One such group of methods relies on the sequence mismatch recognition capabilities of the MutS protein to specifically bind to sequence mismatches in synthetic DNA duplexes. Selective binding of MutS to error-containing DNA can be used to sieve error-free sequences from those that contain errors Carr et al. These methods usually immobilize MutS to a solid matrix material and then purify column-bound error-containing DNA sequences from unbound material error-reduced. Error-containing heteroduplex DNA can also be sieved using enzymes that recognize and cut the DNA duplex at the site of the base mismatch Young and Dong ; Fuhrmann et al.
The use of endonuclease enzymes or enzyme cocktails , which recognize and cleave DNA heteroduplexes at the sites of mismatches, has been shown to be highly effective at reducing synthesis-related errors in synthetic genes allowing for time and material savings such that in some cases the treated genes can be used directly in functional assays without cloning and sequence verification Dormitzer et al.
An alternative to enzyme-mediated error correction techniques is the direct sequencing of oligonucleotides and assembled synthons using NextGen sequencing techniques.
Although more expensive on a per-sequence basis than enzyme-based error correction techniques, NextGen sequencing techniques offers tremendous multiplexing capabilities in which thousands of sequence reads can be taken on multiple oligonucleotides or synthons simultaneously.
In one such application, Matzas et al. Kim et al. In a similar approach, Schwartz et al. Recently, they have refined this approach to improve the multiplexing capacity of microarray-synthesized DNA to pairwise assemble — bp synthons from a single oligonucleotide pool and used bar-coded primers to selectively isolate sequence-verified individuals Klein et al. All of these methods point to ways in which NextGen technologies can be used to improve the quality of synthetic DNA.
In addition, because sequence-based approaches evaluate collections of individual molecules of DNA, they are suitable for the sequence verification of synthetic DNA, which may contain regions of sequence degeneracy such as libraries for directed evolution.
Currently, each of these methods are limited by the sequence read-length capabilities of the NextGen instrument used and by sequencing-related errors. However, as NextGen instrumentation and techniques continue to improve, these limitations will become less significant and allow for the accurate verification of longer pieces of synthetic DNA. Concomitant advances in assembling DNA into longer and longer pieces have led to methods to construct large enzyme complexes Kodumal et al.
Methods to assemble synthons have been developed to assemble synthons and larger DNA assemblies in both in vitro Gibson et al. Although restriction enzyme-based cloning techniques have been the de rigueur choice for manipulating DNA constructs for a couple of decades and were the basis of early BioBrick and similar assembly methods Shetty et al. Seam-less assembly and cloning methods available to the modern DNA jockey include Gibson assembly Gibson et al. Which DNA assembly technique to use is largely a matter of choice, and multiple approaches are often applied in parallel.
Of these methods, Gibson assembly is probably the most commonly used to assemble multiple pieces of DNA together into larger constructs. This method uses a one-pot isothermal technique, which uses an enzyme mixture containing a thermostable DNA polymerase, DNA ligase, and exonuclease to chew-back, anneal and repair adjacent overlapping DNA sequences to assemble the desired construct.
Recent innovations in the design of unique overlapping sequences to direct the assembly process has further expanded the usage of the Gibson assembly method for combinatorial assembly of large DNA sequences Guye et al. One of the hallmarks of synthetic biology is the application of rational design principles to the design and assembly of biological components; however, because it is often difficult to know a priori how well a given DNA construct will work once introduced into a cell, it is often necessary to try several versions of the construct to find which one will work best.
Therefore, a greater emphasis on the modular design of DNA parts enables the assembly of a greater variety of potential constructs through mix-and-match combinatorial assembly of DNA components. In addition to simplifying the overall assembly process, modular design and assembly of DNA components makes automation of the process possible, which can reduce the time, labor, and cost of making and testing multiple constructs.
Most of the aforementioned assembly methods can be used for the assembly in vitro, and Gibson assembly has been applied to the assembly of DNA segments multiple kilobases in length Gibson et al. In another such example, Gibson assembly was used to assemble the Efficient assembly of even larger synthetic DNA segments can also be performed in vivo by using the homologous recombination capabilities of the yeast Saccharomyces cerevisiae.
In an example of the exceptional ability of yeast to assemble exogenous DNA into larger assemblies from overlapping synthons or subassemblies, researchers at the J.
Craig Venter Institute have successfully used yeast to assemble multiple 0. Each of the aforementioned assembly techniques could be automated to further increase the throughput for constructing larger synthetic DNAs and enable the exploration and testing of large biological hypotheses. Synthetic DNA is central to the development of methods to engineer biology and when combined with the massive amounts of sequence data being generated by NGS efforts will contribute to the advancement of synthetic biology toward applications heretofore unimaginable.
To date, there have been a handful of moonshot demonstrations such as the complete synthesis of an entire yeast chromosome Annaluru et al. These examples, when combined with numerous projects in which synthetic DNA has been used to evaluate functional biological components Salis et al. To enable larger-scale engineering efforts and to democratize the use of synthetic biology techniques and design principles, recent innovations have sought to lower the cost of synthesizing DNA oligonucleotides and their subsequent assembly into synthetic constructs ready for use.
Figure 4. The strategy for the synthesis of the vip3aI gene using overlap extension PCR. For the successive PCR methods, five reactions each containing 12 oligonucleotides were carried out using 1. To obtain the full length of the bp vip3aI gene, the DNA fragments, four bp segments and one bp segment produced from the five PCR reactions, were mixed and used to assemble the template for the second PCR reaction.
The second PCR reaction was performed with the two outermost oligonucleotides as primers. Figure 1S shows the steps involved in the synthesis of the vip3aI gene using 90mer oligonucleotides. The PCR reaction for the gene assembly reaction was essentially the same as for the 60mer oligonucleotides, except that the extension time was 1 min 20 s in the first step.
For the single-step successive PCR procedure, sixty groups of 60mer oligonucleotides were added into a reaction tube, with 1. The PCR conditions for the gene assembly reaction using thirty-four 90mer oligonucleotides Figure 2 were essentially the same as for the 60mer oligonucleotides Figure 2S.
To obtain the full length of the bp vip3aI gene, four bp and one bp products from the PCR reactions were mixed and used as the template for the second PCR reaction, with the two outermost oligonucleotides as primers. Figure 3S shows the steps of synthesizing the vip3aI gene using 90mer oligonucleotides. An identical PCR protocol was used for the synthesis and assembly of the vip3aI gene using 60mer oligonucleotides, except that the extension time of the first cycle was 1 min 20 s.
Analyses of clones and DNA sequences The molecular cloning of the synthesized DNA fragments was performed according to the standard procedures AY and the sequence has For the synthetic gene used, all codons were optimized for the expression in Pichia pastoris 27 , The sequences for potential hairpin structures and motifs containing consecutive ATs were altered by using degenerating codons Figure 5S.
We synthesized sixty oligonucleotides that were 60mer in length and were named a1 to a60 Figures 1 and 6S. Five DNA blocks [named blocks 1, 2, 3 and 4 bp each and block 5 bp ; see Figure 1 ] were assembled and amplified Figure 5a , lanes 1 to 5.
The PCR was carried out to synthesize the full-length bp vip3aI gene using a mixture of the five DNA blocks as the template and the two outermost oligonucleotides, a1 and a60, as the primers. The full-length vip3aI gene fragment obtained from the PCR reaction is shown in Figure 5b lanes 1 and 2. We also synthesized two bp blocks and one bp block lanes 6 to 8 of Figure 5a using thirty-four oligonucleotides that were 90 nt in length and named b1 to b34 Figures 1S and 7S. The full-length bp vip3aI gene was assembled and amplified using a mixture of blocks 1 to 3 as the template and the b1 and b34 oligonucleotides as primers.
Figure 5. View large Download slide A Four and three blocks were easily amplified using five groups of 60 and 90 nt oligonucleotides separately by two-step successive PCR. Lane 1: bp block; lanes 2 to 5: four bp blocks; lane 6: bp block; lanes 7 and 8: bp blocks.
Lane 1: the full-length vip3aI gene amplified using 60mer oligonucleotides by single-step successive PCR; lane 2: the full-length vip3aI gene amplified using 90mer oligonucleotides by single-step successive PCR; lanes 3 and 4: the amplified vip3aI gene using 30 pmol of the two outermost primers.
We synthesized sixty oligonucleotides that were 60 nt in length, named c1 to c60 Figures 2 and 8S , and thirty-four oligonucleotides that were 90 nt in length, named d1 to d34 Figures 2S and 9S.Our experience is that the single-step PCR method is long for synthesizing a DNA fragment that is shorter. The strategy for the synthesis of the vip3aI gene using overlap extension PCR. Selective binding of MutS to error-containing DNA can be used to synthesis dna sequences from mymathlab homework answer key that contain formation of heteroduplexed DNA at positions that contain errors. Each unique synthon would be given a unique set of flanking subpool primer sequences such that the oligonucleotides oligonucleotides using a unique enzyme recognition sequence flanking the amplified from the synthesis pool for subsequent assembly. Following amplification of the subpool evolutions, the priming sequences are accurate by restriction sequence of the amplified dsDNA necessary to assemble any given synthon would be selectively was 1 min 20 s.
Following assembly, a synthetic DNA can be thought of as a population of sequences containing a mixture of correct and incorrect sequences.
The outermost priming sequences allow for amplification of the array-synthesized oligonucleotide pool, which has been cleaved from the chip surface following synthesis. What started with a synthetic tRNA gene more than 40 years ago has led to the recent synthesis of the first minimal bacterial genome Hutchison et al.
The amplified subpools can then be further subdivided into assembly oligonucleotide pools by additional unique priming sites included in the oligonucleotide flanking sequences. With these two groups of oligonucleotides, a bp PCR product, the full-length vip3aI gene, was obtained using the single-step PCR method, but the yields of the products were very low Figure 5c , lanes 1 and 2. In addition to simplifying the overall assembly process, modular design and assembly of DNA components makes automation of the process possible, which can reduce the time, labor, and cost of making and testing multiple constructs. Positions that contain mutations within these heteroduplexes can be acted on by proteins, which specifically recognize sequence mutations in DNA. Kim et al. Also, we have compared the PTDS method side by side with several previously published methods with regard to the error rates, costs and DNA product quality.
Craig Venter Institute have successfully used yeast to assemble multiple 0.
What started with a synthetic tRNA gene more than 40 years ago has led to the recent synthesis of the first minimal bacterial genome Hutchison et al. B Synthons can also be synthesized off-chip by first cleaving the oligonucleotide pools from the array. Each unique synthon would be given a unique set of flanking subpool primer sequences such that the oligonucleotides necessary to assemble any given synthon would be selectively amplified from the synthesis pool for subsequent assembly. For the synthetic gene used, all codons were optimized for the expression in Pichia pastoris 27 ,
However, as NextGen instrumentation and techniques continue to improve, these limitations will become less significant and allow for the accurate verification of longer pieces of synthetic DNA. Sequences that are unknowingly toxic to the host cells used for intermediate manipulations of the DNA e.
Synthetic DNA is central to the development of methods to engineer biology and when combined with the massive amounts of sequence data being generated by NGS efforts will contribute to the advancement of synthetic biology toward applications heretofore unimaginable. The full-length bp vip3aI gene was assembled and amplified using a mixture of blocks 1 to 3 as the template and the b1 and b34 oligonucleotides as primers. Recent innovations in the design of unique overlapping sequences to direct the assembly process has further expanded the usage of the Gibson assembly method for combinatorial assembly of large DNA sequences Guye et al.
Advanced Search Abstract Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression.
Another method first introduced by Kosuri et al. The strategy for the synthesis of the vip3aI gene using overlap extension PCR. Also, in many cases, it is highly desirable to use a chemical synthesis method to modify coding sequences to achieve high expression levels.