DNA Printer
A new method of "DNA printing" will allow you to create a gene in one day
Yulia Vorobyova, Vesti
Create a new gene in one day or even in a few hours? Until recently, it seemed an unthinkable task, but the new technology has made the dream of many scientists a reality.
(This is a bit exaggerated. A simple calculation shows that the synthesis of a chain of thousands of nucleotides using the technique described below will take about 20 hours – VM.)
A team from the University of California and the Lawrence Berkeley National Laboratory has presented a technique that allows you to simulate the process of DNA synthesis.
The authors of the development explain that the question of creating DNA has been studied for more than 40 years. The traditional approach uses nucleotides – the building blocks of DNA, of which there are only four types – adenine, guanine, cytosine and thymine. They need to be added in a certain sequence to an oligonucleotide – a short base fragment of DNA. In this way, you can build up a full-fledged chain.
However, this method involves the use of toxic organic reagents. In addition, due to the high probability of errors, the DNA chain created in this way is limited to only 200 bases. On the scale of existing natural genetic codes, this number can be called negligible. To assemble even a small gene, researchers have to synthesize it in parts, and then "stitch" them together, it takes a lot of time and considerable costs.
Young scientists from the USA proposed a fundamentally new approach, which was called "DNA printing" for its speed and principle of operation.
"If you're a mechanical engineer, it's great to have a 3D printer that can print out a part overnight so you can check it out the next morning. If you are a researcher or bioengineer, and you have a tool that simplifies DNA synthesis, you can quickly test your ideas and explore new ones. I think this will lead to a lot of innovations," says co–author Dan Arlow.
Together with his colleague Sebastian Palluk, who was invited from Germany, he worked on methods of using a DNA synthesizing enzyme that is contained in cells of the immune system and allows adding nucleotides to an existing DNA molecule. An important condition: the process must take place in water, because it is in such an environment that DNA is most stable.
According to bioengineers, the new method improves the accuracy of work, and as a result, it is possible to create DNA strands up to several thousand bases long. This is the size of a full-fledged average gene.
"We have developed a new way of DNA synthesis using a mechanism that nature itself uses to create DNA. This approach is promising because enzymes have evolved over millions of years to achieve high "chemical precision," Palluk notes.
He explains that most cells in living organisms, as a rule, do not synthesize DNA from scratch. They copy it with the help of many different enzymes called polymerases, based on the templates laid down in the cell at its birth.
Back in the 60s, scientists discovered an unusual polymerase that does not rely on an existing DNA matrix, but randomly adds nucleotides to genes. In this way, antibodies of the immune system with millions of genetic variations are created. Thanks to this mechanism, they learn to attack a variety of pathogens.
The enzyme in question is known as terminal deoxynucleotidyltransferase (TdT). It is able to add all four types of nucleotides to the DNA chain, increases up to 200 bases per minute, and its use minimizes the number of adverse reactions.
But there is an important caveat: TdT adds new letters to the genetic code in random order. Researchers have been looking for control methods to create the desired sequences for a long time, and now a solution has been found.
Arlow and Palluk started by adding chemical groups to the nucleotides that act as "stop signals". As a result, the enzyme could attach only one desired nucleotide to the base DNA fragment. After that, the DNA strand was treated with a special compound that washed off the "stop group" and prepared for the attachment of a new base.
But even in this case, the researchers faced difficulties: it turned out that TdT is very picky. The attachment of the bases modified by "stop groups" took a very long time, so the team slightly changed the approach.
They added a blocking chemical group not to the nucleotide, but to the TdT enzyme itself. Then it was bound to nucleotides, and they were added to the base DNA fragment, the oligonucleotide. As a result, the enzyme, attaching the base to the DNA molecule, remained bound and blocked the formation of any additional copies by itself.
After the DNA molecule receives a new base, it remains only to "cut the binding cable" to release the enzyme, and unlock the end of the strand to add a new nucleotide that also contains its enzyme, the authors write.
This approach turned out to be less costly (bacterial and yeast cultures shared the TdT enzyme with scientists), as well as fast. Attaching a new nucleotide to the base molecule takes from 10 to 20 seconds, and it takes another minute to unlock the end of the chain to add a new base.
However, the authors have yet to improve the accuracy of the technology. So far, it is 98%, while classical methods of DNA synthesis are 99% accurate. According to experts, to create a "correct" DNA molecule with a thousand bases, 99.9% accuracy will be required.
If specialists manage to achieve such indicators, the new method will make a real revolution in synthetic biology, as well as give the opportunity to pack giant arrays of data into compact DNA archives.
In addition, the rearrangement of the genes of microorganisms will allow the creation of new medicines, as well as fuel.
More details about this work are described in an article published in the journal Nature Biotechnology (Palluk et al., De novo DNA synthesis using polymerase-nucleotide conjugates).
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