In spite of these selleck kinase inhibitor accomplishments, the time and cost of synthesizing such molecules have somewhat limited the use of DNA as a current research tool. Another significant drawback in this technology has been the significant error rate of synthetic DNA sequences [87]. The reduction and correction of errors are, thus, essential for the synthesis of long DNA molecules. The correction of these errors is, however, very time-consuming and expensive. There are several approaches to develop error-free sequences in synthesized populations of DNA. These methods may include, but are not limited to, physical separation which

may apply the use of metals to chelate partially denatured purine bases and allow elimination of errors [88] or PCR-based approaches such as hairpin PCR, which completely separates genuine Epigenetics inhibitor mutations from polymerase mis-incorporations. Hairpin PCR operates by converting a DNA sequence to a hairpin following ligation

of Selleck Rabusertib oligonucleotide caps to DNA ends. Conditions are such to allow a DNA hairpin to be efficiently PCR‐amplified so that during DNA synthesis, the polymerase copies both DNA strands in a single pass. Consequently, when a mis-incorporation occurs, it forms a mismatch following DNA amplification and is distinguished from genuine mutations that remain fully matched [89]. Sequential errors have also been removed using ‘selective destruction’ methods. Smith and Modrich employed the use of MutH, MutL and MutS mismatch repair proteins under double-strand cleavage conditions, followed by isolation of uncleaved product by size selection. This technique has allowed them to reduce the number of

mutations in PCR products and reduce errors [90]. In another instance, Young and colleagues combined dual asymmetrical PCR and overlap extension PCR, which enables any Orotidine 5′-phosphate decarboxylase DNA sequence to be synthesized error free. For PCR-based purification methods, gel electrophoresis and cloning is performed. However, the existing approaches are not well suited for error removal in long synthetic DNA sequences where virtually all members in the population contain multiple errors [91] as shown in Figure 12. Figure 12 Mismatch repair mechanism of synthetic DNA to produce error-free DNA. Representation of an inter-strand repair mechanism which involves mismatch repair, excision repair, and homologous recombination [91]. New approaches in the production of error-free DNA exploit the use of self-assembly and natural error correction proteins. Among these proteins, celery I nuclease enzyme (CEL I; Surveyor, Transgenomic, Inc., Omaha, USA) endonuclease has been very useful [92]. Hughes and colleagues [92] found CEL I to be a reasonably effective at reducing synthetic DNA errors up to six times.