Scientists have developed a nanopore-based sequencing by synthesis (Nano-SBS) technology that they claim is far more accurate than existing nanopore-based methods at reading each of the four different bases in a DNA molecule. The key to the new technique is the use of PEG-labeled nucleotides in the strand synthesis process. Effectively, the technology identifies the sequence of bases in a growing strand of DNA not by detecting the nucleotides themselves as they pass through a nanopore, but by measuring the current changes caused by the passage of each of four different PEG-based tags, explain the technique’s developers at Columbia University.
The basic concept of nanopore-based sequencing is built around the fact that, when a nanopore is immersed in a conducting fluid and a voltage is applied across it, an electric current is generated by the flow of ions through the pore. When a molecule such as a nucleotide passes through the pore, it causes a momentary and unique change in the current, and it’s this disturbance that is detected. Unfortunately, the chemical similarity of the four bases in a strand of DNA means that the current disturbances caused by each as they pass through the pore is also very similar, and existing nanopore-based sequencing methods don’t demonstrate 100% base-call accuracy: not every nucleotide will be identified correctly as carrying an A, C, T, or G base.
To try and get around this problem the Columbia team, working with colleagues at the National Institute of Standards and Technology (NIST), developed an approach that effectively uses large, more easily differentiated PEG molecules as surrogates for the four different bases. This is possible because the 5′-terminal phosphate position of a nucleotide can be modified using such tags without any effect on subsequent recognition by the polymerase that orchestrates the addition of units to a lengthening DNA strand.
The researchers exploited this to generate four nucleotides that each had a different, base-specific PEG tag attached. The nucleotide analogues were then used in the construction of a DNA strand along a template by a polymerase that was immobilized right next to the nanopore entrance. As each nucleotide analog was added sequentially to the growing strand, its PEG-based tag was released and funnelled into the nanopore for detection. Because each of the four different PEG molecules causes a far more distinct current disturbance than the four nucleotides that they represent, the effective base-call accuracy was much improved. “This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single-base resolution,” claim Jingyue Ju, Ph.D., et al.
Usefully, the fact that the tags are large molecules with slow diffusion rates greatly increases their chance of entering the nanopore and producing an ionic current blockade signal, the investigators add. And because the polymerase extension and tag release rate is much slower than the tag interaction time with the pore, there isn’t any need to slow down the transit speed of the tag through the pore, effectively eliminating phasing issues that are inherent to strand sequencing methods.
The team tested their technology using nucleotides carrying four different length PEG-coumarin tags and confirmed both that the nucleotide analogues were efficiently incorporated into the DNA strand during the polymerase reaction, and that the nanopore was able to accurately discriminate between the tags. For their experiments the Columbia investigators used an alpha-hemolysin protein nanopore, but they claim the basic approach could also be applied to other protein or solid-state nanopores. The coumarin label they used for their proof-of-concept studies could also be replaced with other molecules of larger size or different charge to enhance nanopore discrimination even further.
They claim that constructing a large array of nanopores will allow the technology to be used for high-throughput DNA sequencing. “In its full implementation in the future, it should be capable of long, accurate reads, and potentially offer very high-throughput electronic single-molecule DNA sequencing.”
The team’s technology is described in Scientific Reports, in a paper titled “PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis.”