Release date: 2018-06-21
Since the 1970s, researchers have begun research on synthetic DNA. Synthetic DNA is important for the development of pharmaceuticals, fuels and other chemical products. With the demand for custom genes in commercial companies engaged in the production of biomedicine, industrial enzymes, or useful chemicals, the topic of DNA synthesis has become more and more interesting. In addition to commercial applications, researchers also need to purchase synthetic genes and implant them into animals or plants, or to experiment with new disease-based treatments based on CRISPR.
The traditional method of DNA synthesis is to add the nucleotides of the DNA (chemical letters A, G, C, T) one by one to form a chain called an oligonucleotide. However, this method is limited to the direct production of oligonucleotides of about 200 bases in length, because as the length increases, inevitable errors occur in the synthesis process, resulting in a low yield of the correct sequence; This process is costly and very slow and uses a range of toxic organic reagents. If scientists want to synthesize a small gene (usually composed of thousands of nucleotides) in this way, they must be synthesized piece by piece, each length is about 200 bases, and then they are stitched together. together. This is very time consuming and has a high failure rate.
Recently, scientists at the Berkeley, California, and Lawrence Berkeley National Laboratory have invented a new method for synthesizing DNA that can be simpler, faster, and potentially more accurate for DNA synthesis without the use of toxic chemicals. The high accuracy of this technology allows it to produce 10 times longer than the DNA strands produced by existing methods. This research on DNA synthesis was done by PhD student Sebastian Palluk and graduate student Daniel Arlow. In the June 18 issue of Nature-Biotechnology, they detailed the details of this new technology.
The new technology relies on a DNA synthetase found in the cells of the immune system, which naturally has the ability to add nucleotides to DNA molecules in water (DNA is most stable in water). This technology is expected to increase the accuracy, so that the synthetic DNA strand contains thousands of bases, and the length is increased by 10 times to the size of a medium-sized gene.
The way cells synthesize DNA is completely different from the prior art. They have a class of enzymes called polymerases that read single-stranded DNA and then synthesize a complementary strand that binds to it. This feature has led many scientists to dream of writing new DNA by redesigning the polymerase. In the 1960s, scientists discovered an unusual polymerase that could attach new nucleotides to an oligonucleotide chain without following the existing DNA template strand. It is called TdT (terminal deoxynucleotidyl transferase).
Natural TdT does this in order to write new variants of millions of antibody genes that the immune system can choose to attack invaders. It has a very high rate of synthesis and, under natural conditions, it can extend DNA by about 200 bases per minute. But the natural enzymes add new bases randomly, rather than the precisely controlled base sequences that researchers want.
Over the years, many laboratories have tried to use this enzyme to synthesize the desired DNA sequence, but this enzyme is difficult to control. The key question is how to stop the enzyme after adding a nucleotide, and then repeat the process with different nucleotides. In all previous proposals, scientists have attempted to add chemical groups to the four bases of DNA as "termination" signals. Therefore, when TdT adds a modified base to an oligonucleotide of any length, it can hinder the addition of another base. They then cleaned the oligonucleotide at this time and then treated it with another compound for the next extension.
However, TdT does not work well with these modified nucleotides. It is very "critical", for example, in such a system, it takes about an hour to add a modified base, which is very time consuming and difficult to use on a large scale.
In the new approach, two young students solved the problem in a novel way. They start with four separate base pools, each of which has a TdT that is "plugged" to either A, or G, or C, or T. In order for the oligonucleotides to grow, they add a base to one of the pools. When TdT adds a base to the end of the oligonucleotide, it still maintains a tethered state, which effectively prevents any additional enzyme replicas from reacting with the oligonucleotide and leading to further extension. At this time, the oligonucleotide is removed, the tether is cut, the free TdT is washed away, and the oligonucleotide is prepared for the addition of the next base.
This method is not costly because TdT is easily produced in bacteria and yeast. At the same time, it is also very fast. According to the study, most new nucleotides will attach to growing oligonucleotides within 10 to 20 seconds. However, the step of cutting the tether still takes about a minute. Therefore, it may take a long time to synthesize a complete gene.
In the first attempt, they produced 10 base oligonucleotides using engineered TdT enzymes in 10 cycles. The emergence of new methods does not mean that the traditional way of DNA synthesis will be completely abolished. When they analyzed the "product", it was found that about 80% of the DNA molecules had the expected 10 base sequences. This means that the average accuracy of each step is 98%, which is lower than 99% of the traditional method. But this is already a very good result for a problem that has been going on for more than 50 years.
To write oligonucleotides of up to 1000 bases in this way, a single accuracy of 99.9% is required. And this is the goal of the researchers. If this accuracy is achieved, it will not only completely change the writing and testing of new genes in synthetic biology, but also write a large number of databases in DNA.
By directly synthesizing long-chain DNA molecules, the necessity of splicing together oligonucleotides and the cumbersome processes that result therefrom can be greatly reduced. In this case, it is no longer unreachable to directly synthesize the sequence of gene lengths that researchers need in a few days.
Reference source:
Http://news.berkeley.edu/2018/06/18/new-dna-synthesis-technique-promises-rapid-high-fidelity-dna-printing/
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