New Cloning Technique By University of Bayreuth Researchers
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New Cloning Technique By University of Bayreuth Researchers

DNA – the hereditary information carrier in an organism, consists of long chains of nucleotides. To be able to study the purposes depending on the arrangement of these building blocks, DNA molecules have to be inserted into carrier atoms (plasmid-vectors) to be multiplied. The University of Bayreuth researchers have developed a highly effective, fast and inexpensive cloning method that’s flexible enough to be deployed in all areas of biology, biochemistry, and biotechnology.

Most of the molecular cloning techniques that have one thing in common – is the DNA fragments of interest are first incorporated into bigger carrier atoms, the plasmid-vectors. Bacteria are subsequently made to take these vectors bearing the DNA fragments.

Up to now, but these methods have had one significant drawback: Considering the insertion of DNA fragments into the carrier molecule doesn’t necessarily move as smoothly and perfectly as required, just a few, but by no means all, colonies come to have the vectors with the DNA fragments to be reproduced. In order to identify these “success stories, “time-consuming and costly screening has been unavoidable up to now.

The Bayreuth researchers led by Prof. Dr. Stefan Schuster have succeeded in

making this screening redundant. The vector they used is a plasmid that includes a toxic gene. DNA fragments are subsequently integrated to the plasma in such ways as to replace this gene. When it does not succeed, the plaid retains its toxic potential. And when, in turn, this plasmid is taken up by an E. coli-bacterium, its toxic effect sets in, as it interferes with the bacterium’s cell division and consequently its ability to build colonies.

This way, it may be ensured right from the start that only those E. coli-bacteria that do, in fact, contain the DNA fragments will create colonies. They won’t subsequently have to be painstakingly selected.

Dr. David Richter, a lead author of the research said, “The reason why our brand new cloning process is so efficient is that the selection of the bacteria equipped with all the cloned DNA occurs reliably and all by itself.”

In addition, the method introduced in Scientific Reports simplifies the cloning process in another aspect: The scientists also optimized an extract (SLiCE) derived from the cells of E. coli-bacteria to ensure it is particularly suitable as a “paste” to chain together several DNA fragments. Consequently, it’s currently possible to insert all sorts of mixes of DNA fragments into the plasmid — which much more quickly than with previous methods.

“ZeBRα”; an acronym deriving in the scientific conditions for two decisive factors in their job – is the name coined by the Bayreuth research team for their new cloning system. This implies: Bacteria that don’t comprise the DNA fragments to be duplicated don’t form inconvenient colonies at the background. “Redα-Exonuclease” is a component of the E. coli infusion, with which different DNA fragments could be strung together and integrated into the vector.

Building on the study results they have published, the scientists plan to equip their cloning vectors with added functions in the future to make it more useful. Specifically, they intend to further maximize the vector to be able to simplify the transformation of particular organisms or cell lines. Because this type of transfer is also a rare occasion, it’s advantageous if the vector also transports DNA sequences that result in the formation of fluorescent proteins to track the practice of transformation. These proteins can then indicate the effective uptake of these plasmid-bearing DNA fragments into the organisms or cells.

Press release by University of Bayreuth Researchers

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