Featured Article
Monday January 04, 2010
by Catherine Shaffer
As the technology landscape shifts, needs in the area of PCR cloning are shifting with it. Next generation sequencing platforms are displacing PCR cloning applications for sequencing, and there is a greater need for vectors able to clone large fragments. Specialized applications like microsatellite cloning are also gaining popularity.
Invitrogen, part of Life Technologies, has long been a major supplier of PCR cloning products to the life sciences market. One of their most popular products for PCR cloning is the TOPO® cloning system. The technology has gathered more than 10,000 citations in the literature. It allows the cloning of PCR fragments with blunt ends (from a proof-reading DNA polymerase) or A-overhangs (from a Taq polymerase reaction) into a vector in a five-minute bench-top reaction with up to 95% efficiency. The company also offers subcloning, sequencing, and expression vectors that are compatible with the TOPO system. But one of TOPO's greatest attractions is that it is simple and user friendly.
Ease of use is a feature that brings in many nontraditional users. Jaime Humara, PhD, a senior product development manager in Life Technologies Corporation’s molecular biology systems division explains, “There are also more people doing cloning from different fields of science that are not necessarily molecular biologists, and they love the products where the steps are simple. They cut days out of their work, and things work without having to understand the biology behind it.” And the system is reliable. “Even though the technology is a bit over 10 years old, researchers still rely on it and we hear over and over again that if you can't clone something with a TOPO-adapted vector it is because the gene is toxic.”
The Invitrogen TOPO® cloning technology harnesses the activity of DNA topoisomerase I, which in nature functions both as a restriction enzyme and as a ligase. It cleaves and rejoins DNA during Vaccinia virus replication. Topoisomerase I specifically recognizes the pentameric sequence 5´-(C/T)CCTT-3´ and forms a covalent bond with the phosphate group of the 3´ thymidine. It then cleaves one DNA strand, enabling the DNA to unwind. The enzyme then re-ligates the ends of the cleaved strand and releases itself from the DNA. To harness the re-ligating activity of topoisomerase I, TOPO® vectors are provided linearized with topoisomerase I covalently bound to each 3´ phosphate. This enables the vectors to efficiently ligate linear DNA fragments with compatible ends.
Another approach to PCR cloning comes from Delphi Genetics. StabyCloning products are designed for cloning of blunt-ended PCR products. They allow selection not only for the presence of insert, but also its orientation in the vector. Because the technology is a real selection and not screening-based, such as using LacZ, the background is close to zero because all bacteria containing a vector without an insert are killed. The StabyCloning technology is efficient for inserts as small as 1 base and as big as several kilobases, and can be done in the absence of antibiotic.
Because the StabyCloning system is efficient for cloning of small inserts compared to other methods, it is being used successfully for cloning microsatellite loci. Cloning of very small inserts using conventional enzymes results in problems like double sequences or poor inactivation of marker inserts. The Staby system clones these very small inserts reliably.
Another application is the construction of libraries. “Since small or big inserts are not favored using StabyCloning, the libraries are more representative of the DNA samples. Moreover, the number of recombinant clones is higher compared to other kits” says Cedric Y. Szpirer, PhD, cofounder and head of product development at Delphi Genetics.
Delphi is currently working on vectors for protein expression on bacteria and eukaryotes compatible with StabyCloning with easy transfer of the DNA fragment from one vector to another and StabyCloning vectors without any antibiotic resistance gene for industrial use (a current unmet need in the field).
Although products like TOPO and StabyCloning perform well for many types of experiments, the increased pace of development in biotechnology means that there is continuing demand for more efficient technologies for DNA assembly. This is because an increasing number of laboratories outside of the traditional molecular biology markets are using PCR cloning and similar techniques to analyze genes. Technology vendors are scrambling to meet these needs, as well as those of emerging markets such as synthetic biology.