Technology is capable of amazing things, but it doesn’t mean those things are easy. It’s incredible that scientists can produce DNA in a lab, but the process is difficult, lengthy and requires toxic chemicals. Imagine, however, if they could simply print it, the way that you would 3D print anything else. That could be the future, after scientists at UC Berkeley and Lawrence Berkeley National Laboratory developed a new way to synthesize DNA. The method could lead to DNA printers, similar to ordinary 3D printers, that could produce DNA strands that are more accurate and 10 times longer than the strands produced with today’s methods – more quickly and easily, and without the use of toxic chemicals.
“If you’re a mechanical engineer, it’s really nice to have a 3D printer in your shop that can print out a part overnight so you can test it the next morning,” said UC Berkeley graduate student Dan Arlow. “If you’re a researcher or bioengineer and you have an instrument that streamlines DNA synthesis, a ‘DNA printer,’ you can test your ideas faster and try out more new ideas. I think it will lead to a lot of innovation.”
The research was led by Arlow and PhD student Sebastian Palluk, a doctoral student at the Technische Universität Darmstadt in Germany and a visiting student at Berkeley Lab. It is published in a paper entitled “De novo DNA synthesis using polymerase-nucleotide conjugates,” which you can access here. The research was conducted at the Department of Energy’s Joint BioEnergy Institute (JBEI).
“I personally think Dan and Sebastian’s new method could revolutionize how we make DNA,” said Jay Keasling, a UC Berkeley professor of chemical and biomolecular engineering, senior faculty scientist at Berkeley Lab and Chief Executive Officer of JBEI.
Keasling and JBEI scientists specialize specialize in adding genes to microbes, typically yeast and bacteria, to sustainably produce useful products. Palluk came from Germany specifically to work with Arlow in Keasling’s lab.
“We believe that increased access to DNA constructs will speed up the development of new cures for diseases and simplify the production of new medicines,” Palluk said.
The synthesis of DNA is a growing business; companies are ordering custom-made genes so that they can produce chemicals, biologic drugs or industrial enzymes. Researchers purchase synthetic genes to engineer plants and animals or try out new CRISPR-based disease therapies. Some scientists have even researched storing information in DNA, but that would require much larger quantities of DNA than are currently synthesized. All of these applications require that synthesis produce the desired sequence of nucleotides or bases, the building blocks of DNA, in each of millions or billions of copies of DNA molecules.
Current DNA synthesis is limited to producing oligonucleotides about 200 bases long, because errors in the process lead to a low yield of correct sequences as the length increases. To assemble even a small gene, scientists have to stitch together segments of about 200 bases long. The turnaround time for a small gene of 1,500 bases long can be two weeks at a cost of $300, limiting the experiments that scientists can do. Synthetic biologists like Arlow, Palluk and Keasling often insert a dozen different genes at once into a microbe to get it to produce a chemical, and each gene presents its own synthesis problems.
“As a student in Germany, I was part of an international synthetic biology competition, iGEM, where we tried to get E. coli bacteria to degrade plastic waste,” said Palluk. “But I soon realized that most of the research time was spent just getting all the DNA together, not doing the experiments to see if the engineered cells could break down the plastic. This really motivated me to look into the DNA synthesis process.”
The technology developed by Palluk, Arlow, Keasling and their team relies on a DNA-synthesizing enzyme found in cells of the immune system that has the natural ability to add nucleotides to an existing DNA molecule in water, where DNA is most stable. The technology results in increased precision, allowing synthesis of DNA strands several thousand bases long – a medium-sized gene.
“We have come up with a novel way to synthesize DNA that harnesses the machinery that nature itself uses to make DNA,” Palluk said. “This approach is promising because enzymes have evolved for millions of years to perform this exact chemistry.”
Cells create DNA by copying it with the help of several different polymerase enzymes that build on DNA already in the cell. But in the 1960s, scientists discovered a polymerase that doesn’t rely on an existing DNA template but instead randomly adds nucleotides to genes that make antibodies for the immune system. The enzyme, called terminal deoxynucleotidyl transferase (TdT), creates random variation in these genes, resulting in antibody proteins that are better able to attack new types of invaders.
TdT is fast and does not have side-reactions that could affect the resulting molecule. Scientists over the years have tried to use the enzyme to synthesize DNA sequences, but it was hard to control. The key is to find a way to get the enzyme to add one nucleotide and then stop, so that the sequence can be synthesized one base at a time. Previous approaches tried to obtain that control by using modified nucleotides with a special blocking group that prevents multiple additions at once. After the DNA molecules have been extended by a blocked nucleotide, the blocking groups are removed to allow the next addition.
TdT, however, cannot accommodate a blocking group on the nucleotide being added. But Arlow came up with the idea to tether an unblocked nucleotide to TdT, so that after the nucleotide is added, the enzyme remains attached and prevents further additions. After the molecule has been extended, the tether is cut, releasing the enzyme and re-exposing the end for the next addition.
In the first trials, the researchers demonstrated that this technique is not only faster and simpler, but nearly as accurate as other techniques in each step of the synthesis.
“When we analyzed the products using NGS, we were able to determine that about 80 percent of the molecules had the desired 10-base sequence,” Arlow said. “That means, on average, the yield of each step was around 98 percent, which is not too bad for a first go at this 50-plus-year-old problem. We want to get to 99.9 percent in order to make gene-length DNA.”
Once they can reach 99.9 percent fidelity, they can synthesize a 1,000-base-long molecule with a yield of more than 35 percent, which is currently impossible with existing techniques.
“By directly synthesizing longer DNA molecules, the need to stitch oligonucleotides together and the limitations arising from this tedious process could be reduced,” said Palluk. “Our dream is to directly synthesize gene-length sequences and get them to researchers within few days.”
“Our hope is that the technology will make it easier for bioengineers to more quickly figure out how to biomanufacture useful products, which could lead to more sustainable processes for producing the things that we all depend on in the world, including clothing, fuel and food, in a way that requires less petroleum,” said Arlow.
He added, “Our dream is to make a gene overnight. For companies trying to sustainably biomanufacture useful products, new pharmaceuticals, or tools for more environmentally friendly agriculture, and for JBEI and DOE, where we’re trying to produce fuels and chemicals from biomass, DNA synthesis is a key step. If you speed that up, it could drastically accelerate the whole process of discovery.”
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[Source: UC Berkeley / Images: Marilyn Chung, Berkeley Lab]