The reliable and rapid native DNA cloning strategy described here is based on an asymmetric single-tube bridge PCR reaction and intramolecular homologous recombination in E.coli. Asymmetric PCR has two major advantages. The first is the introduction of the outermost oligo (primer P1R), which anneals to the end of the linear fragment and so produces large amounts of fused sequence. This manipulation results in an accurate and highly efficient gene fusion process that enables rapid cloning free of unwanted bases. This process can be used for a wide variety of DNA molecules than previously reported PCR-based gene cloning approaches [7, 14–17]. The second advantage is that the asymmetric reaction ensures that the insert is amplified in a limited amounts while DNA polymerase generates long fused products. The concentration of pR primer was found to be critical to the success of PCR output and colony formation capacity. This was also shown during the cloning of porcine MSTN sequences. This addresses the key question of how to enhance the efficiency in a PCR-based cloning method. In addition, we compared ABI-REC with a two-step PCR-based cloning protocol described previously . Although it remains unknown why we did not find any singe colonies in the two-step protocol, our work demonstrated that ABI-REC is powerful in term of cloning efficiency (Figure 2).
The double resistance reporter assay is a good way to prove the underlying principles of ABI-REC. This model enabled us to investigate the inherent properties of ABI-REC, including the effects of homology and insert length. In this study, we found 20-25 bp homologous arms to be sufficient to produce long fused sequences in PCR and to provoke efficient recombination in bacteria. These short homologous arms allows ABI-REC to be classified as an oligonucleotide-based strategy. In this way, it is cost-effective. We extended the insert length from 1.6 kb to 4 kb, allowing this process to cover a wide range of genes of interest in molecular biology. We predict that longer DNA sequences can be cloned by ABI-REC, in the assumption that the bridge PCR reaction will become optimized with either potent DNA polymerase or a more robust buffering system. This will definitely be investigated in future studies.
Although the double resistance reporter assay is an ideal proof-of-principle model, it is not very versatile in gene engineering. This is largely because, in many cases, only a single resistance marker is present. This makes it difficult to apply ABI-REC to single-marker cloning systems. In other words, it is important to remove the original circular plasmid to diminish background. DpnI is the method of choice for this process because it is capable of cleaving the methylated nucleotides present in plasmid . We studied the association between time required for DpnI digestion and the ratio of positive colonies. We found that with increasing DpnI digestion time, the positive rate shifted from 93% to 100%. This indicates that ABI-REC is nearly zero-background under single-marker conditions with suitable DpnI treatment. A previous study introduced a new logic gene engineering method based on intermolecular (linear plus circular) homologous recombination in bacteria, but this study failed to address the background of methylated circular plasmids in the single marker system . Another previous study also attempted to enhance mutation efficiency by adding limited amounts of circular plasmid in PCR reaction . ABI-REC outperforms these two methods in that removal of circular plasmid and intramolecular recombination reduce the time required for single marker cloning experiments. Given the ultra-high efficiency of ABI-REC, sequencing is required on a very limited number of clones to check for the presence of junction sites. Please note that a dam + E.coli strain like DH5α is required for successful ABI-REC in order to discriminate against methylated and hemimethylated parental plasmids.
The two major findings presented in this work will have practical implications for genetic engineering and transgenic biology. First, ABI-REC described here will be particularly favorable to gene functional studies. It has been intensively documented that widely used ligation-dependent cloning methods introduce unwanted bases into target region and that these bases can cause the inclusion of extraneous amino acids or unexpected regulatory sequences . These unwanted bases can compromise the function of protein of interest or even bring misleading results. ABI-REC takes advantage of homologous recombination in bacteria so that native sequences can be fused into pre-selected sites in target plasmid without the involvement of any heterologous nucleotides. This method is free of unwanted base and is totally independent of availability of restriction sites and thus highly desirable to DNA engineering and protein function analysis. Moreover, ABI-REC is likely to be a high-throughput method because of the compatibility of its primer design. Presumably, a series of porcine MSTN gene regulatory elements could be cloned just by changing the site of P2 sequence, without any other modifications upon the whole procedures. In addition, ABI-REC works independent of the availability of restriction sites or even the knowledge of the entire sequence of chosen DNA molecules. This makes it amenable to the construction of DNA library. For example, the pF and pR primers could be used as adaptors to link to target DNA or cDNA, and then the linked molecules could be fused into recipient plasmids. Therefore, the experimental design could be adapted to automated high-throughput applications favoring mass generation and large-scale screening of mutants. This will greatly reduce the time, difficulty and labor required to create recombinant plasmids.
Second, the porcine MSTN expression cassette is of highly importance to site-specific modification in transgenic animals. In this work, the porcine MSTN expression cassette was proven to work in a synergistic manner. This cassette could be used to express gene of interest as commonly used gene expression unit. Genes of interest could be introduced into the native porcine MSTN locus for in situ expression through gene targeting or ZFN technology . Taking into account the research conducted in MSTN knockout mice , one can conclude that MSTN locus is a safe harbor for genome modification. The present study has identified the docking site for transgene integration. This will be of significance to transgenesis in pigs. Given the high conservation of MSTN across mammals, the gene regulatory elements identified here will also be helpful to the study of other types of transgenic livestocks, such as cattle and sheep. The application of this locus in transgenic large animals is now underway.