Based on the data presented in this paper, we believe that the PTDS method offers a simple, rapid, high-fidelity and low-cost alternative method of synthesizing and assembling long DNA fragments. To perform the PTDS method successfully, we recommend 60mer oligonucleotides with 20 nt sequence overlap for each and a Tm of ~56°C for the first step of the PTDS method. We suggest that 12 oligonucleotides be grouped to synthesize a DNA block that is ~500 bp in length. For the second PCR reaction of the PTDS method, the synthesized DNA blocks are mixed and assembled to produce the template DNA to synthesize a full-length gene using the two outermost oligonucleotides as primers. The concentrations recommended are 1.5 pmol for the inner oligonucleotides and 30 pmol for the outer oligonucleotides for the first PCR reaction. The concentrations recommended for the two outermost oligonucleotides for assembling and amplifying a full-length gene (the second step of the PTDS method) are 30 pmol. For the first step of the PTDS method, PCR can be conducted for 25 cycles with 2.5 U Pfu Taq polymerase (each cycle consisting of 30 s at 90°C, 45 s at 60°C, 50 s at 72°C). For the second PCR reaction, we recommend one cycle at 94°C for 5 min and (2.5 U pyrobest Taq polymerase should be added in this step). We recommend 25 cycles for the PCR reaction, each cycle at 94°C for 30 s, 56°C for 35 s, 72°C for 2–6 min (variable according to the length of the gene to be synthesized).
Our experiments have demonstrated that the PTDS procedures are simple and easy to perform to obtain DNA blocks that are ~500 bp and to assemble the resulting DNA blocks to produce a full-length gene using PCR with the two outermost oligonucleotides. We have demonstrated that the PTDS method is highly suitable for synthesizing DNA fragments that are 0.3–5.0 kb in length. It may also work well for longer DNA fragments, but that possibility needs to be investigated.
We used 25 PCR amplification cycles in the PTDS method, whereas the number of amplification cycles in traditional overlap extension PCR was over 50 using Stemmer's method (17
). In the successive PCR and TBIO methods, the number of amplification cycles was more than 25. The reduced number of PCR amplification cycles for the PTDS method should reduce the error rate, thereby producing DNA with higher fidelity. If errors are revealed through DNA sequencing analysis, we can correct them using the overlap extension method (17
). For both the successive PCR method and the TBIO method, we can use an oligonucleotide that is one of the oligonucleotides already used, but we need to chemically synthesize another, new oligonucleotide. For the PTDS method, both the sense- and antisense oligonucleotides to be used for correction have already been synthesized, which reduces the cost of, and time for, synthesis of new oligonucleotides considerably. The cost-effectiveness of the PTDS method is more obvious when long DNA fragments are synthesized.
DNA polymerase exhibits the lowest error rate among all thermostable DNA polymerases (24
), the enzyme has been the choice for the DNA synthesis and assembly in many of the methods previously described. However, while we have found Pfu
DNA polymerase to be excellent for amplification of DNA fragments that are <1.0 kb, it becomes difficult to synthesize DNA fragments that are >2.0 kb in length. Another high-fidelity DNA polymerase, pyrobest
, is excellent for synthesizing DNA fragments that are >2.0 kb. Thus, for the PTDS method we recommend the Pfu
enzyme for synthesizing 500 or 800 bp DNA blocks in the first step of the method because it has the lowest error rate, while the pyrobest
enzyme is recommended for assembling full-length genes that are >2.0 kb in length.
There are some major differences between the PTDS method and the Smith method (21
). First, the Smith method involves at least four major steps: (i) phosphorylation of oligonucleotides; (ii) Taq
ligase-mediated ligation of the oligonucleotides; (iii) polymerase cycline assembly of ligation products into a full-length gene (35 to 70 cycles) and (iv) PCR amplification of the assembled full-length gene. For the PTDS method, only two major steps are needed: (i) PCR-mediated synthesis of ~500 bp DNA fragments and (ii) PCR-mediated synthesis of the full-length gene. Because fewer steps are involved and because only two PCR enzymes are used for the entire procedure, the PTDS method is much simpler and easier to perform, more rapid (5–7 days for the PTDS method but 14 days for the Smith method) and also less expensive than the Smith method. Second, because the Smith method uses 42mer oligonucleotides while the PTDS method uses 60mer oligonucleotides, the cost associated with the PTDS method can be further reduced. Third, the Smith method uses functional screening to select the clones and in so doing will underestimate the error rates. Also, although functional screening is ideal for selecting clones with the correct DNA sequence, in many cases functional screening is impossible to perform. For instance, if one wants to synthesize a flowering gene that will be expressed in plants, functional screening to select genes with the correct DNA sequence at the gene assembly and synthesis stages is not practical. Thus, the application of the Smith method would be limited to the synthesis of genes whose functions can be easily assayed if the error rate based on non-discriminable clones for the Smith method were high (i.e. higher than for the previously published methods). On the other hand, the low error rate for the PTDS method is based on non-discriminable clones, and the method should therefore be applicable to the synthesis of any gene or DNA fragment regardless of whether its function can be easily assayed or not.
In general, chemical-synthesis-mediated gene assembly should eliminate/reduce secondary structures, contrary repeats and repetitive sequences, and adjust G + C composition. However, in some cases (26
), such as DNA shuffling, these structures, repeats or high G + C composition should be preserved. When using some of the previously published gene synthesis methods, such as TBIO, successive PCR and TDL, it is difficult to synthesize long genes with these types of structures or sequences directly in a single step. On the other hand, based on our experience, the PTDS method, which synthesizes shorter DNA fragments (~500 bp in length) as a first step and then assembles these DNA blocks, can make it easier to synthesize and assemble long genes with complex structures.