DNA is the genetic material of almost all living organisms. Chemically, DNA is a chain of 4 nucleotides and the sequence of their arrangement on a phospho-sugar backbone serves as instructions for the synthesis of proteins. The sequencing of the human genome made it clear that there are around 26000 genes per cell (1, 2). It is essential to understand the functions of each of these genes in order to comprehensively understand cellular physiology. One way researchers try to do so is to artificially synthesize the DNA sequence of a particular gene of interest and study its functions in lab-grown cells in dishes. The average size of a human gene is around 3000 bp (base pairs) long (3), meaning that it has a sequence of 3000 nucleotides as described above. However, the current DNA synthesizing technologies allow only around 200-300 bp of high-fidelity synthesis (4).
Researchers from the University of California and Lawrence Berkeley National Laboratory, USA, have developed a new method to synthesize DNA with high fidelity (5). This new technique relies on a DNA-synthesizing enzyme naturally found in cells of the immune system. It is called terminal deoxynucleotidyl transferase (TdT) and has the ability to add nucleotides to an existing DNA molecule in water, where DNA is most stable. However, unlike regular DNA polymerases it doesn’t require a template DNA molecule. Instead, it randomly adds nucleotides to the end of a growing DNA molecule. Cells use this enzyme to create random variation in genes that make antibodies so that the resulting antibody proteins are better able to target never-before-seen invaders.
The problem with using TdT enzyme to synthesize DNA is that it cannot be stopped from adding the next nucleotide. To get around this problem, the researchers tethered each nucleotide to a molecule of the enzyme (5). So, when the enzyme adds the nucleotide to the chain of DNA, it stays attached to the enzyme which doesn’t allow the addition of the next nucleotide. To continue the reaction, the tether is released and the next nucleotide can be added.
In their first trials – 10 cycles using the engineered TdT enzyme to create a 10-base oligonucleotide – the Berkeley researchers showed that their faster and simpler technique is nearly as accurate in each step of the synthesis as current techniques. They say that the technique promises improved precision, which could allow synthesis of DNA strands 10 times longer, or several thousand bases long – the size of a medium-sized gene.
By directly synthesizing longer DNA molecules, the need to stitch oligonucleotides together and the limitations arising from this tedious process could be reduced. The researchers are working towards developing this technology 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,” one of the researcher said.