By Ben Butkus
Japanese biologists have developed a method that allows the fusion of two DNA sequences from separate cloning vectors using a single PCR reaction, according to a recently published research paper.
The method, called restriction enzyme-assisted mega-primer PCR, or REM-PCR, is similar to an existing method called overlap extension PCR, but is less time-consuming and less prone to unwanted errors, according to the scientists.
The biologists, Nobuhisa Umeki and Taro Uyeda of Japan's National Institute of Advanced Industrial Science and Technology, came up with the technique as part of their research on the cellular slime mold Dictyostelium discoideum.
Specifically, the scientists wanted to express a heterologous protein, mammalian actin, in D. discoideum host cells, and as such, needed to fuse a constitutive promoter sequence in the host cell to the mammalian actin gene, Uyeda told PCR Insider in an e-mail.
Generally, fusing two DNA sequences from different cloning vectors is common in research laboratories, and most commercially available vectors, many of which carry sequences for purification tags or fluorescent proteins, have convenient multi-cloning sites, Uyeda said.
"In those cases, people just add appropriate restriction sites in the primers for PCR," and the process would involve PCR, subcloning of the product into a cloning vector, sequence verification, and subcloning of the gene fragment with appropriate restriction sites into the expression vector, Uyeda said.
However, laboratories often want to fuse two custom DNA sequences, as Umeki and Uyeda did. In these cases, one existing method was to engineer common restriction sites and fuse the two sequences in a standard cloning procedure using restriction enzymes and DNA ligase, followed by two independent PCR reactions.
Meantime, another more rapid method, overlapping PCR, involved two independent PCR reactions followed by a fusion PCR and subcloning of the product into a cloning vector.
Overlapping PCR is the preferred technique, Uyeda said, but still has drawbacks in that the two rounds of PCR make it prone to errors; it requires subcloning of the product into a cloning vector, which takes two to three days; and it requires four primers.
The REM-PCR method, described in a paper published online last week in Analytical Biochemistry, uses only one round of PCR, has the fusion product already in a cloning vector, and only requires two primers as opposed to four, Uyeda noted.
In REM-PCR, the two DNA sequences to be fused are subcloned into cloning vectors in the same orientation to a common sequence; and fusion of the sequences is achieved by mutual priming at the common sequence between the pair of DNA fragments generated by a restriction enzyme and linearly amplified by repetitive priming in the reaction mixture.
Uyeda said that the single PCR run "reduces the time and, more importantly, the risk of unwanted errors [and] represents a substantial advantage over the existing method."
The researchers noted that one of the limitations of REM-PCR is that it requires the two DNA sequences to be fused in the same orientation in the same type of plasmid vector. However, this is not as significant an issue as it might initially seem, Uyeda said.
"[This] does not practically limit the usefulness of this method, because the ColE1sequence is present in virtually all cloning vectors, expression vectors, and shuttle vectors to be amplified in E. coli," Uyeda said. "The ampicillin resistance gene is also shared by many plasmids, further extending the applicability of this method."
He conceded that the requirement that the DNA sequences to be fused must be in the same orientation relative to the common sequence "may be a practical limitation under certain situations;" however, "in many cases when PCR products are subcloned into cloning vectors … both directions are produced with the same probability, allowing for a choice of appropriately directed plasmid. In the case of expression vectors, the DNA sequences should be in the same orientation relative to the [untranslated] sequences provided by the plasmid."
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