DNA sequencing is the most powerful method to reveal genetic variations at the molecular level, leading to a better understanding of our body in physiological settings, and pathological conditions. It is the beginning of the long road towards better diagnostics and personalized medicine. Even though there have been great advances in DNA sequencing technologies there are still many setbacks. These include time-consuming sample preparation, complicated algorithms for data processing, low throughput, high cost, and short read lengths. For example, the Sanger method, which is commonly used for DNA sequencing, typically requires two working days, a PCR step and a very high quantity of nucleic acid fragments to produce a detectable band by gel electrophoresis! It is safe to say that there are still some adjustments to be made. Sanger sequencing is considered to be the first generation of sequencing methods; amplification-based massively parallel sequencing is the second generation; and single-molecule sequencing is the third generation. After the development of three generations, DNA sequencing technology is now entering the era of single-molecule nanopore sequencing technology – the fourth generation.
What Is Nanopore Sequencing?
Nanopore sequencing has been around since the 1990s, when Church et al. and Deamer and Akeson separately proposed that it is possible to sequence DNA using nanopore sensors. The concept is that if each base could produce different ionic current torrents during DNA translocating through a small channel (nanopore), then it would be possible to distinguish between different nucleotides. The pores are usually in a biological membrane, or in a solid-state film, that separates two compartments that contain conductive electrolytes. Electrodes are immersed in each compartment. The resulting electric field causes the electrolyte ions in solution to move through the pore through electrophoresis, generating an ionic current signal. When the pore is blocked, due to the passage of a biomolecule, the current flow is also blocked. You can determine the physical and chemical properties of the target molecules by analyzing the amplitude and duration of the blockades. In sequencing, each nucleotide blocks the channel differently giving a different amplitude and duration of the blockade. This information is converted into DNA sequence information. Nanopore technologies are broadly divided into two categories: biological and solid-state.Applications
Medical Diagnostics
A nanopore-based diagnostic tool offers various advantages:- it detects target molecules at very low concentrations;
- it screens panels of biomarkers or genes;
- provides a fast analysis at low cost;
- and, finally, it eliminates cumbersome amplification and conversion steps, that may introduce biases and errors.