New DNA cleavage technique could lead to more versatile genetic engineering

May 9, 2014
New DNA cleavage technique could lead to more versatile genetic engineering
Figure 1: Quantitative base-induced DNA cleavage (QBIC) is a technique that allows DNA to be cleaved at any thymine site. Credit: lvcandy/iStock/Thinkstock

Genetic engineering of plants, animals and microorganisms such as bacteria typically involves the use of restriction enzymes to 'cut and paste' DNA fragments into certain genetic sequence locations. This process allows scientists to introduce new genes into an organism, but is constrained to specific recognition sequences, limiting the design of recombinant DNA molecules.

A research team led by Hiroki Ueda and colleagues from the Laboratory for Synthetic Biology at the RIKEN Quantitative Biology Center has now developed a chemical-based, non-enzymatic recombination technique that instead uses a DNA base analogue called 5-ethynyluracil to cleave DNA at any site containing the nucleotide thymine.

The technique developed by Ueda and his co-workers, which is called quantitative base-induced DNA cleavage (QBIC), starts with the generation of DNA fragments containing 5-ethynyluracil in place of thymine—two molecules with similar structures. These products are then immersed in an aqueous solution containing methylamine, a derivative of ammonia. In this chemical bath, all the nucleotides containing 5-ethynyluracil become cleaved, introducing gaps near the cleaved ends. The gaps in the resulting DNA fragments create protruding ends that can be inserted into circular DNA molecules known as plasmids. The plasmids can then be inserted into the target organism, such as a bacterial cell, to complete the process.

"Compared with , the QBIC reaction has the advantage that we can freely design the sequences at the protruding termini generated by the DNA cleavage," says Katsuhiko Matsumoto from the research team. "The experimental procedure for DNA concatenation using the QBIC reaction is also simple," he adds. "DNA can be concatenated by the addition and removal of methylamine, hybridized by heating and cooling, and incorporated into an organism—in this case the bacterium Escherichia coli."

Another potential boon of the QBIC method is that it is less sensitive to laboratory conditions than enzyme-based techniques and can be run at room temperature. Being a chemical method, it is also generally cheaper to perform than enzyme-based methods. One limitation of the QBIC method in its present form is that long stretches of DNA can lose their structure after treatment with the methylamine solution, which prevents the two-stranded, helical shape from being restored. Ueda's team is now refining the protocol to extend its ability to handle longer DNA fragments. "If we find a solution to this problem," Matsumoto notes, "the QBIC method would become very attractive for the concatenation of long DNA fragments."

Explore further: New method for mass-producing high-quality DNA molecules

More information: Ikeda, S., Tainaka, K., Matsumoto, K., Shinohara, Y., Ode, K. L., Susaki, E. A. & Ueda, H. R. "Non-enzymatic DNA cleavage reaction induced by 5-ethynyluracil in methylamine aqueous solution and application to DNA concatenation." PLoS ONE 9, e92369 (2014). DOI: 10.1371/journal.pone.0092369

Related Stories

New method for mass-producing high-quality DNA molecules

June 2, 2013

A new method of manufacturing short, single-stranded DNA molecules can solve many of the problems associated with current production methods. The new method, which is described in the scientific periodical Nature Methods, ...

Newly found DNA catalysts cleave DNA with water molecule

August 16, 2009

Better tools for manipulating DNA in the laboratory may soon be possible with newly discovered deoxyribozymes (catalytic DNA) capable of cleaving single-stranded DNA, researchers at the University of Illinois say.

Fast new, one-step genetic engineering technology

May 22, 2013

A new, streamlined approach to genetic engineering drastically reduces the time and effort needed to insert new genes into bacteria, the workhorses of biotechnology, scientists are reporting. Published in the journal ACS ...

Recommended for you

Yeast knockouts peel back secrets of cell protein function

September 26, 2016

Proteins are the hammers and tongs of life, with fundamental roles in most of what happens in biology. But biologists still don't know what thousands of proteins do, and how their presence or absence affects the cell.

How the anthrax toxin forms a deadly 'conveyer belt'

September 26, 2016

Researchers have built a three-dimensional map of the anthrax toxin that may explain how it efficiently transfers its lethal components into the cytoplasm of infected cells. The study, "Structure of anthrax lethal toxin prepore ...

Discovery may benefit farmers worldwide

September 26, 2016

University of Guelph plant scientists have shown for the first time how an ancient crop teams up with a beneficial microbe to protect against a devastating fungal infection, a discovery that may benefit millions of subsistence ...


Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.