A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences

Abstract

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are approximately 500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5-7 days) and suitable for synthesizing long segments of DNA (5-6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.

High-fidelity PCR enzyme with DNA-binding domain facilitates de novo gene synthesis

“…A variety of approaches for DNA synthesis have been developed (Xiong et al 2004(Xiong et al , 2006Yang et al 2012) but the resulting DNA sequences obtained using any of these techniques need to be edited during the following steps. Perhaps the problem-solving approach is to use the new generation polymerases which have higher fidelity.…”

“…But even if well-designed and additionally purified oligonucleotides are used it is not recommended to generate DNA molecules larger than 500 bp by one-step. For the synthesis of longer chains there are several strategies, mainly based on the formation of long sequences from short blocks by the second step of PCR (Xiong et al 2004(Xiong et al , 2006Young and Dong 2004;Li et al 2006). Despite all well known advantages of these methods, an additional editing of obtained DNA sequences is required using OE-PCR, T7 endonuclease I treatment or by other approaches (Ma et al 2012).…”

Human Papilloma Virus Immunogen Creation on the Base of Chimeric Recombinant Protein L2e7

“…Синтез последовательностей гена l2e7 осуществляли методом ПЦР с использованием перекрываю-щихся олигонуклеотидов [42]. В общей слож-ности для синтеза гибридного гена l2e7 длиной 1550 п.н.…”

Protein fabrication automation

“…Furthermore, the use of the SOS vectors obviates the need for PAGE-purified oligonucleotides, providing a significant savings in both cost and effort. Other gene assembly methods require PAGE-purified oligonucleotides to facilitate gene synthesis (Stemmer et al 1995;Strizhov et al 1996;Hoover and Lubkowski 2002;Gao et al 2003;Carr et al 2004;Seyfang and Jin 2004;Tian et al 2004;Xiong et al 2004;Young and Dong 2004).…”