ability to efficiently reuse primers is an important and powerful feature of this software package. the concentration of each component is specified: in the inside-out nucleation reactions where the primary fragments are assembled. It is also capable of limited point-and-click DNA editing and of filtering of sequences for unwanted restriction sites. arbitrary sequences may be specified for inclusion in a gene. so that the primary products may be diluted and added directly to the secondary reactions. GeneFab. we recoded each murine antibody gene using a simplified codon table to ensure only one common E. In short. additional mutation lists may be imported to create variants of a parental sequence. the assembly strategy is set up in FabMgr. It has a graphical interface that provides a view of the sequence. This capability even extends beyond lists of mutations. as additional. which takes into account the number of oligonucleotides in each primary fragment and the number of secondary fragments that will be assembled. oligonucleotide concentrations are set in a gradient where low concentrations are used for the innermost pairs and high concentrations are used for the outermost pairs. thus negating differences in codon usage at a given position and thereby . There are two software components that facilitate the implementation of PFA. Being able to quickly and reliably manufacture numerous. 12.. coli. The combination of these two programs ultimately produces scripts for TECAN robotic workstations. and FabMgr. During our use of PFA. this preserves the reading frame of the synthetic genes and facilitates oligonucleotide reuse (example below). coli codon would be used for each amino acid. and once the assembly scheme has been established. Once a suitable scheme is devised in GeneFab. related genes is a transformative capability for protein engineering. This process was designed to eliminate intermediate purification steps. In addition to the oligonucleotide assembly scheme. The GeneFab software will render a DNA sequence as overlapping fragments and oligonucleotide pairs based on userprovided overlaps and oligonucleotide length constraints. its translation. FabMgr is used to manage variant sequences and maintains a database to enable efficient reuse of shared oligonucleotides. we have recently used the PFA approach to construct numerous single chain antibodies (scFvs) based on sequence analyses of antibody abundances in immune repertoires (Reddy et al.6). provided that the additional sequences are of identical length. we have observed that 80–100 bp oligonucleotides with 30-bp overlaps within fragments and 35-bp overlaps between fragments produce uniformly excellent results for most gene sequences up to approximately 2 kb.

Two. Oligonucleotide reuse was further facilitated by making the sequence of otherwise different genes more uniform. the synthesis of 192 oligonucleotides takes approximately 3 days.302 Randall A. we were able to reuse on average three oligonucleotides out of the 12 required for each gene constructed. The number of total gene variants which may be constructed is in turn limited by the number of fragments required per gene. By employing this design strategy. the number of 96-well plates of oligonucleotides that can be drawn from is limited to the number of spots available on a plate carrier (9-position carriers are common). After the oligonucleotides are synthesized. We typically assemble genes from 80 to 100 nt oligonucleotides. we were ultimately able to further reduce the total number of oligonucleotides needed to assemble the antibody variants by one-third. In addition to TECAN-specific code. All of these variants were paradoxically padded to be a uniform 824 bp in length by adding stuffer sequences onto their 50 ends to enable construction with a uniform overlapping oligonucleotide scheme. We have found that the control provided by in-house synthesis allows for long (> 70 bp) oligonucleotides to be produced with a suitable degree of purity for successful assembly. The primary fragment-generating PCRs are set up and run in an automated fashion on the robot deck. The overall rate of any gene synthesis operation is primarily limited by the time it takes to synthesize the oligonucleotides necessary to construct the genes. the robot will retrieve the plate from the thermocycler. avoiding the generation of a new primers. In the current configuration. Using the Mermade 192 DNA synthesizer. which is capped at 96 as the software currently only processes one plate at a time.. 2010). Hughes et al. and combine the fragments in another plate for assembly of the final sequence. if desired. any additional oligonucleotides needed are dynamically created and databased.and threefragments assemblies are common for genes < 2 kb. they are concentration-normalized to 1 mM and placed on the TECAN robot deck. although commercially sourced oligonucleotides can also be used (and indeed the software will output comma-separated lists suitable for input into a synthesizer or uploading as a commercial order). After aligning the antibody sequences around a common poly-glycine-serine linker. FabMgr outputs a humanreadable file which enumerates each liquid handling operation. The software also automatically generates an oligonucleotide order. Scripts to direct robotic assembly are also generated by FabMgr. dilute the reactions. Upon completion of this reaction. allowing smaller numbers of assemblies to be carried out manually. For example. provided that a plate-handling arm and a suitable PCR machine (controllable through the TECAN software and equipped with a motorized hot top) are available. with an additional half day for the robotically . the antibody genes naturally ranged from 740 to 824 bp due to the variability of the antibody complementary determining regions (Reddy et al. As each specified mutant or additional sequence is parsed by FabMgr. and therefore on the order of 32 or 48 gene variants are created per run.

I.14 and $0. Kleppe. it will be interesting to see whether they remain the province of individual researchers and facilities. (1988). M. A. J. E.. S. Caruthers. Given that the techniques reviewed herein are quickly becoming the cornerstone of modern molecular and synthetic biology methods. as previously occurred with the synthesis of short chemical oligonucleotides and as is currently occurring with the serial sequencing of single DNA templates.08 Mbp genome of the bacterium M. plasmids.. Khorana. G. L. H. Kingsman.. Sgaramella.. and Edwards.. E.25 per base-pair synthesized.. A. K. and viruses can be constructed from scratch with relative ease. M. M. It can be argued that the commoditization of gene synthesis will put all researchers on the same footing. R. However. and our experience strongly suggests that gene fabrication facilities can be successfully developed for many different research settings. REFERENCES Adams. Rajbhandary.. L. . yet recently.. The specialized technical skills. That said oligonucleotide synthesis and enzymatic gene assembly have been optimized to the point where small operons. U. J. D. H. H. These prices are comparable to those charged by commercial operations that enjoy much larger economies of scale. Kumar. Nucleic Acids Res.... or whether they are largely outsourced to commercial enterprises.. Braddock. 8. mycoides (Gibson et al. we have seen the complete combined chemical-enzymatic synthesis of the 1. Conclusions Thirty years ago. Ohtsuka.. and cost required to build a synthetic DNA sequence of this size makes it almost unobtainable to most research laboratories at the current time. the synthesis of DNA sequences multiple kilobases in length would have seemed like an impossibility.Gene Synthesis: Methods and Applications 303 controlled PCR assembly of the oligonucleotides into full-length genes.. it can equally well be argued that such outsourcing may stymie innovation in this area. Buchi. This review and method therefore stands at a crossroads and may either soon be regarded as an anachronism or as a call to arms. Johnson. this extreme example is currently more of an exception to the rule.. This impressive feat demonstrates the power of existing gene synthesis techniques for assembling large DNA sequences from chemically synthesized oligonucleotides. Kingsman. Synthesis of a gene for the HIV transactivator protein TAT by a novel single stranded approach involving in vivo gap repair. M. 16. H. equipment. N. S.. 2010a). We now routinely synthesize up to 50 unique 1–2 kb gene sequences (or several hundred 1–2 kb protein variants) per week at a current cost of between $0.. Agarwal. 4287–4298. Van de Sande. V. K. Gupta.

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