Team:DTU-Denmark-2/Project/introduction

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The emerging and fast growing field of molecular and synthetic biology calls for simpler, faster and more efficient cloning techniques. Conventional cloning techniques include restriction digestion and ligation, which can be both time consuming and complex. So in order to optimize the cloning process to become faster and more efficient, it would be convenient to avoid the use of restriction enzymes. 



In early 1990s, the uracil excision-based (USER) cloning was invented as a ligation-independent cloning technique that could substitute the conventional cloning with restriction enzymes and ligase. This technique was further developed, and a few years later, New England Biolabs (NEB) introduced the USER Friendly Cloning Kit, which was to ensure cloning without the use of restriction enzymes. However, the kit was not compatible with proofreading polymerases, since they stalled when encountering a uracil base in the DNA template, as it is a promutanegic event (1; New England Biolabs, 2004). 

Newer proofreading polymerases have now been developed that can read through the uracils and thus are compatible with the concept of USER cloning. This has opened the door to a more simple and flexible approach to molecular cloning.

USER cloning

The method applies long complementary overhangs on the PCR product(s) as well as the destination vector. Overhangs are designed in the range of 7-15 nucleotides long and selected deoxy thymidine nucleotides are substituted with deoxy uridine nucleotides. The overhangs on the PCR product are custom made and independent of restriction sites. In the USER cloning technique the DNA is treated with USER enzyme that is a mix of DNA glycosidase and DNA glycosylase-lyase endo VIII. This treatment results in a release of the DNA sequence upstream the deoxy uridine nucleotide and the resulting exposed overhangs can anneal to each other to form a stable hybridization product. This allows for a bacterial transformation without prior ligation (1,2). In order to avoid template carry-over after PCR, the PCR product is usually treated with the restriction enzyme DpnI. DpnI cleaves only when its recognition site is methylated, this means that dam methylated plasmid DNA will be cleaved while unmethylated PCR-derived DNA will be left intact (1).

(1) Hussam H. Nour-Eldin, Fernando Geu-Flores, and Barbara A. Halkier. USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories. Methods in Molecular Biology 643.

(2) Nørholm, M. H. H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21 (2010).

Hej Julie, alt herfra ned er jeg i tvivl om jeg synes passer ind på denne side We will develop a standardized cloning system, called “Plug’n’Play with DNA”, where certain categories of biological parts can be gathered. This means that the parts in the form of pre-produced PCR-products are directly mixed with a vector, which makes assembly of an expression vector possible within a few hours. All the parts in the form of PCR-products will be distributed in microtiter plates directly ready for cloning. Furthermore, the “Plug’n’Play” kit will contain a back-up where all parts are contained on a plasmid to ensure amplification of a mutation free template if needed. The simple and easy use of the system will be demonstrated by developing a reporter targeting system for the fungus Aspergillus niger. Furthermore, will we demonstrate its application in mammalian cells.