Several years ago, Alexander Ulrich was frustrated while purifying proteins and cloning plasmids as a graduate student in the lab of Thomas Schwartz at the Massachusetts Institute of Technology (MIT). The main cloning method he was using, known as restriction-free cloning, was generating relatively low product yields due to linear PCR amplification (1,2). "I was really unhappy with the efficiency of the method, so I came up with an idea to make this method more efficient," said Ulrich, who is now studying structural biochemistry at the Free University of Berlin.
The new technique involves two consecutive PCR steps that amplify the template exponentially rather than linearly. In the first step, the insert is exponentially amplified with a forward primer and a reverse primer containing a 5’ overhang that is complementary to a portion of the target vector; in the second step, the amplified insert is used as a megaprimer with a forward and reverse primer to exponentially amplify the target vector, thereby seamlessly integrating the insert into the plasmid. To further improve the time efficiency of the method, the two PCR steps can be combined into one reaction, which is not possible with restriction-free cloning. The downside, however, is a greater chance for abnormal amplifications and increased difficulty in troubleshooting.
Compared with restriction-free cloning, EMP cloning is more efficient, especially for inserts longer than 2.5 kb: it generates higher PCR product amounts, higher colony numbers, and a higher ratio of positive clones over background. And compared with recombination-based cloning techniques, such as sequence- and ligation-independent cloning (SLIC), which do not allow researchers to monitor cloning success before obtaining colonies, EMP cloning would allow them to monitor the success of vector-insert fusion by gel electrophoresis prior to transformation so that they could fix problems at early stages and save time as a result.
"The general problem with regular cloning or classical cloning with restriction enzymes is that you get unwanted amino acids," said Ulrich.
EMP cloning enables seamless insertion of DNA fragments, and it does not leave behind any scars. Seamless cloning allows DNA fragments to be joined precisely so that no unwanted nucleotides are added at the junctions between the fragments. In addition, EMP cloning is very efficient and offers great flexibility in the choice of insert and vector. As a result, the study authors write that it is an ideal tool for any application that requires the generation of plasmid libraries, including expression libraries for structural biology.
"For structural biology, it's very important to get constructs that don't contain any unwanted amino acids, because if you want to determine the structure of the protein and get crystals of the protein, unwanted amino acids could prohibit crystal formation,” said Ulrich. “So, having the chance to control the final product is important."
Win Some, Lose Some
Traditionally, restriction enzyme-mediated ligation has been used to insert DNA fragments into target plasmids, but this type of approach suffers from either low efficiency or a limited availability of suitable restriction sites. Likewise some restriction-free cloning techniques also have limitations, including the dependence on specific sequence elements and the requirement of specific vectors and expensive enzymes. In contrast, homologous recombination techniques do not require specific sequence elements, but cloning success cannot be monitored before obtaining colonies.
While EMP cloning overcomes some problems with past methods, Quinn Lu, who manages a GlaxoSmithKline core facility that generates DNA constructs for research purposes, would have further simplified the protocol by eliminating some of the enzymatic steps following PCR amplification, which together take about two hours. "The reason we have not explored a procedure like EMP is that restriction-free cloning is simpler, and the efficiency is good enough for us," said Lu.
Dominic Esposito, who directs the Protein Expression Laboratory at the Advanced Technology Program (Frederick National Laboratory for Cancer Research and SAIC-Frederick, Inc.)—a core facility that provides protein expression services to the National Cancer Institute and other components of the National Institutes of Health, agrees. "I think this is a custom technique that people might use in a particular situation, but overall this process is pretty laborious in terms of the amount of design that you have to do going into it," he said.
"They sort of play up the quick speed of it, but there's a lot of design and downstream work," said Esposito. "So, whether or not this becomes a popular technique, I'm not sure it's going to be quite high throughput enough."
Before PCR was invented, seamless fusion of DNA fragments was achieved through complicated procedures (4). After the advent of PCR, several innovative methods for seamless gene fusion were developed. The introduction of overlap PCR allowed for any two fragments to be freely joined at any predetermined sequence location, independent of any restriction sites, provided that the fragments can be faithfully amplified. Using this principle, multiple DNA fragments can be spliced together seamlessly, and point mutations, insertions, deletions, and replacements can be introduced into any point of a gene in a seamless fashion. PCR-generated fusion genes can subsequently be cloned into appropriate vectors.
There are several other methods capable of achieving seamless gene fusion (4). For one, site-specific mutagenesis generates point mutations, deletions, insertions, and sequence replacements on plasmid backbones. Meanwhile, type IIS restriction enzymes have been used to generate cohesive ends from PCR fragments for the seamless assembly of genes and viral genomes, and they also allow for a DNA fragment to be fused seamlessly with existing DNA fragments contained in a vector. On the other hand, ligation-independent cloning (LIC) methods do not depend on restriction enzyme digestion and ligation and are amenable to high-throughput experiments. Another approach called homologous recombination allows for the exchange of genetic material between two molecules with homologous sequence regions. Because the location for homologous recombination can be freely chosen, it enables seamless DNA manipulation.
"The advantage of a lot of the seamless cloning technologies, such as LIC and SLIC and some of the restriction-free methods, is that they're highly amenable to high throughput and they're relatively inexpensive," said Esposito.
But in the end, all of these methods rely on the use of PCR to generate the correct ends on the insert or the vector fragments to enable seamless gene fusion or cloning. Even though high-fidelity DNA polymerases have improved the PCR process, the real challenge for improving seamless cloning and other cloning techniques moving forward is still to enhance the robustness of the PCR process. "We have PCR enzymes that have high fidelity and don't make a lot of mistakes, but there is a chance of making mistakes," said Lu. "But with improvements in that technology, that worry goes away gradually."
Esposito agrees that improving PCR is the main challenge lying ahead. "One of the drawbacks to exponential megapriming PCR is that there is a very large amount of PCR going on, and even with high-fidelity polymerases, I'm concerned about how accurate the results are going to be," he said. "The biggest issue is getting high-quality PCR amplification of whatever you're trying to get. And I think that still remains a big black box for a lot of people."
1. Geiser, M., R. Cèbe, D. Drewello, and R. Schmitz. 2001. Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase. BioTechniques 31(1):88-90, 92.
2. Bryksin, A.V., and I. Matsumura. 2010. Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. BioTechniques 48(6):463-5. doi: 10.2144/000113418.
3. Ulrich, A., K.R. Andersen, and T.U. Schwartz. 2012. Exponential Megapriming PCR (EMP) cloning—Seamless DNA insertion into any target plasmid without sequence constraints. PLoS ONE 7(12): e53360. doi:10.1371/journal.pone.0053360.
4. Lu, Q. 2005. Seamless cloning and gene fusion. Trends Biotechnol 23(4):199-207.