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The mixture is then cooled to allow annealing of oligo dimers at their termini all possible products are shown. Round 2: Further iterations of melting, reannealing and extension occur generating the first appearance of the gene product. Further rounds of PCA generate more gene product until the primers are consumed. As shown in Figure 2 , many combinations of dimers can anneal and not all result in successful extension.

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For examples of any more than 4 oligos, the various iterations of annealing and elongation get too complicated to represent diagrammatically. PCA eliminates the need for the whole gene to be synthesised solid phase. Once assembled, the gene product can be amplified by traditional PCR to generate a suitable concentration and purified by filtration to remove any remaining primers. Unfortunately, the process is not without errors.

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Insertion and deletion mutations frameshift mutations can be introduced at the solid phase synthesis stage and chain extension by polymerase risks further substitutions , insertions and deletions. Fortunately, natural DNA repair enzymes exist to correct these errors.

However, since synthetic DNA has no indication of which strand has the correct sequence unlike DNA in vivo , nucleases that rely on positive identification of the correct template strand cannot be used. Instead, it is necessary to use endonucleases that completely excise stretches of DNA that contain a mutation Figure 3. Since PCR cannot discriminate between correct sequences and those with errors, any mutations that have accumulated in the solid phase synthesis or polymerase extension of one gene strand will be matched in the reverse complement during amplification: the mutation is "invisible".

Therefore, the mixture of correct and incorrect gene products is melted and reannealed. It is statistically unlikely that mutations will occur in the same place in the same way on two random strands. As such, random reannealing to a strand which likely doesn't have the complementary mutation generates a mismatch as shown in Figure 3. This mismatch perturbs the secondary structure of the gene. Certain mismatch repair enzymes such as T7 Endonuclease I recognise these structural perturbations and cleave the first, second or third phosphodiester bond 3' to the mismatch dependent on the specific enzyme on both strands.

A single strand exonuclease then chews up the overhanging single stranded DNA 3' to 5'. This process produces dsDNA of varying sizes that all overlap and between them contain the correct sequence spanning the entire gene. Figure 3 Error correction Random errors present in dsDNA sequence will present as mismatches when melted and reannealed to error-free complements.

The resulting single stranded overhangs are removed by an exonuclease and PCA is carried out to re-assemble the various sized fragments into the full gene product. Figure adapted from Trends Biotechnol. While PCA facilitates the synthesis of large genes and genomes, even after error correction the product often has many errors present in the sequence. Gene synthesis by ligation Figure 4 has a lower error rate than PCA, but is limited to the synthesis of smaller genes since the fragment oligos must span the entire sequence of both the sense and antisense strands: this is impractical for longer genes.

However, if the goal is to synthesise a smaller gene Figure 4 Gene synthesis by ligation Oligos that span the entire gene are ligated together to form the gene product. Complete complementarity yields a lower error rate than PCA but ligation-based synthesis is limited by size. Solid phase synthesis generates the constituent fragments which are then purified ready for ligation. These oligos span the entire gene on both strands, missing only the phosphodiester bonds that link neighbouring oligos. Fragments are designed so two adjacent oligos are held together with a complementary template the splint then ligated enzymatically.

Three major methods of ligation-based synthesis exist Figure 5 :. This continues until the entire gene is assembled, which is then cleaved from the support at the end of the synthesis.

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While the error rate of ligation based synthesis is lower, it may still be necessary to perform error correction to yield a legitimate number of viable genes. Furthermore, PCA is the dominant methodology used for artificial gene synthesis. Ligation-based techniques require polyacrylamide gel electrophoresis PAGE purification and 5' phosphorylation, making the whole method costly and labour intensive.

PCA and associated error correction is preferred as these steps are eliminated. PCA and ligation based methods remain by far the most common methods of gene synthesis.

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Once the gene has been synthesised, the resulting solution will contain a mixture of gene strands with correct and incorrect sequences. To purify this mixture, it is necessary to separate out each individual strand and amplify it. This would be a difficult task for traditional DNA purification techniques : the difference between a correct and incorrect sequence may only be a single nucleotide, and separation by length would not discriminate between substitution mutations.

First, the gene strands must be inserted into a vector that allows movement of the gene into the bacterial cell. Plasmids are small circular pieces of DNA found in bacteria that are ancillary to the main bacterial chromosome. They are readily transferred from cell to cell and from environment to cell and undergo replication within the cell. Therefore, plasmids are a useful vector into which the gene product may be inserted for in vivo amplification.

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Insertion is achieved by one of two major methods. Cohesive-ended or "sticky-ended" ligation involves digestion of the plasmid with a restriction endonuclease that linearises the circular DNA by cutting both strands unevenly, leaving single stranded 3' overhangs at each end. The PCR product is also designed with flanking restriction endonuclease sites identical to the plasmid: these are also digested by the same enzyme. The resulting sticky ends are complementary to those of the plasmid so the PCR product and plasmid can anneal and the nicks are ligated by a ligase or other phosphodiester bond-forming enzyme Figure 6.

The PCR product must be engineered with these restriction sites in-built. The PCR product and plasmid overlap and anneal and the nicks are closed by a ligase. So, if the gene is intended to produce a functional nucleic acid or protein product, this will be expressed by the bacteria once transformed. As a result, directionality of insertion into the plasmid is largely unimportant; however unidirectional forms of ligation do exist if required. Blunt-ended ligation involves the digestion of the plasmid with a restriction endonuclease that linearises the circular DNA by cutting both strands evenly, leaving no overhangs at either end.

The PCR product is produced by a polymerase that leaves the ends similarly blunt, and both the plasmid and the gene are combined and ligated Figure 7. Figure 7 Blunt-ended ligation The plasmid is cut at a recognition sequence by a blunt-end forming restriction endonuclease. The PCR product need not undergo any transformation since it is already blunt-ended. The PCR product and plasmid associate and the nicks are closed by a ligase. Blunt-ended ligation is less efficient than cohesive-ended ligation due to the lack of sticky-ends that serve as templates for ligation; however it eliminates the post-PCR digestion step required for cohesive-ended ligation.

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